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PCR-MATE EP MODEL 391 DNA SYNTHESIZER User’s Manual © Copyright 2002, Applied Biosystems. All rights reserved. PCR-MATE, OPC, Aminolink 2, and Syncom are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries. The Polymerase Chain Reaction (PCR) process is covered by Cetus Corporation’s U.S. Patent Number 4,683,202. No license to use the PCR process is given or implied by Applied Biosystems Cetus’ licensing of PCR-MATE. Teflon is a trademark of the E.I. DuPont de Neumors Company. Parafilm is a trademark of the American Can Company. Drierite is a trademark of the W.A. Hammond Drierite Company. Swagelock is a trademark of the Swagelock Company. HOW TO START Begin by reading Section 1, Introduction. It contains vital information and will help you understand how the manual is organized and how to use it. Next, refer to Section 2, Operation, for instructions on how to prepare and start a synthesis. Contents Section 1: Introduction How To Use This Manual .....................................................................................................1-2 General Introduction .............................................................................................................1-3 How to Get Help ...................................................................................................................1-5 Contacting Technical Support .........................................................................................1-5 To Contact Technical Support by E-Mail.........................................................................1-5 Hours for Telephone Technical Support..........................................................................1-5 To Contact Technical Support by Telephone or Fax.......................................................1-6 To Reach Technical Support Through the Internet .........................................................1-8 To Obtain Documents on Demand..................................................................................1-9 User Attentions ...................................................................................................................1-10 Material Safety Data Sheets (MSDS) .................................................................................1-10 Text Conventions ...............................................................................................................1-11 Section 2: Operation Power On .............................................................................................................................2-2 Pre-synthesis Check List ......................................................................................................2-3 How to Prepare Phosphoramidites.......................................................................................2-4 How to Dissolve Phosphoramidites.................................................................................2-5 How to Store Dissolved Phosphoramidites .....................................................................2-6 How to Install Reagent Bottles .............................................................................................2-7 How to Install the Phosphoramidites (bottles 1-5)...........................................................2-7 How to Install Bottles 9-15...............................................................................................2-8 How to Install Bottle 18....................................................................................................2-8 How to Begin a Synthesis ....................................................................................................2-9 How to Install the Column .............................................................................................2-12 Post Synthesis....................................................................................................................2-13 Manual Deprotection and Cleavage ...................................................................................2-13 Introduction....................................................................................................................2-13 Materials and Methods ..................................................................................................2-14 Methods.........................................................................................................................2-16 How to Abort a Synthesis ...................................................................................................2-18 About the Synthesis Reagents and Solvents .....................................................................2-19 i Applied Biosystems How to Store Reagents .................................................................................................2-19 Manual Deprotection and Cleavage Reagents..............................................................2-22 About Deoxyinosine ......................................................................................................2-23 How to Change an Argon Tank ..........................................................................................2-25 How to Change the Waste Bottle .......................................................................................2-25 How to Perform the Flow Test Procedure ..........................................................................2-26 Aminolink 2™ Reagent.......................................................................................................2-30 Introduction....................................................................................................................2-30 Chemical Description ....................................................................................................2-30 How to Use Aminolink 2 .....................................................................................................2-32 Analysis and Purification of Aminolink™-Oligonucleotides ................................................2-32 3'-End-Labeling .............................................................................................................2-32 Applications ........................................................................................................................2-34 General Protocol for Tag/Labeling Substrate ................................................................2-34 How to Use OPC (Oligonucleotide Purification Cartridge) .................................................2-35 Introduction....................................................................................................................2-35 Protocol for Lysine Pre-treatment of Longmers (>70 bases) ........................................2-37 OPC Purification Protocol...................................................................................................2-38 Solutions Needed ..........................................................................................................2-38 References .........................................................................................................................2-40 Section 3: Software Menu Descriptions Introduction...........................................................................................................................3-2 The MAIN and MENU Soft Keys .....................................................................................3-3 Software Abbreviations and Symbols...................................................................................3-4 The Main Menu ....................................................................................................................3-5 Summary of Main Menu Options.....................................................................................3-6 Main Menu Option: DNA Editor ............................................................................................3-7 The Edit Key....................................................................................................................3-8 The Copy Key................................................................................................................3-11 The Print Key.................................................................................................................3-12 Main Menu Option: Start Synthesis ....................................................................................3-13 Main Menu Option: Monitor Synthesis ...............................................................................3-16 Instrument Status: Synthesizing....................................................................................3-17 The Holding Menu .........................................................................................................3-19 The Jump Step Menu ....................................................................................................3-22 The Interrupt Menu........................................................................................................3-24 Main Menu Option: Change Bottles ...................................................................................3-29 Menu Description ..........................................................................................................3-29 ii Applied Biosystems The Change Bottles Menu Keys....................................................................................3-30 Bottle Usage Data .........................................................................................................3-33 Main Menu Option: Cycle Editor.........................................................................................3-34 Introduction....................................................................................................................3-34 The Cycle Editor Menu..................................................................................................3-35 The Edit Key..................................................................................................................3-35 The Base Specifier Field ...............................................................................................3-37 The Copy Key................................................................................................................3-39 Main Menu Option: Manual Control....................................................................................3-40 The Valve Key ...............................................................................................................3-40 The Function Key ..........................................................................................................3-41 Main Menu Option: Fract Pulse ..........................................................................................3-42 Main Menu Option: Procedure Editor .................................................................................3-42 The Edit Key..................................................................................................................3-43 The Copy Key................................................................................................................3-43 The Print Key.................................................................................................................3-44 Main Menu Option: FXN Editor ..........................................................................................3-44 The Edit Key..................................................................................................................3-45 The Print Key.................................................................................................................3-46 Main Menu Option: Power Fail ...........................................................................................3-47 Main Menu Option: Self Test ..............................................................................................3-48 Main Menu Option: Set Clock.............................................................................................3-51 Main Menu Option: Shut Down ..........................................................................................3-52 Section 4: Functions, Cycles and Procedures Introduction...........................................................................................................................4-2 Valves...................................................................................................................................4-2 Functions ..............................................................................................................................4-5 Synthesis Cycle Functions ............................................................................................4-10 Procedure Functions .....................................................................................................4-15 Test Functions...............................................................................................................4-18 Synthesis Cycles ................................................................................................................4-18 More About the .2µM Cycle...........................................................................................4-20 Procedures .........................................................................................................................4-23 The Phosphoramidite Purge Procedure ........................................................................4-23 The Bottle Change Procedure.......................................................................................4-24 The Shut Down Procedure ............................................................................................4-27 The Flow Test Procedure ..............................................................................................4-28 iii Applied Biosystems Section 5: System Description – Hardware Introduction...........................................................................................................................5-2 The Chemical Delivery System ............................................................................................5-3 Pressure ..........................................................................................................................5-4 Reagent and Solvent Reservoirs.....................................................................................5-4 Delivery Valve Blocks......................................................................................................5-6 Vacuum Assist.................................................................................................................5-9 The Column...................................................................................................................5-10 Waste and Venting ........................................................................................................5-11 The Battery.........................................................................................................................5-12 The Controller.....................................................................................................................5-13 Fuses..................................................................................................................................5-14 Section 6: Chemistry for Automated DNA Synthesis Introduction...........................................................................................................................6-2 The Solid Support - CPG......................................................................................................6-5 DNA Synthesis Chemistry Cycle ..........................................................................................6-7 Detritylation .....................................................................................................................6-8 Coupling ........................................................................................................................6-12 Capping .........................................................................................................................6-15 Oxidation .......................................................................................................................6-16 Completion of the Synthesis Cycle.....................................................................................6-18 Manual Deprotection and Cleavage ...................................................................................6-18 Cleavage .......................................................................................................................6-18 Phosphate Deprotection................................................................................................6-18 Base Deprotection.........................................................................................................6-19 Quantitation of the Oligonucleotide ....................................................................................6-20 Storage of the Oligonucleotide ...........................................................................................6-20 Analysis and Purification ....................................................................................................6-21 OPC, Oligonucleotide Purification Cartridge .................................................................6-21 PAGE ............................................................................................................................6-22 HPLC.............................................................................................................................6-24 Alternative Chemistries ......................................................................................................6-24 RNA Synthesis ..............................................................................................................6-24 Hydrogen-Phosphonate Chemistry ...............................................................................6-25 Phosphorothioate DNA..................................................................................................6-26 5' Attachments....................................................................................................................6-27 Fluorescent-dye Linked Sequencing Primers................................................................6-28 iv Applied Biosystems References .........................................................................................................................6-30 Appendix A: Functions, Cycles and Procedures Appendix B: DNA Synthesizer Schematic DNA Synthesizer Plumbing Diagram Appendix C: Synthesis Log Sheet Reagent/Solvent Log Sheet Appendix D: 391 Illustrated Parts List Appendix E: Warranty 391 Pre-Installation Manual Safety First! ......................................................................................................................... E-3 User Attention Words, Material Safety Data Sheets, and Waste Profiles ........................... E-4 User Attention Words ..................................................................................................... E-4 Material Safety Data Sheets (MSDS)............................................................................. E-4 Waste Profiles ................................................................................................................ E-5 Abbreviations, Initializations, and Units ............................................................................... E-6 Introduction.......................................................................................................................... E-7 Chemicals and Accessories ................................................................................................ E-7 Start-Up Chemical Kit..................................................................................................... E-7 Additional Chemicals and Columns................................................................................ E-9 Shipping List................................................................................................................. E-10 User-Supplied Equipment ............................................................................................ E-11 Site Preparation................................................................................................................. E-13 Laboratory Space ......................................................................................................... E-13 Electrical Requirements ............................................................................................... E-14 Power “Quality”............................................................................................................. E-15 Cooling Requirements.................................................................................................. E-16 Argon............................................................................................................................ E-16 v Applied Biosystems Ventilation..................................................................................................................... E-16 Liquid Waste Disposal.................................................................................................. E-17 Operator Training at Installation ................................................................................... E-17 Proof of Performance ................................................................................................... E-17 Printer........................................................................................................................... E-17 Preinstallation Checklist .................................................................................................... E-18 Technical Support ............................................................................................................. E-19 Contacting Technical Support ...................................................................................... E-19 To Contact Technical Support by E-Mail...................................................................... E-19 Hours for Telephone Technical Support....................................................................... E-19 To Contact Technical Support by Telephone or Fax.................................................... E-20 To Reach Technical Support Through the Internet ...................................................... E-22 To Obtain Documents on Demand............................................................................... E-23 AB LIMITED WARRANTY ................................................................................................. E-24 vi Section 1: Introduction How To Use This Manual. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-2 General Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-3 How to Get Help . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-5 User Attentions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-10 Material Safety Data Sheets (MSDS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-10 Text Conventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-11 Applied Biosystems How To Use This Manual This Users Manual is divided into 6 sections; Introduction (1), Operation (2), Software Menu Descriptions (3), Functions, Cycles and Procedures (4), System Description – Hardware (5), and Chemistry for Automated DNA Synthesis (6). In addition, there are five appendices (refer to the Table of Contents). For an overview and a general understanding of the synthesizer, read the General Introduction in this section and the introductions to Sections 3 through 6. Be sure to read the rest of this section for other important information such as how to get help if you have questions or problems. Section 2: Operation, provides step-by-step instructions on how to operate the instrument and perform necessary procedures. Prior to your initial synthesis be sure to read the following parts of this section: Power On, Pre-synthesis Check List, How to Prepare Phosphoramidites, How to Install Reagent Bottles, How to Begin a Synthesis, Post Synthesis, and Manual Deprotection and Cleavage. The remaining parts of this section should be read as needed. Section 3: Software Menu Descriptions, explains how to use each menu option shown on the display screen. A summary of all options appears on page 3-6. Prior to your initial synthesis, be sure to read the Main Menu Option: DNA Editor, Main Menu Option: Start Synthesis, Main Menu Option: Monitor Synthesis and Main Menu Option: Change Bottles. Read about the remaining menu options as you need to use them. Sections 4, 5 and 6 contain important information about how the instrument operates. It is very useful to read through these sections, but they can also be used as a reference guide. Refer to the Table of Contents to locate a specific topic. Each section is further described below. Section 4: Functions, Cycles and Procedures, contains information about valves, functions, synthesis cycles and procedures (e.g., the bottle change procedure). Pages 4-5 and 4-6 describe functions and show an example of how to use the DNA Synthesizer Schematic. Pages 4-18 and 4-19 describe the synthesis cycles. Section 5: System Description – Hardware, describes the components of the synthesizer and explains the chemical delivery system. Section 6: Chemistry for Automated DNA Synthesis, explains the DNA synthesis chemistry. In addition, it contains an overview of oligonucleotide analysis and purification procedures, a description of alternative chemistries and 5' attachments, and information about quantifying and storing oligonucleotides. 1-2 Section 1: Introduction Applied Biosystems General Introduction The Applied Biosystems Model 391 DNA Synthesizer automatically performs all steps for DNA synthesis to produce the highest quality oligonucleotides possible. When used as a system-including AB reagents and columns-the PCR-MATE EP delivers high reliability, ease of operation and efficient use of your time. The Model 391 uses the phosphoramidite method of oligonucleotide synthesis because of its inherently high coupling efficiency and the stability of the starting materials. The synthesis begins with the 3' terminal nucleoside attached to a solid support which is contained within a column (the reaction chamber). Nucleotide bases are added one at a time to the support-bound DNA chain until the sequence is fully synthesized. Solid support synthesis allows excess reagents to be removed by filtration and eliminates the need for purification between base additions. Typically, stepwise yields of 98 to 100% are obtained enabling synthesis of oligonucleotides more than 180 bases in length. With these high coupling efficiencies, the desired sequence is so abundant in shorter syntheses (up to ~30 bases) that you usually do not need to purify the product. A simple desalting is adequate for most applications. When you need to purify longer oligonucleotides, Applied Biosystems offers OPC, the Oligonucleotide Purification Cartridge. This fast and easy-to-use cartridge completely desalts and purifies the DNA product in about 15 minutes. All Applied Biosystems chemicals are pretested to ensure high yield syntheses. Solvents and reagents are packaged in bottles that you attach directly to the synthesizer. All chemicals, except the phosphoramidites, are ready to use. To prepare the phosphoramidites, you only need to dissolve them in anhydrous acetonitrile. Each phosphoramidite, column, reagent and solvent manufactured by Applied Biosystems is unconditionally guaranteed. If you are not completely satisfied by the product (and it is used prior to any applicable expiration date and under the correct operating conditions), it will be replaced at no charge. Reagents and solvents are delivered to the column by a pressure-driven chemical delivery system. The system uses patented zero-dead volume valves which increase reliability, eliminate cross-contamination, and reduce the reagent costs. The Model 391 has menu-driven software designed for simplicity and ease of operation. A 2-line screen displays various options and necessary information about the synthesis or status of the instrument. In response, you select an option and give instructions by pressing the appropriate key on the keyboard. Automated synthesis uses a cycle, consisting of a series of steps, which completes all chemical reactions needed to couple one base. The cycle is repeated until all bases of the oligonucleotide chain are added. Applied Biosystems supplies four cycles that have been thoroughly tested and optimized for use with β-cyanoethyl phosphoramidites. They vary according to how much initial 3' terminal nucleoside is attached to the support (e.g., .2µM, 1µM, 10µM). You decide which one to use based on how much final product you need for your experiments. Section 1: Introduction 1-3 Applied Biosystems The Extended Programmability (EP) feature allows you to create and edit cycles (as well as procedures and functions) optimized for your own needs. And, you can implement alternative chemistries such as RNA synthesis and hydrogen phosphonate synthesis to create nucleotide analogs. When using these chemistries, you only need to load the appropriate reagents and customize a cycle. No hardware changes are needed. The PCR-MATE EP also includes the computer software program, Syncom™. Syncom electronically transfers DNA sequences from an IBM or compatible personal computer (PC) to your synthesizer. With Syncom you can create a vast library of sequences on a PC, then easily transfer a sequence to the instrument for synthesis. The program offers the opportunity to link your Model 391 to a network of computers to provide Laboratory Information Management (LIM) capabilities. Applied Biosystems has been perfecting the science of DNA synthesis on automated instruments since 1982. The PCR-MATE EP is the latest advance, offering the individual laboratory an instrument system that delivers high quality and reliability at an economical price. 1-4 Section 1: Introduction Applied Biosystems How to Get Help Contacting Technical Support You can contact Applied Biosystems for technical support by telephone or fax, by e-mail, or through the Internet. You can order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents 24 hours a day. In addition, you can download documents in PDF format from the Applied Biosystems Web site (please see the section “To Obtain Documents on Demand” following the telephone information below). To Contact Technical Support by E-Mail Contact technical support by e-mail for help in the following product areas: Product Area E-mail address Genetic Analysis (DNA Sequencing) [email protected] Sequence Detection Systems and PCR [email protected] Protein Sequencing, Peptide and DNA Synthesis [email protected] Biochromatography, PerSeptive DNA, PNA and Peptide Synthesis systems, CytoFluor®, FMAT™, Voyager™, and Mariner™ Mass Spectrometers [email protected] LC/MS (Applied Biosystems/MDS Sciex) [email protected] or [email protected] Chemiluminescence (Tropix) [email protected] Hours for Telephone Technical Support In the United States and Canada, technical support is available at the following times: Product Hours Chemiluminescence 8:30 a.m. to 5:30 p.m. Eastern Time Framingham support 8:00 a.m. to 6:00 p.m. Eastern Time All Other Products 5:30 a.m. to 5:00 p.m. Pacific Time Section 1: Introduction 1-5 Applied Biosystems To Contact Technical Support by Telephone or Fax In North America To contact Applied Biosystems Technical Support, use the telephone or fax numbers given below. (To open a service call for other support needs, or in case of an emergency, dial 1-800-831-6844 and press 1.) Product or Product Area Telephone Dial... Fax Dial... ABI PRISM® 3700 DNA Analyzer 1-800-831-6844, then press 8 1-650-638-5981 DNA Synthesis 1-800-831-6844, then press 21 1-650-638-5981 Fluorescent DNA Sequencing 1-800-831-6844, then press 22 1-650-638-5981 Fluorescent Fragment Analysis (includes GeneScan® applications) 1-800-831-6844, then press 23 1-650-638-5981 Integrated Thermal Cyclers (ABI PRISM ® 877 and Catalyst 800 instruments) 1-800-831-6844, then press 24 1-650-638-5981 ABI PRISM ® 3100 Genetic Analyzer 1-800-831-6844, then press 26 1-650-638-5981 BioInformatics (includes BioLIMS, BioMerge™, and SQL GT™ applications) 1-800-831-6844, then press 25 1-505-982-7690 Peptide Synthesis (433 and 43X Systems) 1-800-831-6844, then press 31 1-650-638-5981 Protein Sequencing (Procise Protein Sequencing Systems) 1-800-831-6844, then press 32 1-650-638-5981 PCR and Sequence Detection 1-800-762-4001, then press 1 for PCR, 2 for the 7700 or 5700, 6 for the 6700 or dial 1-800-831-6844, then press 5 1-240-453-4613 Voyager MALDI-TOF Biospectrometry and Mariner ESI-TOF Mass Spectrometry Workstations 1-800-899-5858, then press 13 1-508-383-7855 Biochromatography (BioCAD Workstations and Poros Perfusion Chromatography Products) 1-800-899-5858, then press 14 1-508-383-7855 Expedite Nucleic acid Synthesis Systems 1-800-899-5858, then press 15 1-508-383-7855 Peptide Synthesis (Pioneer and 9050 Plus Peptide Synthesizers) 1-800-899-5858, then press 15 1-508-383-7855 PNA Custom and Synthesis 1-800-899-5858, then press 15 1-508-383-7855 1-6 Section 1: Introduction Applied Biosystems Product or Product Area Telephone Dial... Fax Dial... FMAT 8100 HTS System and Cytofluor 4000 Fluorescence Plate Reader 1-800-899-5858, then press 16 1-508-383-7855 Chemiluminescence (Tropix) 1-800-542-2369 (U.S. 1-781-275-8581 only), or 1-781-271-0045 Applied Biosystems/MDS Sciex 1-800-952-4716 1-650-638-6223 Telephone Dial... Fax Dial... 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Fax Dial... Russia (Moskva) 7 095 935 8888 7 095 564 8787 South East Europe (Zagreb, Croatia) 385 1 34 91 927 385 1 34 91 840 Spain (Tres Cantos) 34 (0)91 806 1210 34 (0)91 806 1206 Sweden (Stockholm) 46 (0)8 619 4400 46 (0)8 619 4401 Switzerland (Rotkreuz) 41 (0)41 799 7777 41 (0)41 790 0676 The Netherlands (Nieuwerkerk a/d IJssel) 31 (0)180 331400 31 (0)180 331409 United Kingdom (Warrington, Cheshire) 44 (0)1925 825650 44 (0)1925 282502 All other countries not listed (Warrington, UK) 44 (0)1925 282481 44 (0)1925 282509 81 3 5566 6230 81 3 5566 6507 Japan Japan (Hacchobori, Chuo-Ku, Tokyo) Latin America Del.A. Obregon, Mexico 305-670-4350 305-670-4349 To Reach Technical Support Through the Internet We strongly encourage you to visit our Web site for answers to frequently asked questions and for more information about our products. You can also order technical documents or an index of available documents and have them faxed or e-mailed to you through our site. The Applied Biosystems Web site address is http://www.appliedbiosystems.com/techsupp To submit technical questions from North America or Europe: Step Action 1 Access the Applied Biosystems Technical Support Web site. 2 Under the Troubleshooting heading, click Support Request Forms, then select the relevant support region for the product area of interest. 3 Enter the requested information and your question in the displayed form, then click Ask Us RIGHT NOW (blue button with yellow text). 4 Enter the required information in the next form (if you have not already done so), then click Ask Us RIGHT NOW. You will receive an e-mail reply to your question from one of our technical experts within 24 to 48 hours. 1-8 Section 1: Introduction Applied Biosystems To Obtain Documents on Demand Free, 24-hour access to Applied Biosystems technical documents, including MSDSs, is available by fax or e-mail or by download from our Web site. To order documents... Then... by index number a. Access the Applied Biosystems Technical Support Web site at http://www.appliedbiosystems.com/techsupp b. Click the Index link for the document type you want, then find the document you want and record the index number. c. Use the index number when requesting documents following the procedures below. by phone for fax delivery a. From the U.S. or Canada, call 1-800-487-6809, or from outside the U.S. and Canada, call 1-858-712-0317. b. Follow the voice instructions to order the documents you want. Note through the Internet for fax or e-mail delivery There is a limit of five documents per request. a. Access the Applied Biosystems Technical Support Web site at http://www.appliedbiosystems.com/techsupp b. Under Resource Libraries, click the type of document you want. c. Enter or select the requested information in the displayed form, then click Search. d. In the displayed search results, select a check box for the method of delivery for each document that matches your criteria, then click Deliver Selected Documents Now (or click the PDF icon for the document to download it immediately). e. Fill in the information form (if you have not previously done so), then click Deliver Selected Documents Now to submit your order. Note There is a limit of five documents per request for fax delivery but no limit on the number of documents you can order for e-mail delivery. Section 1: Introduction 1-9 Applied Biosystems User Attentions Four user-attention words appear in the text of this manual. Each one implies a certain level of observation or action as follows: Note This word is used to call attention to information. IMPORTANT This word is used when information is necessary for proper instrument operation. Caution This word informs you that damage to the instrument could result if you do not comply with this information. WARNING Physical injury to you or other people could result if these required precautions are not taken. Material Safety Data Sheets (MSDS) WARNING 1-10 Some chemicals used with this instrument are considered hazardous. Hazards are prominently displayed on the labels of all hazardous chemicals. In addition, there are MSDS which provide information about physical characteristics, hazards, precautions, first aid, spill cleanup and disposal procedures. The MSDS appear in the User’s Safety Information Section in the front of this manual. Please be sure to familiarize yourself with the information in these documents before attempting to operate the instrument or use the reagents. Additional copies of the MSDS are available from Applied Biosystems at no extra cost. Section 1: Introduction Applied Biosystems Text Conventions Menu displays and keys appear in this manual as follows: 1. Menus are boxed and appear centered on the page: DNA start change cycle more editor synth bottles editor menu 2. When describing what a soft key does, the key is boxed and appears at the left margin: DNA EDITOR Creates, edits and prints up to four DNA sequences 3. When the text refers to a key that you will press, the key appears in capital letters: When you select DNA EDITOR, the screen displays one of the DNA strands and shows the number of bases in that strand. Section 1: Introduction 1-11 Section 2: Operation This section describes step-by-step procedures for operating the Model 391 DNA Synthesizer. If questions arise about the software that are not explained in this section, refer to Section 3 which describes all menus and keys in detail. Before beginning a synthesis, be sure to read the following parts: Pre-synthesis Check List, How to Prepare Phosphoramidites, How to Install Reagent Bottles, How to Begin a Synthesis, Post Synthesis, Manual Deprotection and Cleavage, and How to Store Reagents. Use the rest of the section as you need to. Power On. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-2 Pre-synthesis Check List. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-3 How to Prepare Phosphoramidites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-4 How to Dissolve Phosphoramidites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-5 How to Store Dissolved Phosphoramidites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-6 How to Install Reagent Bottles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-7 How to Begin a Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-9 How to Install the Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-12 Post Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-13 Manual Deprotection and Cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-13 How to Abort a Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-18 About the Synthesis Reagents and Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-19 How to Store Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-19 About Deoxyinosine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-23 How to Change an Argon Tank . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-25 How to Change the Waste Bottle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-25 How to Perform the Flow Test Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-26 Aminolink 2™ Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-30 How to Use Aminolink 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-32 How to Use OPC (Oligonucleotide Purification Cartridge) . . . . . . . . . . . . . . . . . . . . . . . . .2-35 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-40 Applied Biosystems Power On 1. Connect the instrument to a power source and turn the power switch on. The power switch is located on the front of the instrument to the right of bottle position 5. IMPORTANT The argon cylinder must always be connected to the synthesizer before the power is turned on. If this is not done, the vacuum-assist valves will be activated continuously and can overheat and fail causing argon to leak. 2. Select START from the following display: Applied Biosystems 391 DNA Synthesizer PCR-MATE EP Ver. 1.00 start Page 1 of the Main Menu will then be shown: DNA start change cycle more editor synth bottles editor menu The display can be adjusted for optimal viewing by turning the knob at the left of the screen. 2-2 Section 2: Operation Applied Biosystems Pre-synthesis Check List Before beginning a synthesis, be sure to complete the following steps. 1. Check reagent levels of all reservoirs. Empty reservoirs should be replaced with bottles of fresh reagents. From the Main Menu, choose CHANGE BOTTLES and follow the prompts. For further instructions, refer to How to Install Reagent Bottles found later in this section. Be sure bottles are on every position. Since all phosphoramidite reservoirs are pressurize simultaneously with a single valve, all 5 bottles must be attached to the instrument (even if some are empty) to perform synthesis. 2. Check that all alarms are set correctly. Refer to Section 3: Main Menu Option: Change Bottles, for instructions on how to set the alarm. 3. As needed, prepare the phosphoramidites for use by dissolving them in anhydrous acetonitrile. For further instructions, refer to How to Prepare Phosphoramidites later in this section. 4. Check the pressure supply of the argon tank. Check that the low pressure gauge reads 60 psi. Change the tank as it is depleted. Watch it carefully when the high pressure gauge drops below 400 psi. With average synthesizer use, an argon tank should last approximately 3 months. To change an argon tank, refer to the instructions later in this section. 5. Check the waste level. The synthesizer generates 1 to 2 liters of hazardous, halogenated, organic liquid waste per 100 base additions. The waste is collected in a 4-liter bottle supplied by Applied Biosystems. When the waste bottle is full, it must be emptied and the waste disposed of properly. The bottle can be changed prior to a synthesis or when a synthesis is interrupted. To change the waste bottle, refer to the instructions later in this section. 6. Fill the fraction collector with 10- to 15-mL volumetric tubes to collect the trityl cation released at each detritylation step. Use one tube for each base in the sequence. Be sure to label the tubes and align the fraction collector so tube number one collects the first trityl. Section 2: Operation 2-3 Applied Biosystems How to Prepare Phosphoramidites The prepackaged phosphoramidites are bottled as powders and sealed under argon pressure. In this state, they are stable for at least 1 year. To prepare them for use, they are dissolved in anhydrous acetonitrile. Since the phosphoramidites are extremely sensitive to acid, oxygen, and water, you must take special care when dissolving them. The following instructions will help avoid contamination, prevent degradation and ensure high coupling yields. Important factors to consider when dissolving phosphoramidites: 1. Use anhydrous acetonitrile with less than 90 ppm water to dissolve the phosphoramidites. Use of Applied Biosystems anhydrous acetonitrile, Part Number 400060, is strongly recommended. Do not use HPLC grade acetonitrile, its higher water content will decrease coupling efficiency. After opening the acetonitrile, keep it blanketed with argon to avoid contamination with air. 2. When transferring the acetonitrile to a phosphoramidite bottle, use a clean, dry, glass syringe with a needle. Store it in a 100-120°C oven to prevent atmospheric moisture contamination. Keep the syringe dedicated to this use. Use acetonitrile to rinse it, do not use water. Do not contaminate the acetonitrile bottle with traces of phosphoramidites (i.e., do not allow the syringe needle to contact the phosphoramidites). 