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CHAPTER 1 : INTRODUCTION TO FLUORESCENCE
Chapter 1
INTRODUCTION TO FLUORESCENCE
Advantages of fluorescent detection
Fluorescent labelling and staining, when combined with an appropriate
imaging instrument, is a sensitive and quantitative method that is widely
used in molecular biology and biochemistry laboratories for a variety of
experimental, analytical, and quality control applications. Commonly
used techniques, including total nucleic acid and protein quantification,
Western, Northern and Southern blotting, PCR✧ product analysis, and
DNA sequencing, can all benefit from the application of fluorescencebased methods for detection. Fluorescent detection offers a number of
important advantages over other methods, several of which are described
below.
Sensitivity
Fig 1. Fluorescently labelled DNA size
ladders and PCR products loaded in
the same lanes were electrophoretically
separated in a polyacrylamide gel and
imaged using Typhoon™ 8600 scanner.
Fluorescein (green), Cy™3 (yellow),
ROX™ (blue) and Cy5 (red) labels were
used in amounts varying from 0.25 to
5 fmol per band.
Fluorescent probes permit sensitive detection of many biological
molecules. Fluorescent stains and dyes are frequently the most sensitive
option for detection of total DNA, RNA, and protein compared with
traditional colourimetric methods. Many fluorescence applications
approach the sensitivity afforded by radioisotopes.
Multiple-label possibility
With fluorescent labelling, two or more fluorochromes can be detected
separately using optical filters and a fluorochrome separation algorithm.
Therefore components can be labelled specifically and identified
separately in the same sample or lane of a gel (Fig 1). For example,
standards and unknowns used in PCR can be labelled with different
fluorochromes to provide an internal standard for the assay.
Stability
Fluorescently labelled molecules offer several distinct advantages over
radiolabelled molecules with respect to stability. Whereas fluorescent
antibodies, oligonucleotide hybridization probes, and PCR primers
can be stored for six months or longer, antibodies labelled with 125I
become unusable in about a month, and 32P-labelled nucleotides and
oligonucleotides decay significantly in about a week. Because of their
long shelf-life, fluorescently labelled reagents can be prepared in large
batches that can be standardized and used for extended periods.
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