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6.1.2. Composite Search Options
PCR Primers
This option generates optimal PCR primer pairs from a nucleic acid template.
Compatible primer pairs can be selected across the entire active sequence, or
they can be selected from specific regions on the file by setting search ranges.
Setting search ranges also speeds up this and other OLIGO searches. The
stringency of the search can be controlled using the search stringency settings
(global) and/or individual parameter settings in the "Parameters" window. The
following sub-searches are used to find the PCR primers:
• Eliminate Ambiguous Bases; by default, selects non-degenerate primers
• Duplex-free Oligonucleotides; selects primers free of 3’-end and internal dimers
• Highly Specific Oligonucleotides (3’-end Stability); selects moderately stable 3’ends
• Oligonucleotides with GC Clamp; selects oligos with high internal stability
regions
• Oligonucleotides within Selected Tm Limits; selects oligos with certain Tm
range
• Hairpin-free Oligonucleotides
• Eliminate Mono- and Di-Nucleotide Repeats; selects oligos without internal
repeats
• Detect Sequence Repeats; selects oligos without other repeats than the above
option
• Eliminate Frequent Oligonucleotides; primers with most common in GenBank
3’-ends (6- or 7-mers) are eliminated
• Omit High Secondary Structure Regions in the Template; certain sequence
areas will not be amplified due to high secondary structure
• Check Primers/Probe Sequence Constraints; no constraints by the default but
you may ask OLIGO not to choose primers that start with a specific base, for
example
• Restrict the number of G Bases; good for further multiplexing and overall nondimerization
• Eliminate False Priming Oligonucleotides
The sub-search can be extended on other files by the
• Continue Above Search in Other File(s), thus eliminating primers that could also
prime from the other sequences, and the “opposite” search could also be
performed by checking the
• Consensus Primers
and selecting the sequences on which the primers must prime as well. The
subsearches can also be customized by adding or deleting them from the
standard settings or changing the search parameters.
The Search for Compatible Primer Pairs proceeds as follows:
• Search for optimized positive strand primers for PCR
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