3. Add the correct amount of acetonitrile to each phosphoramidite as follows: β-cyanoethyl phosphoramidite A G C T Deoxyinosine volume of acetonitrile (mL) 2.8 5.6 11.2 2.9 5.8 11.6 2.9 5.9 11.8 3.3 6.6 13.2 3.4 weight of phosphoramidite (grams) .25 .5 1.0 .25 .5 1.0 .25 .5 1.0 .25 .5 1.0 .25 molarity (M) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 The above concentrations result in a 10-fold excess of soluble phosphoramidite over support-bound nucleoside. 2-4 Section 2: Operation Applied Biosystems How to Dissolve Phosphoramidites 1. To prepare the phosphoramidite bottle, pull back the aluminum tab in the direction of the arrow. Do not yet remove it, simply expose the septum. Place a needle (any gauge, without a syringe) into the rubber septum. This vents the pressure in the bottle when the anhydrous acetonitrile is added. Venting also prevents accidental splashing when the phosphoramidite bottle is opened and placed on the instrument. 2. Unscrew the cap from the anhydrous acetonitrile bottle and quickly replace it with a clean rubber septum. The acetonitrile is bottled under argon. Since argon is heavier than air, argon should still blanket the acetonitrile after the septum transfer. 3. Remove the syringe/needle from the oven and allow it to cool to room temperature. Pierce the septum of the acetonitrile bottle with the needle and remove the correct amount of acetonitrile. 4. Pierce the septum of the phosphoramidite bottle a few millimeters with the needle/syringe and slowly add the acetonitrile. Make sure the needle does not touch the phosphoramidite powder or solution. When finished, remove both the venting needle and the needle/syringe and gently swirl the bottle to dissolve the phosphoramidites. Once dissolved, you can place them on the instrument. Use the Change Bottles Menu and follow the prompts (see Section 3 for details). Also, refer to How to Install Reagent Bottles, for instructions on attaching the bottles to the instrument. 5. The anhydrous acetonitrile bottle must now be blanketed with argon. To do so, place a needle on the upper male Luer fitting on the front of the synthesizer (remove the column if necessary). Select MANUAL CONTROL from the Main Menu and activate function 2, a reverse flush. Argon will then flow out of the needle. Next, push the acetonitrile bottle onto the needle so that the needle pierces the bottle’s septum. Hold it for 5 seconds and then remove the bottle and the needle simultaneously. The pressure will immediately be released through the needle but the bottle will remain blanketed with argon. Remove the needle from the bottle and continue dissolving additional phosphoramidites as necessary. Once in solution, phosphoramidites are stable for about two weeks. After this time, coupling efficiencies may slowly begin to decrease. If phosphoramidites cannot be used within this time, it is possible to freeze, store, thaw and reuse them. Although the stored phosphoramidites may show some slight loss of activity, they should be adequate for synthesizing sequences of approximately 20 bases. Section 2: Operation 2-5 Applied Biosystems How to Store Dissolved Phosphoramidites If you cannot use the phosphoramidites within about two weeks, you can remove them from the instrument, store them and reuse them later. Do not use the Change Bottles Procedure for this. Instead follow the protocol below. 1. Using MANUAL CONTROL, activate the appropriate functions, F62 to F66, for 10 seconds, then deactivate them. Argon to Reservoirs F 62 Flush to A F 63 Flush to G F 64 Flush to C F 65 Flush to T F 66 Flush to X 2. Remove the bottles (without using the change bottles procedure) and quickly cap with previously unused rubber septa (Aldrich catalog number Z10,074-9). 3. Rinse the delivery lines with acetonitrile by activating the appropriate functions, F71 to F75, for 10 seconds. Flush the lines with argon by activating the appropriate functions, F62 to F66, for 10 seconds. Acetonitrile to Reservoirs F 71 #18 to A F 72 #18 to G F 73 #18 to G F 74 #18 to T F 75 #18 to X Argon to Reservoirs F 62 Flush to A F 63 Flush to G F 64 Flush to C F 65 Flush to T F 66 Flush to X 4. Seal the bottles with Paraffin film, place in a desiccator containing Drierite and put in a freezer at -20°C. 5. When ready to synthesize, thaw the phosphoramidites, remove them from the desiccator and place them on the instrument using the Change Bottles Menu. Be sure to enter an appropriate alarm setting. 2-6 Section 2: Operation Applied Biosystems How to Install Reagent Bottles A reagent bottle can be changed before beginning a synthesis or when an active synthesis has been stopped by setting an interrupt or by the alarm. When removing and replacing bottles, always select CHANGE BOTTLES from page 1 of the Main Menu and follow the prompts. An exception is if you are going to store and later reuse the phosphoramidites (refer to How to Store Dissolved Phosphoramidites for details). WARNING Consider each chemical in the synthesizer potentially harmful. Do not inhale vapors. Work in a well ventilated area. Always use eye protection and wear acid-impermeable gloves and a lab coat. Do not leave any chemicals uncapped. If any chemical is ingested or comes in contact with the eyes, immediately consult a physician. If there is any physical contact, wash immediately with ample water. Refer to About the Synthesis Reagents and Solvents and the MSDS for further instructions about storing and handling each reagent. Note The phosphoramidites, tetrazole and acetonitrile are atmosphere sensitive. Upon opening one of these bottles, quickly place it on the instrument to prevent contamination. How to Install the Phosphoramidites (bottles 1-5) These reservoirs are push-on bottles that fit around a Teflon insert and silicone O-ring to form an airtight seal inside each bottle neck. Pressing the appropriate black button above the bottle position releases the grip on the bottle and allows for its removal and subsequent replacement. When installing a bottle, the button will return to its out position only when the reservoir is correctly engaged. 1. To remove a bottle, firmly pull it straight down while pressing the black button above its receptacle. If the bottle seems to stick, carefully move it side to side while pulling it off. Cover or recap the bottle immediately to minimize vapor release. Wipe the delivery line with a lintfree tissue. 2. Dissolve the phosphoramidites as described in How to Prepare Phosphoramidites found in this section. Next, remove the aluminum cap and the rubber septum from the bottle. Wipe any crystals or drops of reagent off the bottle neck. 3. To install the bottle, firmly push it up around its receptacle while pressing the black button. As necessary, maneuver the bottle into place by carefully moving it side to side while pushing. 4. When the bottle is correctly engaged, release the button and it will return to its out position. If the button remains in, the bottle is not seated properly and must be repositioned. Section 2: Operation 2-7 Applied Biosystems How to Install Bottles 9-15 These reservoirs screw directly into a threaded cap mounted on the synthesizer. 1. To remove a bottle, unscrew it by turning clockwise. Remove the disposable polyethylene insert. Recap it immediately to minimize vapor release. Note The disposable insert forms an airtight seal between each cap assembly and bottle. It is designed for single use and should be replaced with each bottle change. Inserts are supplied at no charge when you order the chemical reagent kits. To order inserts separately, use the following Part Numbers (PN): for 450mL bottles, 400501; for 200mL bottles, 400790. 2. Open the full bottle. Place a new polyethylene insert inside the bottle neck. Screw the bottle snugly into its threaded cap on the instrument by turning it counterclockwise. Note Receptacles 9-15 have a ratchet cap assembly. When attaching these bottles, you cannot overtighten them. How to Install Bottle 18 Reservoir 18, acetonitrile, is a 4-Liter bottle that is placed on the left side of the instrument, inside a protective carrier and metal rack. To replace the bottle, slowly unscrew the cap assembly to release pressure. Remove it and then screw it on a fresh acetonitrile bottle. IMPORTANT Use HPLC or UV grade acetonitrile with a specification of less than 300 ppm of water. Water contamination of even 1000 ppm will lead to a 1 to 2% decrease in coupling efficiency. 2-8 Section 2: Operation Applied Biosystems How to Begin a Synthesis After completing the pre-synthesis check list, perform the following steps to begin a synthesis. As questions arise, refer to Section 3: Software Menu Descriptions, for further instructions and explanations. 1. View Page 1 of the Main Menu: DNA start change cycle more editor synth bottles editor menu DNA-1 0 mer next main 2. From the Main Menu, select DNA EDITOR: Select action for edit copy print Press NEXT until the desired DNA strand is in view, then press EDIT: 5'>_ <3' 0 ( space ) erase menu Enter the sequence (5' to 3') using the A, G, C, T and X keys. Be sure the sequence is correct. If possible, print and verify it. When finished, return to the Main Menu by selecting MENU and then MAIN. Note that any combination of phosphoramidites can be inserted in any position, except the 3'-terminus, for synthesizing mixed-sequence probes. This is done by pressing the left, open parenthesis, ( , typing the desired base keys, and pressing the right, close parenthesis, ). Multiple bases are added to the support-bound nucleoside by simultaneous delivery of the specified phosphoramidites. 3. From the Main Menu, select START SYNTH: Make DNA-1:12 Cycle-1:63 begin nxt DNA nxt cyc Trityl OFF ON/OFF main Configure the synthesis. a. Press NXT DNA until the correct DNA strand to be synthesized is showing. The most recently edited strand will automatically appear. The above example shows DNA-1 which is a 12 base sequence. b. Press NXT CYC until the desired synthesis cycle is shown. When the main power is first turned on, all Applied Biosystems ROM cycles are automatically loaded into the correct RAM cycle location as shown below. For example, to synthesize using the .2µM cycle, Section 2: Operation 2-9 Applied Biosystems Cycle-1:63 should be displayed. (You can create your own cycles by editing a RAM cycle. Once you change a RAM cycle, the edited version will appear in the Start Synthesis Menu. You can, however, copy a ROM cycle back into a RAM cycle location at any time. Refer to Section 3: Main Menu Option: Cycle Editor, for instructions.) ROM synthesis cycle RAM cycle location Total number of steps cycle time (min.) crude yield* (O.D.) (20mer) .2µM Cycle-1 63 5.5 20-25 1µM Cycle-2 64 5.5 100-120 Low** Cycle-3 61 5.5 20-25 10µM Cycle-4 53 25 800-1000 *Yield figures based on a 20mer sequence. Absorbance measured at 260nm. Assuming 33 micrograms/O.D. unit. **The Low cycle is on the .2µM scale. The synthesis cycle, Low, is a low-reagent consumption cycle on the .2µM scale. It uses 33% less phosphoramidites than the .2µM cycle, although its coupling efficiencies are slightly lower, about 97%. “Low” can be used to synthesize sequences less than 50 bases. Sequences less than 30 bases usually only need desalting. Sequences greater that 30 bases should be OPC purified. c. Press ON/OFF until the desired ending method is shown. The 5' terminus of the fully synthesized DNA chain can either remain protected by a dimethoxytrityl group (trityl on), or can be detritylated to yield a 5'-hydroxyl (trityl off). Trityl on is usually selected when purifying by trityl-specific OPC or reverse phase HPLC. Trityl off is usually chosen when purifying by gel electrophoresis or ion exchange HPLC. When all information is displayed correctly, select BEGIN. All bottles will then be checked for usage: Checking bottle usage for base 1 Please Wait . . . 2-10 Section 2: Operation Applied Biosystems According to the alarm you set in the Change Bottles Menu, a message will be displayed if the alarm will be triggered during the synthesis. For example, if bottle 9, tetrazole, will empty in 11 cycles, the screen will read: Bottle 9 will interrupt in 11 cycles begin menu Check the reagent level of the bottle. Replace it with fresh reagent, if necessary. To do this, return to the Main Menu, select CHANGE BOTTLES and follow the instructions. If the bottle is not empty, you can reenter an appropriate alarm setting in the Change Bottles Menu. Check the reagent level of the other bottles and decide whether to replace them now or wait until the synthesis is interrupted by the alarm. When ready to start synthesis, press BEGIN. The screen will show a display similar to: Install (T) COLUMN, then press enter enter menu Place the correct color-coded column on the instrument. This column will correspond to the base at the 3' end of your DNA sequence. Make sure it matches what is displayed on the screen and is the correct synthesis scale. Record the column’s serial number to identify the synthesis. CPG-bound nucleoside A G C T column color-code green yellow red blue Small scale (.2µM) columns have labels with broken colored lines and serial numbers that begin with “2”. One-micromole columns have labels with solid colored lines and serial numbers that begin with “3”. Ten-micromole columns are larger than the others. They have labels with solid colored lines and serial numbers that begin with “3”. Wide-pore CPG columns are recommended when synthesizing oligonucleotides greater than 80 bases. They are available on the .2µM scale only. Wide-pore columns have labels with broken colored lines and serial numbers that begin with “4”. Section 2: Operation 2-11 Applied Biosystems How to Install the Column 1. Remove the aluminum tabs from both ends and tap the ends on a dark surface to check for leaks. If CPG falls out, do not use the column. Any faulty columns will be replaced by Applied Biosystems without charge. 2. Firmly push either end straight up onto the upper male Luer fitting on the instrument. Since the column is symmetrical, it can be attached in any way. 3. Firmly push the lower male Luer fitting straight up into the bottom of the column. The column should fit securely. Do not twist the fittings or the filters may crimp or tear. Once the column is installed, select ENTER. The screen will read: Purge Phosphoramidite lines? no yes menu Choose whether to purge the phosphoramidite and tetrazole delivery lines. Press YES if the instrument has been idle for more than 12 hours (more than 6 hours in humid environments) or if a phosphoramidite reservoir has not been accessed within 12 hours. Press NO if the instrument has been running continuously and if all the phosphoramidites have been accessed within 12 hours. Upon pressing NO or after the purge is finished, the synthesis automatically begins and page one of the Synthesizing Status Menu (one of the Monitor Synthesis Menus) is shown: SYNTHESIZING base 1 of xx bases hold jump intrpt more main In the above menu, “xx” equals the total number of bases in the cycle. Press the MORE key to view the current synthesis cycle step. Press MORE again to view the DNA sequence and the base currently being synthesized. Press MORE again to view the synthesis configuration. From this menu, you can hold or interrupt a step, or jump to another step in the cycle. Refer to Section 3: Main Menu Option: Monitor Synthesis, for details on how to hold, interrupt and jump a step. Notes for after synthesis begins: Following synthesis start, check that the column does not leak during chemical deliveries. Also, note if and when bottles will need replacing. During the synthesis, you can continue to view the Monitor Synthesis Menu or return to the Main Menu to access other ones. During an active synthesis, Self Test, Manual Control and Shut Down cannot be accessed. However, you can access the Cycle Editor during synthesis to change cycle step times. 2-12 Section 2: Operation Applied Biosystems When the synthesis is finished, SYNTHESIS COMPLETE is displayed in the Monitor Synthesis Menu. Post Synthesis 1. Remove the column. To identify it, be sure to record its serial number. 2. Remove the trityl tubes. Visually inspect the trityl solutions. Be sure there are no clear tubes and that the intensity of the orange color gradually decreases. Perform the trityl cation assay to determine the coupling efficiency of the synthesis. Refer to User Bulletin 13 (Revised), Evaluation and Purification of Synthetic Oligonucleotides, for instructions. Once the column and trityl tubes are removed, the instrument is ready to begin another synthesis. 3. Perform the Manual Deprotection and Cleavage Procedure described on the next page. 4. If desired, analyze the crude oligonucleotide mixture by one of the methods, such as HPLC or gel electrophoresis, described in User Bulletin 13 (Revised). 5. Prepare the DNA for use in experiments by removing the ammonia and then desalting. Purify the product, if necessary. Applied Biosystems Oligonucleotide Purification Cartridge (OPC) provides a fast and easy way to purify and desalt in one step. An overview of OPC, and other purification methods, can be found in Chemistry for Automated DNA Synthesis. In addition, refer to How to Use OPC (Oligonucleotide Purification Cartridge) found later in this section. Refer to User Bulletin 13 (Revised) for other detailed instructions about analysis and purification. Manual Deprotection and Cleavage Introduction After the synthesis of a deoxyoligonucleotide on the Model 391, the DNA produced is not yet biologically active. The phosphate groups and base exocyclic amines are protected to prevent side reactions during synthesis. Following synthesis, the protecting groups must be removed. In addition, the DNA must be hydrolyzed or cleaved from the solid support. There are three types of protecting groups: 1. 5' protecting group - dimethoxytrityl (DMT) 2. β-cyanoethyl phosphate protecting groups 3. base protecting groups - benzoyl on dA and dC and isobutyryl on dG The 5'-protecting group, dimethoxytrityl, is attached to each nucleoside phosphoramidite and is cleaved from the growing oligonucleotide chain during each cycle of base addition. You can program the 391 to remove the last DMT to yield a 5' hydroxyl by choosing the ending method Trityl off, or leave the DMT on by choosing the ending method Trityl on. When purifying by polyacrylaSection 2: Operation 2-13 Applied Biosystems mide gel electrophoresis (PAGE)1 or ion exchange HPLC2, the last DMT group should be removed. When purifying by trityl-specific OPC or reverse phase HPLC, the last DMT group should be left on.1 After the synthesis is complete (either Trityl on or Trityl off), remove the column from the instrument. The oligonucleotides are then simultaneously decyanoethylated and cleaved from the support using concentrated ammonium hydroxide. Next, the base protecting groups are removed by the addition of fresh concentrated ammonia and incubation at 55ºC. Materials and Methods The recommended method for manual deprotection and cleavage was developed by Tanaka and Letzinger3 and later adapted for use with Applied Biosystems synthesis columns by Schott.4 The procedure presented here is similar to Dr. Schott’s with minor variations. Reagents, solvents and apparatus The manual deprotection kit (Part Number 400257) accompanying the installation kit contains the following items for the deprotection. 1. Ten 1-mL disposable syringes with Luer fittings (VWR Catalog #BD5602). 2. Ten disposable needles with female Luers (VWR Catalog #BD5167). 3. A number 10 rubber stopper (VWR Catalog #59580-386). 4. Five male-to-male Luer connectors (Alltech Associates Inc. Catalog #86506). Items 1 and 2 are used on a one-time basis and should be ordered in sufficient quantities for regular use. Because it is convenient to perform the deprotection on several oligonucleotides simultaneously, you should purchase more male-to-male Luer connectors. Reagent grade, concentrated ammonium hydroxide is provided in the installation reagent kit. Additional quantities should be purchased from a local supplier. Small DNA collection vials with a rubber-lined screw cap are available from Applied Biosystems (Part No. 400048) or can be purchased from a local source. However, use Teflon™-lined caps with the vials because the rubberlined caps can leach contaminants into the DNA-ammonium hydroxide solution. They can be ordered from Wheaton; Part Number 240408, size 13-425. 2-14 Section 2: Operation Applied Biosystems Figure 2-1. Manual Deprotection Apparatus Section 2: Operation 2-15 Applied Biosystems Methods 1. After the synthesis is complete, remove the column from the instrument and attach one end to the syringe Luer. Connect the male-to-male Luer to the other end of the column. Next, attach the syringe needle to the male-to-male Luer. See Figure 2-1. 2. With the syringe plunger inserted into the syringe barrel to the 0.5 mL mark, insert the needle into some fresh, room temperature ammonium hydroxide. Draw up enough reagent to fill the column but minimize the volume in the syringe. IMPORTANT The concentration of ammonia is critical. Use fresh, concentrated ammonium hydroxide which has been opened less than one month. 3. Insert the needle into the rubber stopper and let stand for 15-30 minutes. This treatment begins cleavage and cyanoethyl deprotection. 4. Expel the ammonia into a small DNA collection vial. There can be positive pressure inside the syringe system which can force some of the ammonia solution out of the needle as it is removed from the stopper. This can be minimized by using ammonium hydroxide at room temperature and by slightly withdrawing the syringe barrel as you remove the needle from the stopper. IMPORTANT Use a tightly sealed DNA collection vial that can withstand positive pressure. The vial must also have a Teflon-lined cap. Rubber-lined caps have contaminants that leach out of the cap liner during base deprotection. Teflon-lined caps can be ordered from Wheaton; Part Number 240408, size 13-425. 5. Repeat the ammonia treatment 3 more times for 15 to 30 minutes each. This ensures complete cleavage and cyanoethyl deprotection. IMPORTANT Remember that the product DNA is now in solution and no longer bound to the support. Save the column until the cleavage is confirmed. 6. To remove the exocyclic amine base protecting groups (benzoyl and isobutyryl), first bring the volume of the crude DNA solution to 3mL with fresh concentrated ammonium hydroxide. Then place the vial of DNA at 55ºC for 8 to 15 hours. Longer treatment is advisable if the ammonium concentration is questionable. This also cleaves the acetyl caps from the failure sequences. 7. After completing deprotection, cool the ammonium hydroxide-DNA solution. a. When purifying by OPC, the oligonucleotide solution is diluted 1:3 with water and then loaded directly on the cartridge with no other preparation needed. For details, refer to How to Use OPC (Oligonucleotide Purification Cartridge) found later in this section. 2-16 Section 2: Operation Applied Biosystems b. If the DMT group was removed previously as a part of the synthesis cycle, the DNA is ready for analysis and/or purification by PAGE or ion exchange HPLC. Analysis and purification procedures are discussed in User Bulletin 13 (Revised).1 c. When purifying by trityl-specific reverse phase HPLC, cool the ammonium hydroxideDNA solution (to prevent losses from bubbling) and remove the ammonia by vacuum. Keep the solution basic to prevent accidental detritylation by adding one drop of distilled triethylamine every 10 minutes. Also, avoid heating the sample. After collection and concentration of the product from reverse phase HPLC, detritylate the dried sample by dissolving it in 200-500 µL of 80% acetic acid. Since the acetic acid is an aqueous solution, the trityl cation will react with water and form tritanol and will not give an orange color. After 20 minutes, add an equal volume of 95% ethanol, and lyophilize the sample. The dried sample is then lyophilized from ethanol until no acetic acid remains. The hydrolyzed DMT group can be removed by methods discussed in User Bulletin 13 (Revised).1 Section 2: Operation 2-17 Applied Biosystems How to Abort a Synthesis When a synthesis is aborted, all valves close and you will not be able to continue the run. A procedure for how to abort a synthesis appears below. If you need further details, refer to The Interrupt Menu in Section 3. Caution Interrupt synthesis at a safe step, such as Cycle Entry or Cycle End. Otherwise, chemicals could be left in valve blocks and delivery lines. Salts could then form preventing subsequent deliveries. The resulting repairs are expensive. To abort synthesis: 1. Press INTRPT from the Monitor Synthesis Menu. 2. From the No Interrupt Set page of the Interrupt Menu, press AHEAD. Set an Interrupt Ahead to occur at the next Cycle Entry or Cycle End step. Refer to table below to determine the exact step number. To interrupt at the next base, do not enter a base number. (Note that if an Interrupt Ahead has already been set, you can change it by typing over existing entries.) Synthesis Cycle .2µM 1µM Low 10µM Cycle Entry step number 36 36 34 37 Cycle End step number 63 64 61 53 3. When the synthesis interrupts, the screen will display the Interrupted Status. Press ABORT. A menu confirming your action appears. 4. Press YES to abort the synthesis. Once you choose YES, the synthesis cannot be resumed. If you abort synthesis at a step other than Cycle Entry or Cycle End, the lines and valve blocks must be rinsed and dried. To do this, select Manual Control and activate the following functions for 10 seconds each: Function 10, “#18 TO WASTE” Function 9, “#18 TO COLUMN” Function 1, “BLOCK FLUSH” Function 2, “REVERSE FLUSH” 2-18 Section 2: Operation Applied Biosystems About the Synthesis Reagents and Solvents All chemicals have been thoroughly tested at Applied Biosystems to ensure repeatedly reliable syntheses. Each reagent and solvent has a specific position on the instrument and is referred to by the number located above each receptacle. Position numbers are also printed on the bottle labels. The numbers are 1-5, 9, 11, 12, 14, 15 and 18. These numbers correspond to those on other Applied Biosystems DNA synthesizers which have 18 positions. WARNING Consider each chemical in the synthesizer potentially harmful. Do not inhale vapors. Work in a well ventilated area. Always use eye protection and wear acid-impermeable gloves and a lab coat. Do not leave any chemicals uncapped If any chemical is ingested or comes in contact with the eyes, immediately consult a physician. If there is any physical contact with a chemical, wash immediately with water. Please refer to the Material Safety Data Sheets (MSDS) in the Safety Data Section for further information about the chemicals. The MSDS provide details about physical characteristics, hazards, precautions, first aid, spill cleanup and disposal procedures. In addition, refer to Section 2: How to Change the Waste Bottle before waste disposal. How to Store Reagents Applied Biosystems chemicals must be stored properly to assure their stability. Improper storage will result in reduced shelf life. All DNA synthesizer reagents, except the iodine solution, are guaranteed for one year from the date of manufacture. The iodine is guaranteed for six months from the date of manufacture. Please follow the storage recommendations below. Store at room temperature away from excessive heat and moisture: • 1-methylimidazole, bottle 12 • Acetic Anhydride, bottle 11 • Trichloroacetic Acid, bottle 14 • Iodine, bottle 15 Store in a desiccator at room temperature not exceeding 25°C: • All phosphoramidites, bottles 1 to 5 • All CPG columns • Bulk CPG • Tetrazole, bottle 9 • Anhydrous Acetonitrile Section 2: Operation 2-19 Applied Biosystems Store refrigerated at 4°C: • Ammonium Hydroxide Note Cooler temperatures may cause crystal formation in the iodine or tetrazole solution which could clog delivery lines. Gentle warming and agitation will dissolve these crystals. Bottles 1 to 5: Phosphoramidites Reservoirs 1 through 4 contain phosphoramidites dissolved in anhydrous acetonitrile. They are used to synthesize single species sequences and mixed sequence probes: Contents adenosine phosphoramidite (A) guanosine phosphoramidite (G) cytosine phosphoramidite (C) thymidine phosphoramidite (T) Reservoir 1 2 3 4 Position 5 is a spare phosphoramidite reservoir and can be used for synthesis with modified bases. It is referred to as the “X port” or “X reservoir”. Many useful derivatives, such as deoxyinosine, are available from Applied Biosystems and can be placed in this position. In addition, Aminolink 2™, a linker for various substrates, can be placed in position 5. IMPORTANT Since all phosphoramidite reservoirs are pressurized simultaneously with a single valve, all five bottles must be attached to the instrument, even if some are empty, to perform a synthesis. Phosphoramidites are extremely sensitive to acid, oxygen and water. Once they are in solution and the protective cap is removed, quickly put them on the instrument to prevent contamination. IMPORTANT Use anhydrous acetonitrile with less than 90 ppm water to dissolve the phosphoramidites. Do not use HPLC grade acetonitrile, its higher water content will decrease coupling efficiency. Anhydrous acetonitrile is available from Applied Biosystems, Part Number 400060. Phosphoramidites are stable in powder form for one year and should be stored at room temperature in a desiccator. Once they are dissolved, they should be used within approximately two weeks. After this time, coupling efficiencies may decrease. If they cannot be used within this time, you can freeze, store, thaw and then reuse the phosphoramidites. However, they may show a loss of activity. For details, see How to Prepare Phosphoramidites and How to Store Dissolved Phosphoramidites found earlier in this section. 2-20 Section 2: Operation Applied Biosystems Bottle 9: Tetrazole Tetrazole (0.5 M) in anhydrous acetonitrile is used as the activator for the phosphoramidites. Once the protective seal is removed, quickly put the tetrazole on the instrument to prevent atmospheric water contamination. IMPORTANT Tetrazole will form a precipitate at approximately 12°C. Do not put precipitated tetrazole on the instrument. Examine each bottle before using it. If the tetrazole has precipitated, warm it slightly until it is in solution. In cold climates, the tetrazole may precipitate in the delivery lines. The resulting repairs are expensive, therefore, keep room temperatures above 18°C. Store tetrazole at room temperature. Bottle 11: Acetic Anhydride Acetic anhydride-lutidine-tetrahydrofuran (THF), (1:1:8) is one half of the capping reagent. Atmospheric water will reduce its efficiency. Upon opening the bottle, quickly place it on the instrument. Use in a well ventilated area, avoid inhalation. Store at room temperature. Bottle 12:1-methylimidazole (NMI) 1-methylimidazole (NMI) is the second half of the capping reagent. Within 24 hours of combining NMI with the acetic anhydride reagent, the solution discolors and becomes viscous. To prevent this, the two reagents are stored in separate reservoirs and are simultaneously added to the column to perform capping. Upon opening the bottle, quickly place it on the instrument. Use in a well ventilated area and avoid inhalation. Store at room temperature. Bottle 14: Trichloroacetic Acid Trichloroacetic acid (TCA) - dichloromethane (3% wt/vol) is the detritylating reagent. Store at room temperature and use with caution. Bottle 15: Iodine Iodine-water-pyridine-THF (0.1M:1:10:40) is the oxidizing reagent. It can be stored for up to 6 months from the date of manufacture. After this time, it may develop a tar (visible when the bottle is turned over) and should not be used. Note the expiration date on the bottle. Any iodine which develops the tar before the expiration date will be replaced, without charge, by Applied Biosystems. Use in a well ventilated area and avoid inhalation. Section 2: Operation 2-21 Applied Biosystems Bottle 18: Acetonitrile HPLC grade acetonitrile is used to wash the support, valve blocks and delivery lines and to remove nucleophiles from the support prior to the coupling step. IMPORTANT Use HPLC or UV grade acetonitrile with a specification of less than 300 ppm of water. Water contamination of even 1000 ppm will lead to a 1 to 2% decrease in coupling efficiency. If unsure of the water content, either check the acetonitrile using the Karl Fischer method of titration (requiring an automatic titration apparatus) or distill it over phosphorous pentoxide followed by redistillation over calcium hydride. Although Applied Biosystems supplies HPLC grade acetonitrile, it may be more economical to purchase it from a local supplier, such as Burdick and Jackson, Part Number 015. Manual Deprotection and Cleavage Reagents Ammonium Hydroxide Concentrated ammonium hydroxide is used to deprotect the DNA and cleave it from the support. Reagent grade ammonium hydroxide should be purchased in 500 mL bottles from a local source. Store it tightly sealed and keep refrigerated. It should not be used for more than one month after opening. Any loss of ammonia concentration decreases its effectiveness, therefore, only open it immediately before use. Use in a fume hood. 2-22 Section 2: Operation Applied Biosystems Reagents and Solvents Bottle Position 1 2 3 4 5 Reagent/Solvent Part Number Deoxyadenosine (dA-bz) phosphoramidite β-cyanoethyl β-cyanoethyl β-cyanoethyl (0.25g) (0.5g) (1.0g) 400600 400330 400326 Deoxyguanosine (dG-ib) phosphoramidite β-cyanoethyl β-cyanoethyl β-cyanoethyl (0.25g) (0.5g) (1.0g) 400601 400331 400327 Deoxycyctosine (dC-bz) phosphoramidite β-cyanoethyl β-cyanoethyl β-cyanoethyl (0.25g) (0.5g) (1.0g) 400603 400332 400328 Deoxythymidine (dT) phosphoramidite β-cyanoethyl β-cyanoethyl β-cyanoethyl (0.25g) (0.5g) (1.0g) 400602 400333 400329 Available for modified bases for example: Deoxyinosine (β-cyanoethyl) (0.25g) 400402 9 11 12 14 15 18 Tetrazole/acetonitrile (180mL) Acetic anhydride/lutidine/THF (180mL) 1-methylimidazole (180mL) Trichloroacetic acid (450mL) Iodine/water/pyridine/THF (200mL) Acetonitrile 4-L* Anhydrous Acetonitrile (for dissolving phosphoramidites)* Ammonium hydroxide 400606 400607 400785 400236 400753 400443 400060 400019 *It may be more economical to purchase ammonium hydroxide and HPLC grade acetonitrile from a local supplier. About Deoxyinosine Deoxyinosine base pairs with A, T and C, which makes it a suitable choice for synthesis where the degree of degeneracy needs to be reduced. A 23-mer with five inosine substitutions used a as a probe was found to bind to a complementary strand, and have similar dissociation temperature as a Section 2: Operation 2-23 Applied Biosystems 17-mer. It is not well understood how deoxyinosine affects the stabilization of the DNA duplex, however. Martin, et al determined the stability of oligodeoxyribonucleotide duplexes with each of the four normal bases using optical techniques for measuring melting temperatures.6 They observed large neighboring-base effects upon the stability of the base pairs between inosine and the normal bases. The results obtained by this group indicate that deoxyinosine reduces the specificity of hybridization probes. Ohtsuka, from the faculty of Pharmaceutical Sciences and Institute for Molecular and Cellular Biology at Osaka, Japan, has found that synthetic oligonucleotides with deoxyinosine residues at ambiguous points are useful as hybridization probes.7 Takahashi, also from Osaka University, used a synthetic probe containing deoxyinosine to isolate the cholecytokinin gene directly from a human genomic library.8 2-24 Section 2: Operation Applied Biosystems How to Change an Argon Tank You can replace an empty argon tank before beginning a synthesis or when a synthesis has been interrupted. Replace the empty cylinder as follows: 1. Shut off the cylinder at the tank and at the needle valve on the regulator. 2. Remove the line that connects the tank to the synthesizer by unscrewing the Swagelock fitting. 3. Remove the regulator from the empty tank, clean the threads on the fittings, and install the regulator on a full tank. 4. Turn the tank on and check for leaks at the connection of the tank to the regulator. 5. Open the needle valve. Note You may hear a hissing sound while the new argon supply is recharging the vacuum assist. WARNING Be sure to anchor the cylinder securely and follow required safety practices when handling gas cylinders. How to Change the Waste Bottle The synthesizer generates 1 to 2 liters of hazardous, halogenated, organic liquid waste per 100 base additions. The waste is collected in a 4-liter polyethylene bottle which is placed on the floor or on a nearby bench lower than the instrument. The bottle can be kept inside a protective carrier to contain accidental spillage. A one-gallon carrier is sufficient and can be purchased from VWR, Part Number 56609-186; or Nalge, Part Number 6501-0010. When the bottle is full, it must be emptied as follows: 1. Unscrew the cap assembly and immediately recap the bottle to prevent the release of vapors. Save and reuse old bottle caps. 2. Discard the waste. Place the liquid in a sealed container labeled “FLAMMABLE,” “POISON B N.O.S.” or absorb in vermiculite, dry sand or earth. Dispose of the waste following applicable government regulations. WARNING Section 2: Operation Synthesizer waste must be disposed of properly and carefully. When handling the waste for disposal, wear gloves and eye protection, and avoid inhalation and skin contact. Refer to the Waste Profile and MSDS in the User’s Safety Information Section for details. 2-25 Applied Biosystems 3. After disposal, securely screw the cap assembly supplied by Applied Biosystems on an empty waste bottle. IMPORTANT The waste bottle is the low pressure side of the delivery system and must always be kept vented to atmosphere. Be sure the vent line is properly routed to a fume hood. If the vent line is blocked, back pressure will be generated which will decrease the deliveries of reagents and solvents. How to Perform the Flow Test Procedure Flow test is an automated procedure used to measure the flow rates through all essential delivery lines. It can be used for routine maintenance, troubleshooting and recalibration of the instrument if flow related problems are suspected. For example, if oligonucleotide quality is low, the flow test will help determine if instrument performance is a factor. If the synthesizer has flow related difficulties, be sure to do the test prior to calling the Applied Biosystems Technical Support Department. The flow test has four parts and takes about one hour to perform. The first part washes and primes the delivery lines with acetonitrile. The second measures the flow of acetonitrile from each bottle position to the lower column Luer fitting. The third measures the flow from bottle 14 to the trityl collection line. In the final part, all the lines are flushed dry with argon enabling reattachment of the reagents. The flow test is not found in the procedure editor and cannot be edited or printed. A copy of the procedure can be found in Appendix A. How to Do the Flow Test Procedure From the Main Menu, select SELF TEST and then press MORE until the display shows: Select a test . . . Version 1.00 flowtst more main Press FLOWTST and the screen will read: Remove all bottles except #18 before you begin flowtst 2-26 begin more main Section 2: Operation Applied Biosystems Follow the instructions and remove all reagents except bottle 18, acetonitrile. Do not use the change bottles procedure. Any reagents that will be reused should be purged with argon and capped tightly. Part 1 Press BEGIN, the procedure will start and the screen will show step one: Step:1 hold WAIT jump 5 of 5 sec intrpt more main The first series of steps, 1 through 14, sequentially rinses the lines for each bottle position. Use a beaker to collect the acetonitrile rinse starting with position 15 and working backwards. Each rinse takes 20 seconds. Watch the time on the screen to know when to move the beaker to the next position. Step 12 delivers acetonitrile to the column. Collect this rinse at the lower Luer fitting (remove the column if necessary). Inspect the type of flow coming from each delivery line. Acetonitrile should flow from positions 15, 14, 12 and 11 and should drip steadily from positions 1 through 5 and 9. If the flows are not as described, retest each faulty position by using the JUMP key. Refer to Section 3: Main Menu Option: Monitor Synthesis for details on how to use JUMP. At step 14, the procedure is interrupted and the screen reads: Step:14 resume INTERRUPT jump abort 1 of 1 sec more main Part 2 This pause is used to prepare for steps 15 through 43, part two of the procedure. During this part the flow rate from each reservoir is measured for 2 minutes at the lower column Luer fitting. Before beginning, attach a clean bottle filled with HPLC grade acetonitrile to each bottle position. A set of clean, empty bottles is shipped with the instrument. Next, remove the column (if necessary) and place a 10-mL graduated cylinder under the lower column Luer fitting. When ready, press RESUME and the procedure will continue. At step 17, acetonitrile will flow from Reservoir 1 (A-phosphoramidite) to the lower Luer for exactly 2 minutes. Carefully collect the flow. A 20-second WAIT step immediately follows. Use this time to read and record the measurement. In addition, empty the cylinder and replace it under the lower Luer for the next reading. If 20 seconds is too short, simply press HOLD during the WAIT step. Then, when you are ready to continue, select RESUME. Repeat this procedure until all mea- Section 2: Operation 2-27 Applied Biosystems surements are taken. Please note that step 40, #18 to waste, is used during manufacturing and does not require measurement. Calculate the flow rate in milliliters per minute. Remember, you are collecting for 2 minutes so you must divide the mL amount by 2 to get mL/min. The correct flow rates are listed below. If they are not within specification, the argon pressure regulator must be adjusted and the flows remeasured. BOTTLE POSITION 1 to 5, 9 11, 12 14 15 18 FLOW RATE (mL/min) 1.90 to 2.10 2.80 to 3.00 2.70 to 3.10 2.80 to 3.00 2.80 to 3.20 Note that since the phosphoramidite reservoirs are pressurized simultaneously, their flow rates should be quite similar. If one flow rate is very different, the delivery line for that phosphoramidite may be clogged. If you suspect this, do not change the pressure setting. Be sure to measure all flow rates and then call the Applied Biosystems Technical Support Department. To adjust the pressure regulator, first remove the left side panel of the instrument. The regulator knob is located to the left of the top of the ballast assembly. The ballast assembly is shown as item 14 in Figure D-2, in Appendix D. To access the regulator gauge, remove the back panel of the instrument. The gauge is shown as item 15a in Figure D-3, in Appendix D. The regulator is usually set at approximately 4.0 psi. If the flow rate is too slow, increase the regulator setting by turning the knob clockwise approximately 1/8th of a turn. Using JUMP, perform the delivery step for the faulty position for at least 90 seconds to equilibrate the pressure. For example, if the A-phosphoramidite flow rate is too slow, increase the pressure as just described and jump to step 17, A to column, for equilibration. Next, using JUMP again, repeat the delivery step (i.e. step 17, A to column) and remeasure the flow rate for 2 minutes. Similarly, if the flow is too fast, decrease the regulator setting by turning the knob counterclockwise about 1/8th of a turn. Next, remove the bottle to relieve the excess pressure and then replace it. As above, equilibrate the bottle pressure for 90 seconds and remeasure the flow rate for two minutes. Once the pressure has been changed, remeasure each position to be sure all flow rates are within specification. If all flow rates cannot be adjusted to specification or if you do not feel comfortable adjusting flow rates, please call the Technical Support Department. Do not use the synthesizer until all flow rates are correct. 2-28 Section 2: Operation Applied Biosystems Part 3 At step 43, the procedure is interrupted again. Use this time to prepare for part three, steps 44 to 46. Connect the upper and lower column Luer fittings with the clear plastic shipping tube provided with the instrument. Place the trityl collection line inside a beaker. Place a 10-mL graduated cylinder next to the beaker. When you are ready, select RESUME. This step is timed for 150 seconds. Use the first 30 seconds to prime the line. Then watching the time on the display, place the trityl collection line inside the 10-mL graduated cylinder and collect the flow for 120 seconds. Calculate the flow rate in milliliters per minute. The flow rate should be 2.30 to 2.60 mL/min. If the flow rate is not correct, there may be a problem with the post-column valve block. If you suspect this, call the Technical Support Department for help. Part 4 The procedure is interrupted again at step 46. In part 4, steps 47 to 59, the delivery lines and valve blocks are flushed dry with argon. Press RESUME and the residual acetonitrile will be collected in each bottle starting at position 15 and going backwards. The test is now complete. Reagents can be reattached using the Change Bottles Menu. As each flow test bottle is removed, wipe the exposed delivery line with a lint-free tissue. Once all chemicals are attached, synthesis can begin. Section 2: Operation 2-29 Applied Biosystems Aminolink 2™ Reagent Introduction Aminolink 2 is a DNA synthesis reagent which affords a convenient method to introduce an aliphatic primary amine at the 5' end of oligonucleotides. This amine can react to form oligonucleotide conjugates with a variety of substrates such as biotin9,10, fluorescent dyes11, EDTA12, or alkaline phosphatase.13 Applications include: • Non radio-labeled hybridization probes9,10,13,14 • Sequence specific cleavage of single stranded DNA12 • Automated DNA sequencing - affinity chromatography Aminolink 2 offers many advantages. Because it is a phosphoramidite, it reacts like conventional nucleoside phosphoramidites. The compound is dissolved in anhydrous acetonitrile and placed on the extra phosphoramidite port (bottle position 5, X). It is activated with tetrazole and couples with the same high efficiency found in standard oligonucleotide synthesis. In addition, when using Aminolink 2 you do not need to modify the synthesis cycle. Other features include: • Solution stability is comparable to nucleoside phosphoramidites (at least 2 weeks). • No special cycles or protocols are required. • Labeling at the 5'-terminal amine does not interfere with hybridization. • Aminolinked oligonucleotides can be easily labeled with a variety of commercially available reagents such as fluorescein isothiocyanate and biotin NHS ester. Chemical Description Aminolink 2 (structure shown in Figure 2-2) generates an amino hexyl phosphate linker with a methoxy phosphate protecting group. Prior to deprotection, the phosphite triester is oxidized to the phosphate triester using standard oxidizing reagents. The amine group is protected by a trifluoroacetyl group, which is removed during standard ammonia deprotection procedures. 2-30 Section 2: Operation Applied Biosystems Figure 2-2. Aminolink 2 — Oligonucleotide Section 2: Operation 2-31 Applied Biosystems How to Use Aminolink 2 Aminolink 2 (Part Number 400808) is supplied in 250 mg quantities. Store the unopened linker desiccated away from heat and moisture. 1. Dilute Aminolink 2 with 3.3 mL of anhydrous acetonitrile following the instructions in How to Prepare Phosphoramidites. (Once diluted and placed on the instrument, the compound is stable for at least 2 weeks.) 2. Place Aminolink 2 on bottle position 5. 3. Using the DNA Editor, enter X (the Aminolink bottle position), as the first base, 5' end of the sequence. 4. Configure the synthesis “Trityl ON” since detritylation is not necessary after Aminolink 2 addition. 5. Deprotect and cleave the Aminolink-oligonucleotide following the instructions in the Manual Deprotection and Cleavage Procedure. Although Aminolink has a methyl phosphate protecting group, thiophenol deprotection is not necessary. Ammonia treatment will demethylate the single methyl phosphotriester group of the Aminolink-oligonucleotide without adversely affecting product purity. Analysis and Purification of Aminolink™-Oligonucleotides Aminolink-oligonucleotides can be analyzed and purified by the same methods used for conventional oligonucleotides. Since the 5'-hydroxyl, usually available for phosphorylation by kinase and ATP, is blocked by the linker, an alternate 3'-end labeling procedure must be used to radiolabel Aminolink-oligonucleotides. To effect a 3'-end label, the enzyme Terminal Deoxynucleotidyl Transferase (TDT), is used. TDT adds a single nucleotide to the 3'-hydroxyl. Because the enzyme would continue propagation beyond the first nucleotide addition, a dideoxynucleotide analogue of [alpha-32P]-dATP is used. The analogue, [alpha-32P]- Cordycepin-5'- triphosphate, does not have a 3'-hydroxyl and therefore further addition is prevented. Below is a protocol for the labeling procedure. 3'-End-Labeling 1. Prepare a stock solution of 10X 3'-end-labeling buffer: 1.4 M Sodium Cacodylate* 10 mM Cobaltous Chloride 1.0 mM Dithiothreitol pH 7.5 with HCl *This chemical is carcinogenic, handle with care. 2-32 Section 2: Operation Applied Biosystems 2. Aliquot 2 pmol of oligonucleotide into an Eppendorf-type tube (1.5 mL), dry in a Speed-Vac and ethanol rinse with 10 µL of 95% Ethanol, dry. 3. Add the following to the tube, while on ice: 1 µL of 10X 3'end-labeling buffer 1 µL of [alpha-32P]-Cordycepin-5'Triphosphate (New England Nuclear #NEN-114, specific activity greater than 5000Ci/mmol) 7 µL of deionized water 1 µL of TDT (ENZO Biochemicals, 5 units per microliter) 4. Spin the tube very briefly in a microcentrifuge to bring contents of tube to the bottom. Incubate sample at 37°C for one hour. 5. Add 25 µL of 1.0 mM EDTA in deionized formamide loading buffer to the sample. Bromophenol Blue and Xylene Cyanol can be added as marker-dyes to the formamide prior to addition to the sample. The sample can be loaded onto an analytical gel in the same manner as described in Applied Biosystems DNA Synthesis User Bulletin No. 13 (Revised), Analysis and Purification of Synthetic Oligonucleotides. The linker retards the migration of the oligonucleotide by approximately one-half of a base in the gel. There is occasionally a radioactive contaminant which is a by product of the labeling procedure. This contaminant is most likely a type of enzyme-Cordycepin complex which co-electrophoreses with the sample and may appear on the autoradiogram as a fuzzy band in the region of a 16-mer. It is recommended that a blank sample containing the constituents of the reaction but without DNA be loaded on the gel so that the contaminant can be compared with the DNA sample. Note that since phosphorylation of the 5-hydroxyl by T4 kinase should be blocked by the presence of the linker, this procedure can be used as a qualitative analysis of the efficiency of Aminolink 2 addition. Anion-exchange HPLC, described in User Bulletin No. 13 (Revised) can be used for Aminolinkoligonucleotide analysis or purification. The inherent problem with using reverse phase: HPLC for purification is that the oligonucleotide and the Aminolink oligonucleotide are not well resolved. Gel purification seems to give better results. Once a dye is attached, reverse phase HPLC may be used to purify the derivatized oligonucleotides. Section 2: Operation 2-33 Applied Biosystems Applications General Protocol for Tag/Labeling Substrate Described below is a generic protocol from which to extrapolate individual application protocols. Note that most commercially available biotinylation reagents come with usage protocols. 1. Prepare a solution of Aminolink-oligonucleotide at a concentration of 1mM or greater in a 50mM-200mM-NaHC03/Na2CO3 Buffer, pH 9. 2. Add a 50-molar excess of the solid tag/labeling substrate (NHS-Biotin, Dye, etc.) and vortex to dissolve. If the tag or DMSO is insoluble in aqueous solutions, it can be dissolved in dimethylformamide before addition to the Aminolink-oligonucleotide. The final concentration of organic solvent in the reaction mixture should not exceed 30%. 3. After several hours reaction time at room temperature, the mixture can be purified by gelexclusion in a G-50 or G-25 Sephadex™ column to remove the excess tag. Use 100mM TEAA as the eluant. Note 2-34 Reactions with fluorescent dyes should be done in the dark. Section 2: Operation Applied Biosystems How to Use OPC (Oligonucleotide Purification Cartridge) Introduction OPC (Part No. 400771) is a rapid purification cartridge used specifically for synthetic DNA. It provides the level of purity required for the common applications of synthetic DNA. Using OPC eliminates the need to purify by time-consuming and labor intensive polyacrylamidge gel electrophoresis (PAGE) or HPLC. If you currently only desalt your synthetic oligonucleotides, you can now use OPC to desalt and purify in less time than it takes to desalt alone. OPC is fast, easy to use and delivers consistent results. The time required to go from the deprotected, crude oligonucleotide to its use in an experiment is greatly reduced. Complete purification requires only 15 minutes. After completing deprotection and cleavage, an aliquot of the crude oligonucleotide, still in ammonia is diluted with water and then loaded directly onto the OPC cartridge. This eliminates the need to evaporate the sample prior to purifying. The remaining portion of the crude synthesis can then be stored in ammonia either refrigerated or at -20°C without harming the DNA. In fact, this is preferable to storing it as an evaporated sample or dissolved in water. The ammonia protects the sample from acidic by products present in the crude. Detritylation is also done on the cartridge, eliminating the need for post-purification work-up. The purified oligonucleotide is then eluted as a trityl-off species in a volatile buffer. Quantification can be easily accomplished and only the volume containing the amount required for the experiment need be evaporated. The reminder can be stored at -20°C for later use. Once dry, the pure oligonucleotide can be redissolved in an appropriated buffer and used in your experiment. The product obtained from OPC is highly-purity DNA that can be used with complete confidence. The OPC purification protocol appears at the end of this section. It successfully purifies sequences up to 70 bases. Note that for sequences 40-70 bases long, you must perform an additional step, number 9a. This step adds several washes with dilute ammonia after detritylation with 2% trifluoroacetic acid. The extra basic wash removes preferentially any shorter sequences that had trityl protecting groups attached and thus co-purified with the desired product. The purification of longer oligomers (>70-mers) using OPC can be more difficult. But, a protocol has been developed by Thomas Horn and Mickey Urdea of Chiron Co.15 in which you pre-treat the oligonucleotides with lysine and then perform the OPC purification procedure. (See the Protocol for Lysine Pre-treatment of Longmers (>70 bases).) The usefulness of the lysine pre-treatment is dependent on the amount of depurination in the oligonucleotide. The greater the depurination, the more useful the procedure might be. Depurination is sequence and reagent dependent. The higher the purine content of a sequence, the greater the chances are of depurination occurring. The higher Section 2: Operation 2-35 Applied Biosystems the reagent purity, the smaller the chances are of depurination occurring. Because of Applied Biosystems high purity reagents, depurination is usually not detectable. More about lysine treatment of longmers (>70 bases) before OPC purification: For long oligomers (even with very high synthesis efficiency) the desired, full length product is a minor component compared to the sum of the failure sequences. Certain side reactions also become more prevalent due to increased exposure to the synthesis reagents. In particular, the acids used for detritylation (trichloroacetic or dichloroacetic acid) can promote cleavage of the purine nucleobases (A and G) from the ribose rings. During synthesis of a long-mer, the cumulative time that the bases near the 3' end are subjected to acid can be more than an hour. During this time apurinic sites can be created. These apurinic sites in the oligonucleotide undergo internucleotide cleavage during ammonia deprotection at 55°C. When the synthesis is conducted Trityl On, in preparation for OPC purification, some of the cleavage products will bear a 5'-trityl group (see Figure 2-3). Apurinic cleavage can generate trityl bearing species that are less than full length. Reverse phase HPLC or OPC purification methods, which depend on trityl selectivity, are then less efficient in purifying the full-length oligo. 3' CPG CPG AGTCAGTGCGCTTAGCCATAAG 3' AGTC*GTGCGCTTAGCCATAAG -2 5' 5' CPG + 3' AGTC-OPO + GTGCGCTTAGCCATAAG 3 DMT (full-length oligo) DMT (oligo with apurinic site) 5' DMT (ammonia cleavage products) Figure 2-3. Thomas Horn and Mickey Urdea of Chiron Co. published a procedure for extending the utility of OPC to the purification of long oligonucleotides15. They found that apurinic sites undergo internucleotide cleavage with 1 molar lysine, without cleavage of the oligonucleotide from the support. (See the Protocol for Lysine Pre-treatment of Longmers (>70 bases).) The DMT bearing cleavage fragment is washed away, leaving a non-DMT bearing fragment bound to the support. Following ammonia cleavage and deprotection, they used the OPC purification protocol. This was effective in purifying oligomers up to 118 bases in length. Although depurination is a valid concern during DNA synthesis of long oligonucleotides, the degree of depurination encountered during synthesis is highly sequence and reagent dependent. Since 2-36 Section 2: Operation Applied Biosystems the 3' bases of an oligo (the initial couplings) have the greatest reagent exposure, varied purine content in this area will generate varied potential for depurination. Another factor in depurination is the purity of the acid used for detritylation. Contaminants, such as water or HCl, in the trichloroacetic or dichloroacetic acid will greatly promote depurination. Applied Biosystems supplies high purity trichloroacetic and dichloroacetic acid reagents. In a study involving the synthesis of a 72-mer, the use of Applied Biosystems reagents did not promote detectable depurination. Analysis of the 72mer synthesis, OPC purified with and without prior lysine treatment, showed no detectable purity difference. These results may not apply to all synthesis sequences and lengths, but the study demonstrates Applied Biosystems commitment to synthesis reagent and product quality. Protocol for Lysine Pre-treatment of Longmers (>70 bases) 1. Synthesize the oligonucleotide Trityl ON. 2. Dry the column by reverse flushing (function 2) for 60 seconds with argon or allow the column to air dry. 3. Remove the phosphate protecting groups: Connect a syringe (with plunger) to one end of the column. In a fume hood, fill another syringe with the following solution, attach it to the synthesis column and fill the column with the solution. For cyanoethyl phosphoramidites: add 1 ml of t-butylamine:pyridine (1:9 v/v) and treat at room temperature for 1 hour (t-butylamine; Aldrich B8,920-5 and pyridine; Aldrich 27,040-7). Periodically circulate the solution by alternately depressing the syringe plungers. Note Gently depress the syringe plungers; otherwise you risk breaking the filters that hold the CPG. 4. Remove the solution and wash 5 times with acetonitrile. 5. Allow the column to air dry or blow the column dry with argon. 6. Prepare a 1M lysine solution pH = 9 (adjust pH with NaOH if necessary) (L-lysinemonohydrochloride; Sigma # L5626, Aldrich # L460-5). 7. Place 2 ml of the lysine solution into a 16 X 100 mm test tube. Connect a single syringe to one side of the synthesis column and a male-to-male luer fitting (AB p/n 110127) the other. Insert the column-syringe-luer apparatus into the test tube and draw the lysine solution into the column. Incubate at 55°C for 90 minutes by inserting the test tube and contents into a heating block. The lysine solution should be periodically circulated (every 30 minutes). Note The luer fitting of the syringe must be located in the center of the syringe barrel to allow easy insertion into the test tube. 8. After incubation in the lysine solution, wash the column 5 times with water. 9. Complete the Manual Deprotection and Cleavage Procedure described earlier in this section. Then follow the OPC purification protocol. Section 2: Operation 2-37 Applied Biosystems OPC Purification Protocol Solutions Needed • HPLC grade acetonitrile, 5 mL • 2.0 M triethylamine acetate (Part No. 400613), 5 mL • Deionized water, 1 mL • Dilute ammonium hydroxide, 15 mL (1:10 dilution of conc. ammonium hydroxide in deionized water) • 2% trifluoroacetic acid in deionized water, 10 mL (Neat TFA Part No. 400137) • 20% v/v acetonitrile in deionized water, 1 mL 1. After completion of a Trityl On synthesis, cleave the oligonucleotide from the support and deprotect following the Manual Deprotection and Cleavage Procedure found earlier in this section. 2. Connect an all-polypropylene syringe (Aldrich Zl 1686-6) to one end of the OPC column and a male-to-male Luer tip to the other end. See Figure 2-4. Make sure all fittings are snug. 3. Flush the cartridge with 5 mL HPLC grade acetonitrile, followed by 5 mL 2.0 M triethylamine acetate. Note Keep the flow rate at 1 to 2 drops per second for all reagent additions. 4. Dilute the ammonium hydroxide solution containing the cleaved, deprotected, “trityl-on” crude oligonucleotide with one-third volume of deionized water. Figure 2-4. 5. Load 20 - 40 OD’s of the above solution into the syringe and then gently push it through the cartridge, saving the eluted fraction. Reload this fraction, and again push it through the cartridge.This will load 1 to 5 OD units of the trityl oligonucleotide (depending on length, sequence, and synthesis quality) onto the cartridge. Save this final eluted fraction in ammonia at -20°C, it can be put through another cartridge until exhausted of trityl oligonucleotide. 6. Flush cartridge with 3 x 5 mL dilute ammonium hydroxide. 7. Flush cartridge with 2 x 5 mL deionized water. 2-38 Section 2: Operation Applied Biosystems 8. Detritylate the OPC-bound oligonucleotide with 5 mL of the 2% trifluoroacetic acid solution. Gently push ~1mL through the cartridge, incubate for 5 minutes, then flush the remaining TFA solution through the cartridge. 9. Flush cartridge with 2 x 5 mL deionized water. 10.(For sequences ≥40 bases, add this step) Flush cartridge with 1 x 5 mL dilute ammonium hydroxide, then 2 x 5 mL deionized water. 11. Elute the purified, detritylated oligonucleotide by flushing the cartridge with 1 mL of the 20% acetonitrile solution. 12.Evaporate to dryness an aliquot of the eluate (step 10) and dissolve in water to determine the OD units at A-260. 13.Store any unused OPC-purified oligonucleotide as a dry solid at -20°C. Section 2: Operation 2-39 Applied Biosystems References 1. --------------, Evaluation and Purification of Synthetic Oligonucleotides, Applied Biosystems User Bulletin 13 (Revised) (1987). 2. Gait, M.J., and Sheppard, R.C., Nucleic Acids Research 4, 4391 (1977). 3. Tanaka, T. and Letsinger, R.L., Nucleic Acids Research 10, 3249 (1982). 4. Schott, M.E., A simple manual method for oligonucleotide synthesis, American Biotechnology Laboratory 3, 20-23 (1985). 5. McBride, L.J., Eadie, J.S., Efcavitch, J.W., and Andrus, W.A., Base modification and the phosphoramidite approach, Proceedings of the 7th International Round Table on Nucleosides, Nucleotides and Their Biological Applications, (1986). 6. Martin, F.M., Castro, N.M., Aboula-ela, F., Tinoco, I., Base Pairing Involving Deoxyinosine: Implications for Probe Design, Nucleic Acids Research 13, 8927-8938, (1985) 7. Ohtsuka, E., Matsuki, S., Ikehara, M., Takahashi, Y., and Matsubara, K., An Alternate approach to Deoxyoligonucleotides Hybridization Probes by Insertion of Deoxyinosine at Ambiguous Codon Positions, J. Biol. Chem. 260, (5), 2605-2608, (1985) 8. Takahashi, Y., Kato, K., Hayashizaki, Y., Wakabayashi, T., Ohtsuka, E., Matsuka, S., Ikehara, M., and Matsubura, K., Molecular Cloning of Human Cholecytokinin Gene by Use of a Synthetic Probe Containing Deoxyinosine, PNAS 82, 1931-1935, (1985) 9. B.C.F. Chu and L.E. Orgel, DNA 4 327-331 (1985). 10.A. Chollet and E.H. Kawashima, Nucleic Acids Research 13 11. L.M. Smith et al., Nature 321 1529-1541 (1985). 647-679 (1986). 12.G.B. Dreyer and P.B. Dervan. Proc. Natl. Acad. Sci. USA 82 13.E. Jablonski, et al., Nucleic Acids Research 14 14.B.A. Connolly, Nucleic Acids Research 15 968-972 (1985). 6115-6128 (1986). 3131-3137 (1987). 15. Horn, T. and Urdea, M. S., Nuc. Acids Res. 16 (24), 11559-11574 (1988). 2-40 Section 2: Operation Section 3: Software Menu Descriptions This section describes each menu in detail. The most frequently used menus are the DNA Editor, Start Synthesis, Monitor Synthesis, and Change Bottles. Be sure you carefully read about these menus. You can use this section while viewing the synthesizer. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-2 Software Abbreviations and Symbols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-4 The Main Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-5 Summary of Main Menu Options. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-6 Main menu options in detail: Main Menu Option: DNA Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-7 Main Menu Option: Start Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-13 About the Ending Method: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-14 Main Menu Option: Monitor Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-16 Instrument Status: Synthesizing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-17 The Holding Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-19 The Jump Step Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-22 The Interrupt Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-24 Main Menu Option: Change Bottles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-29 About the Alarm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-31 Bottle Usage Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-33 Main Menu Option: Cycle Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-34 The Base Specifier Field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-37 Main Menu Option: Manual Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-40 Main Menu Option: Fract Pulse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-42 Main Menu Option: Procedure Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-42 Main Menu Option: FXN Editor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-44 Main Menu Option: Power Fail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-47 Main Menu Option: Self Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-48 Flow Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-50 Main Menu Option: Set Clock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-51 Main Menu Option: Shut Down . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-52 Applied Biosystems Introduction The Model 391 software is “menu driven”. Menus and pages of menus are shown on a 2-line liquid crystal display (LCD). The menus present various options and necessary information about the synthesis or status of the instrument. In response, you select an option and give instructions by pressing the appropriate key on the keyboard. Figure 3-1. The Keyboard The keyboard has several labeled keys (0-9; A, G, C, T, X; delete; left and right arrows) and 5 “soft keys” (directly below the display). The labels and actions of the soft keys change depending on the information displayed directly above them. Pressing one of the gray keys labeled 0 through 9, enters a numeric value at the cursor position. 0-9 A C G T Pressing the appropriate gray key, labeled A, G, C, T or X, enters a base in a DNA sequence. These keys operate when viewing the DNA Editor Menu. X ---> <--- To make entries or deletions, the cursor must be in the correct position and often must be moved. Pressing the right (→) or left (←) arrow key moves the cursor one position in the arrow’s direction. Continuously pressing an arrow repeats the action. In menus that have multiple entry areas, arrows also move the cursor from one entry area to another. 3-2 Section 3: Software Menu Descriptions Applied Biosystems Pressing the delete key erases an entry at the cursor position. When the cursor is not under an entry, the deletion is made to the left of the cursor. Continuously pressing delete will repeatedly erase entries. DELETE In addition to using the delete key to erase numerical entries, mistakes can be corrected by moving the cursor to the incorrect number and typing over it. The MAIN and MENU Soft Keys The MAIN and MENU keys appear in the far right positions of most displays. MAIN Pressing the MAIN always returns to Page 1 of the Main Menu. MENU Pressing MENU returns to the display which was previously shown. MENU can be selected repeatedly until the MAIN key appears. Section 3: Software Menu Descriptions 3-3 Applied Biosystems Software Abbreviations and Symbols 3-4 # number 1µM one-micromole β-cyanoethyl synthesis cycle .2µM two-tenth micromole β-cyanoethyl synthesis cycle 10µM ten-micromole β-cyanoethyl synthesis cycle #1 A-PHOS bottle change for reservoir 1 containing adenosine phosphoramidite (as seen in Change Bottles) #1 BOTTLE CHANGE bottle change procedure for reservoir 1 containing adenosine phosphoramidite (as seen in the Procedure Editor Menu) C1 Cycle-1 CLR clear Fract pulse fraction collector pulse time Fxn function Imediate immediate Intrpt interrupt Low low consumption, two-tenth micromole β-cyanoethyl synthesis cycle Nxt cyc next cycle Nxt DNA next DNA strand #PP PHOS PURGE phosphoramidite purge procedure Prev previous Proc procedure RAM random access memory ROM read only memory #SD SHUT DOWN shut down procedure Std standard Synth synthesis Section 3: Software Menu Descriptions Applied Biosystems The Main Menu Once the instrument is connected to a power source and the power is turned on, the following display appears: Applied Biosystems 391 DNA Synthesizer PCR-MATE EP Ver. 1.00 start Select START and the screen will display Page 1 of the Main Menu which presents five options: DNA start change cycle more editor synth bottles editor menu Selecting MORE displays Page 2 of the Main Menu and shows additional options: manual start proc fxn more control pulse editor editor menu Selecting MORE again displays Page 3 of the Main Menu and shows additional options: power self set shut more fail test clock down menu Selecting MORE again returns to Page 1 of the Main Menu. All other menus or menu pages are accessed by selecting the appropriate key from Page 1, 2 or 3 of the Main Menu. To move from one menu (e.g., DNA EDITOR) to another (e.g., START SYNTH) requires returning to the Main Menu by pressing the MAIN key. Note that sometimes, MENU is shown instead of MAIN. Pressing MENU will return to the previous menu. Continue to press MENU until the MAIN key is displayed. Section 3: Software Menu Descriptions 3-5 Applied Biosystems Summary of Main Menu Options DNA EDITOR Creates, edits and prints up to four DNA sequences. START SYNTH Initiates the steps to begin a synthesis. From this menu, you specify the cycle, ending method and DNA strand you want to synthesize. MONITOR SYNTH Displays the status of the instrument during a synthesis. Use it to identify which base is being added and which step and function are being performed. Use Monitor Synth to interrupt or abort a synthesis and to manipulate the synthesis cycle by using the HOLD and JUMP keys. In addition, during synthesis the ending method can be changed from page 4 of this menu. Note When the instrument is ready to begin a synthesis, START SYNTH is displayed on Page 1 of the Main Menu. However, during a synthesis, MONITOR SYNTH is shown in that position. CHANGE BOTTLES CYCLE EDITOR MANUAL CONTROL 3-6 Provides a procedure for removing empty reservoirs and replacing them with bottles of fresh reagents. It also shows bottle usage and provides an alarm to alert you that reagent levels are low. Creates, edits and prints synthesis cycles. Used to manually activate functions and to open and close individual valves. FRACT PULSE Changes the pulse time which advances the fraction collector. PROC EDITOR Used to create, edit and print the bottle change, phosphoramidite purge and shut down procedures. Section 3: Software Menu Descriptions Applied Biosystems FXN EDITOR Used to create and edit user functions and to print the standard, user and test functions. POWER FAIL Used to program the instrument to automatically restart synthesis following a power outage or to interrupt the synthesis until you restart or abort it. SELF TEST Verifies the correct operation of the electronic components of the instrument. Access the Flow Test procedure. SET CLOCK Used to enter the current time and date. SHUT DOWN Prepares the instrument for long term storage by washing and drying all chemical pathways. Main Menu Option: DNA Editor The DNA EDITOR is used to create, edit and print up to four DNA sequences. The sequences or strands are named DNA-1, DNA-2, DNA-3 and DNA-4. When DNA EDITOR is selected, the screen displays one of the DNA strands and shows the number of bases in that strand. For example, the following menu shows DNA-1 which currently has zero bases: Select action for edit copy print DNA-1 0-mer next main Follow the prompt and select an action for DNA-1: EDIT Select to create, edit or delete DNA strand designated in the menu. COPY Select to copy the designated DNA strand into any other DNA strand. PRINT When printer is connected to the instrument, select this option to print the designated DNA strand. NEXT Select to access the next DNA strand for subsequent editing, copying, and/or printing. Section 3: Software Menu Descriptions 3-7 Applied Biosystems The Edit Key When EDIT is selected, the display reads: 1 -5'>_ ( <3' 0 space ) erase menu The 5' and 3' positions are clearly marked and the cursor is to the right of 5'>. The number at the top left corner of the screen signifies which DNA strand is being shown (i.e. 1 = DNA-1). The number at the top right comer of the screen indicates the base position number. It equals zero when there are no entries and increases by one as each base is entered. Also, it displays the base number corresponding to the cursor position. For example, when the cursor is under C in the sequence 5'AGCT 3', C is shown as base number 3: 5'>AGCT <3' 3 IMPORTANT Following standard convention, The DNA sequence is entered 5' to 3'. The DNA Editor Menu displays the 5' terminus as Base 1 and subsequent bases are numbered 5' to 3'. However, the DNA strand is actually synthesized 3' to 5'. To enter a sequence, begin by pressing the gray key (labeled A, G, C, T or X) which corresponds to the 5' terminus. The base will appear on the screen, the cursor will move one position to the right and the base position number will increase to one. The display will read: 1 -5'>A_ ( <3' 1 space ) erase menu Continue to enter the sequence (5' to 3') until it is the specified length. When finished, check it carefully to be sure it is correct. If possible, print the sequence to verify it. ( ) Use parentheses to insert multiple bases in one position for synthesizing mixed sequence probes. This is done by selecting the left “open” parenthesis, “( ”, typing any combination of bases A, G, C, T or X; and then selecting the right “close” parenthesis, “)”. For example, to insert AGCT in the third position of the sequence 5'AA 3': 3-8 Section 3: Software Menu Descriptions Applied Biosystems select ( 5'>AA_ <3'; ; 5'>AA(_ <3'; press A, G, C, T ; 5'>AA(AGCT_ select ) ; 5'>AA(AGCT)_ <3'; <3'; continue entering the sequence Multiple bases are added to the support-bound nucleotide by simultaneous delivery of the specified phosphoramidites. They can be placed in any position except the 3' terminus. SPACE Inserts a space to the left of the cursor position. For example, to insert a space following C in the sequence 5'AGC 3': 5'>AGC_ <3' press space 5'>AGC _ <3' To insert a space between C and T in the sequence 5'AGCTAG 3': 5'>AGCTAG <3' press space 5'>AGC TAG <3' Spaces inserted every three bases allow the sequence to be entered as triplet codons. Also, placing spaces at specified intervals simplifies verification of the sequence. This is for convenience only and has no effect on the synthesis. ERASE Select to delete all entries from the DNA strand being edited. Upon selecting ERASE, the following display appears: 1 - To erase DNA strand, press yes. no yes menu YES Deletes the DNA strand designated in the menu and returns to the previous menu. NO Returns to the previous menu without deleting the sequence. Section 3: Software Menu Descriptions 3-9 Applied Biosystems How to Insert a Base Insertions are made at or to the left of the cursor. For example, to insert T in the third position in the sequence 5'AAA 3': 5'>AAA <3' 5'>AATA <3' 5'>AAAT_ <3' select T To insert T in the fourth position in the sequence 5'AAA3': 5'>AAA_ <3' select T Continuously pressing the button corresponding to a base, will repeatedly insert that base. How to Delete a Base Most deletions are made at the cursor position. For example, to delete C from the sequence 5'AGCT 3': 5'>AGCT <3' select delete 5'>AGT <3' When the cursor is at the 3'-end of the sequence, the deletion is made to the left of the cursor. For example, to delete T from the sequence 5'AGCT 3': 5'>AGCT_ <3' select delete 5'>AGC_ <3' Multiple bases within parentheses are deleted when the cursor is on either parenthesis. For example, to delete AGCT from the third position in the sequence 5'AA(AGCT)TTT 3': 5'>AA(AGCT)TTT <3' select delete 5'>AATTT <3' To erase a single base within parentheses place the cursor on the unwanted base and press delete. For example, to delete C from the sequence 5'AA(AGCT)TTT 3': 5'>AA(AGCT)TTT <3' select delete 5'>AA(AGT)TTT <3' Continuously pressing delete will repeatedly erase entries. 3-10 Section 3: Software Menu Descriptions Applied Biosystems How to Change a Base To change a base, you first insert the desired base, then delete the unwanted base. For example, to change 5' AATG 3' to 5' AACG 3': 5'> AATG <3' select C 5'> AACTG <3' select delete 5'> AACG <3' You can enter up to 250 characters (a combination of bases, spaces and parentheses) to create a single sequence. The screen shows a maximum of 28 characters at a time. When additional characters are entered, the display shifts to the left and the base(s) at the 5' position will no longer be seen. To observe parts of a sequence not displayed, press the right or left arrow key (→ or ←) to move the cursor toward the area you want to see. Note Once a synthesis has begun, the DNA sequence which is currently being synthesized cannot be changed. The DNA Editor Menu can be accessed during an active synthesis and new DNA sequences can be created for use in subsequent syntheses. Entering a new sequence will not affect the one currently being made even if the active strand is erased or edited. The Copy Key The COPY key is used to transfer an exact duplicate of one DNA strand (e.g. DNA-1) into another DNA strand (e.g. DNA-2). This feature is useful when performing consecutive syntheses of similar but not identical sequences. For example, if DNA-1 has a 60 base sequence, the sequence can be copied into DNA-2. DNA-2 can then be edited instead of having to enter the entire sequence. To transfer a copy of DNA-1 into DNA-2, the first page of the DNA editor should read: Select action for edit copy DNA-1 print 60-mer next Choose COPY and the menu will state that the sequence in DNA-1 will be copied into another DNA strand (in this example, DNA-2): Copy DNA-1 into enter Section 3: Software Menu Descriptions DNA-2 0-mer next menu 3-11 Applied Biosystems If DNA-1 will be copied into a different location, select NEXT until the correct DNA strand is shown. In this example, DNA-2 has zero bases. However, the copy command will over-write any DNA sequence currently in DNA-2. Select ENTER to execute the copy command. The DNA Edit Menu will then be displayed showing the sequence in DNA-2 and enabling any necessary editing. Note that the DNA sequence will not only be in DNA-2, but will also remain in DNA-1. 2-5'>AGC AGC AGC AGC AGC AGC AGC <3' 60 ( space ) erase menu The Print Key The printout from the DNA Editor not only includes the DNA sequence, but also lists the total number of bases, the molecular weight, the base composition, and the time and date. Note that base X and mixed-base sites are not included in the molecular weight calculation. The following example shows a DNA Editor printout. DNA SEQUENCE 1 3-12 NUMBER OF BASES: BASES USED DALTONS: TIME DATE: 10 A=2 C=2 2644 10:00 06/01/89 5' > AGCTXXAGCT <3' G=2 T=2 X=2 Section 3: Software Menu Descriptions Applied Biosystems Main Menu Option: Start Synthesis This section describes the software of the start synthesis menu. For more details on how to begin a synthesis refer to Section 2: Pre-synthesis Check List and How to Begin a Synthesis. When the instrument is ready to begin a synthesis, the START SYNTH (synthesis) key is displayed on Page 1 of the Main Menu. Upon selection, the screen shows the Configure Synthesis Menu. Use it to choose the DNA strand to be made and the synthesis cycle and ending method to be performed: DNA-1:12 Make begin nxt DNA Cycle-1:63 nxt cyc Trityl OFF ON/OFF main The above example shows DNA-1 which has a 12 base sequence (DNA-1:12), Cycle-1 which has 63 steps (Cycle-1:63) and the ending method which is trityl off. Note that the most recently edited DNA strand and cycle are automatically displayed. If the menu shows the correct information, synthesis can begin. Any incorrect information must be changed using the following keys: NXT DNA Specifies the subsequent DNA strand. Continue to press this key until the correct DNA strand is displayed. NXT CYC Specifies the subsequent synthesis cycle. Continue to press this key until the correct cycle is displayed. Note When the main power is first turned on, the Applied Biosystems ROM cycles are automatically loaded into the correct RAM cycle locations as shown on the next page. For example, to synthesize using the .2µM cycle, simply display Cycle-1:63. (You can create your own cycles by editing a RAM cycle. Once you change a cycle, the edited version will appear in the Start Synthesis Menu. Refer to The Cycle Editor Menu for instructions.) ROM cycle name RAM Cycle location Number of steps .2µM Cycle-1 63 1µM Cycle-2 64 Low Cycle-3 61 10µM Cycle-4 53 Section 3: Software Menu Descriptions 3-13 Applied Biosystems ON/OFF Specifies the desired ending method. When one ending method is shown, pressing ON/OFF will display the other. About the Ending Method: The 5' terminus of the fully synthesized DNA chain can either remain protected by a dimethoxytrityl group (trityl on), or can be detritylated to yield a 5'-hydroxyl (trityl off). Trityl on is usually selected when purifying by trityl-specific OPC or reverse phase HPLC. Trityl off is usually chosen when purifying by gel electrophoresis or ion exchange HPLC. Note If trityl on is specified, the DNA can be manually detritylated by a 15-minute treatment with 80% acetic acid at room temperature. This is done following synthesis after base deprotection. When all synthesis information is displayed correctly, press BEGIN. Once pressed, you can no longer change the DNA strand and the synthesis cycle selection. However, cycle step times can be edited during synthesis by using the Cycle Editor. Although the ending method is designated before beginning a run, it can be changed during a synthesis until the final addition cycle is complete. This is done from Page 4 of the Monitor Synthesis Menu. After the last step of the final base addition, the ending method cannot be changed. After you select BEGIN, the screen reads: Checking bottle usage for base 1 Please wait . . . Each reagent will be checked for usage and for when it will need replacing. According to alarms that you must set in the Change Bottles Menu, the screen will show if and when the synthesis will be interrupted by the alarm. For example, if an oligomer 20 bases long will be synthesized, and tetrazole (Bottle 9) will empty after performing 11 cycles of base addition, the screen will read: Bottle 9 will interrupt in 11 cycles begin menu You then have the option of allowing the alarm to be triggered (interrupting the synthesis after eleven base additions) or changing the bottle immediately. To replace the bottle, return to the Main Menu and choose the Change Bottles Menu. When bottle usage has been checked and synthesis is ready to start, press BEGIN. 3-14 Section 3: Software Menu Descriptions Applied Biosystems After pressing BEGIN, the display will show which column you need to place on the instrument. For example, if T is the 3' terminus of DNA-1, the screen will read: Install (T) Column, then press enter enter menu Follow the instructions and install a (T) column. Be sure to use the column size corresponding to the synthesis cycle. Remember to record the serial number to identify the synthesis. After installing the column, select ENTER and the display will read: Purge Phosphoramidite lines? no yes menu IMPORTANT You can press the MENU key from any of the above menus to cancel the start of the synthesis. The display will then show the Configure Synthesis Menu allowing you to begin again. The above menu gives the option of purging the tetrazole and phosphoramidite delivery lines prior to beginning a synthesis. A purge refills these lines with fresh reagents. This is strongly recommended when the instrument has been idle for more than 12 hours (more than 6 hours in humid environments) or if one of the phosphoramidite reservoirs has not been accessed within 12 hours. Over this time oxygen and atmospheric water can penetrate the delivery lines and cause failures of the fast addition reaction. Answer the question, do you want to purge the phosphoramidite delivery lines? Choose YES or NO. NO Bypasses the purge steps. Synthesis automatically begins and the menu displaying the synthesizing status is shown. NO is usually chosen when the instrument has been running continuously and all phosphoramidites have been accessed within 12 hours. YES Purges the phosphoramidite lines. The screen then shows the step number being performed and the time remaining for that step: Phos purge step seconds Section 3: Software Menu Descriptions 1 9 of 9 steps. stop 3-15 Applied Biosystems When all steps are finished, the synthesis automatically begins and the menu displaying the synthesizing status is shown. STOP Ends the purge by performing steps 8 and 9 of the procedure which rinse the reagent valve blocks. The following menu is then shown: Rinsing . . . step seconds 8 of 9 steps 5 stop Once you stop the purge, the configure synthesis menu is shown and a new synthesis can begin. Main Menu Option: Monitor Synthesis When you select MONITOR SYNTH (synthesis), the instrument status is displayed and one of the following five menus is shown. Synthesizing status is shown during an active synthesis: SYNTHESIZING base x of xx bases hold jump intrpt more main (x = the number of the base currently being coupled as counted from the 3'-terminus, xx = the total number of bases in the sequence) Interrupted status is shown when a run has been temporarily stopped mid-synthesis: INTERRUPTED at base x of xx bases resume jump abort more main Holding status is shown when an individual synthesis step is being executed indefinitely: HOLDING base x of xx bases resume 3-16 jump intrpt more main Section 3: Software Menu Descriptions Applied Biosystems Synthesis Complete is shown when a synthesis is finished: SYNTHESIS COMPLETE main Synthesis Aborted is shown after permanently ending a run mid-synthesis: SYNTHESIS ABORTED main Instrument Status: Synthesizing When you press MONITOR SYNTHESIS during a run, Page 1 of the Synthesizing Status Menu is displayed: SYNTHESIZING base x of xx bases hold jump intrpt more main It shows the total number of bases in the sequence (xx) and the number of the base currently being synthesized as counted from the 3' terminus (x). IMPORTANT Although the DNA sequence is entered 5' to 3', the DNA chain is synthesized 3' to 5'. In all menus selected from Monitor Synthesis, the 3' terminus is shown as base number 1 and subsequent bases are numbered 3' to 5'. Summary of Synthesizing Status Menu Options HOLD When you press HOLD, the current step will be executed indefinitely. See The Holding Menu for details. JUMP Allows you to perform an out-of-sequence step, before or after the current step, and then continue with the synthesis. See The Jump Step Menu for details. INTRPT Allows you to stop a synthesis immediately and then continue it, program a future interrupt or abort a run. See The Interrupt Menu for details. Section 3: Software Menu Descriptions 3-17 Applied Biosystems Displays additional information about the synthesis. See below. MORE Synthesizing Status Menu options in detail The MORE key: Selecting MORE from page 1 of the Synthesizing Status Menu displays Page 2. It shows additional information about the synthesis including which step and function are being performed, and the total time of duration and time remaining for that step. For example, during step 1 shown below, acetonitrile (bottle # 18) is delivered to waste for a total of 5 seconds with 3 seconds remaining until completion. Step:1 hold #18 TO WASTE jump intrpt 3 OF 5 SEC more main Selecting MORE again displays Page 3 which shows the base currently being coupled (as denoted by the blinking box) and which bases will be subsequently added. For example, in the sequence 5'AAA AAA AAA GCT 3' shown below, C is currently being coupled, G will be added next which is followed by A and so on. The number in the upper left corner signifies which DNA strand is being synthesized (for example: 1 =DNA-1). 1-5'>AAA AAA AAA GCT hold Note jump <3' intrpt more main The hold, jump and intrpt keys operate from pages 1, 2 and 3 of the synthesizing status menu. Selecting MORE again displays Page 4 which shows the DNA strand being synthesized (e.g. DNA1, a 12-base sequence), the cycle being performed (e.g. Cycle-1 which has 63 steps) and the ending method (e.g. trityl off). Making ON/OFF DNA-1:12 Cycle-1:63 Trityl OFF more main The ON/OFF key operates during synthesis to change the ending method from Trityl OFF to Trityl ON and vice versa. Following the last step of the final base addition, the ending method cannot be changed. 3-18 Section 3: Software Menu Descriptions Applied Biosystems Selecting MORE again returns to Page 1 of the Synthesizing Status Menu. The Holding Menu When you select HOLD, the step currently being executed will continue indefinitely. During a Hold, all activated valves remain open. The Hold can be used to increase the delivery time of a reagent. The Holding Menu and its relationship to the Synthesizing Status Menu are shown in Figure 3-2. Upon pressing HOLD, the screen will read: HOLDING base x of xx bases resume jump intrpt more main Select MORE for additional synthesis information. Note that the time remaining for the step freezes when you press HOLD. STEP:1 resume #18 TO WASTE jump intrpt 3 OF 5 SEC more main Select MORE again for further information: 1-5'>AAA AAA AAA GCT resume jump <3' intrpt more main Select MORE again for further information: Making ON/OFF Note DNA-1:12 Cycle-1:63 Trityl OFF more main The resume, jump and intrpt keys operate from Pages 1, 2 and 3. The ONIOFF key operates from Page 4. IMPORTANT When a chemical delivery step is on hold, the extra reagent consumption is not recorded in bottle usage and is not included in the alarm tabulation. RESUME Releases the Hold on the step and continues the synthesis. Section 3: Software Menu Descriptions 3-19 Applied Biosystems JUMP Shows the Jump Step Menu, the hold is not yet released. Using Jump Step, the synthesis can be continued at any point in the cycle before or after the step currently on Hold. The Hold is released when the jump is activated. Jump Step allows parts of the chemical cycle to be skipped or repeated. Refer to The Jump Step Menu for further instructions. INTRPT Displays the first page of the Interrupt Menu. When you interrupt immediately, it releases the Hold on the step and stops the synthesis. The synthesis will not continue until the interrupt is removed. For further instructions, refer to The Interrupt Menu. MAIN 3-20 Returns to the Main Menu. This will not affect the Holding status. Section 3: Software Menu Descriptions Figure 3-2. The Holding Menu Applied Biosystems Section 3: Software Menu Descriptions 3-21 Applied Biosystems The Jump Step Menu Using JUMP, the synthesizer can execute any step in the cycle before or after the current step being performed. Jumping to out-of-sequence steps enables you to repeat or skip parts of the chemical cycle. The jump step menu and its relationship to the Synthesizing Status Menu are shown in Figure 3-3. When you select JUMP, the step being executed will continue indefinitely, like pressing HOLD. The screen will display the Jump Step Menu showing the current step, for example, step 1, #18 to waste: Enter step #1 enter #18 TO WASTE next prev menu Follow the prompt and enter the step number to be performed next. The number and the corresponding function description will appear on the screen, be sure it is correct. Then, press ENTER: ENTER Continues synthesis from the specified step. The screen will then display Page 2 of the Synthesizing Status Menu showing the new step being performed. If you press ENTER without specifying a step number, the synthesis will continue from the same step. IMPORTANT Jumping to step 1 increments to the next base. For example, when synthesizing base 2 and you jump to step 1, the synthesis will proceed with step 1 of base 3. To remain within the base 2 cycle, jump to step 2 or any subsequent step. During synthesis, if you jump over Cycle Entry, the cycle counter which records bottle usage will not increment. NEXT Displays the subsequent step and its function description. Continue to press NEXT until the desired step is shown. PREV Displays the preceding step and its function description. Continue to press PREV until the desired step is shown. MENU 3-22 Upon selecting MENU, a jump will not occur. The step being held will be released, the synthesis will resume and the display will return to the synthesizing status menu. Section 3: Software Menu Descriptions Applied Biosystems Figure 3-3. The Jump Step Menu Section 3: Software Menu Descriptions 3-23 Applied Biosystems The Interrupt Menu Selecting INTRPT (interrupt) allows you to stop a run at a specified step mid-synthesis. Using Interrupt, a synthesis can be aborted, stopped immediately and then continued or programmed to stop at any future step in the synthesis. Once a run has been interrupted, a jump step can also be performed. The set of interrupt menus and their relationship to the Synthesizing Status Menu are shown in Figure 3-4. IMPORTANT Interrupt synthesis at a safe step, such as Cycle Entry or Cycle End, so that the DNA is left chemically stable. Upon selecting INTERRUPT, one of two menu pages will be displayed. (This does not yet interrupt the synthesis.) 1. When an interrupt has been programmed to occur in the future, the screen shows when it will happen. For example: Interrupt at step 1 ahead imediat base 2 clear menu 2. When an interrupt has not been programmed, the screen reads: No interrupt set ahead Note imediat clear menu The IMEDIAT (immediate) and MENU keys operate from both pages. AHEAD only operates from the “no interrupt set” page and CLEAR only from the “interrupt at step_ base_” page. Interrupt Ahead AHEAD After pressing AHEAD from the “no interrupt set” page, the screen displays: Interrupt at step 0 ahead imediat base 0 clear menu By choosing AHEAD, you can program an interrupt to occur at a future step anytime during the synthesis. This is done by entering the step number and base number where the synthesis should 3-24 Section 3: Software Menu Descriptions Applied Biosystems stop. If no base is specified, the interrupt will occur at the next base. To enter the values, move the cursor as necessary and type the desired numbers. For example, to stop the synthesis the moment it reaches Step 1 of the first addition cycle, enter “1” next to step and “2” next to base. (Remember, the support-bound nucleotide is base 1. Base 2 is coupled during the first addition cycle.) The screen will read: Interrupt at step 1 ahead imediat base 2 clear menu When the synthesis actually reaches this point, it will stop and the menu displaying the Interrupted Status will be shown. Only one interrupt can be set at a time. As soon as one has been programmed, an asterisk (*) will appear when viewing the Synthesizing and Holding Status Menus (:intrpt*:) which serves as a reminder. Once set, an Interrupt Ahead may be changed by typing over the existing entries. CLEAR Erases an interrupt. MENU Returns to Page 1 of the Synthesizing Status Menu. Interrupt Immediate IMEDIAT Closes all valves instantly and stops the synthesis. The screen will display the interrupted Status and will show when the synthesis was stopped. For example: Interrupted at base 2 of 12 bases resume Section 3: Software Menu Descriptions jump abort more main 3-25 Applied Biosystems Select MORE for additional information. Note that the time remaining for the current step freezes when a synthesis is immediately interrupted. STEP 1 resume #18 TO WASTE jump abort 3 of 5 SEC more main Select MORE for further information: 1-5'>AAA AAA AAA GCT resume jump <3' abort more main Select MORE for further information: Making ON/OFF DNA-1:12 Cycle- 1:63 Trityl OFF more main To summarize the above example: The synthesis was interrupted (by pressing IMEDIAT) when it was coupling base number 2 which is C. It was stopped after performing step 1 (#18 to waste) for 2 seconds (3 seconds remain). There are a total of 12 bases in this sequence (DNA-1:12). Cycle-1 is being performed and the ending method is trityl off. IMPORTANT Interrupt synthesis at a safe step, such as Cycle Entry or Cycle End, so that the DNA is left chemically stable. Note 3-26 The resume, jump and abort keys operate from Pages 1, 2 and 3. The ON/OFF key operates from Page 4. RESUME Continues synthesis from the beginning of the interrupted step. The screen then returns to the Synthesizing Status Menu. JUMP Shows the Jump Step Menu allowing you to continue the synthesis before or after the interrupted step. The synthesis remains interrupted until the jump is activated. Refer to The Jump Step Menu for instructions. ABORT Select to end a synthesis prematurely. This might be done if a sequence was entered incorrectly. When pressed, the display reads: Section 3: Software Menu Descriptions Applied Biosystems To abort synthesis, press yes. no yes main Follow the instructions and choose yes or no. YES Ends the synthesis permanently. Once you press YES, the synthesis cannot be resumed. The display will then read: Synthesis Aborted main NO Prevents aborting the synthesis. The interrupt is not affected and the display returns to the Interrupted Status Menu. Section 3: Software Menu Descriptions 3-27 Figure 3-4. The Interrupted Menu Applied Biosystems 3-28 Section 3: Software Menu Descriptions Applied Biosystems Main Menu Option: Change Bottles The Change Bottles Menu provides a procedure for removing empty reservoirs and replacing them with bottles of fresh reagents. In addition, it displays bottle usage information and provides an alarm to alert you that reagent levels are low. Upon selecting CHANGE BOTTLES, the screen will read: #1 A-PHOS next alarm: 0 prev change cycle: 0 main Menu Description A reservoir number and an abbreviated description of its contents are displayed. The above example shows Bottle 1 (#1) which contains A-phosphoramidite. The screen also shows the alarm and cycle (or bottle usage) information for the reservoir being viewed. The Cycle Setting Cycle: displays the number of cycles a reservoir has been accessed. During a synthesis, the software tabulates which bottles are accessed during each cycle of base addition. Tabulation occurs when Function 33, cycle entry, has been reached. If a bottle has been used (at least once or more than once) during that cycle, the cycle counter increases by one. When performing the bottle change procedure, the counter is automatically reset to zero when a bottle is removed and replaced. If a phosphoramidite is partially used, stored frozen and then reused, the cycle counter should be reset. Simply move the cursor to the right of cycles: and enter up to a 3-digit number. The Alarm Setting Alarm: displays the number of cycles which will trigger the alarm. You need to set this number according to the guidelines described in the following pages. (See About the Alarm and Table 3-1, Bottle Usage Data.) To set the alarm, move the cursor to the right of alarm: and enter up to a 3-digit number. Note If the alarm is set to zero, it will not function. Section 3: Software Menu Descriptions 3-29 Applied Biosystems The Change Bottles Menu Keys NEXT Displays the cycle and alarm information for the subsequent reservoir. When bottle 18 is being viewed, selecting NEXT will show bottle 1. PREV Displays the cycle and alarm information for the preceding bottle. When bottle 1 is being viewed, selecting PREV (previous) will show bottle 18. CHANGE Initiates the steps for the bottle change procedure. About the Bottle Change Procedure The bottle change procedure is used to remove empty reservoirs and replace them with bottles of fresh reagents. It can be done before beginning a synthesis or when an active synthesis has been interrupted either manually or by the alarm. This process is especially important for preventing oxygen and water contamination of atmosphere-sensitive phosphoramidites and tetrazole. In summary, the procedure begins by removing old reagent from the delivery line and forcing it back into the reservoir by an acetonitrile wash. The bottles are then removed and replaced. Next, argon purges the reservoir’s headspace to eliminate air and the delivery line is then refilled with fresh reagent. For a complete description of the bottle change procedures, see Section 4. To begin the procedure, press CHANGE. The screen will read: Enter bottle # to change: 1 enter press enter menu Follow the instructions and enter the bottle number that you want to change. Note that the screen will automatically display the bottle number which was showing in the previous menu. If a different reservoir needs to be changed, type the appropriate number and it will appear on the screen. Be sure it is the correct number as printed on the bottle label and above its receptacle. Next, press ENTER and the steps which prepare the bottle for removal will begin. The screen will display which bottle is being changed, the step number being performed and the time remaining for that step. For example, if the A-phosphoramidite reservoir is empty and needs replacing; enter “1” (unless 1 is already displayed), press ENTER and the screen will read: Changing # 1 A-PHOS STEP 1 OF 9 STEPS 3-30 10 sec stop Section 3: Software Menu Descriptions Applied Biosystems Select to halt the current step and return to the previous menu. Stop has the same function in all Change Bottles displays. STOP Note Do not reuse the phosphoramidites or tetrazole after these steps are completed because the reservoirs now contain HPLC grade acetonitrile from bottle 18. If you are going to remove partially used phosphoramidites, store them and reuse them later; the bottle change procedure should not be performed. See Section 2 for instructions on How to Store Dissolved Phosphoramidites. When these steps are completed, the screen displays: Change bottle 1 A-PHOS enter then enter stop Follow the prompt, Change Bottle 1 A-PHOS. Remove the empty reservoir and upon opening the fresh bottle, quickly place it on the instrument. (For details on How to Install Reagent Bottles, refer to Section 2.) After the reservoir is correctly installed, press ENTER. The reservoir is then prepared for chemical deliveries and the cycle counter is reset to zero. The screen will now display the step number being performed and the time remaining for that step. Changing # 1 A-PHOS STEP 6 OF 9 STEPS 5 sec stop When the procedure is finished, the display returns to the first menu in this series enabling you to change an additional bottle. To return to the Main Menu, select MAIN. Now the synthesis can either begin by selecting START SYNTH or continue by releasing the interrupt (select MONITOR SYNTH and press RESUME), as needed. About the Alarm The alarm is triggered when a bottle has been used a specified number of cycles. To operate effectively, the alarm must be set correctly. If set too high, the reservoir could run dry and the synthesis could fail. Chemical consumption can vary with each instrument due to different pressure regulator settings and use of different cycle scales. Bottle usage data is provided in Table 3-1 as a guideline for setting the alarm. This data shows the average number of uses (or cycles) obtainable per bottle. When ini- Section 3: Software Menu Descriptions 3-31 Applied Biosystems tially setting the alarm, use values which are 5 to 7 uses less than these numbers and adjust as necessary. IMPORTANT Since consumption rates will vary from instrument to instrument, it is important to visually monitor reagent consumption for the first several syntheses. Once you determine the usage, the alarms can be effectively set. Note that the number of uses obtainable per bottle will change depending on which cycle is being performed (e.g., .2µM, lµM, Low or 10µM). Longer chemical delivery steps result in greater reagent consumption and fewer uses per bottle. Consider these factors when setting the alarm. Phosphoramidites used during the phosphoramidite purge procedure are not tabulated in the cycle counter. This process uses less than one half the amount of phosphoramidite used in a coupling step of a one-micromole synthesis. In addition, manual deliveries (using the Manual Control Menu), deliveries during a hold step, and acetonitrile used during the bottle change procedure are not tabulated in the cycle counter. When synthesizing mixed-sequence probes, each phosphoramidite which is delivered is tabulated in the cycle counter. When the alarm is triggered, the instrument will finish the current addition cycle, interrupt the synthesis at Cycle Entry and sound an alarm. A blinking “A” will be shown in the bottom right corner of any menu being viewed. Press the MENU key until the display reads: Bottle Empty Alarm!!! Change bottle to clear alarm. main Select MAIN to return to the Main Menu and then select CHANGE BOTTLES. Once the Change Bottles Menu is showing, the alarm message will be cleared. However, the synthesis will still remain interrupted. Proceed with the bottle change procedure as described earlier in the section. When finished, return to the Main Menu, select MONITOR SYNTH and continue the synthesis by selecting RESUME. If the alarm has been triggered but the bottle is not yet empty, the synthesis can be continued by clearing the interrupt (by choosing MONITOR SYNTH and then RESUME). The alarm message can be cleared by simply viewing the Change Bottles Menu. If this is done, it is important to reset the alarm to an appropriate number. 3-32 Section 3: Software Menu Descriptions Applied Biosystems Bottle Usage Data Table 3-1. Bottle Usage Data Synthesis Scale 0.2µM 1.0µM (cycles) (cycles) Reagent Phosphoramidites 0.25 grams: A G C T Low (cycles) 10.0µM (cycles) 18 18 18 20.7 12.5 12 12.3 13.7 28 27 28 31 2 2 2 2.25 0.5 grams A G C T 37.5 36.5 37 41.5 25 24 24.5 27.5 56 54.5 55.5 62 4 4 4 4.5 1.0 grams A G C T 75 73 74 83 50 48 49 55 112 109 111 124 8 8 8 9 Tetrazole (180 mL) 411 343 561 101 Acetic anhydride (180 mL) 732 610 732 183 NMI (180 mL) TCA (450 mL) 732 190 610 190 732 190 183 74 Iodine (200 mL) 486 486 486 119 Acetonitrile (4 L) 803 803 803 156 Section 3: Software Menu Descriptions 3-33 Applied Biosystems Main Menu Option: Cycle Editor Introduction Use the Cycle Editor to create, edit and print synthesis cycles. Four fully programmable, synthesis cycle locations are available in RAM (Random Access Memory). They are named Cycle-1 through Cycle-4. RAM cycles can be created or edited according to your needs. Immediately before synthesis, you select the RAM cycle you want to use from the Start Synth Menu. Applied Biosystems also supplies four cycles which are permanently stored in ROM (Read Only Memory). These ROM cycles are optimized for synthesizing on the .2-micromole, one-micromole and ten-micromole scales. They are named .2µM, 1µM and 10µM respectively. The fourth cycle is named Low. It is a low reagent-consumption cycle on the .2-micromole scale which uses 33% less phosphoramidites. Cycles are further explained in Section 4. To synthesize using a ROM cycle, it must be transferred to a programmable RAM cycle location (i.e. Cycle-1, 2, 3 or 4). Only the RAM cycles appear as choices in the Start Synthesis Menu; .2µM, 1µM, Low and 10µM cycles do not appear as options. Note The first time the main power is turned on, the Applied Biosystems ROM cycles are automatically loaded into the RAM cycle locations as shown below. For example, to synthesize using the .2µM cycle, simply display Cycle-1:63 in the Start Synthesis Menu. ROM synthesis cycle RAM cycle location Total Number of steps cycle time (min.) crude yield* (O.D.) (20mer) .2µM Cycle-1 63 5.5 20-25 1µM Cycle-2 64 5.5 100-120 Low** Cycle-3 61 5.5 20-25 10µM Cycle-4 53 24 800-1000 *Yield figures based on a 20mer sequence. Absorbance measured at 260nm. Assuming 33 micrograms/O.D. unit. **The Low cycle is on the .2µM scale. After the ROM cycles are transferred to RAM cycle locations, they will be stored there indefinitely until you change or delete them. Once transferred, they can be edited as desired. Once you edit a cycle, the edited version will be stored in RAM and will appear in the Start Synthesis Menu. If the 3-34 Section 3: Software Menu Descriptions Applied Biosystems main power is turned off, the edited version will still be stored in RAM. If you want to transfer a ROM cycle back into a RAM location, use the COPY key. The Cycle Editor Menu Upon selecting CYCLE EDITOR, the display shows a cycle name and the number of steps in that cycle. If a cycle has zero steps, it has not been defined. The following example shows Cycle-1 which has 63 steps due to the automatic transfer of the .2µM cycle: Select action for: edit Cycle- 1:63 steps copy print next main Follow the prompt and select an action for Cycle-1. EDIT Select to create, modify or review the steps of the designated cycle. The EDIT key will not function when viewing the ROM cycles (.2µM, 1µM, Low, 10µM). To edit these cycles, transfer them to a programmable, RAM cycle location using the COPY key. COPY Select to duplicate the designated cycle into any programmable, RAM cycle location (1, 2, 3 or 4). PRINT Select to print the designated cycle. NEXT Select to view the subsequent cycle name and the number of steps in that cycle. Press NEXT until the desired cycle is shown. The cycles will appear in the following order, .2µM, 1µM, Low, l0µM, Cycle-1, Cycle-2, Cycle -3, Cycle-4. The Edit Key Upon selecting EDIT, the screen will display step zero and the total number of steps in the cycle: C1- 0 of 63 steps in Cycle- 1 next Section 3: Software Menu Descriptions prev insert delete menu 3-35 Applied Biosystems An abbreviated cycle name appears on the top left of the display to signify the cycle being viewed (C1 = Cycle-1). Step zero (-0) is not an active step and is not performed during synthesis. Because all new steps are added after existing ones, it appears so that step 1 can be inserted, if necessary. NEXT Select to view the subsequent step. When viewing the last step, pressing NEXT will show step zero. PREV Select to view the preceding step. When viewing step zero, pressing PREV (previous) will show the last step. INSERT Select to insert a step in an existing cycle or to enter steps when creating new cycles. DELETE Erases the step that is currently showing. IMPORTANT All steps in the cycle are erased by selecting DELETE when step “0” is showing. When DELETE is chosen, a menu confirming the action appears: Press yes to delete all steps in Cycle-1 yes menu Press YES to erase the cycle. Press MENU to avoid erasure and to return to the previous menu. Editing an Existing Cycle When viewing step zero of an existing cycle, press NEXT and step 1 appears. It shows the function number and description, and the step time. In addition, it shows that the step will be active for the bases which are listed. C1-1 next Fxn:10 prev #18 TO WASTE insert t=2 delete AGCTX menu The above display shows Cycle-1 (C1), step 1 (-1) and function 10 (Fxn:10) which is #18 TO WASTE. The step will occur for 2 seconds (t=2) and will be performed when synthesizing all bases (AGCTX). 3-36 Section 3: Software Menu Descriptions Applied Biosystems When viewing this menu, the current step can be edited by changing the function, the time and the bases. To view another step in the cycle, place the cursor under the current step number, type the new number and two new menu options (JUMP and UNDO) will be shown. Press JUMP and the screen will show the information pertaining to the new step. Press UNDO to return to the original step. NEXT and PREV can also be used to view subsequent and preceding steps. To change the function, move the cursor under the existing function number and type the desired number over it. The function description corresponding to the new number will automatically be displayed. Similarly, to change the step time, move the cursor to the existing time and type a new one over it. Time is displayed in seconds with a maximum entry of 999 seconds. The function number and time can also be erased by placing the cursor under the existing number and pressing the gray delete key on the fixed keyboard. The Base Specifier Field When a base is listed in the base specifier field (located in the top right corner of the display), the step will be active when synthesizing that base. Since most syntheses are performed so that all steps are active for all bases, every base (AGCTX) is automatically listed for each step and therefore needs no adjustment. However, to execute a step for only certain bases, the unwanted bases must be deleted from the base specifies field. To indicate that a step should not be performed for a particular base, first move the cursor to the unwanted base (e.g. X). When the cursor is under a base, two new menu options, YES and NO, appear: C1-1 next Fxn:10 prev #18 TO WASTE insert t=2 yes AGCTX no NO Erases the specified base and the step will not occur when synthesizing that base. YES If a base has already been erased, move the cursor to the base position and select YES. The base will then reappear and the step will occur when synthesizing that base. To display the DELETE and MENU keys again, move the cursor under the time, step or function number. Section 3: Software Menu Descriptions 3-37 Applied Biosystems Creating a New Cycle/How to Insert Steps The INSERT key is used to add steps to existing cycles and to create entirely new cycles. To create a new cycle, begin by viewing step zero of the desired programmable, RAM cycle (e.g. Cycle-4): C4-0 of 0 steps in Cycle- 4 next prev insert delete menu IMPORTANT All steps of an existing cycle will be erased by pressing DELETE while observing step zero. Select INSERT and the display will read: C4-1 step ok Fxn:_ AGCTX exit To define the first step, move the cursor next to Fxn:_, and enter the correct function number. The corresponding function description will automatically appear. If an invalid function is entered, Bad Function is displayed. Once a function has been entered, the symbol for the step time (t=) appears. Move the cursor next to t= and enter the desired time. If the step should occur for every base, all bases should be showing in the base specifier field. Once you enter the correct information, press either EXIT or STEP OK. STEP OK Select to enter the step in the cycle and to remain in the insert mode. The subsequent step can then be defined and entered. Choose STEP OK when inserting several consecutive steps. EXIT Inserts the step in the cycle and returns to the previous menu. Choose EXIT after completing all insertions or when inserting a single step. If neither a valid function nor the time has been entered, pressing EXIT will return to the previous menu without inserting the step. How to Correct Mistakes To correct mistakes, type over the incorrect entry or erase it by pressing the delete key on the fixed keyboard. Once a step has been inserted, it can only be erased by viewing the step and selecting the DELETE soft key. 3-38 Section 3: Software Menu Descriptions Applied Biosystems IMPORTANT When creating new cycles, they must contain Function 33, Cycle Entry, and Function 34, Cycle End, for non-zero times (e.g., 1 second) In addition, Cycle Entry should appear before Cycle End. Adding a step to an existing cycle is done in the same manner as just described. Note that all new steps are inserted after existing ones. For example, when step 8 is shown and insert is selected, the new step will become step 9. All subsequent steps are renumbered accordingly. IMPORTANT During an active synthesis, only step times can be edited. Any new times will be effective immediately and will be used during the current synthesis as well as in subsequent syntheses. The Copy Key The COPY key is used to transfer an exact duplicate of one cycle (1, 2, 3, 4, .2µM, 1µM, Low or l0µM) into a programmable RAM cycle (1, 2, 3 or 4). For example, to modify the .2µM cycle, select NEXT from the first page of the Cycle Editor until the menu reads: Select action for: Cycle .2µM 63 steps edit copy print next main Choose COPY and the message will state that the .2µM cycle will be copied into another cycle location (in this example Cycle-2): Copy Cycle .2µM to: Cycle-2: 0 steps enter next menu If .2µM should be copied into a different location, press NEXT until the desired cycle is shown. In this example, Cycle-2 has zero steps. However, if Cycle-2 were already defined, it would not need erasing prior to copying. Copying will overwrite any cycle. Select ENTER to execute the copy command. The editor will then be displayed showing step zero of the new Cycle-2 and enabling any changes. C2-0 of next Section 3: Software Menu Descriptions 63 steps in Cycle-2 prev insert delete menu 3-39 Applied Biosystems Main Menu Option: Manual Control Use the Manual Control Menu to manually activate functions and to open and close individual valves. IMPORTANT Before opening any valves, refer to the DNA synthesizer schematic or the plumbing diagram (Appendix B) and the function list (Appendix A) to be sure the correct flow path will be formed. Also refer to Section 4: Functions, Cycles and Procedures, and Figure 4-4. Manual control will not operate during a synthesis unless you interrupt the run. Upon selecting MANUAL CONTROL, the screen will display: ON: valve VALVE FXN ALL OFF fxn all off main Select to activate a valve. Select to activate a function. Immediately closes all valves. The Valve Key To open or close valves, first press VALVE and the screen will display: Enter valve #_ on 3-40 and press on or off. off ON Opens the valve. OFF Closes the valve. menu Section 3: Software Menu Descriptions Applied Biosystems MENU Erases a valve number after it was entered and returns to the previous menu. Press MENU if VALVE was accidently selected from the previous display. To activate the valve, follow the instructions on the screen and enter a valve number. To erase a mistake, without returning to the previous menu, move the cursor to the unwanted number and press the delete key on the fixed keyboard. Next, follow the prompt and press ON or OFF. When you select ON, the valve is activated. The screen returns to the previous menu and shows the activated valve. ON: 1 valve fxn all off main A maximum of eight valves can be opened simultaneously by repeating this procedure (i.e., Press VALVE, enter the valve number, then press ON). Select ALL OFF to immediately close all valves. To close a single valve, select VALVE, enter the appropriate valve number and press OFF. The screen will then display the previous menu and show the valves still remaining open. The Function Key Upon choosing FXN (function), the screen will display: Fxn # ? _ on press on or off off next prev menu ON Choose to activate the function. OFF Select to deactivate the function and return to the previous menu. NEXT Shows the subsequent function number and its description. When the last function is being shown, selecting NEXT will display Function 1. PREV Shows the preceding function number and its description. When Function 1 is being shown, selecting PREV will display the last function. Section 3: Software Menu Descriptions 3-41 Applied Biosystems MENU Deletes the designated function number, closes all valves if a function was activated and returns to the previous menu. Follow the prompt, FXN # ?, and type a function number. The number and its description will be shown. For example, when 9 is entered, Function 9, #18 to column is displayed: Fxn # 9 on #18 TO COLM off next press on or off prev menu If you enter an invalid function number, that area of the screen will be blank. Next, press ON or OFF. When you choose ON, the function is activated and the screen returns to the previous menu which shows the active function. The function can be deactivated by pressing ALL OFF or OFF. Also, a valve or function will be deactivated automatically upon returning to the Main Menu. Only one function can operate at a time. When a second one is activated, the first is automatically deactivated. However, when a function is on, up to eight additional individual valves may be opened. Main Menu Option: Fract Pulse Use the Fract Pulse Menu (fraction collector pulse) to change the pulse time which advances the fraction collector. When selected, the screen reads: Fraction collector pulse time: 100 msec main Applied Biosystems uses a standard pulse time of 100 milliseconds. You can change the time by moving the cursor to the unwanted number and inserting a new one over it. When finished, return to the Main Menu by selecting MAIN. Main Menu Option: Procedure Editor Use the Proc (procedure) Editor Menu to create, edit and print the phosphoramidite purge, shut down and bottle change procedures. Standard procedures, further described in this section, are automatically used during instrument operation. You can, however, edit standard procedures or create new ones. Note that the flow test procedure, which is accessed from self test, cannot be edited or printed. 3-42 Section 3: Software Menu Descriptions Applied Biosystems When you select PROC EDITOR, the display shows an abbreviated name for the phosphoramidite purge procedure, #PP PROS PURGE: Edit/print procedure: #PP PHOS PURGE edit copy print next main Select NEXT to view the subsequent procedure name. The display will then show #1 BOTTLE CHANGE signifying the bottle change procedure for reservoir 1 which contains A-phosphoramidite. Continuing to press NEXT will show the names for all eleven bottle change procedures and then will display #SD SHUT DOWN signifying the shut down procedure. Press NEXT until the desired procedure is showing. Select an action (edit, copy or print) for the designated procedure. The Edit Key Select this option to edit standard procedures or to create new ones. When chosen, the screen shows step zero of the designated procedure: PP-0 of 9 steps in PHOS PURGE next prev insert delete menu The Procedure Editor operates in the same way as the Cycle Editor. Refer to The Cycle Editor Menu for complete instructions. Once you change a standard procedure, the new user version will automatically be used during subsequent operations and will appear in the editor. To return to the standard version, copy it into the editor using the COPY key. The Copy Key Select this option to copy the designated standard procedure into the editor. Copying is only necessary to restore the standard procedure once it has been edited or deleted. When you press COPY, the screen reads: Copy standard version to: #PP PHOS PURGE yes Section 3: Software Menu Descriptions menu 3-43 Applied Biosystems Choose YES to execute the copy command. Step zero of the procedure will then be shown in the editor. Once the standard version is restored, it will automatically be used during subsequent operations. The Print Key When a printer is connected to the instrument, select this option to print the designated procedure. When you choose PRINT, the display reads: Select print version: #PP PHOS PURGE user USER STD std menu Prints the procedure currently in the editor. Prints the designated standard procedure. If the standard procedure has not been edited, it will print when you press USER or STD. Main Menu Option: FXN Editor Use the FXN (function) Editor to create and edit user functions and to print lists of functions. You can create new functions not already in memory. These user functions are defined by specifying a set of valves to be opened when the function is activated. Up to eight user functions, called F 92 USER A through F 99 USER H, can be created. Like standard functions, they can be activated from Manual Control, inserted into any synthesis cycle via the Cycle Editor and into any procedure via Procedure Editor. Note Refer to the DNA Synthesizer Schematic (Appendix B) when creating user functions and check that the correct flow path will be created. Also refer to Section 4: Functions, Cycles and Procedures, and Figure 4-4. The Function Editor is also used to print the standard, test and user functions. Standard functions include F 1 through 90, 108 and 109 and are used in the synthesis cycle, the phosphoramidite purge, the bottle change and the shut down procedures. Test functions include F 101 through F 107 and are used to perform the flow test. Note that the standard and test functions are stored in the permanent memory and cannot be edited. 3-44 Section 3: Software Menu Descriptions Applied Biosystems Upon selecting FXN EDITOR from Page 2 of the Main Menu, the display will read: Select action for function(s): edit print main Follow the prompt and select an action: Choose to create and modify user functions. EDIT Prints the complete list of standard, user and test functions when a printer is connected to the instrument. PRINT The Edit Key When you press EDIT, the screen displays the first user function which is F 92 User A: 92 User A: next #_ on # off all off menu NEXT Shows the subsequent user function. # ON Enters a specified valve in the function. For instructions, refer to the example below. Note The “# on” key enters the valve number but does not activate the valve. # OFF ALL OFF Deletes a valve number from the function. Deletes all valves listed for the function. Example To create a function which would open valves 1, 13 and 17, first press the gray key labeled 1. The screen will then display the valve number in both the “# on” and “# off” keys: Section 3: Software Menu Descriptions 3-45 Applied Biosystems 92 User A: next #1_ on #1 off all off menu Next, press the “#1_ on” key and the specified valve number will be entered and shown on the top line of the display (92 User A: 1). Continue to enter all the valves to be activated by this function, up to a maximum of 10 valves. To delete a valve from the function, type the valve number (e.g., 1) and press “#1 off”. To clear all valves simultaneously, simply select the ALL OFF key. The Print Key Upon selecting PRINT, the screen will display: Select functions to print: std user test menu Follow the prompt and select an option: STD 3-46 Prints the standard functions, F 1 through F 90. USER Prints the user functions, F 92 User A through F 99 User H. TEST Prints the test functions, F 101 through F 107. Note that F108 and F109 are printed as test functions, but are used in cycles and procedures. Section 3: Software Menu Descriptions Applied Biosystems Main Menu Option: Power Fail The Model 391 automatically resumes synthesis after a power failure. Using the Power Fail Menu, You can program the instrument to remain interrupted and therefore not resume synthesis following a power outage. When a power failure occurs during an active synthesis, the synthesis will be interrupted immediately. The instrument cannot operate until the main power returns. During the outage, all synthesis parameters are retained in memory. These parameters include the DNA sequences; user defined cycles, procedures and functions; bottle usage information and the time. A maximum power fail time can be set so that if a failure occurs for less than the specified time, the synthesis will automatically resume when the main power returns. If a failure occurs for longer than the specified time, the synthesis will remain interrupted and can only continue when you clear the interrupt. After selecting POWER FAIL, the screen will read: Enter maximum power fail time: 0 min (0 means always continue) main To enter the maximum power fail time, move the cursor as necessary and insert the desired value (up to a 3-digit number). Mistakes can be corrected by typing over an incorrect number. Note When zero is entered, the synthesis will always continue when the main power returns regardless of the duration of the outage. Example 1: If the maximum time is 20 minutes and a power failure occurs for less than that, an alarm will sound for 15 seconds when the main power resumes. The synthesis will then automatically continue and the following menu will appear. Applied Biosystems 391 DNA Synthesizer PCR-MATE EP Note Ver. 1.00 start A When the instrument is not synthesizing, another menu may appear. Section 3: Software Menu Descriptions 3-47 Applied Biosystems The blinking “A” in the bottom right corner signifies the alarm has been triggered. To stop the ringing, if necessary, and to view more information, select START and the display will read: Down Time 7/08 14:20:34 POWER FAIL!! Up Time 7/08 15:45:44 main A The down time is the time the power failed. The up time is the time the main power resumed. Select MAIN to clear this display and return to the Main Menu. If desired, select Monitor Synth from the Main Menu to view the Synthesizing Status Menu and information about the current step being performed. Example 2: If the maximum power fail time is 20 minutes and a power failure has occurred for longer than that, the synthesis will remain interrupted when the main power returns. The alarm will sound and the screen will show the two menus mentioned in Example l. However, you must decide whether to resume or abort the synthesis. This is done by selecting MONITOR SYNTH to view the Interrupted Menu and then pressing RESUME or ABORT. It is helpful to view page 2 of the Interrupted Menu to determine which step was interrupted. For example, if the synthesis was stopped during detritylation, the oligonucleotides would be exposed to acid from the time the power failed. This could cause depurination resulting in some intrachain cleavage during base deprotection. If the exposure was for several hours, it would be best to abort the synthesis and begin another one. Note that if you abort synthesis at a step other than Cycle Entry or Circle End, the valve blocks and delivery lines must be rinsed. Refer to Section 2: How to Abort a Synthesis, for details. Main Menu Option: Self Test Use the Self Test Menu to verify the correct operation of the electronic components of the instrument and to access the flow test procedure. Self test should be performed routinely, approximately once every few months. In addition, it should be done if the instrument malfunctions. The easiest way to perform a self test is by pressing the option ALL. If the test passes, the top line of the display will read PASSED. If a FAILED message is shown, contact the Applied Biosystems Technical Support Department. Upon choosing SELF TEST, the screen will read: Select a test . . . all 3-48 battery Version 1.00 valves more main Section 3: Software Menu Descriptions Applied Biosystems ALL BATTERY VALVES Performs the self test on the battery, the valves, the relays, ROM (read only memory) and RAM (random access memory). When testing these components, it is simpler to press ALL instead of each option individually. Tests for the proper functioning of the battery. Tests for the proper operation of all valves. Select MORE for additional test options: Select a test . . . relays RELAYS ROM Version 1.00 RAM more main Tests for the proper operation of the fraction collector relays. ROM Tests for the proper operation of the permanent memory or the Read Only Memory. RAM Tests for the proper operation of the Random Access Memory or the user defined memory. Select MORE for additional test options: Select a test . . .tr.235 keys display Version 1.00 clock more main Select MORE for additional test options: Select a test . . . tones repeat Version 1.00 RESET more main The KEYS, DISPLAYS, CLOCK and TONES key are only used during manufacturing. REPEAT Section 3: Software Menu Descriptions 3-49 Applied Biosystems Repeats the self test for the battery, valves, relays, ROM and RAM until the STOP key is pressed. RESET Clears the clock setting and RAM including any stored DNA sequences and user defined cycles, procedures and functions. After pressing RESET, the ROM cycles are reloaded into the RAM cycle locations. When you press RESET, a menu confirming the action appears: RESET erases ALL user entries/edits and resets the time ---> RESET menu Press RESET to clear all RAM. Press MENU to retain all user memory and to return to the previous menu. Select MORE from page 4 of the Self Test Menu to view an additional test option: Select a test . . . Version 1.00 flowtst more main Flow Test FLOWTST Choose to perform the flow test procedure which measures flow rates through all essential delivery lines. Before doing this procedure, refer to Section 2: How to Perform the Flow Test Procedure, for complete details. When you choose FLOWTST, the following display is shown: Remove all bottles except #18 before you begin flowtest begin more main Follow the instructions and remove all the reagent bottles except bottle 18, acetonitrile. Be sure to properly reseal any chemicals that you will reuse. Press BEGIN and the flow test procedure will automatically start. The display will then show the current step being performed and its corresponding time: Step: 1 hold 3-50 WAIT jump intrpt 5 of 5 sec more main Section 3: Software Menu Descriptions Applied Biosystems This menu is identical to Monitor Synthesis. For details on how to use the HOLD, JUMP and INTRPT keys, refer to the Monitor Synthesis Menu found earlier in this section. Note that the MORE key does not operate from this menu. While performing the flow test, you can access the Main Menu by pressing MAIN. To view the flow test procedure again, select MONITOR SYNTH from the Main Menu. Steps 1 through 14 sequentially rinse and prime the lines for each bottle position. Use a beaker to collect the acetonitrile rinse starting with position 15 and working backwards. At step 14 the procedure is interrupted and the screen reads: Step: 14 INTERRUPT resume jump abort 1 of 1 sec more main The RESUME, JUMP and ABORT keys operate as they do in Monitor Synthesis. This pause is used to prepare for steps 15 through 43. At this time, place bottles filled with acetonitrile on all positions. When ready, press RESUME to continue the procedure. Carefully measure the flow from each bottle beginning with position 1, Adenosine phosphoramidite. After the final measurement, the procedure is interrupted at step 43. Use this pause to prepare to measure the flow from bottle 14 through the trityl collection line. When ready, press RESUME and collect the flow. At step 46, the procedure is interrupted again. The last series of steps flushes the delivery lines with argon. When ready, press RESUME. Once completed, chemicals can be reattached using the Change Bottles Menu and synthesis can begin. Main Menu Option: Set Clock Use the Set Clock Menu to enter the current time and date. The time is used to record when a power failure occurred and to provide dates on printouts. After selecting SET CLOCK, the display will show: Time: : Date: / / main Move the cursor to the right of time: and enter the current time, then to the right of date: and enter the date. The date is entered as month/day/last two digits of the year. For example, if the time is ten A.M. on July 8, 1989, the display should read: Section 3: Software Menu Descriptions 3-51 Applied Biosystems Time: 10:00 Date: 07/08/89 main To correct mistakes, move the cursor to the incorrect entry and type a new one over it. Note The clock will continue to update the time, although the time shown on the menu will remain fixed. Main Menu Option: Shut Down Use this menu to prepare the instrument for long term storage. The shut down procedure removes all reagents in the delivery lines and washes and dries all chemical pathways. The procedure takes about 17 minutes and is further described in Section 4. Upon selecting SHUT DOWN, the screen reads: Replace 9, 11, 12, 14, 15 with empty bottles enter main Follow the prompt and remove bottles 9, 11, 12, 14 and 15 and replace them with clean, empty bottles. Removing the reservoirs prevents HPLC grade acetonitrile from contaminating reagents which could be reused at a later date. If the phosphoramidites will be reused, their reservoirs should be removed as well. When finished, press ENTER and the display will show the step number being performed and the time remaining for that step: Shutdown step 1 of 29 steps seconds: 60 -total time= 17 min. stop During these steps, old reagents are removed from the lines by acetonitrile washes. The lines are then flushed dry. When all steps are completed, the screen will read: Remove all bottles, wipe all lines. enter 3-52 stop Section 3: Software Menu Descriptions Applied Biosystems Follow the prompt and take all bottles off the instrument and the wipe all delivery lines with a lintfree tissue. When finished, press ENTER. The screen will then show the steps being performed (additional argon flushes): Shutdown step x of 29 steps seconds: -total time= 17 min. stop When finished, the screen will read: Place clean bottles on all positions main To protect the exposed delivery lines, place clean, empty bottles on all positions. STOP Halts the procedure. Section 3: Software Menu Descriptions 3-53 Section 4: Functions, Cycles and Procedures This section contains information essential to understanding functions, synthesis cycles and procedures. Be sure to read the Introduction, Valves, Functions (pages 4-5 to page 4-6) and Synthesis Cycles (pages 4-18 to 4-20). The rest of the section can be used as needed. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-2 Valves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-2 Functions Synthesis Cycle Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-10 Procedure Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-15 Test Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-18 Synthesis Cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-18 Procedures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-23 The Phosphoramidite Purge Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-23 The Bottle Change Procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-24 The Shut Down Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-27 The Flow Test Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-28 Applied Biosystems Introduction Automated DNA synthesis requires chemical deliveries to specified destinations on the instrument, such as the column. These deliveries are controlled by electrically activating solenoid valves to open and close creating various pathways through the Model 391. Each solenoid valve is assigned a number which can be used to open or close it. A valve or set of valves opened or closed simultaneously to perform a specific delivery or task is a function. For example, function 9, #18 to column, delivers acetonitrile to the column. Function 9 opens valves 1, 12, 16 and 17 simultaneously. A function which is programmed to occur for a specified amount of time is a step. (e.g., Function 9 can be activated for 10 seconds.) A series of steps programmed to perform a chemical process repeatedly is a cycle. Synthesis cycles contain all the steps necessary for the efficient addition of one base. The cycle is repeated until every base in the sequence is added. Procedures are a series of steps programmed to complete a task, such as changing a reagent bottle. Upon command, the steps are performed once and are not repeated. There are four procedures: bottle change, phosphoramidite purge, shut down and flow test. Valves The Model 391 has 24 solenoid valves (numbered 0 to 23) which are opened and closed electrically and are controlled through the microprocessor. Refer to Section 5: Delivery Valve Blocks, for a physical description of the valves. During a synthesis, valves are automatically opened to create the correct chemical pathways and allow all necessary reagent and gas deliveries. You can also manually operate individual valves by using the Manual Control Menu. This is helpful when troubleshooting and can be done before synthesis or when a synthesis is interrupted. All valves with descriptions of what they control are listed next. Refer to Figure 4-1. DNA Synthesizer Schematic, to view the placement of the valves. Valves 0 to 11 control the flow of the following reagents (or argon) from their reservoirs to the reagent valve blocks: 4-2 Section 4: Functions, Cycles and Procedures Applied Biosystems Valve: 0 1 2 3 4 5 6 7 8 9 10 11 Controls the flow of: argon acetonitrile (bottle 18) iodine (bottle 15) TCA (bottle 14) NMI (bottle 12) acetic anhydride (bottle 11) tetrazole (bottle 9) contents of reserve 5 T-phosphoramidite (bottle 4) C-phosphoramidite (bottle 3) G-phosphoramidite (bottle 2) A-phosphoramidite (bottle 1) Valve: 12 13 14 15 16 Description: Directs flow between the reagent valve blocks and the column Directs flow from the reagent valve block to the waste bottle Controls the flow of argon to the column valve block Directs flow from the column valve block to the trityl collection port Directs flow from the column valve block to the waste bottle Valves 17-20, 22, 23 are pressure valves which control the flow of argon to the following reagent reservoirs: Valve: 17 18 19 20 22 23 Argon to: acetonitrile (bottle 18) iodine (bottle 15) TCA (bottle 14) all phosphoramidite reservoirs simultaneously NMI and acetic anhydride (bottles 12 and 11) simultaneously tetrazole (bottle 9) Valve 21 Description Vents all phosphoramidite reservoirs simultaneously Note that for flow to actually occur, several valves must be opened simultaneously as explained in the function descriptions, next. Section 4: Functions, Cycles and Procedures 4-3 Applied Biosystems Figure 4-1. 4-4 DNA Synthesizer Schematic Section 4: Functions, Cycles and Procedures Applied Biosystems Functions A valve or set of valves opened simultaneously to perform a specific delivery or task is a function. Functions 1 through 90, 108 and 109 provide all necessary operations for synthesis. Seven test functions, 101 to 107, are used during the flow test procedure. In addition, up to eight user functions, numbered 92 to 99, can be created using the Function Editor Menu. Refer to Figure 4-3 for a complete list of all functions. Functions are automatically performed during a synthesis and require no intervention. When a function is activated, the correct valves automatically open. After a specified time, the function is deactivated and the valves close. Using the Manual Control Menu, functions can also be manually activated prior to synthesis or when a synthesis is interrupted. This can be useful when troubleshooting. Functions are given abbreviated names to describe the action they perform. For example, Function 9 (F9) is named “#18 to column”. It delivers the contents of bottle #18, acetonitrile, to the column. Functions can be grouped according to their action (see Figure 4-4). Note that a single function may appear in more than one category. Many of the functions are used during the synthesis cycle and the remainder are used during the bottle change, phosphoramidite purge, shut down and flow test procedures. During synthesis, you can check which function is being performed by viewing the Monitor Synthesis Menu. DNA synthesis requires various reagents to be delivered to a specified destination. A reagent can flow from its reservoir to the waste bottle, or through the column and then to the waste bottle or the trityl collection port. Functions have been defined to perform each necessary delivery. To achieve flow of a reagent, specific valves must open simultaneously so that the following occurs: 1. The reservoir is pressurized; 2. The pathway from the reservoir to the valve block is opened; and 3. An exit is provided out of the valve block (i.e. to waste, or to the column and then to waste or the trityl collection port). The pathway created by activating a function can be traced using the 391 DNA synthesizer schematic, Figure 4-1. For example, Function 9 (#18 to column) delivers acetonitrile to the column. Activating Function 9 does the following: 1. Opens Valve 17 to allow argon to flow into the acetonitrile reservoir to pressurize it; 2. Opens Valve 1 to open the pathway between the reservoir and the valve block; 3. Opens Valve 12 to open the pathway from the valve block to the column; and 4. Opens Valve 16 to provide an exit (the waste bottle). Section 4: Functions, Cycles and Procedures 4-5 Applied Biosystems When all four valves are open, the argon pressure will force the acetonitrile out of the reservoir, through the reagent valve blocks, through the column and out of the synthesizer to the waste bottle. When Function 9 is deactivated, all valves close and the flow stops. The pathway created by activating Function 9 is traced using the 391 DNA synthesizer schematic and is shown in Figure 4-2. Figure 4-2. 4-6 Schematic representing the flow of Function 9 (#18 to column) which opens valves 1, 12, 16 and 17. Section 4: Functions, Cycles and Procedures Figure 4-3. 391 FUNCTION LIST VERSION: TIME: DATE: FUNCTION NUMBER 1 2 4 5 6 7 9 10 13 14 16 17 19 22 28 31 32 33 34 43 51 52 53 54 55 56 59 60 61 62 63 64 65 66 69 70 71 72 73 74 75 78 81 82 84 85 86 87 88 89 90 1.00 04:27 05/11/89 FUNCTION NAME FUNCTION VALVE LIST BLOCK FLUSH REVERSE FLUSH WAIT ADVANCE FC WASTE PORT WASTE BOTTLE #18 TO COLM #18 TO WASTE #15 TO COLUMN #14 TO COLUMN CAP PREP INTERRUPT B+TET TO COLUMN CAP TO COLUMN PHOS PREP RCDR ON RCDR OFF CYC ENTRY CYC END #18 PREP TET PREP A TO WASTE G TO WASTE C TO WASTE T TO WASTE X TO WASTE CAP A TO WASTE CAP B TO WASTE TET TO WASTE FLUSH TO A FLUSH TO G FLUSH TO C FLUSH TO T FLUSH TO X FLUSH TO TET FLUSH TO #18 #18 TO A #18 TO G #18 TO C #18 TO T #18 TO X #18 TO TET #15 TO WASTE #14 TO WASTE #18 TO 14 #18 TO 15 FLUSH TO 14, 15 #18 TO 11 #18 TO 12 FLUSH TO 11, 12 TET TO COLUMN 0, 13, 14, 16 12, 13, 14 Section 4: Functions, Cycles and Procedures 1, 12, 16, 17 1, 13, 17 2, 12, 16, 18 3, 12, 16, 19 22 6, 12, 16, 20, 23 4, 5, 12, 16, 22 20, 23 17 23 11, 13, 20 10, 13, 20 9, 13, 20 8, 13, 20 7, 13, 20 5, 13, 22 4, 13, 22 6, 13, 23 0, 11, 21 0, 10, 21 0, 9, 21 0, 8, 21 0, 7, 21 0, 6 0, 1 1, 11, 17, 21 1, 10, 17, 21 1, 9, 17, 21 1, 8, 17, 21 1, 7, 17, 21 1, 6, 17 2, 13, 18 3, 13, 19 1, 3, 17 1, 2, 17 0, 2, 3 1, 5, 17 1, 4, 17 0, 4, 5 6, 12, 16, 23 4-7 FUNCTION NUMBER 101 102 103 104 105 106 107 108 109 FUNCTION NUMBER 92 93 94 95 96 97 98 99 4-8 FUNCTION NAME FUNCTION VALVE LIST A TO COLUMN G TO COLUMN C TO COLUMN T TO COLUMN X TO COLUMN #11 TO COLUMN #12 TO COLUMN FLUSH TO TRIT FLUSH THRU COL 11, 12, 16, 10 10, 12, 16, 20 9, 12, 16, 20 8, 12, 16, 20 7, 12, 16, 20 5, 12, 16, 20 4, 12, 16, 22 0, 12, 15 0, 12, 16 FUNCTION NAME FUNCTION VALVE LIST USER A USER B USER C USER D USER E USER F USER G USER H Section 4: Functions, Cycles and Procedures Applied Biosystems Figure 4-4. MODEL 391 DNA SYNTHESIZER FUNCTIONS CONTROL 4 - Wait 5 - Advance Fraction Collector 6 - Waste to Port 7 - Waste to Bottle 17 - Interrupt 31 - Recorder On 32 - Recorder Off 33 - Cycle Entry 34 - Cycle End DELIVER TO COLUMN 9 - #18 to Column 13 - #15 to Column 14 - #14 to Column 19 - Base + Tetrazole to Column 22 - Cap to Column 101 - A to Column 102 - G to Column 103 - C to Column 104 - T to Column 105 - X to Column 90 - TET to Column 106 - #11 to Column 107 - #12 to Column RINSE AND FLUSH 1 - Block Flush 2 - Reverse Flush 9 - #18 to Column 10 - #18 to Waste 108 - Flush to Trit 109 - Flush thru Col PREPARE REAGENTS 16 - Cap Prep 28 - Phosphoramidite Prep 43 - #18 Prep 51 - Tetrazole Prep Section 4: Functions, Cycles and Procedures PRIME DELIVERY LINES 52 - A to Waste 53 - G to Waste 54 - C to Waste 55 - T to Waste 56 - X to Waste 59 - Cap A to Waste 60 - Cap B to Waste 61 - Tetrazole to Waste 81 - #15 to Waste 82 - #14 to Waste ARGON TO RESERVOIRS 62 - Flush to A 63 - Flush to G 64 - Flush to C 65 - Flush to T 66 - Flush to X 69 - Flush to Tetrazole 70 - Flush to #18 86 - Flush to 14, 15 89 - Flush to 11, 12 ACETONITRILE TO RESERVOIRS 71 - #18 to A 72 - #18 to G 73 - #18 to C 74 - #18 to T 75 - #18 to X 78 - #18 to Tetrazole 84 - #18 to 14 85 - #18 to 15 87 - #18 to 11 88 - #18 to 12 4-9 Applied Biosystems Synthesis Cycle Functions This part of Section 4 categorizes the functions and explains what each function does. Functions which deliver a reagent to the column The following five functions provide for flow of a reagent (driven by argon pressure) from its reservoir, through the valve block, through the column and ultimately to the waste bottle. Since the column is the site of all chemical reactions necessary for synthesis, these deliveries are critical. FUNCTION NUMBER NAME VALVES F9 #18 TO COLUMN 1, 17, 12, 16 (Acetonitrile is delivered to the column; frequently used during synthesis to wash the column and support to remove traces of reagents before a chemical step.) F 13 #15 TO COLUMN 2, 12, 16, 18 (Iodine is delivered to the column; used to oxidize the DNA following capping) F 14 #14 TO COLUMN 3, 12, 16, 19 (TCA is delivered to the column; used to detritylate the support-bound oligonucleotides prior to coupling) F 19 BASE + TET TO COLM X, 12, 16, 20, 23, 6 X = any combination of 7, 8, 9, 10, 11 (According to the sequence, the correct phosphoramidite(s) and tetrazole are |simultaneously delivered to the column to perform the coupling reaction.) F 22 CAP TO COLM 4, 5, 12, 16, 22 (Both capping reagents, acetic anhydride (cap A) and NMI (cap B), are simultaneously delivered to the column; used following coupling to terminate or cap unreacted oligonucleotide chains.) F 90 TET TO COLUMN 6, 12, 16, 23 (Tetrazole is delivered to the column; activates phosphoramidites for coupling reaction.) 4-10 Section 4: Functions, Cycles and Procedures Applied Biosystems Functions which control external events The following three functions do not activate solenoid valves, but control electrical switches. They are used to give electrical signals to a fraction collector or a chart recorder. FUNCTION NUMBER NAME SWITCH ACTION F5 ADVANCE FRAC. COLLECT PULSE F 31 RECORDER ON RELAY CLOSE F 32 RECORDER OFF RELAY OPEN Function 5, advance frac. collect (fraction collector), provides a 100-millisecond pulse to Terminals 1 and 2 located on the terminal strip at the left rear of the instrument. When the terminals are connected to a fraction collector, this pulse moves the tube rack one position. The pulse time can be changed by using the Fract Pulse Menu. The fraction collector is used to collect the trityl cation released during each detritylation step. Function 31, recorder on, closes an electrical relay or switch and activates an external chart recorder. Function 32, recorder off, and deactivates the chart recorder. The chart recorder should be connected to Terminals 3 and 4 on the rear of the instrument to receive the proper signal. When connected to a spectrophotometer with a flow cell, a chart recorder can be used to automate trityl assays. However, these readings will not be quantitative because the trityl solution is too concentrated. In addition to controlling a chart recorder, these functions can be used to activate and deactivate a heater. However, be sure not to run any of the heater’s current through the terminals. Contact the Applied Biosystems Technical Support Department for further information. Functions which manipulate chemical pathways The following two functions control the pathway of the column effluent: FUNCTION NUMBER NAME VALVES F6 WASTE TO PORT SUBSTITUTE 15 FOR 16 (Sends the column effluent to the trityl collection port) F7 WASTE TO BOTTLE DEFAULT TO 16 (Sends the column effluent to the waste bottle) Section 4: Functions, Cycles and Procedures 4-11 Applied Biosystems When Function 6 (waste to port) is activated, all subsequent functions using Valve 16 will have Valve 15 opened instead. All deliveries to the column will then exit through the trityl collection port. This is done prior to TCA delivery so that the trityl cation released during detritylation can be sent to a fraction collector for subsequent analysis. To return the flow of the column effluent to the waste bottle, Function 7 (waste to bottle) is activated. This signals the controller to open Valve 16 again (default to Valve 16). Functions which prepare a reagent for delivery The following functions pressurize reagent reservoirs to prepare them for delivery. A prep (preparation) function opens the gas valve to the reservoir allowing argon to flow to the headspace of the bottle. After several seconds, the bottle reaches the correct pressure necessary for proper delivery. These functions are used for reagents which are delivered simultaneously and in equal volumes. FUNCTION NUMBER NAME VALVES F 16 CAP PREP 22 (Capping reagents prep (preparation); pressurizes the acetic anhydride and NMI reservoirs simultaneously.) F 28 PHOSPHORAMIDITE PREP 20, 23 (Phosphoramidite preparation; pressurizes the tetrazole and all phosphoramidite reservoirs simultaneously.) Function 43 and Function 51 are used during the bottle change procedure after fresh tetrazole and acetonitrile are placed on the instrument. They pressurize the reservoir and blanket it with argon. F 43 #18 PREP 17 F 51 TET PREP 23 4-12 Section 4: Functions, Cycles and Procedures Applied Biosystems Functions used to rinse or flush the chemical pathways Four functions are frequently used throughout the synthesis to clear the valve blocks, the column and the interconnecting delivery lines. They are performed prior to a chemical delivery to remove residual reagent from a previous delivery. Two of the functions, #18 to Column and #18 to Waste, rinse the pathways with acetonitrile. The other two, Block Flush and Reverse Flush, flush the pathways to remove all liquid. A flush uses argon pressure to force the liquid to the waste bottle. FUNCTION NUMBER NAME VALVES F1 BLOCK FLUSH 0, 13, 14, 16 (Removes any solvent or reagent from the reagent valve blocks and the column valve block; argon enters the three valve blocks simultaneously and forces all liquid to the waste bottle.) F2 REVERSE FLUSH 12, 13, 14 (Removes any reagent or solvent from the column and support; argon flows from the column valve block, through the column, into the reagent valve block and then to waste. This forces the reagent out of the column in the reverse direction of normal flow (i.e. it drains the column from the top to the bottom.) F9 #18 TO COLUMN 1, 17, 12, 16 (Acetonitrile is delivered to the column. This rinses the column and support to remove traces of reagent. Argon pressure forces the acetonitrile from its reservoir, then through the reagent valve block, the column, the column valve block and finally to the waste bottle.) F 10 #18 TO WASTE 1, 17, 13 (Acetonitrile is delivered to the waste bottle. Argon pressure forces acetonitrile from its reservoir, through the reagent valve blocks to rinse them thoroughly and then to the waste bottle.) During synthesis, these four functions are often performed sequentially. For example, function 10 can be activated followed by F 9, F 2 and F 1: Function 10 Rinses the reagent valve blocks Function 9 Rinses the column and the column valve block Function 2 Removes the acetonitrile from the column Function 1 Removes the acetonitrile from the reagent valve block and the column valve block Section 4: Functions, Cycles and Procedures 4-13 Applied Biosystems Two other functions, 108 and 109, flush the column with argon from the bottom to the top. F 108 FLUSH TO TRIT 0, 12, 15 (Used following detritylation to flush remaining traces of trityl cation to the trityl collection port. Produces more accurate trityl assay results.) F 109 FLUSH THRU COL 0, 12, 16 (Argon flows through the column from the bottom to the top and exits at the waste bottle.) Functions which prime the reagent valve blocks Three functions are used to fill or prime the reagent valve blocks prior to column delivery of the reagent. Argon pressure drives the reagent from its reservoir through the valve block and to the waste bottle. These functions are also used during the bottle change procedure. FUNCTION NUMBER NAME VALVES F 61 TET TO WASTE 6, 13, 23 (Delivers tetrazole to the waste bottle; fills the reagent valve block prior to coupling.) F 81 15 TO WASTE 2, 13, 18 (Delivers iodine to the waste bottle; fills the reagent valve block prior to oxidation.) F 82 14 TO WASTE 3, 13, 19 (Delivers TCA to the waste bottle; fills the reagent valve block prior to detritylation.) Miscellaneous functions FUNCTION 4 WAIT Function 4 is a pause which is used following a chemical delivery (such as base + tetrazole to column) to allow the reagents to remain in the column so the reaction can be completed. FUNCTION 33 CYCLE ENTRY This function signals the controller to perform the first set of chemical steps necessary for base addition (detritylation). Note that this does not mean the first numerical step of the cycle. FUNCTION 34 4-14 CYCLE END Section 4: Functions, Cycles and Procedures Applied Biosystems At the end of each cycle, this function signals the controller to perform Step 1 of the cycle which begins the coupling reaction. Upon completion of all base additions, the cycle will finish at “cycle entry” if the DNA is to remain trityl on. If the ending method is trityl off, detritylation will be performed and the cycle will finish at “cycle end”. FUNCTION 17 INTERRUPT This function is used during the bottle change and shut down procedures. It provides a pause when reagent bottles are being removed and replaced. Procedure Functions The following functions are used during the bottle change, phosphoramidite purge and shut down procedures. The bottle change procedure is used to remove empty reservoirs and replace them with fresh reagents. The phosphoramidite purge procedure fills the phosphoramidite and tetrazole delivery lines with fresh reagent prior to beginning a synthesis. The shut down procedure prepares the instrument for storage. It removes all reagents from the delivery lines, and washes and dries all chemical pathways. Functions which prime delivery lines When a bottle of fresh reagent is placed on the instrument, the delivery line from the reservoir to the valve block must be primed or filled with the reagent. To prime the line, argon pressure forces the chemical from its reservoir, through the reagent valve blocks and to the waste bottle. Functions 52-56 and 61 are also used during the phosphoramidite purge procedure. Prior to synthesis, these functions fill the phosphoramidite and tetrazole lines with fresh reagent. In addition, Functions 61, 81 and 82 are used during the synthesis cycle to fill the reagent valve block prior to column delivery. The following functions deliver the contents of the designated reservoir to the waste bottle. For example, Function 52, A to waste, delivers adenosine phosphoramidite to the waste bottle. Section 4: Functions, Cycles and Procedures 4-15 Applied Biosystems FUNCTION NUMBER NAME VALVES F 52 F 53 F 54 F 55 F 56 F 59 A TO WASTE G TO WASTE C TO WASTE T TO WASTE X TO WASTE CAP A TO WASTE (acetic anhydride to waste) CAP B TO WASTE (NMI to waste) TET TO WASTE #15 TO WASTE #14 TO WASTE 11, 13, 20 10, 13, 20 9, 13, 20 8, 13, 20 7, 13, 20 5, 13, 22 F 60 F 61 F 81 F 82 4, 13, 22 6, 13, 23 2, 13, 18 3, 13, 19 Functions which deliver acetonitrile to a reservoir Before removing an empty phosphoramidite or tetrazole bottle, the old reagent in the delivery line is forced back into the reservoir and the line is rinsed. This is done during the bottle change procedure by delivering acetonitrile through the reagent valve block to the empty reservoir. In addition, functions delivering acetonitrile to bottle positions 11, 12, 14 and 15 are used during the shut down and flow test procedures to remove the reagents from the delivery lines. The following functions deliver acetonitrile to the designated reservoir. For example, Function 71, #18 to A, delivers acetonitrile to the adenosine phosphoramidite reservoir. FUNCTION NUMBER NAME VALVES F 71 F 72 F 73 F 74 F 75 F 78 F 84 F 85 F 87 F 88 #18 TO A #18 TO G #18 TO C #18 TO T #18 TO X #18 TO TET #18 TO 14 #18 TO 15 #18 TO 11 #18 TO 12 1, 11, 17, 21 1, 10, 17, 21 1, 9, 17, 21 1, 8, 17, 21 1, 7, 17, 21 1, 6, 17 1. 3. 17 1, 2, 17 1, 5, 17 1, 4, 17 4-16 Section 4: Functions, Cycles and Procedures Applied Biosystems Functions which deliver argon to a reservoir These functions are used during the bottle change procedure for the phosphoramidite, tetrazole, and acetonitrile reservoirs. After acetonitrile removes old reagent from the delivery line, the acetonitrile is removed by an argon flush. Argon travels through the reagent valve block (by opening Valve 0) and into the reservoir which forces the acetonitrile into the bottle. The phosphoramidite bottles share a single-vent valve which is open during this step. In addition, functions that deliver argon to bottle positions 11, 12, 14 and 15 are used during the shut down and flow test procedures. The functions below deliver argon to the designated reservoir. For example, Function 62, Flush to A, delivers argon to the adenosine phosphoramidite reservoir. FUNCTION NUMBER NAME VALVES F 62 FLUSH TO A 0, 11, 21 F 63 FLUSH TO G 0, 10, 21 F 64 FLUSH TO C 0, 9, 21 F 65 FLUSH TO T 0, 8, 21 F 66 FLUSH TO X 0, 7, 21 F 69 FLUSH TO TET 0, 6 F 70 FLUSH TO #18 0, 1 F 86 FLUSH TO 14, 15 0, 2, 3 F 89 FLUSH TO 11, 12 0, 4, 5 Section 4: Functions, Cycles and Procedures 4-17 Applied Biosystems Test Functions Test functions are used to perform the flow test procedure.The procedure is used during instrument manufacturing, installation and troubleshooting. If desired, test functions can be inserted into a synthesis cycle using the Cycle Editor or a procedure using the Procedure Editor. The following functions deliver the contents of the designated bottle to the column. FUNCTION NUMBER NAME VALVES F 101 A TO COLUMN 11, 12, 16, 20 F 102 G TO COLUMN 10, 12, 16, 20 F 103 C TO COLUMN 9, 12, 16, 20 F 104 T TO COLUMN 8, 12, 16, 20 F 105 X TO COLUMN 7, 12, 16, 20 F 90 TET TO COLUMN 6, 12, 16, 23 F 106 #11 TO COLUMN 5, 12, 16, 22 F 107 #12 TO COLUMN 4, 12, 16, 22 (Note that when you print the functions, F108 and F109 are listed with the test functions. When categorizing, they are included with functions that rinse and flush chemical pathways.) Synthesis Cycles A function performed for a specified time is called a step. During synthesis, steps are arranged in a particular order to create a synthesis cycle. The cycle completes all operations necessary for one base addition and is repeated until DNA of desired length is fully synthesized. Applied Biosystems supplies four synthesis cycles which are optimized for use with β-cyanoethyl phosphoramidites. They are stored permanently in the ROM cycle locations of the Cycle Editor. They cannot be changed or deleted. The cycles are used for synthesizing on the ten-micromole, onemicromole and small scales (0.2 micromole) and are named “10µM”, “1µM” and “.2µM” respectively. A low reagent consumption cycle on the 0.2µM scale has also been developed which uses 33% less phosphoramidites. This cycle is called “Low”. When the instrument’s main power is first turned on, all ROM cycles (.2µM, IµM, Low, 10µM) are automatically loaded into the four RAM cycle locations of the Cycle Editor. See Table 4-1 for the exact locations. Once in a RAM location, any cycle can be edited and all changes will be stored in memory even if the power is turned off. In the Start Synthesis Menu, you must choose a RAM cycle 4-18 Section 4: Functions, Cycles and Procedures Applied Biosystems for use in DNA synthesis, the ROM cycles do not appear as options. Note that at any time, you can copy a ROM cycle into a RAM cycle location by using the Cycle Editor. You can also create entirely new cycles in any RAM location. ROM synthesis cycle RAM cycle location Total Number of steps cycle time (min.) crude yield* (O.D.) (20mer) .2µM Cycle-1 63 5.5 20-25 1µM Cycle-2 64 5.5 100-120 Low** Cycle-3 61 5.5 20-25 10µM Cycle-4 53 25 800-1000 *Yield figures based on a 20mer sequence. Absorbance measured at 260nm. Assuming 33 micrograms/O.D. unit. **Low is a low consumption cycle on the .2µM scale. Table 4-1. Applied Biosystems Synthesis Cycles The 0.2-µMol scale provides sufficient oligomers for most applications. Using this scale, the expected crude yield in optical density units (O.D. or odu, A260 nm.) equals the number of bases in the sequence. For example, synthesis of a 20-mer typically yields approximately 20 O.D. of crude oligonucleotide. When larger quantities of DNA are needed, the 1µM or l0µM cycle can be used. With the 1µM cycle, synthesis of a 20-mer will yield about 100 O.D. of crude oligonucleotide (1µM crude yield, O.D. = length x 5). Synthesizing a 20-mer with the l0µM cycle will yield about 800 odu of crude DNA. The steps in each cycle have optimized delivery times which ensure completion of each chemical reaction, yet an entire synthesis cycle is complete in 5.5 minutes. (The cycle time for l0µM is 25 minutes.) The 0.2µM cycle cost is about 45% less than the 1µM cycle cost. The “Low” cycle costs are about 25% less than the 0.2µM cost. All cycles are listed in Appendix A. In addition, the .2µM cycle is shown in Figure 4-5. Each cycle consists of the required number of steps which are listed with the corresponding function number, name and step time. To determine the purpose of each step, refer to the explanation of Synthesis Cycle Functions found earlier in this section. Section 4: Functions, Cycles and Procedures 4-19 Applied Biosystems More About the .2µM Cycle The four essential chemical reactions necessary for synthesis are listed below with their corresponding cycle step numbers: 1. 2. 3. 4. Detritylation Coupling Capping Oxidation .2µM Steps 36-63 Steps 1-15 Steps 16-21 Steps 22-35 Although each reaction requires different treatment, the following generalizations can be made: Prior to the chemical reaction: • The valve blocks, the column and the interconnecting delivery lines are rinsed and flushed dry; and • The reagent reservoir(s) is prepared for delivery. To perform the chemical reaction: • The reagent(s) is delivered to the column, often followed by a Wait step to complete the reaction. 4-20 Section 4: Functions, Cycles and Procedures Applied Biosystems Figure 4-5. STEP NUMBER 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 .2µΜ β−cyanoethyl synthesis cycle FUNCTION # NAME 10 9 2 1 28 90 19 90 19 90 19 90 4 16 2 1 22 4 10 2 1 81 13 10 4 2 1 10 9 2 9 2 9 2 1 33 10 9 2 1 5 6 82 14 108 14 #18 TO WASTE # 18 TO COLM REVERSE FLUSH BLOCK FLUSH PHOS PREP TET TO COLUMN B+TET TO COLM TET TO COLUMN B+TET TO COLM TET TO COLUMN B+TET TO COLM TET TO COLUMN WAIT CAP PREP REVERSE FLUSH BLOCK FLUSH CAP TO COLUMN WAIT #18 TO WASTE REVERSE FLUSH BLOCK FLUSH #15 TO WASTE #15 TO COLUMN #18 TO WASTE WAIT REVERSE FLUSH BLOCK FLUSH #18 TO WASTE #18 TO COLM REVERSE FLUSH #18 TO COLM REVERSE FLUSH #18 TO COLM REVERSE FLUSH BLOCK FLUSH CYC ENTRY #18 TO WASTE #18 TO COLM REVERSE FLUSH BLOCK FLUSH ADVANCE FC WASTE PORT #14 TO WASTE #14 TO COLUMN FLUSH TO TRIT #14 TO COLUMN Section 4: Functions, Cycles and Procedures STEP TIME 2 15 5 3 3 3 2 2 2 2 2 2 15 3 5 3 10 5 3 5 3 3 10 5 15 5 3 5 10 5 10 5 10 5 3 1 3 10 5 3 1 1 3 10 1 10 A Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes ACTIVE FOR BASES G C T X Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes (continued →) 4-21 Applied Biosystems Figure 4-5. STEP NUMBER 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 4-22 .2µΜ β−cyanoethyl synthesis cycle (Continued) FUNCTION # NAME 108 14 108 14 108 14 108 14 108 9 108 7 1 9 2 1 34 FLUSH TO TRIT #14 TO COLUMN FLUSH TO TRIT #14 TO COLUMN FLUSH TO TRIT #14 TO COLUMN FLUSH TO TRIT #14 TO COLUMN FLUSH TO TRIT #18 TO COLM FLUSH TO TRIT WASTE BOTTLE BLOCK FLUSH #18 TO COLM REVERSE FLUSH BLOCK FLUSH CYC END STEP TIME 1 10 1 10 1 10 1 10 1 10 8 1 3 20 5 3 1 A Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes ACTIVE FOR BASES G C T X Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Section 4: Functions, Cycles and Procedures Applied Biosystems Procedures The Phosphoramidite Purge Procedure The phosphoramidite purge procedure fills all phosphoramidite and tetrazole delivery lines (from the reservoir to the reagent valve block) with fresh reagent. This process uses less than one-half the amount of phosphoramidite used in a coupling step of a one-micromole synthesis. A purge is done prior to beginning a synthesis when the instrument has been idle for more than 12 hours (more than 6 hours in humid environments) or if one of the phosphoramidite reservoirs has not been accessed within 12 hours. Over this time, oxygen and atmospheric water can penetrate the delivery lines and cause failures in the first coupling. A phosphoramidite purge eliminates this problem and helps ensure a successful synthesis. IMPORTANT Not performing a phosphoramidite purge when it is needed will cause a synthesis to fail. When in doubt, it is best to purge since it may save a synthesis. Before beginning a synthesis, you must choose whether to perform the procedure. Refer to Section 3: Main Menu Option: Start Synthesis for further information. Note that a phosphoramidite purge is not included in the bottle cycle counter which is used to tabulate the alarm. As shown below, the procedure consists of nine steps. If desired, the steps can be changed using the Procedure Editor Menu. The Phosphoramidite Purge Procedure STEP NUMBER 1 2 3 4 5 6 7 FUNCTION NUMBER FUNCTION NAME TIME SECONDS F 28 PHOS PREP 10 (pressurizes the phosphoramidite and tetrazole reservoirs prior to delivery) F 52 A TO WASTE 4 F 53 G TO WASTE 4 F 54 C TO WASTE 4 F 55 T TO WASTE 4 F 56 X TO WASTE 4 F 61 TET TO WASTE 4 (Steps 2-7 deliver the contents of the designated reservoir to the waste bottle. Fills the delivery lines and the reagent valve block) Section 4: Functions, Cycles and Procedures 4-23 Applied Biosystems STEP NUMBER 8 9 FUNCTION NUMBER FUNCTION NAME TIME SECONDS F 10 #18 TO WASTE 7 (Removes the phosphoramidite and tetrazole left in the reagent valve block. Rinses the valve block) F1 BLOCK FLUSH 10 (Removes acetonitrile from the reagent valve blocks) The Bottle Change Procedure Bottle change procedures are used to remove empty reservoirs and replace them with bottles of fresh reagents. They can be performed before beginning a synthesis or when a synthesis is interrupted either manually or by the alarm. This process is especially important for preventing oxygen and water contamination of atmosphere-sensitive phosphoramidites and tetrazole. A procedure exists for all 11 bottles and each can be edited using the Procedure Editor. Copies of the procedures can be found in Appendix A. 4-24 Section 4: Functions, Cycles and Procedures Applied Biosystems The Phosphoramidite Bottle Change Procedure STEP NUMBER FUNCTION NUMBER FUNCTION DESCRIPTION TIME SECONDS 1 2 F1 BLOCK FLUSH 5 F 10 18 TO WASTE 7 (The reagent valve blocks are cleared by F1, block flush, for 5 seconds and rinsed by F 10, #18 to waste, for 7 seconds.) 3 A-PHOS, F 71 18 TO BOTTLE 5 G-PHOS, F 72 C-PHOS, F 73 T-PHOS, F 74 X-PHOS, F 75 (Old reagent in the delivery line is removed and forced back into the reservoir by an acetonitrile wash via delivering #18 to the designated bottle for 5 seconds.) 4 A-PHOS, F 62 FLUSH TO BOTTLE 10 G-PHOS, F 63 C-PHOS, F 64 T-PHOS, F 65 X-PHOS, F 66 (An argon flush clears the lines for 10 seconds) 5 INTERRUPT, F 17 (Remove the bottle, place the new bottle on the instrument) If you are going to remove the phosphoramidites and save them for later use, do not complete these steps or HPLC grade acetonitrile will contaminate the bottles. Refer to Section 2 for instructions on how to store dissolved phosphoramidites. 6 A-PHOS, F 62 FLUSH TO BOTTLE 5 G-PHOS, F 63 C-PHOS, F 64 T-PHOS, F 65 X-PHOS, F 66 (Argon purges the headspace of the phosphoramidite reservoirs to eliminate air.) 7 A-PHOS, F 52 BOTTLE TO WASTE 4 G-PHOS, F 53 C-PHOS, F 54 T-PHOS, F 55 X-PHOS, F 56 TETRAZOLE, F 61 (The delivery line is refilled (primed) with fresh reagent by delivering the contents of the designated bottle to waste for 10 seconds.) 8 F 10 18 TO WASTE 7 9 F1 BLOCK FLUSH 10 (The reagent valve blocks are rinsed by F 10, #18 to waste, for 7 seconds and cleared by F 1, block flush, for 10 seconds.) Section 4: Functions, Cycles and Procedures 4-25 Applied Biosystems Similarly, the bottle change for acetonitrile (Bottle 18) begins by performing F 1, block flush, for 5 seconds and F 10, #18 to waste, for 7 seconds. The bottle is then removed and replaced. Finally, fresh acetonitrile is delivered to waste (F 10 for 7 seconds) and the reagent valve block is cleared by a block flush, F 1, for 10 seconds. Both capping reagents (cap A, acetic anhydride, Bottle 11; cap B, NMI, Bottle 12) use the same procedure. The valve blocks are cleared and rinsed by F 1 for 5 seconds and F 10 for 7 seconds. The bottles are then removed and replaced. Next, argon pressurizes the reservoir by F 16, cap prep, for 5 seconds. Then, the contents of the bottles are delivered to waste to prime the lines (using F 59 for acetic anhydride and F 60 for NMI for 5 seconds). Finally, the valve blocks are rinsed and cleared by F 10 for 7 seconds, and F 1 for 10 seconds. The TCA (Bottle 14) and iodine (Bottle 15) reservoirs use the same bottle change procedure. The valve blocks are cleared and rinsed by F 1 for 5 seconds and F 10 for 7 seconds. The bottles are then removed and replaced. Next, the delivery lines are primed for 5 seconds (F 82 for TCA; F 81 for iodine). Finally, the valve blocks are rinsed and cleared by F 10 for 7 seconds and then F 1 for 10 seconds. 4-26 Section 4: Functions, Cycles and Procedures Applied Biosystems The Shut Down Procedure The shut down procedure prepares the instrument for long term storage. It removes all reagents in the delivery lines and washes and dries all chemical pathways. To perform this procedure, select Shut Down from the main menu and follow the instructions in Section 3. STEP NUMBER FUNCTION NUMBER FUNCTION DESCRIPTION TIME SECONDS 1 2 3 4 5 6 7 F1 BLOCK FLUSH 5 F 10 18 TO WASTE 5 F 71 #18 TO A 60 F 72 #18 TO G 60 F 73 #18 TO C 60 F 74 #18 TO T 60 F 75 #18 TO X 60 (Old phosphoramidites in the delivery lines are removed and forced back into the reservoirs by an acetonitrile wash.) 8 F 62 FLUSH TO A 60 9 F 63 FLUSH TO G 60 10 F 64 FLUSH TO C 60 11 F 65 FLUSH TO T 60 12 F 66 FLUSH TO X 60 (The lines are cleared by an argon flush to each bottle) 13 10 #18 TO WASTE 10 14 78 #18 TO TET 60 15 87 #18 TO 11 60 16 88 #18 TO 12 60 17 84 #18 TO 14 60 18 85 #18 TO 15 60 (Reagents in these delivery lines are removed and forced back into the reservoirs by an acetonitrile wash) 19 69 FLUSH TO TET 60 20 89 FLUSH TO 11, 12 60 21 86 FLUSH TO 14, 15 60 (The lines are cleared by an argon flush to each bottle) 22 10 18 TO WASTE 10 23 1 BLOCK FLUSH 5 24 17 INTERRUPT 1 (F 17 Provides a pause to remove all bottles and wipe the lines) 25 62 FLUSH TO A 5 26 63 FLUSH TO G 5 27 64 FLUSH TO C 5 28 65 FLUSH TO T 5 29 66 FLUSH TO X 5 30 70 FLUSH TO 18 10 (Provides an additional argon flush to clear the phosphoramidite and acetonitrile lines) 31 1 BLOCK FLUSH 10 Section 4: Functions, Cycles and Procedures 4-27 Applied Biosystems The Flow Test Procedure The flow test procedure is used to measure flow rates through all essential delivery lines. It can be used for routine maintenance, troubleshooting and recalibration of the instrument if you suspect flow related problems. To perform this procedure, select SELF TEST from the Main Menu. Then, press MORE until the FLOWTST option is shown. Further instructions appear in Section 3: Main Menu Option: Self Test and Section 2: How to Perform the Flow Test Procedure. This procedure is not found in the procedure editor and cannot be edited or printed. A copy of the flow test procedure can be found in Appendix A. The flow test has four parts and takes about one hour to perform. The first part, steps 1-14, washes and primes the delivery lines with acetonitrile. The second, steps 15- 43, measures the flow of acetonitrile from each bottle position to the lower column Luer fitting. The third part, steps 44-46, measures the flow from bottle 14 to the trityl collection line. In the final part, steps 47-59, all the lines are flushed dry with argon enabling reattachment of the reagents. 4-28 Section 4: Functions, Cycles and Procedures Section 5: System Description – Hardware This section describes the components of the Model 391 DNA Synthesizer. Use it as a reference guide to familiarize yourself with the synthesizer and its operations. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-2 The Chemical Delivery System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-3 Pressure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-4 Reagent and Solvent Reservoirs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-4 Delivery Valve Blocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-6 Vacuum Assist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-9 The Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-10 Waste and Venting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-11 The Battery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-12 The Controller . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-13 Fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-14 Applied Biosystems Introduction The major components of the Model 391 Automated DNA Synthesizer are shown in Figure 5-1. All chemical steps required for DNA synthesis take place within a reaction chamber, called the column. The column contains the 3'-terminal nucleoside which is covalently attached to a support. The DNA chain is built by adding one base at a time to the support-bound nucleoside. All reagents and solvents necessary for synthesis are accessed by the chemical delivery system. In this system, a set of solenoid valves opens to create a pathway for chemical flow. Pressure regulated argon forces the chemicals to flow from their reservoirs through the pathway (consisting of one or more valve blocks and delivery lines) and then to the column. After completing a chemical step, the column effluent flows to either the waste bottle, or the trityl collection port to collect the dimethoxytrityl cation for analysis. The controller directs and initiates all synthesizer activity and consists of a microprocessor, the software, a keyboard, a display screen and associated electronics. Information is displayed on a twoline screen and you give instructions by pressing the appropriate key on the keyboard. 5-2 Section 5: System Description – Hardware Applied Biosystems The Chemical Delivery System The flow of reagents, solvents and gas through the Model 391 is controlled by a positive-pressure chemical delivery system. The system components include a regulated pressure source, eleven reagent and solvent reservoirs, delivery valves, a column, a vented waste bottle and delivery lines which interconnect the components. All inner surfaces of the chemical delivery system are made of inert materials. Figure 5-1. Major Components of the Model 391 Section 5: System Description – Hardware 5-3 Applied Biosystems Pressure System pressure is provided by pre-purified (99.998%) argon. Argon’s high density and low oxygen contamination make it preferable to nitrogen. An argon cylinder is connected to the inlet port at the right rear of the Model 391 using ¼-inch oxygen-impermeable tubing and a gas-tight connector supplied by Applied Biosystems. The pressure regulator on the tank is set to 60 psi. This high pressure argon is used to operate the vacuum-assist system (described later in this section) and to supply argon to a regulator in the synthesizer. IMPORTANT The argon cylinder must always be connected to the synthesizer before the power is turned on. If this is not done, the vacuum-assist valves will be activated continuously and can overheat and fail causing argon to leak. An empty argon cylinder can be replaced before beginning a synthesis or when an active synthesis is interrupted. Refer to Section 2: How to Change an Argon Tank. Argon entering the synthesizer travels through a 10-micron particle filter to a pressure regulator. The regulator delivers argon to specific pressure valves used to pressurize the reagent and solvent reservoirs. (Pressure valves are further discussed in Section 4: Valves, and are shown in Figure 41.) In addition, the regulator supplies argon to the column valve block and the reagent valve blocks. The phosphoramidites have one pressure valve which supplies argon to a manifold pressurizing all five phosphoramidite bottles simultaneously. For proper pressurization, bottles must be attached to all five positions, even if some are empty. The two capping reagents (1-methylimidazole, NMI, and acetic anhydride) share a pressure valve which channels the argon to a tee to pressurize both bottles. All other reagent reservoirs (tetrazole, trichloroacetic acid, iodine and acetonitrile) are pressurized using a single valve for each bottle. The regulator is adjusted so that acetonitrile from bottle 18 has a flow rate of 2.80 to 3.20 mL/min measured at the lower Luer fitting of the column. The pressure gauge should read approximately 4.0 psi. For details on how to adjust flow rates, refer to Section 2: How to Perform the Flow Test Procedure. Reagent and Solvent Reservoirs Each reservoir has a unique position on the instrument and is referred to by the number located above each receptacle. Position numbers are also printed on the bottle labels. The position or reservoir numbers are 1-5, 9, 11, 12, 14, 15, and 18. These numbers correspond to those on other Applied Biosystems DNA synthesizers which have 18 positions. Reservoirs 1-5 are pushed upward around a Teflon insert and a silicone O-ring to form an airtight seal inside each bottle neck. Pressing the appropriate black button above the bottle position releases 5-4 Section 5: System Description – Hardware Applied Biosystems the grip on the bottle and allows for its removal and subsequent replacement. When replacing a bottle, the button will return to its out position only when the reservoir is correctly engaged. Bottles 9, 11, 12, 14 and 15 screw snugly into a threaded cap mounted on the synthesizer. A disposable polyethylene insert forms an airtight seal between each cap and reservoir. Note Receptacles 9-15 have a ratchet cap assembly. When attaching these bottles, you cannot overtighten them. Reservoir 18, containing acetonitrile, is a 4-Liter bottle that does not attach directly to the instrument. Instead, you place it inside a protective carrier and put it into a metal rack that attaches to the left side of the instrument. A cap assembly, which includes the delivery and gas lines, screws onto the bottle and connects to the synthesizer. The cap has a teflon insert and a silicone rubber gasket. Applied Biosystems provides the cap assembly, the carrier, the rack and one bottle of acetonitrile. The Pressure and Delivery Lines Each bottle has an argon pressure line and a delivery line entering through the cap insert. For the phosphoramidites, bottles 1-5, the pressure line also functions as a vent line. As shown in Figure 52, the argon line remains above the liquid level while the delivery line extends to the bottom of the bottle. Upon opening the correct set of valves, the reservoir headspace is pressurized by argon and the liquid is pushed into the delivery line and flows to its destination. Figure 5-2. Reagent Reservoir Since all phosphoramidite reservoirs are pressurized simultaneously by a single valve, bottles must be attached to all 5 positions (even if some are empty) or the argon will escape out any exposed pressure line. Also, bottles should be placed on all positions to keep the lines clean. Notice that all lines are color coded. Phosphoramidite delivery lines are red (0.5mm ID), while the other reagent delivery lines are blue (0.8mm ID). All pressure lines are blue (0.8mm ID). Section 5: System Description – Hardware 5-5 Applied Biosystems Venting In addition to being pressurized, the phosphoramidites are vented using the pressure/vent line. When fresh phosphoramidites, which are atmosphere-sensitive, are placed on the instrument, they are purged with argon to eliminate air. A purge delivers gas through the delivery line. As the gas is passed through the bottle, the air escapes out the pressure/vent line. This is performed automatically by the bottle change procedure. Delivery Valve Blocks The chemical delivery system includes two reagent valve blocks and one column valve block.The valve blocks control gas and chemical flows to the column and exit ports. Figure 5-3 shows a schematic representation of all three valve blocks. 5-6 Section 5: System Description – Hardware Applied Biosystems Figure 5-3. Valve Blocks Schematic Section 5: System Description – Hardware 5-7 Applied Biosystems The first reagent valve block is an eight-port block which controls the delivery of the following solvents and reagents: tetrazole acetic anhydride 1-methylimidazole trichloroacetic acid iodine acetonitrile bottle 9 bottle 11 bottle 12 bottle 14 bottle 15 bottle 18 Each of the above reagents flows from its reservoir through the first reagent valve block and then through the second which directs the flow to the column or waste. The first reagent valve block also controls the delivery of argon through both reagent valve blocks. The second reagent valve block, an eight-port block, controls the delivery of the phosphoramidites to the column or waste. It also directs the flow from the first reagent valve block to the column or waste. The column valve block, a four-port block, directs the column effluent to either the waste or the trityl collection port. It also controls the argon gas used to remove or flush the reagents from the column and the column valve block. The design of the valve blocks provides zero-dead volume. Delivery lines feed into each valve block and connect to the common pathway in the valve block manifold via a manifold inlet line and a solenoid-controlled diaphragm valve. See Figure 5-4. The delivery lines enter the valve block through the manifold inlet lines. Passage between the manifold inlet line and the common pathway of the valve block is accomplished by an open solenoid valve. When a valve opens, the solenoid piston pulls away from a diaphragm located under the piston. With vacuum assist, an open solenoid will cause the diaphragm to form a 2-µL domed chamber. The domed chamber creates a passageway between the inlet line and the common pathway. The common pathway zigzags through the valve block manifold and passes other closed valves which are unaffected by the flow. The direction of flow is determined by the pressures on either side of the valve block. During proper operation, the flow will be toward the column or one of the exit ports. 5-8 Section 5: System Description – Hardware Applied Biosystems Figure 5-4. The Valve Block Vacuum Assist For proper valve block operation, it is crucial that each diaphragm forms a domed chamber. The vacuum assist creates a vacuum on the solenoid side of the diaphragm to form a domed chamber each time a solenoid valve opens. An aspirator pump provides vacuum assist to each valve block. The aspirator pump requires that regulated argon enter the synthesizer at a gauge pressure of approximately 60 psi. The argon regulator should be connected to the instrument with the ¼-inch tubing supplied by Applied Biosystems. IMPORTANT Do not use 1/8-inch tubing, it is too narrow to deliver the correct volume of gas. The vacuum gauge should always read between 14 in. and 18 in. Hg. When the gauge drops below 14 in. Hg. (once every 4-10 hours), a valve opens to allow high pressure argon to flow to the aspirator pump. The gas is then diverted out of the pump and creates a Venturi vacuum when a second Section 5: System Description – Hardware 5-9 Applied Biosystems valve opens to the vacuum-assist plumbing. If the gauge pressure drops below 14 in. Hg. more than once every 4 hours, there is a leak in the system. Contact the Applied Biosystems Technical Support Department for repair instructions. The Column The initial support-bound nucleoside is contained in a disposable column which has two silica-retaining filters, two end fittings and two caps (Figure 5-5). All parts, except the aluminum caps, are made of inert materials. The retaining filters are porous Teflon held in place by a circular ridge on the inside of the column. Under pressure from the metal cap, the end fittings press against the ridge to form a tight seal around the edge of each filter. Tear-off aluminum tabs protect the filters during shipping. The inlet and outlet are female Luer fittings designed to mate with the male Luer fittings on the instrument. The column is symmetrical (i.e. no top or bottom, no front or back) and can be attached to the male Luer fittings in any way. Each column is color coded to show the initial nucleoside and has a unique serial number. Column A G C T Color code green yellow red blue The normal flow into the column is from the bottom. By sending the liquid stream upward, the CPG particles are lifted and maintained in a fluidized state. The flow rates of the solvents and reagents have been set to achieve proper mixing of the particles. 5-10 Section 5: System Description – Hardware Applied Biosystems Figure 5-5. The Column Waste and Venting The ultimate destination of most chemical deliveries is the waste bottle. The bottle is a free-standing, four-liter polyethylene container which should be placed on the floor near the synthesizer or on a nearby bench lower than the instrument. The bottle can be kept inside a protective bottle carrier to contain accidental spillage. A one-gallon carrier is sufficient and can be purchased from VWR, Part Number 56609-186, or Nalge, Part Number 6501-0010. Section 5: System Description – Hardware 5-11 Applied Biosystems WARNING Synthesizer waste must be handled and disposed of properly and carefully. Depending on the synthesis cycle used, the instrument generates 1 to 2 liters of hazardous, halogenated organic liquid waste per 100 cycles of operation. When handling the waste for disposal, wear gloves, a lab coat and eye protection, and avoid inhalation and skin contact. Place the liquid in a sealed container labeled “FLAMMABLE”, “POISON B.N.O.S.” or absorb in vermiculite, dry sand or earth. Dispose of properly according to the appropriate local government regulations. A waste line and vent line enter the cap of the waste bottle. Be sure this cap is always securely tightened. The waste line carries liquid waste from the synthesizer to the bottle. Be sure the line slopes downward toward the bottle and has no troughs that can collect waste and block the line. The vent line carries gaseous waste to a suitable exhaust such as a fume hood. Applied Biosystems supplies a 25-foot length of tubing to connect the vent line to a fume hood. Proper venting allows operation of the Model 391 on an open lab bench. IMPORTANT Prevent condensation from collecting in the vent line by continuously sloping the tubing upward toward the fume hood. The tubing should not be horizontal or have troughs that will form collecting points. Condensation will eventually cause erratic deliveries and can even stop the flow. IMPORTANT The waste bottle is the low pressure side of the delivery system and must always be vented to atmosphere. If it is not, back pressure will be generated which will decrease the deliveries of reagents and solvents. The Battery A lithium battery is provided in the Model 391. When the main power is turned off (either intentionally or during a power failure), all synthesis parameters are retained. These parameters include the DNA sequences; user-defined cycles, procedures and functions; and bottle usage information. If a power failure occurs during an active synthesis, the synthesis will be interrupted and can only be resumed when the main power returns. The battery also maintains the synthesizer’s internal clock. If a power outage does occur, the clock records the time the power failed and the time it was restored. A synthesis will then automatically resume if the elapsed time of the failure is less than the time you entered in the Power Fail Menu. Refer to Section 3: Main Menu Option: Power Fail, for further instructions. 5-12 Section 5: System Description – Hardware Applied Biosystems The battery’s voltage is checked during the system self-test of the electronic components. It should operate for several years. When it needs replacing, the display screen will show a battery low message. WARNING The used lithium battery must be replaced and disposed of properly. For further information, consult the Applied Biosystems Technical Support Department. The Controller The controller directs and initiates all synthesizer activity. Its major components are the software, the microprocessor, the display screen, the keyboard and associated electronics. The software defines all necessary operations for synthesis and is interpreted and executed by the microprocessor. The software is contained within a removable memory cartridge which plugs into the rear of the instrument. Updated software supplied by Applied Biosystems can be easily installed by removing the old cartridge and replacing it with the new one. IMPORTANT Before removing the memory cartridge, be sure the main power is off. Synthesis information is shown on a two-line liquid crystal display (LCD). The display can be adjusted for optimal viewing by turning the knob at the left of the screen. You interact with the instrument by selecting keys on the keyboard. The keyboard consists of five soft keys (located below the screen) and several labeled keys (0-9; A, G, C, T, X; delete; and left and right arrows). The labels and actions of the soft keys change depending on the information displayed directly above them. The software is “menu-driven” where menus and pages of menus present various options and necessary information about the synthesis or status of the instrument. In response, you select an option and give instructions by pressing the appropriate key. For a complete description of all menus, refer to Section 3. To perform automated synthesis, the software uses a cycle consisting of a series of steps which complete all chemical reactions. For further information about cycles, see Section 3: Main Menu Option: Cycle Editor, and Section 4: Synthesis Cycles. Proper operation of the controllers electronic components are verified by a system self test. For further instructions, refer to Section 3: Main Menu Option: Self Test. Section 5: System Description – Hardware 5-13 Applied Biosystems IMPORTANT A fan on the rear of the instrument distributes air through the electronics for cooling and should not be obstructed. Fuses The Model 391 has two power fuses. They are located at the rear of the instrument to the left of the fan and directly above the power line. Although the fuses have a very long life, they can stop operating. For example, if the 391 has no power (i.e. the fan is not working or the LCD screen is blank), a bad fuse may be causing the problem and will need replacing. Before concluding there is a bad fuse, however, check if the instrument is properly connected to a functional power source. Extra fuses are included in the spare parts kit. For further information call the Applied Biosystems Technical Support Department. 5-14 Section 5: System Description – Hardware Section 6: Chemistry for Automated DNA Synthesis Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-2 The Solid Support - CPG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-5 DNA Synthesis Chemistry Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-7 Detritylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-8 Monitoring the trityl cation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-9 Depurination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-11 Coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-12 Phosphoramidites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-12 Capping. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-15 Oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-16 Completion of the Synthesis Cycle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-18 Manual Deprotection and Cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-18 Cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-18 Phosphate Deprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-18 Base Deprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-19 Quantitation of the Oligonucleotide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-20 Storage of the Oligonucleotide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-20 Analysis and Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-21 OPC, Oligonucleotide Purification Cartridge. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-21 PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-22 HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-24 Alternative Chemistries. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-24 RNA Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-24 Hydrogen-Phosphonate Chemistry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-25 Phosphorothioate DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-26 5' Attachments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-27 Fluorescent-dye Linked Sequencing Primers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-28 Amino Link 2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-28 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-30 Applied Biosystems Introduction DNA synthesis is quite simple in concept. A reactive 3' phosphorous group of one nucleoside is coupled to the 5' hydroxyl of another nucleoside. The former is a monomer, delivered in solution. The latter is immobilized on a solid support. An internucleotide linkage is thus formed. Three other chemical reactions are necessary to prepare the growing chain of DNA for the next coupling. In this way a synthesis cycle is conducted, adding one nucleoside monomer at a time. The desired sequence and length are defined by the operator on the synthesizer.1 When the chain is complete, the crude DNA (oligonucleotide) must be cleaved from the support and deprotected. Further purification is usually advised. When the complete operation becomes routine, the synthesis of oligonucleotides becomes reliable and their biological activity is assured. Since most laboratories are not interested in this as a research project, the goals are for DNA synthesis to become cheaper, faster, better, easier, and more flexible. This chapter will help you understand the synthesis chemistry and how you can attain these goals. The phosphoramidite method of oligonucleotide synthesis is the chemistry of choice for most laboratories because of efficient and rapid coupling and the stability of the starting materials.2 The synthesis is performed with the growing DNA chain attached to a solid support so that excess reagents which are in the liquid phase can be removed by filtration.3 Therefore, no purification steps are required between cycles. This support material is a form of silica, controlled-pore-glass (CPG) beads.4 The particle size and the pore size have been optimized for liquid transfer and mechanical strength. The synthesis cycle is depicted in Figure 6-1. The starting material is the solid support derivatized with the nucleoside which will become the 3'-hydroxyl end of the oligonucleotide. As shown in Figure 6-2, the nucleoside is bound to the solid support through a linker attached at the 3'-hydroxyl. The 5'-hydroxyl is blocked with a dimethoxytrityl (DMT) group. The first step of the synthesis cycle is treatment of the derivatized solid support with acid to remove the DMT group (Figure 6-4). This frees the 5'-hydroxyl for the coupling reaction. (Figure 6-5). An activated intermediate is created by simultaneously adding the phosphoramidite nucleoside monomer and tetrazole, a weak acid, to the reaction column. The tetrazole protonates the nitrogen of the phosphoramidite, making it susceptible to nucleophilic attack. This intermediate is so reactive that addition is complete within 30 seconds. As shown in Figure 6-5, the phosphoramidite is blocked at the 5'-OH with the dimethoxytrityl group. The next step, capping, terminates any chains which did not undergo addition. Since the unreacted chains have a free 5'-OH, they can be terminated or capped by acetylation. These unreacted chains are also called “failure products”. Capping is done with acetic anhydride and 1-methylimidazole.5 Since the chains which reacted with the phosphoramidite in the previous step are still blocked with the dimethoxytrityl group, they are not affected by this step. Although capping is not required for DNA synthesis, it is highly recommended because it minimizes the length of the impurities and thus facilitates product identification and purification (Figure 6-7). 6-2 Section 6: Chemistry for Automated DNA Synthesis Applied Biosystems The internucleotide linkage is then converted from the phosphite to the more stable phosphotriester. Iodine is used as the oxidizing agent and water as the oxygen donor. This reaction is complete in less than 30 seconds (Figure 6-8). After oxidation, the dimethoxytrityl group is removed with a protic acid, either trichloroacetic or dichloroacetic acid. The cycle is repeated until chain elongation is complete. At this point, the oligonucleotide is still bound to the support with protecting groups on the phosphates and the exocyclic amines of the bases A, G, and C. The oligonucleotide is cleaved from the support by a one-hour treatment with concentrated ammonium hydroxide. Ammonia treatment also removes the cyanoethyl phosphate protecting groups. The crude DNA solution in ammonium hydroxide is then treated at 55°C for 8 to 15 hours to remove the protecting groups on the exocyclic amines of the bases (Figure 6-9). Note that synthesis can be performed using methyl, or the newer cyanoethyl phosphoramidites.6 These two versions of synthesis monomers differ only by the protecting group on the phosphorous oxygen. The synthesis chemistry and the resulting oligonucleotide are the same with both. Excellent results are obtained with either one.7 The primary difference is that when using methyl phosphoramidites, thiophenol treatment is required to deprotect the internucleotide methyl phosphotriester groups at the end of synthesis. Thiophenol is foul smelling and toxic. In addition, this adds an extra 30-60 minutes to deprotection time. For these reasons, use of cyanoethyl phosphoramidites is strongly recommended. Methyl phosphoramidites, since they offer no advantage or unique utility, are no longer available from Applied Biosystems. Section 6: Chemistry for Automated DNA Synthesis 6-3 Applied Biosystems Figure 6-1. 6-4 The Synthesis Cycle Section 6: Chemistry for Automated DNA Synthesis Applied Biosystems The Solid Support - CPG The Model 391 DNA Synthesizer uses a solid phase synthesis chemistry in which the growing DNA chain remains covalently attached to an insoluble support. All reagents and solvents flow through the support which is contained within a synthesis column. The support used for DNA synthesis is Controlled-Pore-Glass (CPG).4 When used with the Model 391 and other Applied Biosystems reagents, CPG produces coupling efficiencies of 98 to 100%, as measured by the trityl cation assay. This enables the synthesis of oligomers up to 175 bases in length.8 CPG is a porous, non-swelling particle which is about 150 microns in diameter and has 500Å pores. A wide-pore CPG support (1000Å) is also available and should be used when synthesizing oligonucleotides greater than 60 bases8. CPG is covalently derivatized with one of the four nucleosides (A, G, C or T), see Figure 6-2. The reactive groups on these nucleosides are blocked or protected to prevent unwanted side reactions. They are all blocked at the 5'-hydroxyl with a dimethoxytrityl group. As shown in Figure 6-3, the exocyclic amines on adenosine (A) and cytosine (C) are protected by a benzoyl group (Abz, Cbz), and the exocyclic amine on guanosine (G) by an isobutyryl group (Gib). Thymidine does not need a protecting group since the thymine ring does not participate in the synthesis chemistry. As shown in Figure 6-2, CPG has a linker attached to its surface via a siloxane bond. All free silanol groups are capped to prevent side reactions. The support is then derivatized by covalently attaching the 3'-hydroxyl of the nucleoside to the linker via a succinate ester bond. The bond is base labile and allows for removal of the DNA from the support with ammonia. After synthesis is complete the oligonucleotide is quantitatively cleaved with a free 3'-hydroxyl. The loading of nucleoside as measured by DMT release is 27 to 30 micromoles per gram of 500Å support. Pre-filled columns contain 0.2 µmol, 1 µmol or 10 µmol of initial nucleoside. The 0.2 µmol scale provides sufficient amounts of purified oligomers for most applications. The 1 µmol scale is used when greater quantities of DNA are needed. The 10 µmol scale is useful for physical studies, such as X-ray crystallography or nmr. Wide-pore CPG supports are only available on the 0.2-micromole level. The loading of nucleoside is lower, about 15 µmol/gm of 1000Å support. For the synthesis of long oligonucleotides (“bigmers”), it has been shown that a lower loading of nucleoside and larger CPG pore size are critical requirements for success. Section 6: Chemistry for Automated DNA Synthesis 6-5 Applied Biosystems Typical quantities of oligonucleotide obtained from the different synthesis scales are given below: synthesis cycle .2µM crude yield* (O.D.) (20mer) 20-25 1µM 100-120 Low** 20-25 10µM 800-1000 *Yield figures based on a 20mer sequence. Absorbance measured at 260nm. Assuming 33 micrograms/O.D. unit. **Low is a low consumption cycle on the .2gM scale. With automated synthesis, the DNA is built from 3' to 5'. Before beginning a synthesis, one of four support-bound nucleosides (A, C, G or T) contained within a column is placed on the instrument. This nucleoside is the 3'-terminus of the sequence. Figure 6-2. 6-6 The DMT protected nucleoside is attached to the controlled pore glass (CPG) support (B = Base, A, G, C, T). Section 6: Chemistry for Automated DNA Synthesis Applied Biosystems Figure 6-3. Protected exocyclic base amines. Adenosine (A) and cytosine (C) are protected by a benzoyl group (Bz), and guanosine (G) by an isobutyryl group (Ib). Thyrnidine (T) does not need a protecting group. DNA Synthesis Chemistry Cycle Each cycle of base addition consists of four steps: 1. Detritylation 2. Coupling 3. Capping 4. Oxidation. These reaction steps are repeated in the above order until all bases are added. Following synthesis, the DNA chain must be manually cleaved and deprotected from the solid support. Each step will be discussed in detail. Section 6: Chemistry for Automated DNA Synthesis 6-7 Applied Biosystems Detritylation The first step in oligonucleotide synthesis is to remove the acid-labile, dimethoxytrityl (DMT) protecting group on the 5'-hydroxyl of the support-bound nucleoside. As shown in Figure 6-4 treatment with the protic acids, trichloroacetic acid (TCA) or dichloroacetic acid (DCA), will deprotect or detritylate the 5'end. This will yield a reactive 5'-hydroxyl which can couple with a phosphoramidite during the following addition step. The two acids can be used interchangeably and are both available from Applied Biosystems. Dichloroacetic acid is a weaker acid and may yield lower levels of depurination in the synthesis of long oligonucleotides. However, this observation has not been consistently corroborated. For convenience, in this manual the detritylation acid will exclusively be referred to as TCA. Detritylation in more detail Immediately before detritylation, the CPG support is washed with acetonitrile to eliminate traces of the preceding reagent. Next, TCA (bottle 14) is delivered to the column to cleave the trityl group. Detritylation under anhydrous conditions, is a reversible reaction. The DMT cation is highly reactive and can re-tritylate any reactive nucleophile. Detritylation is driven to completion by the removal of the DMT cation from the synthesis column. Therefore, detritylation is conducted by continuous delivery of TCA and elution of the DMT cation. Unlike the other reactions in the cycle, there is no wait period for detritylation. Any residual TCA must be removed by an acetonitrile wash to prevent detritylation of the incoming phosphoramidite. If the phosphoramidite monomer becomes detritylated, an unwanted dimer can form in solution and then couple to the support-bound nucleotide chain. Continued chain propagation would result in some sequences being longer than the product, making purification difficult. Following both acetonitrile washes, the remaining solvent is forced out of the column by an argon reverse flush - argon passes through the column from top to bottom and pushes the liquid to waste. For a summary of these steps, see Table 6-1. 6-8 Section 6: Chemistry for Automated DNA Synthesis Applied Biosystems Table 6-1. Detritylation Steps STEP DESCRIPTION PURPOSE acetonitrile (bottle 18) delivery to column wash column and support, remove traces delivery of preceding reagent argon reverse flush remove residual acetonitrile from column TCA (bottle 14) delivery to detritylate support-bound nucleoside column acetonitrile (bottle 18) delivery to column wash column, remove traces of TCA argon reverse flush remove residual acetonitrile from column Note The complete synthesis cycle contains steps to wash and flush the valve blocks and delivery lines. Figure 6-4. Detritylation. TCA or DCA is used to remove the DMT from the 5'end. This leaves a 5' hydroxyl to react with the incoming phosphoramidite in the coupling step. Quantitating the released trityl cation indicates the step-wise yield and can be used to monitor the instrument’s performance. Monitoring the trityl cation When the dimethoxytrityl, also referred to as DMT or trityl, protecting group is cleaved from the nucleotide, it exists as a cation (Figure 6-4). When in acid solution, this cation is relatively stable and produces a brilliant orange color. It has an absorbance maximum at about 498 nm and extinction Section 6: Chemistry for Automated DNA Synthesis 6-9 Applied Biosystems coefficient of about 70,000 in most solvents, such as acetonitrile or dichloromethane. It is easily detected and quantitated spectrophotometrically. To quantitate the trityl cation released at each detritylation step, the column effluent from the TCA delivery and the subsequent wash with acetonitrile is collected. Instead of the effluent flowing into the waste bottle, it is channelled through the trityl collection port into tubes in a fraction collector. The Model 391 cycles provide for the collection of trityl effluent fractions and include a signal to advance a fraction collector (user provided). Next, the absorbance of the fractions is measured to quantify the trityl released in each addition cycle. Since the trityl solution is very concentrated, it must be diluted before quantitating or significant errors in the readings will occur. Typically, the trityl fraction is brought up to a volume of 10 mL with 0.1 M p-toluenesulfonic acid in acetonitrile. The trityl yield is then used to calculate the coupling efficiency of each addition step by dividing the next step absorbance value from the previous step value. From this data, an overall stepwise yield can be determined and the expected product yield can be estimated. The molar amount of DMT cation can be calculated using Beer’s Law: A = eCl where: A - absorbance e - extinction coefficient C - concentration l - path length overall yield = coupling yield number of couplings For more details, see the trityl assay procedure discussed in User Bulletin 13 (Revised) in the appendix. Monitoring the trityl cation is very important, but the results must be interpreted with caution. The trityl assay is only an indirect measure of synthesis efficiency. Certainly, high trityl cation yields (>98%) must be present for a good synthesis. However, high trityl yields can be present when a poor synthesis occurs. The reason for this discrepancy is that although this chemistry is highly refined, it is not perfect. Unwanted side-reactions do occasionally occur. Some of these side-reactions contribute to the trityl cation released each cycle. In particular, a low level of extraneous chain growth other than the desired oligonucleotide occurs. Sites besides the 5' hydroxyl group participate in coupling (e.g., imperfectly capped sites on the support or branched sites on the nucleic bases). Other scenarios for imperfect results is if detritylation or capping is incomplete. This could happen if the synthesizer is not operating correctly, the cycle is not optimized, or the reagents are wet or impure. These occasions generate species other than the desired product which release the trityl cation. Low trityl cation yields (<98%) always predict a less than optimal synthesis. In practical terms, 6-10 Section 6: Chemistry for Automated DNA Synthesis Applied Biosystems trityl yields of less than 95% will not allow identification or purification of even short oligonucleotides. For the above reasons the trityl assay must be viewed only as a preliminary, yet convenient, monitor of the synthesizer’s performance. The assay is useful to aid in the early detection and diagnosis of instrument related problems. Many laboratories monitor their trityl fractions only by visual inspection. With experience, a failed synthesis is detected this way. Evaluation of the oligonucleotide by PAGE or HPLC is much more informative than the trityl assay. Synthesizer or reagent problems can only be adequately diagnosed by these methods, which are direct analyses of the product. Depurination Trichloroacetic acid is a very effective protic acid detritylating agent. However, in the presence of protic acids, amine-protected purines (Abz and Gib) are susceptible to depurination (removal of the purine from its sugar). The chemical mechanism is initial protonation at N-7 of the purine ring, causing increased lability of the ribose 1' - purine N-9 bond. Cleavage of adenine and guanine bases yields a 1' hemiacetal ribose ring, the result of depurination. Oligonucleotides which contain apurinic sites are cleaved during the ammonium hydroxide treatment. Cleavage occurs at the internucleotide bonds on the 3' hydroxyl side of the apurinic deoxyribose (This is similar in effect to the chemistry of Maxam-Gilbert sequencing). Each purine in the oligonucleotide chain is exposed to acid at each detritylation step. Purines near the 3' end will have the longest cumulative exposure time and a greater chance for depurination. It has been reported that the 5' terminal purine is more susceptible to depurination than an internucleotide purine with a 3' and a 5' phosphorous.9 The quantity of DNA fragments generated by apurinic ammoniolysis is usually insignificant. If the synthesis is conducted Trityl On, for purification by OPC or Trityl On HPLC, the 5' end fragment of apurinic ammoniolysis will bear a DMT group and may complicate purification. In practice however, using the AB reagents and cycles optimized for DNA synthesis, depurination is not detectable or significant except for long oligonucleotides (>80 bases) which are purine rich near the 3' end.10 To minimize depurination, each treatment with TCA should not be extended beyond the times specified in the cycles. IMPORTANT Do not stop a synthesis while the DNA is exposed to TCA. Do not increase TCA delivery times. Section 6: Chemistry for Automated DNA Synthesis 6-11 Applied Biosystems Coupling Phosphoramidites Phosphoramidites (shown in Figure 6-5) are chemically modified nucleosides which are used as the building blocks for synthesizing DNA. They are added to the support-bound nucleotide chain one at a time until all bases in the sequence are coupled. The cyanoethyl phosphoramidite nucleosides have the following functional groups: 1. A diisopropylamino on a 3' trivalent phosphorous moiety.11 The phosphoramidite is very stable and is made highly reactive by the activator, tetrazole. 2. A β-cyanoethyl protecting group on the 3' phosphorous moiety. This group prevents side reactions and aids in solubility of phosphoramidites. It is removed upon completion of the synthesis by using ammonia. In deprotection, ammonia acts as a base to remove a proton on the methylene group bearing the nitrile group. This anion is formed only in low concentration, but rapidly fragments by a β-elimination reaction to form acrylonitrile and the deprotected internucleotide phosphodiester group. Acrylonitrile then reacts irreversibly with ammonia to form 3-aminopropionitrile, an inert compound. 3. A dimethoxytrityl (DMT) protecting group on the 5' hydroxyl. The DMT is removed during each detritylation step leaving a reactive 5' hydroxyl available for coupling an incoming phosphoramidite. 4. A benzoyl protecting group on the exocyclic amines of A and C (Abz, Cbz), and an isobutyryl protecting group on the exocyclic amine of G (Gib). These amide groups prevent side reactions and are removed upon completion of the synthesis with ammonia. Since thymidine is unreactive and does not contain an exocyclic amine moiety, it is not protected. Figure 6-5. 6-12 Structure of guanosine cyanoethyl phosphoramidite Section 6: Chemistry for Automated DNA Synthesis Applied Biosystems Coupling Before beginning the coupling step, the support is made anhydrous and free of nucleophiles (e.g. water) by an extensive wash with acetonitrile. Any extraneous nucleophiles will compete with the support-bound 5' hydroxyls for the activated phosphoramidite and will decrease coupling efficiency. The column is then dried by an argon reverse flush to remove residual acetonitrile. Tetrazole, the phosphoramidite activator, is next delivered to the column. According to the oligonucleotide sequence, one or more of the phosphoramidites (bottles 1 to 5) and tetrazole (bottle 9) are then simultaneously delivered to the column. Depending on the synthesis cycle, alternate deliveries of tetrazole and then base-plus-tetrazole are repeated up to 3 times. When these reagents mix, the mild acid, tetrazole, (pKa = 4.8, Ref.8) transfers a proton to the nitrogen of the diisopropyl group on the 3' phosphorous (See Figure 6-6). This protonated amine makes a very good leaving group upon nucleophilic attack by the tetrazole to form a tetrazolyl phosphoramidite.12 This is the reactive intermediate which forms the internucleotide phosphite with the support bound 5' hydroxyl. A molar excess of tetrazole ensures that the phosphoramidite will be activated. The excess of phosphoramidite relative to free 5' hydroxyl ensures that the reaction is nearly quantitative (over 98% coupling). The coupling steps are summarized in Table 6-2. Mixed sequence probes are synthesized by simultaneous delivery of up to 5 bases (AGCTX) and tetrazole with near equivalent coupling. The four nucleoside phosphoramidites have slightly different reactivities, as all different molecules must.13 The cyanoethyl phosphoramidites follow the reactivity order of T > G > C > A. When all four are delivered simultaneously, their representation will be (normalized to 100%): T - 30% G - 26% C - 24% A - 20% These values are slightly dependent on cycle, location of the site in the oligonucleotide, age of the phosphoramidite solutions, etc.They have a range of about 3% because of these variables. Section 6: Chemistry for Automated DNA Synthesis 6-13 Applied Biosystems Table 6-2. Coupling Steps STEP DESCRIPTION PURPOSE acetonitrile (bottle 18) delivery to column remove nucleophiles, render support anhydrous argon reverse flush remove residual acetonitrile, dry column tetrazole (bottle 9) delivery to column deliver activator tetrazole + phosphoramidite (bottles 1 to 5) delivery to column activate phosphoramidite, begin coupling reaction tetrazole delivery to column continue coupling tetrazole + phosphoramidite delivery to column continue coupling wait complete coupling reaction argon reverse flush remove the tetrazole and phosphoramidite IMPORTANT These deliveries are critical. Under-delivery causes low coupling efficiency. Overdelivery wastes reagents. 6-14 Section 6: Chemistry for Automated DNA Synthesis Applied Biosystems Figure 6-6. The coupling step. Phosphoramidites and tetrazole are delivered to the column which contains the support-bound nucleotide. The diisopropylamine is protonated and displaced by tetrazole. When the 5' OH couples to the phosphorous, a 5' to 3' internucleotide linkage is created. Capping Because coupling is not always quantitative, a small percentage (up to 2%) of support-bound nucleotides can fail to undergo addition. These truncated, or failure sequences, will remain attached to the support. If they remain in the hydroxyl form, they can propagate in subsequent coupling steps. Failure sequences with one less base than the product would then be generated making isolation of the product more difficult. Capping the remaining free hydroxyls by acetylation eliminates this problem. The capped failure sequences are then prevented from participating in the rest of the synthesis reactions. To cap, equal volumes and equimolar amounts of two binary reagents, acetic anhydride (bottle 11) and 1-methylimidazole5, NMI, (bottle 12), are simultaneously delivered to the column. As shown in Figure 6-7, the reagents mix and create a powerful acetylating agent. The two reagents need to Section 6: Chemistry for Automated DNA Synthesis 6-15 Applied Biosystems be segregated since the active acetylating agent is unstable. This agent reacts at the 5' hydroxyls rendering these moieties unreactive for the remainder of the synthesis. The excess reagents are then removed by an argon reverse flush. The capping time required to acetylate the 1 or 2% unreacted 5' hydroxyls is very brief, only a few seconds. It is important to minimize this time to prevent loss of cyanoethyl groups from the internucleotide linkages and to prevent base modification by-products. The efficiency of shorter capping time and the capping/oxidation order have been extensively demonstrated in studies at Applied Biosystems.14 Figure 6-7. Capping of unreacted chains. The capping reagents, acetic anhydride and 1methylimidazole (NMI), are used to terminate unreacted chains by acetylating the 5' hydroxyl groups. Oxidation The newly formed internucleotide linkage is a phosphite (trivalent phosphorous) triester. The phosphite linkage is unstable and is susceptible to acid and base cleavage. Therefore, immediately after capping, the trivalent phosphite triester is oxidized to a stable pentavalent phosphate triester. This is shown in Figure 6-8. Oxidation follows capping to eliminate the possibility of traces of water from the oxidizing solution causing acetic anhydride to form acetic acid during capping. This would expose the oligonucle- 6-16 Section 6: Chemistry for Automated DNA Synthesis Applied Biosystems otides to acid as well as make capping less effective. Also, scientists at Applied Biosystems have elucidated the complex chemical pathway whereby a small side-reaction, the phosphitylation of the 0-6 position of guanosine can be minimized when capping immediately follows coupling.5,15 The enzymatic digestion/base composition assay on oligonucleotides made with different cycle order; capping then oxidation and oxidation then capping, shows markedly different results. The Applied Biosystems standard; capping then oxidation, gives far less, usually undetectable, amounts of basemodified nucleosides. Cycles with oxidation then capping give high levels of the mutagenic modified nucleoside, 2,6-diaminopurine. Iodine is used as a mild oxidant in a basic tetrahydrofuran (THF) solution with water as the oxygen donor. When the iodine-water-pyridine-THF mixture (bottle 15) is delivered to the column, an iodine-pyridine complex forms an adduct with the trivalent phosphorous. This adduct is decomposed by water with production of a pentavalent phosphotriester internucleotide group. This is an extremely fast reaction, being quantitative in 30 seconds. The iodine solution is removed by an argon reverse flush and several acetonitrile washes, each followed by an argon reverse flush. Other oxidizing agents such as sulfur16 can be used in place of oxygen to create nucleotide phosphate analogs or to introduce radioactive atoms. IMPORTANT Do not stop the synthesis while the phosphorous is unoxidized. Figure 6-8. Oxidation of the trivalent phosphorous. The unstable trivalent phosphorous of the newly formed internucleotide linkage is oxidized to a stable pentavalent phosphorous using an iodine solution. Section 6: Chemistry for Automated DNA Synthesis 6-17 Applied Biosystems Completion of the Synthesis Cycle Following oxidation, a cycle of nucleotide addition is complete. The 5' terminus of the oligomer is protected by the dimethoxytrityl group. DNA synthesis continues by removing the 5' trityl and repeating another cycle of base addition. This is done until DNA of specified length has been fully synthesized. Immediately after completing the synthesis, a trityl group may still be attached to the 5' terminus, according to the user’s option. The ending method instructions may be programmed for the trityl group to remain attached (trityl on) or be cleaved (trityl off). The oligonucleotides are usually detritylated when purifying by gel electrophoresis or ion exchange HPLC. The trityl groups are usually left intact when purifying by OPC or trityl-specific reverse phase HPLC. The ending method (trityl on or trityl off) is generally chosen before beginning a synthesis. Manual Deprotection and Cleavage When the synthesis is finished, the product and capped failure sequences (still attached to the support) exist as phosphate-protected, base-protected phosphotriesters. Complete deprotection is necessary to produce biologically active DNA. In addition, the oligonucleotides must be cleaved from the support. These steps are performed manually after removing the column from the instrument. For complete instructions, refer to the Manual Deprotection and Cleavage in Section 2. Cleavage Following synthesis, the DNA remains covalently attached to the support. The diester oligonucleotides are then cleaved from the support by a one hour treatment with fresh, concentrated ammonium hydroxide. The double syringe method17 is a convenient technique for cleavage. As seen in Figure 6-9, the cleavage occurs at the base-labile ester linkage between the linker of the support and the 3' hydroxyl of the initial nucleoside. The cleaved DNA has a free 3' hydroxyl. The DNA, now in solution, is collected in a vial fitted with a Teflon-lined cap. The vial contains the crude mix (product and failure sequences) of base protected oligonucleotides in ammonium hydroxide. Then, the protecting groups on the exocyclic amines of A, G and C must be removed (see Base Deprotection). Phosphate Deprotection The cyanoethyl protecting groups are removed by treatment with ammonium hydroxide. This occurs at the same time as cleavage making phosphate deprotection very quick and simple. See Figure 6-9. 6-18 Section 6: Chemistry for Automated DNA Synthesis Applied Biosystems Base Deprotection The benzoyl and isobutyryl base protecting groups are removed by placing the vial of DNA at 55 ºC for 8 to 15 hours. This also cleaves the acetyl caps from the failure sequences. Base deprotection is an ammoniolysis reaction, where ammonia is a nucleophile, attacking the carbonyl of the amide protecting groups. For effective treatment, use fresh, concentrated ammonium hydroxide during cleavage. To ensure no decrease in ammonia concentration, store the reagent in a refrigerator, tightly capped. Discard thirty days after opening. IMPORTANT Use a rightly sealed DNA collection vial that can withstand positive pressure. The vial must also have a Teflon-lined cap. Rubber-lined caps have contaminants that leach out of the cap liner during deprotection. Teflon-lined caps can be ordered from Wheaton; Part Number 240408, size 13-425. After completing deprotection, cool the ammonium hydroxide-DNA solution on ice to prevent losses from bubbling. Then remove the ammonia by vacuum. Ammonia is much easier to transfer at lower temperature than at room temperature. When purifying by OPC or trityl-specific reverse phase HPLC, keep the solution basic to prevent accidental detritylation. To do this, add one drop of distilled triethylamine every 10 minutes. Also, avoid heating the sample. If the 5' DMT group has been left on, it can be removed manually by treatment with 80% acetic acid/water for 20 minutes at room temperature. The acid is then diluted with ethanol and removed by vacuum, followed by several rinses with ethanol. This procedure is usually done after reverse phase HPLC or before radioactively labeling the 5' end prior to analysis by gel electrophoresis. The deprotected, detritylated DNA has a free 5' and 3' hydroxyl and is biologically active. Desalting and purification may be necessary before use in experiments. An overview of these procedures is described later in this section. Details can be found in User Bulletin 13 (Revised) in the appendix of this manual. Section 6: Chemistry for Automated DNA Synthesis 6-19 Applied Biosystems Figure 6-9. Deprotection and cleavage of β-cyanoethyl protected oligonucleotides. Treatment with concentrated ammonium hydroxide removes the β-cyanoethyl protecting groups and cleaves the oligonucleotides from the support. The benzoyl and isobutyryl base protecting groups (X) are removed by heating at 55°C in ammonia for 8 to 15 hours. Quantitation of the Oligonucleotide Nucleic acids of any variety are most easily quantitated by UV spectroscopy, measuring at or near their UV absorbance maxima, about 260 nm. A dilute aqueous solution of 1 ml or less, depending on the cuvette size, is measured by either scanning a region of about 200-350 nm or a single wavelength measurement. A scan of an oligonucleotide will show broad absorbance with a maxima near 260 nm. Using Beer’s law, the concentration of the solution and absolute quantity can be calculated. As a useful approximation, 1 optical density unit (odu) of single-stranded oligonucleotide consists of about 33 micrograms, by mass. An approximation to relate absorbance to molar quantities is that a micromole of oligonucleotide has a number of odu equal to 10 times the number of bases. For example, a micromole of a 20mer would be 200 odu. Storage of the Oligonucleotide Most applications for synthetic oligonucleotides require less DNA than a synthesis provides. Fortunately, oligonucleotides can be stored easily, with little or no degradation for long periods of time. It is probably most convenient to store them refrigerated as a solution, in either a crude or purified state. The media may be concentrated ammonia, used to cleave and deprotect the crude oligonucleotide, water, or dilute buffer or salt. Typical aqueous media may contain ethanol, acetonitrile, tri6-20 Section 6: Chemistry for Automated DNA Synthesis Applied Biosystems ethylammonium acetate (TEAA), EDTA, etc. It is important to keep the oligonucleotides cold to minimize degradation and bacterial growth. Alternatively, oligonucleotides may be stored dried as a pellet in a clean, dry vessel, such as a microcentrifuge tube. The solution used to elute purified oligonucleotides from OPC, 20% acetonitrile, is a convenient and stable storage media. When stored by these means, oligonucleotides are stable for over a year. Avoid solutions that are mutagenic, oxidizing or outside the pH range of 3-12. Analysis and Purification This section reviews the most common methods for purification of synthetic oligonucleotides. HPLC (high performance liquid chromatography) and PAGE (polyacrylamide gel electrophoresis) can provide a high level of purity, but require initial capital investments and are labor intensive and time consuming. A short oligonucleotide (<30 bases) made with typically high synthesis efficiency (>98% average DMT yield/cycle) may only require a less stringent purification, with efficient desalting and removal of non-nucleoside synthesis by-products. These methods are elaborated in detail in User Bulletin No. 13 (Revised). OPC, Oligonucleotide Purification Cartridge The Oligonucleotide Purification Cartridge (OPC, AB part # 400771) was designed specifically for rapid, easy purification of synthetic oligonucleotides.18,19 The method is based on a small, syringemounted cartridge containing an adsorbent material with an affinity for DMT oligonucleotides. The ammonia solution of the crude DMT oligonucleotide (10-20 crude odu) is applied directly to the cartridge. The DMT oligonucleotide product is retained. By-products, failure sequences not bearing a DMT group, and other impurities are not retained and are eluted. The DMT group of the OPCbound oligonucleotide is removed with a mild acid solution, then the purified oligonucleotide is eluted (typically 1-5 odu) with about 1 ml of a 20% acetonitrile solution. The entire operation requires 15-20 minutes, and several OPC purifications can be conducted in parallel. Efficient, reliable purifications are achieved with oligonucleotides up to about 70 bases. A recent report has shown that OPC purification is effective with oligonucleotides longer than 70 bases when a preliminary treatment with a lysine solution is conducted, after synthesis and before ammonia treatment.20 Lysine acts to cleave apurinic sites, a major source of DMT bearing failure sequences. This purifying operation allows OPC to be effective even with long oligonucleotides. Several features of this operation are noteworthy. No sample preparation is required. The OPC material is stable to concentrated ammonia. The ammonia solution provides a denaturing media, eliminating secondary structure and hydrogen-bonding, which would allow co-elution of partially complementary failure sequences. The DMT group is detached and retained in the cartridge. Thus, Section 6: Chemistry for Automated DNA Synthesis 6-21 Applied Biosystems the purified, fully deprotected oligonucleotide is eluted in a small volume of 20% acetonitrile in water, completely desalted and ready for use. PAGE Polyacrylamide gel electrophoresis (PAGE) is a widely used method for the analysis and purification of oligonucleotides.21 When a mixture of charged molecules are exposed to an electric field, they will migrate with velocities determined primarily by their mass to charge ratios. This ratio changes linearly with the log of the molecular weight of DNA of differing chain lengths. This allows a very ordered progression of oligonucleotides with mobility decreasing as the length increases. Proper PAGE technique can provide resolution, and preparative isolation, of single base length differences. Oligonucleotide samples for PAGE are typically prepared by drying a known odu amount (by measuring absorbance) and redissolving in media such as formamide/1X TBE: 9/1 or 7M urea, which are denser than the 1X TBE running buffer. These media and the gel matrix (containing 7M urea) should be denaturing to ensure disruption of secondary structure and hydrogen-bonding. The most desirable format for PAGE of oligonucleotides is the slab gel. There are many commercial devices which essentially consist of a sandwich of two glass plates held apart by two side spacers. The thickness of the spacers determine the gel thickness. For UV shadowing, .75 - 1.5 mm is convenient. The acrylamide solution is poured in between and allowed to polymerize. A comb is also inserted, which forms wells where the oligonucleotide is loaded. Each well forms a vertical lane during the electrophoresis. The plate length should be about 16-40 cm, depending on oligonucleotide length. The width depends only on the number of samples to be run. Dyes, such as bromophenol blue and xylene cyanol are run either in the samples or by themselves as indicators of the migration distance of the oligonucleotides. The acrylamide concentration of the gel matrix determines the velocity of the oligonucleotides. A range of 8-20% is typical. Longer oligonucleotides require lower acrylamide concentration. When the dyes indicate the appropriate migration distance of the oligonucleotide product, the power is turned off and the gel sandwich is disassembled. There are several methods of analysis: UV shadowing, staining, and radiolabelling/autoradiography. UV shadowing This is the simplest, easiest, and introduces the fewest artifacts into the analysis. Depending on oligonucleotide length, from 0.5 to 10 odu are loaded in a well. After electrophoresis, the gel is transferred from the plates to a clear plastic wrapped, fluorescent TLC plate, most conveniently plasticbacked, 20x20cm. The gel is visualized with a UV lamp at short wavelength. The bands appear dark 6-22 Section 6: Chemistry for Automated DNA Synthesis Applied Biosystems against a fluorescent green background. A permanent record can be made by photography through a green filter. Staining Many dyes and intercalating agents, such as methylene blue, ethidium bromide, Stains-All, and others, will visualize oligonucleotides in a polyacrylamide matrix. After electrophoresis, the gel is transferred to a pan containing the staining agent and let soak for some time period. This technique is more sensitive than UV shadowing, but is more time-consuming and uncertain than UV shadowing. Radiolabelling This is the most sensitive, but most laborious method of analysis of oligonucleotides. Typically about 0.01 odu is phosphorylated enzymatically with T4 polynucleotide kinase and gamma-32P ATP to give 5'32P phosphorylated oligonucleotides. Alternatively, 35S ATP can be used, and the oligonucleotides can be labelled at either the 5'or 3' termini. The radiolabelled samples are electrophoresed on the gel. The gel is wrapped in plastic and exposed to X-ray film in a dark room. An autoradiogram is generated in a time period ranging from minutes to days, depending on the specific activity of the ATP and other radiolabelling parameters. The film has a finite capacity for development and exposure time must be carefully monitored, usually by taking several exposures. The gel pattern on the film may be quantitated by densitometry. Product identification is obvious when the appropriate tracking dye or oligonucleotide standard is also present on the gel. By any of these techniques, the gel pattern of electrophoresis of an oligonucleotide can be very diagnostic about the course of synthesis. Many synthesizer and reagent problems can be diagnosed by the appearance and relative amounts of the “failure bands”. Also, PAGE has a distinct advantage in that many samples can be analyzed and purified concurrently. The equipment is relatively inexpensive and easy to maintain. For bigmers, PAGE is usually the most efficient purification method and the only analytical method. The primary disadvantages of PAGE are that it is labor intensive and dependent on good technique. Oligonucleotides can be purified by locating the product band by UV shadowing and excising the gel material therein with a clean razor blade. The gel material which is removed should be free of failure bands, most typically the lower N-1 band. In a preparative electrophoresis run, the product is run further, on a thicker gel, than in an analytical run, to maximize the separation of the product band from the N-1 band. The excised gel fragment(s) is soaked in an elution buffer. The gel debris is then removed by a desalting method, such as with an OPC cartridge. Section 6: Chemistry for Automated DNA Synthesis 6-23 Applied Biosystems HPLC High Performance Liquid Chromatography (HPLC) is another efficient method which combines quantitative analysis and purification of oligonucleotides. One of the advantages of HPLC is a high level of automation. Systems are available which allow for repetitive programmed injection, analysis, and data manipulation and storage. Two different types of column adsorbents are popular for oligonucleotides; reverse-phase and ion-exchange. Reverse-phase adsorbents discriminate by the hydrophobic differences between oligonucleotides of varying lengths and sequences. When the 5' DMT is on the oligonucleotide, this group is dominant in its interaction with the support adsorbent. Reverse-phase columns also can adequately resolve DMT off oligonucleotides with sufficient capacity. The mobile phase is typically a volatile buffer such as 0.1M triethylammonium acetate. The oligonucleotides are eluted with a gradient of increasing organic solvent, such as acetonitrile. These conditions are non-denaturing. Occasionally, certain sequences can exhibit unpredictable HPLC elution patterns, caused by inter- or intramolecular secondary structure and hydrogen-bonding effects. Ion-exchange adsorbents elute oligonucleotides based on increasing charge, i.e. chain length. An increasing salt gradient in the mobile phase is used to displace the oligonucleotide phosphate anions. The salt anions, such as ammonium sulfate or sodium phosphate, and the DNA pair with the adsorbent-bound cation, usually alkylated ammonium species. Since ion-exchange analysis separates only on the basis of increasing charge, the desired product oligonucleotide will always elute after the lesser charged failure sequences. The high salt mobile phase also provides a denaturing media. Both of these factors allow easy product identification. Preparative ion-exchange HPLC requires a final desalting operation, most efficiently conducted with OPC. Consult the chapter on HPLC in User Bulletin 13 (Revised), found in the appendix, for more details. Alternative Chemistries In addition to the phosphoramidite chemistry method to prepare “normal” phosphodiester oligonucleotides, recently many alternative chemistries have been demonstrated on AB synthesizers. These other products include; synthetic RNA, phosphate-analog oligonucleotides, and chemically derivatized oligonucleotides which can be covalently attached to other molecules. RNA Synthesis RNA oligoribonucleotides can be synthesized on the Model 391.22,23 Using the 2' silyl 5' DMT cyanoethyl phosphoramidite RNA monomers, the only cycle change is to increase the coupling wait time to 600 seconds. The RNA monomers bear a large 2' silyl protecting group and therefore require 6-24 Section 6: Chemistry for Automated DNA Synthesis Applied Biosystems longer time to couple. The details from Applied Biosystems User Bulletin No. 47 apply for the Model 391 with a few, simplifying refinements: The ammonia/ethanol : 3/1 solution of crude RNA oligo (DMT OFF) is heated overnight at 55 ºC, dried, treated with 10 microliters (per odu of crude RNA oligo) of 1 molar tetrabutylammonium fluoride/THF for 6 hours. An equivalent volume of 0.1M TEAA is added, mixed, and evaporated to near dryness. 1 ml of 0.1 M TEAA is again added, mixed and applied to an OPC cartridge, following the desalting protocol: 1. pre-wash the OPC cartridge with 5 ml acetonitrile, then 5 ml TEAA 2. load the RNA oligo, recycling it through the cartridge twice, at 1-2 drops/second 3. wash the OPC cartridge with 5 ml 0.1M TEAA, then 10 ml water 4. elute the desalted oligo (up to 50 ODU) with 1 m1 50% acetonitrile This OPC procedure replaces a long and troublesome Sephadex column to remove the tetrabutylammonium salts which complicate further analysis and purification. The RNA oligo is then ready for PAGE or HPLC purification/analysis. RNase degradation is not a significant problem, except when handling less than 0.1 odu. Note RNA synthesis monomers are available from Peninsula Laboratories, 611 Taylor Way, Belmont, CA 94002, USA Telephone: 1-650-592-5392 FAX: 650-595-4071 PEN LABS BLMT. Figure 6-10. RNA phosphoramidites Hydrogen-Phosphonate Chemistry The hydrogen-phosphonate chemistry is useful and effective for preparing either “normal” phosphodiester oligonucleotides or phosphate-analog oligonucleotides.24 User Bulletin No. 44 provides full details on using the synthesis reagents and the post-synthesis protocols. Although the cycle is similar, in that it contains coupling, capping25, and detritylation steps, all reagents26 are different Section 6: Chemistry for Automated DNA Synthesis 6-25 Applied Biosystems than those used for the phosphoramidite method. The synthesis efficiency is routinely less than the phosphoramidite method, typically 95-96% trityl yields. The primary advantage to the hydrogenphosphonate chemistry is the flexibility for one-step, post-synthesis conversion of the internucleoside hydrogen-phosphonate groups to a variety of phosphate species27, such as phosphodiesters, phosphoramidates, phosphotriesters, and most importantly, the phosphorothioates. Figure 6-11. hydrogen-phosphonate monomers Phosphorothioate DNA When the hydrogen-phosphonate synthesis of an oligonucleotide is done, the column may be removed and a sulfurizing reagent introduced by the double-syringe method.17,28 All the internucleoside hydrogen-phosphonates are rapidly converted to phosphorothioates. After regular ammonia cleavage/deprotection, oligonucleotides bearing phosphorothioate linkages behave chemically similarly to “normal” phosphodiester DNA. They show the same electrophoresis and HPLC behavior. This class of phosphate-analog DNA has shown activity in anti-sense translation arrest experiments. In particular, both a sequence, and non-sequence, specific effect has been observed in inhibiting in vitro viral replication of HIV.29 6-26 Section 6: Chemistry for Automated DNA Synthesis Applied Biosystems CONVERSION OF HYDROGEN-PHOSPHONATE TO PHOSPHATE ANALOGS Figure 6-12. 5' Attachments In the last several years many applications have been identified and developed for the covalent attachment of small molecules to oligonucleotides. These molecules include fluorescent dyes30, biotin31, proteins32, and other species that allow the identification of oligonucleotides in biological systems. In addition, oligonucleotides can be derivatized to allow attachment to solid supports.33 In this way, for example, an affinity matrix can be constructed to purify a sequence complementary to the support-bound sequence. The attachment can be made at several sites on the oligonucleotide. However, this site must not impair hybridization or confer chemical instability. For these reasons and for synthesis ease and efficiency, the 5' terminus is usually the preferred location for derivatization of an oligonucleotide. Section 6: Chemistry for Automated DNA Synthesis 6-27 Applied Biosystems Fluorescent-dye Linked Sequencing Primers Amino Link 2 The 5' fluorescent dye labelled sequencing primers used for the Applied Biosystems 370A DNA Sequencer consist of three parts; the oligonucleotide primer, a linker, and a fluorescent dye. The linker bears a highly nucleophilic primary amine group which reacts with the electrophilic N-hydroxy succinimide group of the fluorescent dye. This linker group is created with AMINOLINK 2 (AB Part No. 400808).34 Aminolink 2 is a phosphoramidite molecule with a six carbon chain and a protected amine group. (Figure 6-13). This reagent is handled and used like a phosphoramidite nucleoside. Activation with tetrazole forms an active intermediate that couples to the 5' hydroxyl terminus of the support-bound oligonucleotide, in the final coupling cycle. Oxidation and ammonia cleavage/deprotection yields the aminolink-oligonucleotide, in solution. Coupling to the fluorescent dye-NHS ester, or other electrophilic species, is conducted in a homogeneous solution. 6-28 Section 6: Chemistry for Automated DNA Synthesis Applied Biosystems Figure 6-13. Aminolinked oligonucleotide (in solution) Section 6: Chemistry for Automated DNA Synthesis 6-29 Applied Biosystems References 1. Efcavitch, J.W., “Automated System for the Optimized Chemical Synthesis of Oligodeoxyribonucleotides”, Macromolecular Sequencing and Synthesis, Selected Methods and Applications, pages 221-234, Alan R. Liss, Inc. (1988). 2. Beaucage, S.L. and Caruthers, M.H., Tetrahedron Letters 22, 1859-1862 (1981). Beaucage, S.L. and Caruthers, M.H., United States Patent #4,668,777 3. Matteucci, M.D. and Caruthers, M.H., J. Am. Chem. Soc., 103, 3185 (1981) Matteucci, M.D. and Caruthers, M.H., United States Patent #4,458,066 4. Adams, S.P., Kavka, K.S., Wykes, E.J., Holder, S.B., and Galluppi, G.R., J. Am. Chem. Soc., 105, 661 (1983). 5. Eadie, J.S. and Davidson, D.S., Nucleic Acids Research, 15, 8333-8349, (1987). Farrance, I.K., Eadie, J.S., and Ivarie, R., Nucleic Acids Research, 17, 1231-1245, (1989). 6. Sinha, N.D., Biernat, J., and Koster, H., Tetrahedron Letters, 24, 5843-5846 (1983). 7. McBride, L.J., Eadie, J.S., Efcavitch, J.W., and Andrus, W.A., Nucleosides and Nucleotides, 6, 297-300 (1987). Yeung, A.T., Dinehart, W.J. and Jones, B.K., Nucleic Acids Research, 16, 4539-4554 (1988). 8. Efcavitch, J.W., McBride, L.J. and Eadie, J.S., Biophosphates and Their Analogues Synthesis, Structure, Metabolism and Activity, pages 65-70, Bruzik, K.S. and Stec, W.J. (Eds), Proceedings of the 2nd International Symposium on Phosphorus Chemistry Directed Towards Biology, Lodz, Poland, 1986. 9. Tanaka, T. and Letsinger, R.L., Nucleic Acids Research, 10, 3249-3260, (1982). 10.Efcavitch, J.W. and Heiner, C., Nucleosides and Nucleotides, 4, 267 (1985). 11. McBride, L.J. and Caruthers, M.H., Tetrahedron Letters, 24, 245-248 (1983). 12.Dahl, O., Phosphorus and Sulfur, 18, 201-204 (1983). Dahl, B.H., Nielsen, J. and Dahl, O., Nucleic Acids Research, 15, 1729-1743, (1987). 13.Zon, G., Gallo, K.A., Samson, C.J., Shao, K., Summers, M.F. and Byrd, R.A., Nucleic Acids Research, 15, 8181-8196, (1985). 14.Applied Biosystems Nucleic Acid Research News, Issue No. 7, October 1988 “The Effect of the Synthesis Cycle on the Chemical Authenticity of Synthetic DNA”. 15.Bauer, B.F. and Holmes, W.M., Nucleic Acids Research, 15, 812 (1989). 16.Stec, W.J., Zon, G., Egan, W. and Stec, B., J. Am. Chem. Soc., 106, 6077-6079 (1984). 17.The DOUBLE-SYRINGE METHOD for manual ammonia cleavage: a. Attach an empty luer tip syringe, with plunger fully inserted, into one end of the synthesis column. b. Load 2 ml of conc. ammonia in another luer tip syringe and attach to the other end of the column 6-30 Section 6: Chemistry for Automated DNA Synthesis Applied Biosystems c. Holding a syringe in each hand, carefully inject the reagent through the column to the empty syringe and return the reagent through the column several times. d. Allow it to stand for at least one hour at room temperature. e. Drain all of the reagent into one syringe, detach and eject contents carefully into an appropriate vial for heating to achieve complete deprotection. 18.L.J. McBride, McCollum, C., Davidson, S., Efcavitch, J.W., Andrus, A., and Lombardi, S.J., Biotechniques, 6, No. 4, 362-367 (1988) “A New, Reliable Cartridge for the Rapid Purification of Synthetic DNA”. Applied Biosystems User Bulletin No. 51 “OPC Purification of Long Oligonucleotides”. 19.“Rapid Purification, Desalting, and Detritylation of Synthetic Oligonucleotides”, McCollum, C., Davidson, S., McBride, L.J., Efcavitch, J.W., and Andrus, A., poster, FASEB, Las Vegas, 1988. 20.Horn, T. and Urdea, M.S., Nucleic Acids Research, 16, 11559-11571, (1988). 21.Efcavitch, J.W., “The Electrophoresis of Synthetic Oligonucleotides”, Gel Electrophoresis of Synthetic Oligonucleotides, 2nd Ed., IRL Press, Practical Approach Series, in press. 22.Applied Biosystems User Bulletin No. 47 “RNA Synthesis” April 1988. 23.Usman, N., Ogilvie, K.K., Jiang, M.-Y., and Cedergren, R.J., J. Am. Chem. Soc., 109, 78457854 (1987). Ogilvie, K.K., Damha, M.J., Usman, N., and Pon, R.T., Pure & Applied Chem., 59, 325-330 (1987). Ogilvie, K.K., Usman, N., Jiang, M, -Y., and Cedergren, R.J., Proc. Natl. Acad. Sci., 5764 (1988). 24.Froehler, B.C. and Matteucci, M.D. Tetrahedron Letters, 27, 469-472 (1986). Froehler, B.C., Ng, P.G., and Matteucci, M.D. Nucleic Acids Research, 14, 5399-5407 (1986). Garegg, P.J., Lindh, I., Regberg, T., Stawinski, J., and Stromberg, R., Tetrahedron Letters, 27, 4051-4057 (1986). 25.Andrus, W.A., Efcavitch, J.W., and McBride, L.J., United States Patent #4,816,571, “Chemical Capping By Phosphitylation During Oligonucleotide Synthesis”. 26.Andrus, A., Efcavitch, J.W., McBride, L.J., and Giusti, B, Tetrahedron Letters, 29, 861-864 (1988). 27.Froehler, B.C., Tetrahedron Letters, 27, 5575-5578 (1986). 28.Andrus, A. and Zon, G., Nucleic Acids Research Symposium Series No. 20, 121-122 (1988). Applied Biosystems User Bulletin No. 44 “Oligonucleotide Synthesis with HydrogenPhosphonate Monomers”. 29.Matsukura, M., Shinozuka, K., Zon, G., Mitsuya, H., Reitz, M., Cohen, J.S., and Broder, S., Proc. Natl. Acad. Sci. USA, 84, 7706-7710 (1987). Stein, C.A., Subsinghe, C., Shinozuka, K. and Cohen, J.S., Nucleic Acids Research, 16, 3209-3221 (1988). 30.Smith, L.M. et al, Nature, 321, 674-679 (1986). Section 6: Chemistry for Automated DNA Synthesis 6-31 Applied Biosystems 31.Chu, B.C.F. and Orgel, L.E., DNA, 4, 327-331 (1985). Chollet, A. and Kawashima, E.H., Nucleic Acids Research, 15, 1529-1541 (1985). Agrawal, S., Christodoulou, C. and Gait, M.J., Nucleic Acids Research, 14, 6227-6245 (1986). 32.Li, P., Medon, P.P., Skingle, D.C., Lanser, J.A. and Symons, R.H., Nucleic Acids Research, 15, 5275-5287 (1987). Chu, B.C.F. and Orgel, LE., Nucleic Acids Research, 16, 3671-3691 (1988). Zuckermann, R.N., Corey, D.R. and Schultz, P.G., 1614-1615 (1988). Zuckermann, R.N. and Schultz, P.G., J. Am. Chem. Soc., 110, 6592-6594 (1988). 33.Ghosh, S.S. and Musso, G.F., Nucleic Acids Research, 15, 5353-5372 (1987). 34.Applied Biosystems User Bulletin No. 49. “Aminolink 2”. 6-32 Section 6: Chemistry for Automated DNA Synthesis Appendix A: Functions, Cycles and Procedures MODEL 391 PCR-MATE™ DNA SYNTHESIZER FUNCTIONS CONTROL 4 - Wait 5 - Advance Fraction Collector 6 - Waste to Port 7 - Waste to Bottle 17 - Interrupt 31 - Recorder On 32 - Recorder Off 33 - Cycle Entry 34 - Cycle End DELIVER TO COLUMN 9 - #18 to Column 13 - #15 to Column 14 - #14 to Column 19 - Base + Tetrazole to Column 22 - Cap to Column 101 - A to Column 102 - G to Column 103 - C to Column 104 - T to column 105 - X to Column 90 - Tet to Column 106 - #11 to Column 107 - #12 to Column PRIME DELIVERY LINES 52- A to Waste 53- G to Waste 54 - C to Waste 55 - T to Waste 56 - X to Waste 59 - Cap A to Waste 60 - Cap B to Waste 61 - Tetrazole to Waste 81 - # 15 to Waste 82 - # 14 to Waste ARGON TO RESERVOIRS 62 - Flush to A 63 - Flush to G 64 - Flush to C 65 - Flush to T 66 - Flush to X 69 - Flush to Tetrazole 70 - Flush to #18 86 - Flush to 14,15 89 - Flush to 11,12 ACETONITRILE TO RESERVOIRS RINSE AND FLUSH 1 - Block Flush 2 - Reverse Flush 9 - #18 to Column 10 - #18 to Waste 108 - Flush to Trit 109 - Flush thru Col PREPARE REAGENTS 16 - Cap Prep 28 - Phosphoramidite Prep 43 - # 18 Prep 51 - Tetrazole Prep 71 - #18 to A 72 - #18 to G 73 - #18 to C 74 - #18 to T 75 - #18 to X 78 - #18 to Tetrazole 84 - #18 to 14 85 - #18 to 15 87 - #18 to 11 88 - #18 to 12 Applied Biosystems 391 FUNCTION LIST VERSION: TIME: DATE: 1.00 04:27 05/11/89 FUNCTION NUMBER FUNCTION NAME FUNCTION VALVE LIST BLOCK FLUSH REVERSE FLUSH WAIT ADVANCE FC WASTE PORT WASTE BOTTLE #18 TO COLM #18 TO WASTE #15 TO COLUMN #14 TO COLUMN CAP PREP INTERRUPT B+TET TO COLUMN CAP TO COLUMN PHOS PREP RCDR ON RCDR OFF CYC ENTRY CYC END #18 PREP TET PREP A TO WASTE G TO WASTE C TO WASTE T TO WASTE X TO WASTE CAP A TO WASTE CAP B TO WASTE TET TO WASTE FLUSH TO A FLUSH TO G FLUSH TO C FLUSH TO T FLUSH TO X FLUSH TO TET FLUSH TO #18 #18 TO A #18 TO G #18 TO C #18 TO T #18 TO X #18 TO TET #15 TO WASTE #14 TO WASTE #18 TO 14 #18 TO 15 FLUSH TO 14, 15 #18 TO 11 #18 TO 12 FLUSH TO 11, 12 TET TO COLUMN 0, 13, 14, 16 12, 13, 14 1 2 4 5 6 7 9 10 13 14 16 17 19 22 28 31 32 33 34 43 51 52 53 54 55 56 59 60 61 62 63 64 65 66 69 70 71 72 73 74 75 78 81 82 84 85 86 87 88 89 90 A-4 1, 12, 16, 17 1, 13, 17 2, 12, 16, 18 3, 12, 16, 19 22 6, 12, 16, 20, 23 4, 5, 12, 16, 22 20, 23 17 23 11, 13, 20 10, 13, 20 9, 13, 20 8, 13, 20 7, 13, 20 5, 13, 22 4, 13, 22 6, 13, 23 0, 11, 21 0, 10, 21 0, 9, 21 0, 8, 21 0, 7, 21 0, 6 0, 1 1, 11, 17, 21 1, 10, 17, 21 1, 9, 17, 21 1, 8, 17, 21 1, 7, 17, 21 1, 6, 17 2, 13, 18 3, 13, 19 1, 3, 17 1, 2, 17 0, 2, 3 1, 5, 17 1, 4, 17 0, 4, 5 6, 12, 16, 23 Appendix A: Functions, Cycles and Procedures Applied Biosystems 391 FUNCTION LIST VERSION: TIME: DATE: 1.00 03:16 05/10/89 FUNCTION NUMBER FUNCTION NAME 92 93 94 95 96 97 98 99 USER A USER B USER C USER D USER E USER F USER G USER H FUNCTION NUMBER FUNCTION NAME FUNCTION VALVE LIST 101 102 103 104 105 106 107 108 109 A TO COLUMN G TO COLUMN C TO COLUMN T TO COLUMN X TO COLUMN #11 TO COLUMN #12 TO COLUMN FLUSH TO TRIT FLUSH THRU COL 11, 12, 16, 20 10, 12, 16, 20 9, 12, 16, 20 8, 12, 16, 20 7, 12, 16, 20 5, 12, 16, 20 4, 12, 16, 22 0, 12, 15 0, 12, 16 Appendix A: Functions, Cycles and Procedures FUNCTION VALVE LIST A-5 Applied Biosystems 391 CYCLE/PROCEDURE NAME: NUMBER OF STEPS: TIME: DATE: STEP NUMBER 1 .2µM 63 04:22 05/11/89 FUNCTION # NAME 1 10 #18 TO WASTE 2 9 # 18 TO COLM 3 2 REVERSE FLUSH 4 1 BLOCK FLUSH 5 28 PHOS PREP 6 90 TET TO COLUMN 7 19 B+TET TO COLM 8 90 TET TO COLUMN 9 19 B+TET TO COLM 10 90 TET TO COLUMN 11 19 B+TET TO COLM 12 90 TET TO COLUMN 13 4 WAIT 14 16 CAP PREP 15 2 REVERSE FLUSH 16 1 BLOCK FLUSH 17 22 CAP TO COLUMN 18 4 WAIT 19 10 #18 TO WASTE 20 2 REVERSE FLUSH 21 1 BLOCK FLUSH 22 81 #15 TO WASTE 23 13 #15 TO COLUMN 24 10 #18 TO WASTE 25 4 WAIT 26 2 REVERSE FLUSH 27 1 BLOCK FLUSH 28 10 #18 TO WASTE 29 9 #18 TO COLM 30 2 REVERSE FLUSH 31 9 #18 TO COLM 32 2 REVERSE FLUSH 33 9 #18 TO COLM 34 2 REVERSE FLUSH 35 1 BLOCK FLUSH 36 33 CYC ENTRY 37 10 #18 TO WASTE 38 9 #18 TO COLM 39 2 REVERSE FLUSH 40 1 BLOCK FLUSH 41 5 ADVANCE FC 42 6 WASTE PORT 43 82 #14 TO WASTE (Continued on next page) A-6 Page STEP TIME A 2 15 5 3 3 3 2 2 2 2 2 2 15 3 5 3 10 5 3 5 3 3 10 5 15 5 3 5 10 5 10 5 10 5 3 1 3 10 5 3 1 1 3 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes ACTIVE FOR BASES G C T X Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Appendix A: Functions, Cycles and Procedures Applied Biosystems 391 CYCLE/PROCEDURE NAME: NUMBER OF STEPS: TIME: DATE: STEP NUMBER 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 2 .2µM 63 04:22 05/11/89 FUNCTION # NAME 14 108 14 108 14 108 14 108 14 108 14 108 9 108 7 1 9 2 1 34 Page #14 TO COLUMN FLUSH TO TRIT #14 TO COLUMN FLUSH TO TRIT #14 TO COLUMN FLUSH TO TRIT #14 TO COLUMN FLUSH TO TRIT #14 TO COLUMN FLUSH TO TRIT #14 TO COLUMN FLUSH TO TRIT #18 TO COLM FLUSH TO TRIT WASTE BOTTLE BLOCK FLUSH #18 TO COLM REVERSE FLUSH BLOCK FLUSH CYC END Appendix A: Functions, Cycles and Procedures STEP TIME A 10 1 10 1 10 1 10 1 10 1 10 1 10 8 1 3 20 5 3 1 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes ACTIVE FOR BASES G C T X Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes A-7 Applied Biosystems 391 CYCLE/PROCEDURE NAME: NUMBER OF STEPS: TIME: DATE: STEP NUMBER 1µM 64 04:23 05/11/89 FUNCTION # NAME 1 10 #18 TO WASTE 2 9 # 18 TO COLM 3 2 REVERSE FLUSH 4 1 BLOCK FLUSH 5 28 PHOS PREP 6 90 TET TO COLUMN 7 19 B+TET TO COLM 8 90 TET TO COLUMN 9 19 B+TET TO COLM 10 90 TET TO COLUMN 11 19 B+TET TO COLM 12 90 TET TO COLUMN 13 4 WAIT 14 2 REVERSE FLUSH 15 1 BLOCK FLUSH 16 16 CAP PREP 17 22 CAP TO COLUMN 18 4 WAIT 19 10 #18 TO WASTE 20 2 REVERSE FLUSH 21 1 BLOCK FLUSH 22 81 #15 TO WASTE 23 13 #15 TO COLUMN 24 10 #18 TO WASTE 25 4 WAIT 26 2 REVERSE FLUSH 27 1 BLOCK FLUSH 28 10 #18 TO WASTE 29 9 #18 TO COLM 30 2 REVERSE FLUSH 31 9 #18 TO COLM 32 2 REVERSE FLUSH 33 9 #18 TO COLM 34 2 REVERSE FLUSH 35 1 BLOCK FLUSH 36 33 CYC ENTRY 37 10 #18 TO WASTE 38 9 #18 TO COLM 39 2 REVERSE FLUSH 40 1 BLOCK FLUSH 41 5 ADVANCE FC 42 6 WASTE PORT 43 82 #14 TO WASTE (Continued on next page) A-8 Page 1 STEP TIME 2 15 5 3 3 3 3 2 3 2 3 2 15 5 3 3 12 8 3 5 3 3 10 5 15 5 3 5 10 5 10 5 10 5 3 1 3 10 5 3 1 1 3 A Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes ACTIVE FOR BASES G C T X Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Appendix A: Functions, Cycles and Procedures Applied Biosystems 391 CYCLE/PROCEDURE NAME: NUMBER OF STEPS: TIME: DATE: STEP NUMBER 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 1µM 64 04:23 05/11/89 FUNCTION # NAME 14 108 14 108 14 108 14 108 14 108 14 108 9 108 7 1 2 9 2 1 34 Page 2 #14 TO COLUMN FLUSH TO TRIT #14 TO COLUMN FLUSH TO TRIT #14 TO COLUMN FLUSH TO TRIT #14 TO COLUMN FLUSH TO TRIT #14 TO COLUMN FLUSH TO TRIT #14 TO COLUMN FLUSH TO TRIT #18 TO COLM FLUSH TO TRIT WASTE BOTTLE BLOCK FLUSH REVERSE FLUSH #18 TO COLM REVERSE FLUSH BLOCK FLUSH CYC END Appendix A: Functions, Cycles and Procedures STEP TIME 10 1 10 1 10 1 10 1 10 1 10 1 10 8 1 3 1 20 5 3 1 A Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes ACTIVE FOR BASES G C T X Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes A-9 Applied Biosystems 391 CYCLE/PROCEDURE NAME: NUMBER OF STEPS: TIME: DATE: STEP NUMBER Low 61 04:24 05/11/89 FUNCTION # NAME 1 10 #18 TO WASTE 2 9 # 18 TO COLM 3 2 REVERSE FLUSH 4 1 BLOCK FLUSH 5 28 PHOS PREP 6 90 TET TO COLUMN 7 19 B+TET TO COLM 8 90 TET TO COLUMN 9 19 B+TET TO COLM 10 90 TET TO COLUMN 11 4 WAIT 12 16 CAP PREP 13 2 REVERSE FLUSH 14 1 BLOCK FLUSH 15 22 CAP TO COLUMN 16 4 WAIT 17 10 #18 TO WASTE 18 2 REVERSE FLUSH 19 1 BLOCK FLUSH 20 81 #15 TO WASTE 21 13 #15 TO COLUMN 22 10 #18 TO WASTE 23 4 WAIT 24 2 REVERSE FLUSH 25 1 BLOCK FLUSH 26 10 #18 TO WASTE 27 9 #18 TO COLM 28 2 REVERSE FLUSH 29 9 #18 TO COLM 30 2 REVERSE FLUSH 31 9 #18 TO COLM 32 2 REVERSE FLUSH 33 1 BLOCK FLUSH 34 33 CYC ENTRY 35 10 #18 TO WASTE 36 9 #18 TO COLM 37 2 REVERSE FLUSH 38 1 BLOCK FLUSH 39 5 ADVANCE FC 40 6 WASTE PORT 41 82 #14 TO WASTE 42 14 #14 TO COLUMN 43 108 FLUSH TO TRIT (Continued on next page) A-10 Page 1 STEP TIME 2 15 5 3 3 3 2 2 2 2 15 3 5 3 10 5 3 5 3 3 10 5 15 5 3 5 10 5 10 5 10 5 3 1 3 10 5 3 1 1 3 10 1 A Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes ACTIVE FOR BASES G C T X Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Appendix A: Functions, Cycles and Procedures Applied Biosystems 391 CYCLE/PROCEDURE NAME: NUMBER OF STEPS: TIME: DATE: STEP NUMBER 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 Low 61 04:24 05/11/89 FUNCTION # NAME 14 108 14 108 14 108 14 108 14 108 9 108 7 1 9 2 1 34 Page 2 #14 TO COLUMN FLUSH TO TRIT #14 TO COLUMN FLUSH TO TRIT #14 TO COLUMN FLUSH TO TRIT #14 TO COLUMN FLUSH TO TRIT #14 TO COLUMN FLUSH TO TRIT #18 TO COLM FLUSH TO TRIT WASTE BOTTLE BLOCK FLUSH #18 TO COLM REVERSE FLUSH BLOCK FLUSH CYC END Appendix A: Functions, Cycles and Procedures STEP TIME 10 1 10 1 10 1 10 1 10 1 10 8 1 3 20 5 3 1 A Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes ACTIVE FOR BASES G C T X Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes A-11 Applied Biosystems 391 CYCLE/PROCEDURE NAME: NUMBER OF STEPS: TIME: DATE: STEP NUMBER A-12 10µM 53 04:25 05/11/89 FUNCTION # NAME 1 10 2 9 3 2 4 9 5 2 6 9 7 2 8 1 9 28 10 61 11 19 12 4 13 2 14 1 15 16 16 22 17 4 18 10 19 2 20 1 21 81 22 13 23 4 24 10 25 2 26 1 27 10 28 9 29 2 30 9 31 2 32 9 33 2 34 9 35 2 36 1 37 33 38 10 39 9 40 2 41 1 42 5 43 6 (Continued next page) Page 1 #18 TO WASTE # 18 TO COLM REVERSE FLUSH #18 TO COLM REVERSE FLUSH #18 TO COLM REVERSE FLUSH BLOCK FLUSH PHOS PREP TET TO WASTE B+TET TO COLM WAIT REVERSE FLUSH BLOCK FLUSH CAP PREP CAP TO COLUMN WAIT #18 TO WASTE REVERSE FLUSH BLOCK FLUSH #15 TO WASTE #15 TO COLUMN WAIT #18 TO WASTE REVERSE FLUSH BLOCK FLUSH #18 TO WASTE #18 TO COLM REVERSE FLUSH #18 TO COLM REVERSE FLUSH #18 TO COLM REVERSE FLUSH #18 TO COLM REVERSE FLUSH BLOCK FLUSH CYC ENTRY #18 TO WASTE #18 TO COLM REVERSE FLUSH BLOCK FLUSH ADVANCE FC WASTE PORT STEP TIME A 3 50 45 50 45 50 45 3 3 6 55 15 45 3 3 40 5 3 45 3 3 50 15 3 45 3 3 50 45 50 45 50 45 50 45 3 1 3 80 45 3 1 1 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes ACTIVE FOR BASES G C T X Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Appendix A: Functions, Cycles and Procedures Applied Biosystems Page 2 391 CYCLE/PROCEDURE NAME: NUMBER OF STEPS: TIME: DATE: STEP NUMBER 44 45 46 47 48 49 50 51 52 53 10µM 53 04:25 05/11/89 FUNCTION # NAME 82 14 2 1 10 9 2 1 7 34 #14 TO WASTE #14 TO COLUMN REVERSE FLUSH BLOCK FLUSH #18 TO WASTE #18 TO COLM REVERSE FLUSH BLOCK FLUSH WASTE BOTTLE CYC END Appendix A: Functions, Cycles and Procedures STEP TIME 3 160 45 3 3 80 45 3 1 1 A Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes ACTIVE FOR BASES G C T X Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes A-13 Applied Biosystems 391 CYCLE/PROCEDURE NAME: NUMBER OF STEPS: TIME: DATE: Standard #1 BOTTLE CHANGE 9 04:29 05/11/89 STEP NUMBER FUNCTION # NAME 1 2 3 4 5 6 7 8 9 1 10 71 62 17 62 52 10 1 A-14 Page 1 BLOCK FLUSH #18 TO WASTE #18 TO A FLUSH TO A INTERRUPT FLUSH TO A A TO WASTE #18 TO WASTE BLOCK FLUSH STEP TIME 5 7 10 10 1 5 4 7 10 Appendix A: Functions, Cycles and Procedures Applied Biosystems 391 CYCLE/PROCEDURE NAME: NUMBER OF STEPS: TIME: DATE: Page 1 Standard #2 BOTTLE CHANGE 9 04:29 05/11/89 STEP NUMBER FUNCTION # NAME 1 2 3 4 5 6 7 8 9 1 10 72 63 17 63 53 10 1 BLOCK FLUSH #18 TO WASTE #18 TO G FLUSH TO G INTERRUPT FLUSH TO G G TO WASTE #18 TO WASTE BLOCK FLUSH Appendix A: Functions, Cycles and Procedures STEP TIME 5 7 10 10 1 5 4 7 10 A-15 Applied Biosystems 391 CYCLE/PROCEDURE NAME: NUMBER OF STEPS: TIME: DATE: Standard #3 BOTTLE CHANGE 9 04:29 05/11/89 STEP NUMBER FUNCTION # NAME 1 2 3 4 5 6 7 8 9 1 10 73 64 17 64 54 10 1 A-16 Page 1 BLOCK FLUSH #18 TO WASTE #18 TO C FLUSH TO C INTERRUPT FLUSH TO C C TO WASTE #18 TO WASTE BLOCK FLUSH STEP TIME 5 7 10 10 1 5 4 7 10 Appendix A: Functions, Cycles and Procedures Applied Biosystems 391 CYCLE/PROCEDURE NAME: NUMBER OF STEPS: TIME: DATE: Page 1 Standard #4 BOTTLE CHANGE 9 04:30 05/11/89 STEP NUMBER FUNCTION # NAME 1 2 3 4 5 6 7 8 9 1 10 74 65 17 65 55 10 1 BLOCK FLUSH #18 TO WASTE #18 TO T FLUSH TO T INTERRUPT FLUSH TO T T TO WASTE #18 TO WASTE BLOCK FLUSH Appendix A: Functions, Cycles and Procedures STEP TIME 5 7 10 10 1 5 4 7 10 A-17 Applied Biosystems 391 CYCLE/PROCEDURE NAME: NUMBER OF STEPS: TIME: DATE: Standard #5 BOTTLE CHANGE 9 04:30 05/11/89 STEP NUMBER FUNCTION # NAME 1 2 3 4 5 6 7 8 9 1 10 75 66 17 66 56 10 1 A-18 Page 1 BLOCK FLUSH #18 TO WASTE #18 TO X FLUSH TO X INTERRUPT FLUSH TO X X TO WASTE #18 TO WASTE BLOCK FLUSH STEP TIME 5 7 10 10 1 5 4 7 10 Appendix A: Functions, Cycles and Procedures Applied Biosystems 391 CYCLE/PROCEDURE NAME: NUMBER OF STEPS: TIME: DATE: Page 1 Standard #9 BOTTLE CHANGE 6 04:30 05/11/89 STEP NUMBER FUNCTION # NAME 1 2 3 4 5 6 1 10 17 61 10 1 BLOCK FLUSH #18 TO WASTE INTERRUPT TET TO WASTE #18 TO WASTE BLOCK FLUSH Appendix A: Functions, Cycles and Procedures STEP TIME 5 7 1 4 7 10 A-19 Applied Biosystems 391 CYCLE/PROCEDURE NAME: NUMBER OF STEPS: TIME: DATE: Standard #11 BOTTLE CHANGE 7 03:18 05/10/89 STEP NUMBER FUNCTION # NAME 1 2 3 4 5 6 7 1 10 17 16 59 10 1 A-20 Page 1 BLOCK FLUSH #18 TO WASTE INTERRUPT CAP PREP CAP A TO WASTE #18 TO WASTE BLOCK FLUSH STEP TIME 5 7 1 5 5 7 10 Appendix A: Functions, Cycles and Procedures Applied Biosystems 391 CYCLE/PROCEDURE NAME: NUMBER OF STEPS: TIME: DATE: Page 1 Standard #12 BOTTLE CHANGE 7 03:18 05/10/89 STEP NUMBER FUNCTION # NAME 1 2 3 4 5 6 7 1 10 17 16 60 10 1 BLOCK FLUSH #18 TO WASTE INTERRUPT CAP PREP CAP B TO WASTE #18 TO WASTE BLOCK FLUSH Appendix A: Functions, Cycles and Procedures STEP TIME 5 7 1 5 5 7 10 A-21 Applied Biosystems 391 CYCLE/PROCEDURE NAME: NUMBER OF STEPS: TIME: DATE: Standard #14 BOTTLE CHANGE 6 03:19 05/10/89 STEP NUMBER FUNCTION # NAME 1 2 3 4 5 6 1 10 17 82 10 1 A-22 Page 1 BLOCK FLUSH #18 TO WASTE INTERRUPT #14 TO WASTE #18 TO WASTE BLOCK FLUSH STEP TIME 5 7 1 5 7 10 Appendix A: Functions, Cycles and Procedures Applied Biosystems 391 CYCLE/PROCEDURE NAME: NUMBER OF STEPS: TIME: DATE: Page 1 Standard #15 BOTTLE CHANGE 6 03:19 05/10/89 STEP NUMBER FUNCTION # NAME 1 2 3 4 5 6 1 10 17 81 10 1 BLOCK FLUSH #18 TO WASTE INTERRUPT #15 TO WASTE #18 TO WASTE BLOCK FLUSH Appendix A: Functions, Cycles and Procedures STEP TIME 5 7 1 5 7 10 A-23 Applied Biosystems 391 CYCLE/PROCEDURE NAME: NUMBER OF STEPS: TIME: DATE: Standard #18 BOTTLE CHANGE 5 03:19 05/10/89 STEP NUMBER FUNCTION # NAME 1 2 3 4 5 1 10 17 10 1 A-24 Page 1 BLOCK FLUSH #18 TO WASTE INTERRUPT #18 TO WASTE BLOCK FLUSH STEP TIME 5 7 1 7 10 Appendix A: Functions, Cycles and Procedures Applied Biosystems 391 CYCLE/PROCEDURE NAME: NUMBER OF STEPS: TIME: DATE: Page 1 Standard PHOS PURGE 9 03:17 05/10/89 STEP NUMBER FUNCTION # NAME 1 2 3 4 5 6 7 8 9 28 52 53 54 55 56 61 10 1 PHOS PREP A TO WASTE G TO WASTE C TO WASTE T TO WASTE X TO WASTE TET TO WASTE #18 TO WASTE BLOCK FLUSH Appendix A: Functions, Cycles and Procedures STEP TIME 10 4 4 4 4 4 4 7 10 A-25 Applied Biosystems 391 CYCLE/PROCEDURE NAME: NUMBER OF STEPS: TIME: DATE: Standard SHUTDOWN 31 03:18 05/10/89 STEP NUMBER FUNCTION # NAME 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 1 10 71 72 73 74 75 62 63 64 65 66 10 78 87 88 84 85 69 89 86 10 1 17 62 63 64 65 66 70 1 A-26 Page 1 BLOCK FLUSH # 18 TO WASTE # 18 TO A # 18 TO G # 18 TO C # 18 TO T #18 TO X FLUSH TO A FLUSH TO G FLUSH TO C FLUSH TO T FLUSH TO X # 18 TO WASTE # 18 TO TET # 18 TO 11 # 18 TO 12 # 18 TO 14 # 18 TO 15 FLUSH TO TET FLUSH TO 11, 12 FLUSH TO 14, 15 # 18 TO WASTE BLOCK FLUSH INTERRUPT FLUSH TO A FLUSH TO G FLUSH TO C FLUSH TO T FLUSH TO X FLUSH TO #18 BLOCK FLUSH STEP TIME 5 5 60 60 60 60 60 60 60 60 60 60 10 60 60 60 60 60 60 60 60 10 5 1 5 5 5 5 5 10 10 Appendix A: Functions, Cycles and Procedures Applied Biosystems Flow test procedure STEP 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 Appendix A: Functions, Cycles and Procedures FUNCTION WAIT # 18 TO 15 # 18 TO 14 # 18 TO 12 # 18 TO 11 # 18 TO TET #18 TO X #18 TO T #18 TO C #18 TO G #18 TO A #18 TO COLM # 18 TO WASTE INTERRUPT WAIT PHOS PREP A TO COL WAIT G TO COL WAIT C TO COL WAIT T TO COL WAIT X TO COL WAIT TET PREP TET TO COL WAIT CAP PREP # 11 TO COL WAIT # 12 TO COL WAIT # 14 TO COL WAIT # 15 TO COL WAIT # 18 PREP # 18 TO WASTE WAIT #18 TO COL INTERRUPT WASTE PORT TIME 5 40 20 20 20 20 20 20 20 20 20 20 20 1 5 10 120 20 120 20 120 20 120 20 120 20 10 120 20 10 120 20 120 20 120 20 120 20 10 120 20 120 1 1 A-27 Applied Biosystems Flow test procedure (continued) STEP 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 A-28 FUNCTION #14 TO COL INTERRUPT BLOCK FLUSH WASTE BOTTLE FLUSH TO #18 FLUSH TO 14, 15 FLUSH TO 11, 12 FLUSH TO TET FLUSH TO X FLUSH TO T FLUSH TO C FLUSH TO G FLUSH TO A REVERSE FLUSH BLOCK FLUSH TIME 150 1 60 1 60 60 60 60 60 60 60 60 60 60 60 Appendix A: Functions, Cycles and Procedures Appendix B: DNA Synthesizer Schematic DNA Synthesizer Plumbing Diagram Applied Biosystems Figure B-1. DNA Synthesizer Schematic Appendix B: DNA Synthesizer Schematic DNA Synthesizer Plumbing Diagram B-3 Applied Biosystems Figure B-2. DNA Synthesizer Plumbing Diagram B-4 Appendix B: DNA Synthesizer Schematic DNA Synthesizer Plumbing Diagram Appendix C: Synthesis Log Sheet Reagent/Solvent Log Sheet SYNTHESIS LOG SHEET SEQUENCE NAME: SEQUENCE: DATE: USER: Nucleoside 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Absorbance of Trityl Solution Micromoles REAGENT/SOLVENT LOG MONTH: #1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 NOTES #2 #3 #4 #5 #9 #11 #12 REAGENT/SOLVENT LOG MONTH: #14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 NOTES #15 #18 ARGON WASTE Appendix D: 391 Illustrated Parts List Applied Biosystems Photo Number 1 2 3 4 5 6 7a 7b 7c 8 9 10 11 12 13 14 14a 14b 15 15a 15b 16 17 Part Number Description 601379 200023 000951 602279 000592 601440 200019 630008 630003 400790 630004 400501 110026 600513 600574 600309 600511 600792 600497 200325 160003 601861 160010 160005 000198 000276 000928 110003 600507T (old) Power switch assembly Rocker switch, on-off Drip tray Keyboard display assembly, Model 391 Luer connector, male, 1/4-28, F Lower luer line, 1/16-in. o.d. x 0.5-mm i.d. x 5-in Luer connector, 2-in. tube Bottle, 10 mL Bottle, 8 oz (200 mL) Bottle seals, 200 mL, box of 10 Bottle, 16 oz (450 mL) Bottle seals, 450 mL Bulkhead union, 1/4 T - 1/4 T (argon inlet) Particle filter assembly 24-V pressure valve assembly Mini-vacuum pump assembly 24-V vacuum valve assembly Vacuum-assist cable assembly Vacuum ballast assembly Vacuum switch Vacuum gage, 30 in. of mercury Regulator gage assembly Gage, 0-15 psi Pressure regulator, 0-15 psi Vent manifold fitting (3) Vent manifold sleeve Waste manifold, 3-port Elbow, 1/8 MP-1/4T, teflon Model 381A power supply shown Voltage Select Modules 17a 17 17 600284 600286 600288 600289 601960 (not shown) 600284 600286 600288 600289 602590 (not shown) Appendix D: 391 Illustrated Parts List 100-V module 120-V module 220-V module 240-V module Model 391 power supply 100-V module 120-V module 220-V module 240-V module Model 391 power supply D-3 Applied Biosystems Photo Number Part Number 18 19 19a 19b 19c 19d 19e 20 21 22 23 24 25 26 27 254184 254185 254187 254186 254239 600509T 600811 601087 601864 295014 100170 100167 600501 600500 601862 600344 601927 601947 295007 295018 Description Power Cords 295074 200213 295078 28 29 30 D-4 601854 601929 100176 200215 003020 110019 120 V (50/60 Hz) 220 V (50 Hz) 240 V (50 Hz) 240 V (60 Hz) 100 V (50/60 Hz) Valve control PCB assembly (analog) Controller PCB assembly (digital, behind panel) Cable assembly, 20 pin (to analog PCB) Cable assembly, 40 pin (to keyboard) Relays Battery +5-V supply Delivery valve assembly, 8 port (2) Delivery valve assembly, 4 port Pressure/vent valve assembly Solenoid valve assembly, Angar (7) Base/tetrazole delivery line (6) Capping/reagent delivery line (5) Fuse, 0.75 A (all voltage options) Fuse, 10-A slow-blow, 1/4 in. x 1-1/4 in. (100 V and 120 V): two fuses for part No. 600507T power supply; two fuses for part No. 601960T power supply Fuse, 5A, slow-blow (100 V and 120 V): two fuses for part No. 602590T power supply Fuse, 5A, slow-blow, 25 mm (220 V and 240 V): two fuses for part No. 600507T power supply; two fuses for part No. 601960T power supply Fuse, 3A, slow-blow (220 V and 240 V); two fuses for part No. 602590T power supply PCR-MATE™ memory module, version 1.00 PCR-MATE™ EP memory module, ver.1.00 Fan, 3.15 in. x 3.15 in. x 1.5 in. Fan filter, 80 mm Manifold, 8 port, teflon Plug, 1/4-28 teflon Appendix D: 391 Illustrated Parts List Applied Biosystems Photo Number Part Number Description Bottle Positions 1-5 31 32 600192 200082 601852 221014 Bottle receptacle assembly (pushbutton type) Knob, slide, black Insert/tube assembly, 1-5 O-ring, Kalrez Bottle Positions 9, 11, 12, and 15 33 34 35 36 110130 601946 001208 003558 003560 002571 37 001208 003559 003560 002571 601945 “Tee,” 1/4-28, teflon (Nos. 11 and 12 pressure) Insert assembly, 8 oz (2-line) Ratchet cap receptacle mount (outer shell) Ratchet receptacle, 8 oz Ratchet receptacle lid Wave spring, nickel-plated Bottle Position 14 38 Ratchet cap receptacle mount (outer shell) Ratchet receptacle, 16 oz Ratchet receptacle lid Wave spring, nickel-plated Insert assembly, 16 oz (2-line) Bottle Position 18 39 602458 (not shown) 002268 (not shown) 40 140041 (not shown) 002170 (not shown) 41 601865 (not shown) Cap assembly, 4 L, 1/8-in. T, 2 line Gasket, 1.38-in. o.d. x 0.88-in. i.d. x 0.030-in. high, EPR Safety carrier, 4 L Bottle rack, 4 L Waste Bottle Appendix D: 391 Illustrated Parts List Waste cap assembly, 4 L, 1/4-in. T D-5 Applied Biosystems Figure D-1. Model 391 PCR-MATE™ DNA Synthesizer, Front View D-6 Appendix D: 391 Illustrated Parts List Applied Biosystems Figure D-2. Model 391 PCR-MATE™ DNA Synthesizer, Left-Side View Appendix D: 391 Illustrated Parts List D-7 Applied Biosystems Figure D-3. Model 391 PCR-MATE™ DNA Synthesizer, Rear View D-8 Appendix D: 391 Illustrated Parts List Applied Biosystems Figure D-4. Model 391 PCR-MATE™ DNA Synthesizer, Controller PCB, Memory Module, and Cabling Appendix D: 391 Illustrated Parts List D-9 Appendix E: Warranty 391 Pre-Installation Manual Safety First!. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-3 User Attention Words, Material Safety Data Sheets, and Waste Profiles . . . . . . . . . . . . . . E-4 Abbreviations, Initializations, and Units. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-6 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-7 Chemicals and Accessories. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-7 Start-Up Chemical Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-7 Additional Chemicals and Columns. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-9 Shipping List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-10 User-Supplied Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-11 Site Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-13 Laboratory Space . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-13 Electrical Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-14 Power “Quality” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-15 Cooling Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-16 Argon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-16 Ventilation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-16 Liquid Waste Disposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-17 Operator Training at Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-17 Proof of Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-17 Printer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-17 Preinstallation Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-18 Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-19 Contacting Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-19 To Contact Technical Support by E-Mail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-19 Hours for Telephone Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-19 To Contact Technical Support by Telephone or Fax . . . . . . . . . . . . . . . . . . . . . . . . . . E-20 To Reach Technical Support Through the Internet . . . . . . . . . . . . . . . . . . . . . . . . . . . E-22 To Obtain Documents on Demand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-23 AB LIMITED WARRANTY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-24 Applied Biosystems Safety First! Safety is everyone’s concern. Your laboratory has specific safety practices and policies designed to protect laboratory personnel from the potential hazards, both obvious and hidden, that are present. The AB service engineer assigned to your laboratory has been trained to safely install your new AB instrument and to comply with your safety rules. We require our engineers to take the necessary time to familiarize themselves with the locations of potential hazards, exits, and safety equipment and steps to be taken during an evacuation or other emergency. We request that a representative from your laboratory be in the vicinity and available to our engineer at all times while he or she is onsite. We believe, as a minimum, available safety equipment should consist of a fire extinguisher (Halon™), eye wash, safety shower, eye and hand protection, adequate ventilation, and protection from sources of radiation (lasers, radioisotopes, contaminated equipment, radioactive wastes, etc.) that may be present in the area where our engineer will be working. We make this request to ensure that both your laboratory and our service personnel have a safe environment to work in during the installation and maintenance of your AB instrument system. Completing this part of the preinstallation procedure as well as the other requirements presented in this manual will help to ensure both the successful installation and safe use of your equipment. Appendix E: Warranty 391 Pre-Installation Manual E-3 Applied Biosystems User Attention Words, Material Safety Data Sheets, and Waste Profiles User Attention Words Four “user attention words” appear in the text of Applied Biosystems documents. Categorically, each word implies a particular level of observation or action as follows: Note This word is used to call attention to information. IMPORTANT This information is given because it is necessary for correct operation of the instrument. Caution This word informs the user that damage to the instrument could occur if the user does not comply with this information. WARNING Physical injury to the user or other persons could occur it these required precautions are not taken. Material Safety Data Sheets (MSDS) WARNING E-4 Some chemicals used with this instrument are hazardous. Hazards are prominently displayed on the labels of all hazardous chemicals. In addition, the MSDS, provided in the User’s Safety Information section of this manual, give information on physical characteristics, hazards, precautions, first aid, spill clean-up, and disposal procedures. Please familiarize yourself with information in the MSDSs before handling the reagents or operating the instrument. Additional free copies of MSDSs are available from Applied Biosystems upon request. Appendix E: Warranty 391 Pre-Installation Manual Applied Biosystems Waste Profiles WARNING Some chemicals collected in the waste bottle after a normal cycle may be hazardous and/or require special handling. The waste profiles, presented in the User’s Safety Information section of this manual, give percentage compositions of the reagents and inform the user about waste disposal, which must always be in accord with local, state, and federal regulations. Additional free copies of the waste profiles are available from Applied Biosystems on request. Appendix E: Warranty 391 Pre-Installation Manual E-5 Applied Biosystems Abbreviations, Initializations, and Units AB Btu/h CGA cm DNA ea EP fpm ft g gal h HPLC Hz i.d. in. kg kPa kVA L lb M m m3 mL mm mol. wt MSDS nm No. o.d. oz P/N par. PCR ppm psi V ºC ºF µm µmol E-6 = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = Applied Biosystems British thermal units/hour Compressed Gas Association centimeter deoxyribonucleic acid each extended programmability feet per minute feet gram gallon hour high performance liquid chromatography hertz inside diameter inch kilogram kiloPascal kilovolt-amperes liter pound molar meter cubic meters milliliter millimeter molecular weight material safety data sheet nanometer number outside diameter ounce part number paragraph polymerase chain reaction parts per million pounds per square inch volt degrees Celsius degree Fahrenheit micron micromole Appendix E: Warranty 391 Pre-Installation Manual Applied Biosystems Introduction This manual will help you prepare for the installation of your Model 391 or Model 391 with extended programmability (EP) DNA Synthesizer. The first part describes (1) items that will be shipped with the instrument and (2) chemicals and equipment you should obtain before you receive the instrument. The second part of the manual describes site preparation. Careful attention to these requirements will simplify the installation procedure and ensure that the instrument works correctly. The site preparation section concludes with a Preinstallation Checklist. The Model 391/391 EP is available with four voltage options. It is important that the internal voltage setting for your instrument agree with your site voltage. Please see “Shipping List” on page 10 for specific information. If you purchased an Applied Biosystems installed instrument: Please do not unpack the crate. until your local Applied Biosystems service representative arrives to make the installation. The Start-Up Chemical Kit will be shipped separately and should be unpacked upon arrival. This kit contains sufficient chemicals and reagents to demonstrate that the instrument is operating correctly. If additional DNA will be synthesized immediately following installation, additional chemicals should be ordered. If you purchased a user installed instrument (U.S. only): The User Installation Manual will be sent to you, Please be aware that you will need to order reagents for the installation. We recommend that you order the Start-Up Chemical Kit (P/N 400971), which contains sufficient chemicals and reagents for demonstrating that the instrument operates correctly. The kit contains additional items for post-synthesis processing of the oligonucleotide. If additional DNA will be synthesized immediately following installation, additional chemicals should also be ordered. Chemicals and Accessories Start-Up Chemical Kit If an Applied Biosystems service engineer will install your Model 391 or 391 EP, a Start-Up Chemical Kit will be sent to you for the installation. The kit contains chemicals and reagents (enough for at least 100 base additions), four synthesis columns, and a manual deprotection kit. The Start-Up Chemical Kit will be used during installation to verify instrument performance. Appendix E: Warranty 391 Pre-Installation Manual E-7 Applied Biosystems If you have purchased a user-installed instrument (U.S. only), you must order the Start-Up Chemical Kit, P/N 400971. The contents of the Start-Up Chemical Kit are listed below: Part No. Description Quantity 001753 Seal, 16-oz bottle 2 002267 Seal, 8-oz bottle 5 200140 Septum, 13 mm, rubber 2 400257 Manual deprotection kit 1 400330 CE-phosphoramidite A, 0.5 g 1 400331 CE-phosphoramidite G, 0.5 g 1 400332 CE-phosphoramidite C, 0.5 g 1 400333 CE-phosphoramidite T, 0.5 g 1 400443 Acetonitrile, 4 L, Burdick & Jackson 1 400445 Trifluoroacetic acid, 10 mL 1 400613 Triethylamine acetate, 2 M, 200 mL 1 400771 Oligo purification cartridge 1 400852 400953 Kit, B-CE large bottle reagents (includes the following items) 1 400060 Anhydrous acetonitrile, 30 mL 2 400236 Trichloroacetic acid, 450 mL 2 400501 16-oz bottle seals, 10/pkg 1 400606 Tetrazole/acetonitrile, 180 mL 1 400607 Acetic anhydride, 180 mL 1 400753 Iodine/water/pyridine, 200 mL 1 400785 1-methylimidazole, 180 mL 1 400790 200-mL bottle seals, 10/pkg 1 Small-scale A CPG column 1 400954 Small-scale C CPG column 1 499955 Small-scale G CPG column 1 400956 Small-scale T CPG column 1 Note E-8 The only additional reagent you will need for installation is concentrated analytical grade ammonium hydroxide. Because ammonium hydroxide is a common laboratory reagent, Applied Biosystems does not supply it in some countries, including the United States. If you do not have ammonium hydroxide in your laboratory, you can order it from Baker Laboratories (Concentrated reagent, 29.8%, Baker P/N 9721.1). Appendix E: Warranty 391 Pre-Installation Manual Applied Biosystems The Start-Up Chemical Kit includes a manual deprotection kit (P/N 400257) that contains the following: Part No. Description 200243 1-mL disposable syringes with luer fitting Quantity 10 200244 1.5-in. disposable needles with female luer 10 200245 No. 10 rubber stopper 1 110127 Male-to-male luer connector (Alltech 86506CTFE) 5 400048 DNA collection vial 10 140048 Teflon lined vial cap 10 With the exception of the No. 10 rubber stoppers and the luer connectors, this equipment is used on a one-time basis and should be ordered in sufficient quantities for regular use. In addition, when possible, it is convenient to carry out the deprotection on several compounds simultaneously, necessitating additional male-to-male luer connectors. Additional Chemicals and Columns The supplies included in the Start-Up Chemical Kit will not last beyond the installation period. It is strongly recommended that an additional supply be ordered before installation. If you wish to order chemicals and columns for additional syntheses, please contact your local salesperson or call Applied Biosystems. All phosphoramidites, reagents, and synthesis columns should be ordered from Applied Biosystems with two exceptions: • Ammonium hydroxide for the cleavage/deprotection step. Order concentrated analytical grade reagent. Purchase it in 500-mL bottles, keep it sealed tightly, and store it at 4 °C. Do not use it for more than two weeks after you have opened a bottle. • Acetonitrile (HPLC-grade) for bottle position 18. The Model 391 or 391 EP is equipped to handle a 4-L bottle. The water content must be less than or equal to 300 ppm (0.003%). We have had success with Burdick & Jackson acetonitrile (B&J P/N 015-4). If you are unsure of the water content of a local supplier’s acetonitrile, or if you know that the water content is too high, you can control or avoid this problem by (1) checking the acetonitrile by Karl Fischer titration or (2) distilling HPLC-grade acetonitrile over P2O5. Redistill over CaH2 to remove acid. Appendix E: Warranty 391 Pre-Installation Manual E-9 Applied Biosystems Shipping List The instrument will be shipped in a crate with the following accessory items: Part No. Description Quantity 000160 Ferrule, 1/16 in., teflon 2 000161 Bushing, 1/16 in., plastic 2 002170 4-L Bottle 1 002268 Gasket, EPR 1 100072 Wrench, 1/4 in. x 5/16 in. 1 110005 Connector, 1/4 MP-1/4 T, brass 1 110096 Nut, 1/4 in. brass 1 110097 Ferrule, swage, 1/4 in. 1 140024 4-L waste bottle 1 140041 4-L bottle safety carrier 1 200270 Solvent inlet filter, 40 µm 1 221014 O-ring, Karlez 1 225016 Tubing, 1/4-in. o.d., polypropylene 225075 Tubing, Tygon 10 ft (3 m) 400501 Bottle seals, 16 oz, 10/pkg 1 400790 Bottle seals, 200 ml, 10/pkg 1 600223 Trityl collection tubing assembly 1 600235 Fraction collector cable 1 601865 4-L waste cap assembly 1 3 in. (76 mm) In addition a plug/voltage kit from the list below will be included with your instrument. Part No. Description Voltage and Frequency 401202 Plug/voltage kit for US/Canada 120V (60 Hz) 401203 Plug/voltage kit for Continental Europe 220V (50 Hz) 401204 Plug/voltage kit for United Kingdom 240V (50 Hz) 401205 Plug/voltage kit for Australia 240V (50Hz) 401201 PIug/voltage kit for Japan 100V (50/60Hz) Verify that the plug/voltage kit you received is the correct one for your installation site. E-10 Appendix E: Warranty 391 Pre-Installation Manual Applied Biosystems The 391 EP PCR-MATE will be shipped with these additional items. Part No. Description 254125 RS232 cable, 6 ft (1.8 m) Quantity 1 254132 Null modem 1 254136 RS232 cable, 10 ft (3 m) 1 602022 PC syncom disk 1 User-Supplied Equipment All customer-supplied items (for trityl analysis and post-synthesis processing of your oligonucleotides) are listed below and should be obtained before the installation. The two most critical items are a fraction collector and a spectrophotometer. Although measuring trityl cation release is only an approximate indicator of synthesis success, it is a convenient assay, and so we use it rather than more meaningful HPLC or capillary electrophoresis analysis to confirm instrument performance. The following items are supplied by the user: • Fraction Collector To monitor synthesis efficiency, a fraction collector must be adjacent to the synthesizer to collect the trityl solution from the trityl collection port. Any commercially available fraction collector with an external advance switch will suffice. We recommend that at least one hundred 10-15 mL volumetric tubes be available. The fraction collector cable and trityl delivery line are packed with the instrument. The fraction collector must be placed within 30 cm (12 in.) of the left side of the instrument. WARNING The trityl effluent is composed of trichloroacetic acid and acetonitrile. Before you handle chemicals for the instrument, read the material safety data sheets (MSDS) and waste profile in Appendices A and B of this preinstallation manual. Always follow the safety precautions (eye protection, clothing, etc.) presented in the MSDS. Dispose of waste in accord with all local, state, and federal health and environmental regulations and laws. • Spectrophotometer To measure absorbance of the diluted trityl solutions you will need a visible spectrophotometer capable of reading at a wavelength of 530 nm. Also required are 1-cm cuvettes and a 0.1-M toluene sulfonic acid/acetonitrile solution for dilution. • Accessories for Reconstituting Phosphoramidites 1. Glass syringes (2, 5 or 10 mL) kept in an oven at 100-110 °C (212-230 °F). These are used to dispense anhydrous acetonitrile into the phosphoramidite bottles. Appendix E: Warranty 391 Pre-Installation Manual E-11 Applied Biosystems 2. Syringe needles (20 gage) that fit the glass syringes and are 5-10 cm (2-4 in.) long (Aldrich P/N Z11-711-0). 3. Rubber septa are used to seal the 30-mL bottles of anhydrous acetonitrile (Aldrich P/N Z10-074-9). • Heating Bath or Block The laboratory must have access to a heating bath or block to complete the manual base deprotection of the synthetic oligonucleotides. The temperature of this bath or block is typically set at 55-60 °C (131-140 °F). • Sample Concentrator The ammonium hydroxide deprotection solution is evaporated before they DNA is used or purified. Any device capable of removing an aqueous solution is acceptable. • Toluenesulfonic Acid Purchase the 500-g bottle of p-toluenesulfonic acid monohydrate, 99% (P/N T-3751) from Sigma Chemical Co., St. Louis, MO 63178. This is used to dilute the trityl cation. WARNING P-toluenesulphonic acid is hazardous and must be handled with great caution. Refer to the Sigma Chemical Co. MSDS to familiarize yourself with its hazards and the precautions that must be taken. All procedures must be conducted in a correctly operating fume hood. Use of eye protection, appropriate rubber gloves, and safety clothing is imperative. • Ammonium Hydroxide As mentioned under “Start-Up Chemical Kit” on page 7, you must purchase concentrated analytical grade ammonium hydroxide (preferably in 500-mL bottles) for deprotection. This reagent is not included in the Start-Up Chemical Kit. WARNING E-12 Ammonium hydroxide is hazardous and must be handled with great caution. Refer to the supplier’s MSDS to familiarize yourself with its hazards and the precautions that must be taken. All procedures must be conducted in a correctly operating fume hood. Use of eye protection, appropriate rubber gloves, and safety clothing is imperative. Appendix E: Warranty 391 Pre-Installation Manual Applied Biosystems Site Preparation Laboratory Space Allocating sufficient laboratory space (Figure E-1) is critical for successful installation and troublefree maintenance. A sturdy freestanding table with locking wheels is strongly preferred over a lab bench because it allows access to the rear of the instrument. Specifically, we recommend an open bench with plastic laminate top, which can be ordered from Bay Products, 8701 Torresdale Avenue, Philadelphia, PA 19136 (800) 523-3434 or (215) 338-7300. The Bay Products catalog number is 6303B34. Sufficient room must be provided for the user-supplied external fraction collector and the 4-L acetonitrile bottle container which will be positioned to the left of the synthesizer. A space within approximately 5 ft (1.5 m) of the instrument must also be provided for safely securing an argon gas cylinder (size 1A). The dimensions and height of the Model 391 or 391 EP are as follows: Width Depth Height Weight 52 cm 38 cm 41 cm 29 kg (20.5 in.) (15.0 in.) (16.0 in.) (65 lb) Figure E-1. Laboratory Space Requirement Appendix E: Warranty 391 Pre-Installation Manual E-13 Applied Biosystems Electrical Requirements Power The Model 391 or 391 EP requires a dedicated electrical line with a circuit breaker (Figure E-2) and a power rating of at least 1.25 kVA. The outlet must be located close to the instrument. The supplied power cord is 8 ft (2.5 m). Grounding Certain types of electrical noise are exaggerated by poor or improper electrical round connections. To prevent these problems, the installation site should have a dedicated 1.25-kVA power line and isolated ground between the instrument and building main electrical service, as depicted in Figure E-2. E-14 Appendix E: Warranty 391 Pre-Installation Manual Applied Biosystems Figure E-2. Dedicated Power Line Power “Quality” Line Voltage Fluctuation The line voltage must be within 5% of the nominal figures. High or low voltage conditions will have an adverse effect on electronic components of the instrument. In areas where the supplied power is subject to fluctuations exceeding the above limits, a power line regulator is strongly suggested. Please consult your local Applied Biosystems service representative if you suspect a problem. Voltage Spikes Short-duration, high-voltage spikes can be caused by other devices (such as large motors) using the same power source or by outside influences such as lightning. If your laboratory environment contains devices that are electronically “noisy,” or if you are in an area with frequent electrical storms, a line conditioner may greatly enhance reliability of your system. The line conditioner must have a rating of at least 0.5 kVA. Several sources of line conditioners are as follows: • Best Power, P. 0. Box 280, Necedah, WI 54646 - Tel: (608) 565-7200 • RTE Deltec, 2727 Kurtz St, San Diego, CA 92120 - Tel: (619) 291-4211 • Elgar, 9250 Brown Deer Rd, San Diego, CA 92121 - Tel: (619) 450-0085 Appendix E: Warranty 391 Pre-Installation Manual E-15 Applied Biosystems Cooling Requirements The Model 391 or 391 EP generates a maximum of 600 Btu/h. While there are no special cooling requirements, a constant laboratory temperature between 16-22 °C (60-72 °F) is recommended. Avoid placing the instrument close to heaters or cooling ducts. Temperatures below 16 ºC (60 ºF) must be avoided because they will cause the tetrazole to precipitate from solution. Argon A regulated cylinder (size 1A) of prepurified (99.998%) argon must be connected to the rear panel of the synthesizer. The instrument has a gas input that accepts 1/4-in.-o.d. Parflex™ tubing with appropriate Swagelok® tube fittings (1/4-in. nut and ferrule). A 10-ft length of Parflex™ tubing (1/4-in. o.d.) and the necessary Swagelok® fittings to connect this tubing to the instrument are provided. The argon tank requires a two-gauge regulator with a CGA (Compressed Gas Association) 580 argon cylinder adapter on the inlet side and a Swagelok® fitting that accepts a 1/4-in.-o.d. tube. The primary gauge (0-3000 psi; 0-25,000 kPa recommended) measures tank pressure, and the secondary gauge (0-200 psi; 0-2000 kPa recommended) measures regulated pressure. The regulator must be obtained by the user prior to installation. Typical argon consumption is approximately 3.0 m3 every four weeks. Size 1A cylinders have a capacity of 8.2 cm3 and should last approximately three months. The tank should be regulated to approximately 65 psi (450 kPa). WARNING Compressed gas cylinders must be safely attached to the wall or bench by means of approved restraints. Failure to do so could cause the cylinders to fall over and explode, which could result in physical harm. Turn off or cap the cylinders when they are not in use. Ventilation Gaseous waste is vented from the waste bottle. Your facility must be equipped with a ventilated laboratory chemical fume hood or the equivaent to remove harmful vapors. The waste vent should be routed to the chemical exhaust system by one of the methods shown in the User’s Safety Information section at the beginning of this manual. E-16 Appendix E: Warranty 391 Pre-Installation Manual Applied Biosystems Liquid Waste Disposal WARNING Please review the waste profile for information on contents of the Model 391 and 391 EP liquid waste so that it can be disposed of in accordance with all local, state, and federal health and environmental regulations and laws. Please also read the MSDS to determine the protective equipment required to work with and dispose of these chemicals. The waste profile and MSDS are in the User’s Safety Information section at the beginning of this manual. Operator Training at Installation (Applied Biosystems Service Installed Instruments Only) An important part of in-laboratory installation is operator training. Those persons who will be operating the Model 391 or 391 EP should set aside two uninterrupted days to work with the Applied Biosystems service representative. If this is not possible, the installation should be rescheduled. Proof of Performance Unless previous arrangements have been made, an oligonucleotide of less than 25 bases will be synthesized to demonstrate instrument performance. It is necessary to have the fraction collector, spectrophotometer, and p-toluenesulfonic acid solution prepared so that the trityl assay can be performed. The passing on-site specification is 98.0% (±0.5%) average coupling yield. Printer A printer option consisting of a Hewlett-Packard Think Jet™ printer and interface cable is available from Applied Biosystems. The appropriate part number depends on the site voltage as follows: P/ N 400815, 100 Vac; P/N 400816, 120 Vac; P/N 400817, 220 Vac; and P/N 400818, 240 Vac. The interface cable (P/N 100157) may be ordered separately. Although the printer uses any standard 81/2 x 11-in. (European size A4) single or fanfold paper, best print quality is ensured by using H-P specified Ink Jet paper. Additional paper stock is available from Hewlett-Packard: Part No. 92261 M: Ink Jet paper, 500 sheets, single sheets Part No. 92261 N: Ink Jet paper, 2500 sheets, fanfold Appendix E: Warranty 391 Pre-Installation Manual E-17 Applied Biosystems Preinstallation Checklist The following checklist is designed to ensure that all necessary preparations have been made prior to installation of the Model 391 or 391 EP. If there are any questions concerning the installation procedure or preparations, please contact your local Applied Biosystems service representative. Check (✔) if ready 1. Preinstallation manual read and understood Chemicals and Equipment: 2. Correct plug/voltage kit_ received 3. Start-Up Chemical kit received 4. Additional reagents, including concentrated ammonium hydroxide and acetonitrile (4 L) ordered. 5. Fraction collector 6. Spectrophotometer 7. Accessories for reconstituting phoshoramidites 8. Heating block 9. Sample concentrator 10. Toluenesulfonic acid 11. All material safety data sheets (MSDS) and waste profile material read and understood Site Preparation: E-18 12. Correct laboratory space 13. Constant laboratory temperature 14. Argon cylinder, size 1A (regulator and adapter) 15. Correct ventilation 16. Waste disposal method established Appendix E: Warranty 391 Pre-Installation Manual Applied Biosystems Technical Support Contacting Technical Support You can contact Applied Biosystems for technical support by telephone or fax, by e-mail, or through the Internet. You can order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents 24 hours a day. In addition, you can download documents in PDF format from the Applied Biosystems Web site (please see the section “To Obtain Documents on Demand” following the telephone information below). To Contact Technical Support by E-Mail Contact technical support by e-mail for help in the following product areas: Product Area E-mail address Genetic Analysis (DNA Sequencing) [email protected] Sequence Detection Systems and PCR [email protected] Protein Sequencing, Peptide and DNA Synthesis [email protected] Biochromatography, PerSeptive DNA, PNA and Peptide Synthesis systems, CytoFluor®, FMAT™, Voyager™, and Mariner™ Mass Spectrometers [email protected] LC/MS (Applied Biosystems/MDS Sciex) [email protected] or [email protected] Chemiluminescence (Tropix) [email protected] Hours for Telephone Technical Support In the United States and Canada, technical support is available at the following times: Product Hours Chemiluminescence 8:30 a.m. to 5:30 p.m. Eastern Time Framingham support 8:00 a.m. to 6:00 p.m. Eastern Time All Other Products 5:30 a.m. to 5:00 p.m. Pacific Time Appendix E: Warranty 391 Pre-Installation Manual E-19 Applied Biosystems To Contact Technical Support by Telephone or Fax In North America To contact Applied Biosystems Technical Support, use the telephone or fax numbers given below. (To open a service call for other support needs, or in case of an emergency, dial 1-800-831-6844 and press 1.) Product or Product Area Telephone Dial... Fax Dial... ABI PRISM® 3700 DNA Analyzer 1-800-831-6844, then press 8 1-650-638-5981 DNA Synthesis 1-800-831-6844, then press 21 1-650-638-5981 Fluorescent DNA Sequencing 1-800-831-6844, then press 22 1-650-638-5981 Fluorescent Fragment Analysis (includes GeneScan® applications) 1-800-831-6844, then press 23 1-650-638-5981 Integrated Thermal Cyclers (ABI PRISM ® 877 and Catalyst 800 instruments) 1-800-831-6844, then press 24 1-650-638-5981 ABI PRISM ® 3100 Genetic Analyzer 1-800-831-6844, then press 26 1-650-638-5981 BioInformatics (includes BioLIMS, BioMerge™, and SQL GT™ applications) 1-800-831-6844, then press 25 1-505-982-7690 Peptide Synthesis (433 and 43X Systems) 1-800-831-6844, then press 31 1-650-638-5981 Protein Sequencing (Procise Protein Sequencing Systems) 1-800-831-6844, then press 32 1-650-638-5981 PCR and Sequence Detection 1-800-762-4001, then press 1 for PCR, 2 for the 7700 or 5700, 6 for the 6700 or dial 1-800-831-6844, then press 5 1-240-453-4613 Voyager MALDI-TOF Biospectrometry and Mariner ESI-TOF Mass Spectrometry Workstations 1-800-899-5858, then press 13 1-508-383-7855 Biochromatography (BioCAD Workstations and Poros Perfusion Chromatography Products) 1-800-899-5858, then press 14 1-508-383-7855 Expedite Nucleic acid Synthesis Systems 1-800-899-5858, then press 15 1-508-383-7855 Peptide Synthesis (Pioneer and 9050 Plus Peptide Synthesizers) 1-800-899-5858, then press 15 1-508-383-7855 PNA Custom and Synthesis 1-800-899-5858, then press 15 1-508-383-7855 E-20 Appendix E: Warranty 391 Pre-Installation Manual Applied Biosystems Product or Product Area Telephone Dial... Fax Dial... FMAT 8100 HTS System and Cytofluor 4000 Fluorescence Plate Reader 1-800-899-5858, then press 16 1-508-383-7855 Chemiluminescence (Tropix) 1-800-542-2369 (U.S. 1-781-275-8581 only), or 1-781-271-0045 Applied Biosystems/MDS Sciex 1-800-952-4716 1-650-638-6223 Telephone Dial... Fax Dial... Outside North America Region Africa and the Middle East Africa (English Speaking) and West Asia (Fairlands, South Africa) 27 11 478 0411 27 11 478 0349 South Africa (Johannesburg) 27 11 478 0411 27 11 478 0349 Middle Eastern Countries and North Africa (Monza, Italia) 39 (0)39 8389 481 39 (0)39 8389 493 Eastern Asia, China, Oceania Australia (Scoresby, Victoria) 61 3 9730 8600 61 3 9730 8799 China (Beijing) 86 10 64106608 86 10 64106617 Hong Kong 852 2756 6928 852 2756 6968 Korea (Seoul) 82 2 593 6470/6471 82 2 593 6472 Malaysia (Petaling Jaya) 60 3 758 8268 60 3 754 9043 Singapore 65 896 2168 65 896 2147 Taiwan (Taipei Hsien) 886 2 22358 2838 886 2 2358 2839 Thailand (Bangkok) 66 2 719 6405 66 2 319 9788 Europe Austria (Wien) 43 (0)1 867 35 75 0 43 (0)1 867 35 75 11 Belgium 32 (0)2 712 5555 32 (0)2 712 5516 Czech Republic and Slovakia (Praha) 420 2 61 222 164 420 2 61 222 168 Denmark (Naerum) 45 45 58 60 00 45 45 58 60 01 Finland (Espoo) 358 (0)9 251 24 250 358 (0)9 251 24 243 France (Paris) 33 (0)1 69 59 85 85 33 (0)1 69 59 85 00 Germany (Weiterstadt) 49 (0) 6150 101 0 49 (0) 6150 101 101 Hungary (Budapest) 36 (0)1 270 8398 36 (0)1 270 8288 Italy (Milano) 39 (0)39 83891 39 (0)39 838 9492 Norway (Oslo) 47 23 12 06 05 47 23 12 05 75 Poland, Lithuania, Latvia, and Estonia (Warszawa) 48 (22) 866 40 10 48 (22) 866 40 20 Portugal (Lisboa) 351 (0)22 605 33 14 351 (0)22 605 33 15 Appendix E: Warranty 391 Pre-Installation Manual E-21 Applied Biosystems Region Telephone Dial... Fax Dial... Russia (Moskva) 7 095 935 8888 7 095 564 8787 South East Europe (Zagreb, Croatia) 385 1 34 91 927 385 1 34 91 840 Spain (Tres Cantos) 34 (0)91 806 1210 34 (0)91 806 1206 Sweden (Stockholm) 46 (0)8 619 4400 46 (0)8 619 4401 Switzerland (Rotkreuz) 41 (0)41 799 7777 41 (0)41 790 0676 The Netherlands (Nieuwerkerk a/d IJssel) 31 (0)180 331400 31 (0)180 331409 United Kingdom (Warrington, Cheshire) 44 (0)1925 825650 44 (0)1925 282502 All other countries not listed (Warrington, UK) 44 (0)1925 282481 44 (0)1925 282509 81 3 5566 6230 81 3 5566 6507 Japan Japan (Hacchobori, Chuo-Ku, Tokyo) Latin America Del.A. Obregon, Mexico 305-670-4350 305-670-4349 To Reach Technical Support Through the Internet We strongly encourage you to visit our Web site for answers to frequently asked questions and for more information about our products. You can also order technical documents or an index of available documents and have them faxed or e-mailed to you through our site. The Applied Biosystems Web site address is http://www.appliedbiosystems.com/techsupp To submit technical questions from North America or Europe: Step Action 5 Access the Applied Biosystems Technical Support Web site. 6 Under the Troubleshooting heading, click Support Request Forms, then select the relevant support region for the product area of interest. 7 Enter the requested information and your question in the displayed form, then click Ask Us RIGHT NOW (blue button with yellow text). 8 Enter the required information in the next form (if you have not already done so), then click Ask Us RIGHT NOW. You will receive an e-mail reply to your question from one of our technical experts within 24 to 48 hours. E-22 Appendix E: Warranty 391 Pre-Installation Manual Applied Biosystems To Obtain Documents on Demand Free, 24-hour access to Applied Biosystems technical documents, including MSDSs, is available by fax or e-mail or by download from our Web site. To order documents... Then... by index number a. Access the Applied Biosystems Technical Support Web site at http://www.appliedbiosystems.com/techsupp b. Click the Index link for the document type you want, then find the document you want and record the index number. c. Use the index number when requesting documents following the procedures below. by phone for fax delivery a. From the U.S. or Canada, call 1-800-487-6809, or from outside the U.S. and Canada, call 1-858-712-0317. b. Follow the voice instructions to order the documents you want. Note through the Internet for fax or e-mail delivery There is a limit of five documents per request. a. Access the Applied Biosystems Technical Support Web site at http://www.appliedbiosystems.com/techsupp b. Under Resource Libraries, click the type of document you want. c. Enter or select the requested information in the displayed form, then click Search. d. In the displayed search results, select a check box for the method of delivery for each document that matches your criteria, then click Deliver Selected Documents Now (or click the PDF icon for the document to download it immediately). e. Fill in the information form (if you have not previously done so), then click Deliver Selected Documents Now to submit your order. Note There is a limit of five documents per request for fax delivery but no limit on the number of documents you can order for e-mail delivery. Appendix E: Warranty 391 Pre-Installation Manual E-23 Applied Biosystems AB LIMITED WARRANTY Applied Biosystems (“AB”) warrants to the Customer that, for a period ending on the earlier of one year from completion of installation or 15 months from the date of shipment to the Customer (the “Warranty Period”), the AB Model 391 purchased by the customer (the “Instrument”) will be free from defects in material and workmanship, and will perform in accordance with the performance specifications contained in the applicable AB product literature (the “Specifications”). This Warranty does not apply to the Instrument’s valves, reagent lines, or performance, unless the Customer uses only reagents and solvents supplied by AB or expressly recommended by AB, or to any damages caused by reagents or solvents not supplied by AB, even though recommended by AB. This Warranty does not extend to any Instrument or part thereof that has been subjected to misuse, neglect or accident, that has been modified or repaired by anyone other than AB or that has not been used in accordance with the instructions contained in the Instrument Operator’s Manual. Nor does this Warranty cover any Customer-installable consumable parts for the Instrument that are listed in the Instrument Operator’s Manual. AB’s obligation under this warranty is limited to repairs or replacements that AB deems necessary to correct covered defects or failures of which AB is notified prior to expiration of the Warranty Period. All repairs and replacements under this Warranty shall be performed by AB onsite at the Customer’s location at AB’s expense. No agent, employee, or representative of AB has any authority to bind AB to any affirmation, representation, or warranty concerning the Instrument that is not contained in the printed product literature or this Warranty Statement. Any such affirmation, representation or warranty made by any agent, employee, or representative of AB shall not be binding on AB. AB shall not be liable for any incidental, special, or consequential loss, damage or expense directly or indirectly arising from the use of the Instrument. AB makes no warranty whatsoever in regard to products or parts furnished by third parties; such products or parts will be subject to the warranties, if any, of their respective manufacturers. This Warranty is limited to the original Customer and is not transferable. THIS WARRANTY IS THE SOLE AND EXCLUSIVE WARRANTY AS TO THE INSTRUMENT AND IS EXPRESSLY IN LIEU OF ANY OTHER EXPRESS OR IMPLIED WARRANTIES, INCLUDING, WITHOUT LIMITATION, ANY IMPLIED WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE AND OF ANY OTHER OBLIGATION ON THE PART OF AB. E-24 Appendix E: Warranty 391 Pre-Installation Manual Headquarters 850 Lincoln Centre Drive Foster City, CA 94404 USA Phone: +1 650.638.5800 Toll Free: +1 800.345.5224 Fax: +1 650.638.5884 Worldwide Sales Offices Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches into 150 countries on six continents. For international office locations, please call our local office or refer to our web site at www.appliedbiosystems.com. www.appliedbiosystems.com Applera Corporation is committed to providing the world’s leading technology and information for life scientists. Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses. Printed in the USA, 06/2002 Part Number 900937 Rev. D an Applera business