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Input Target Genes

MSA to find Conserved regions

Candidate Primers Sequence Heuristics

Primer 3 Hybridization Tm’s, Primer Dimers

MFOLD Test Primer folding

Background Sequences

BLAST Check Specificity

Rank Designs

227 Figure 5. Traditional primer design approach. MSA: Multiple sequence alignment.

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4.3 Modern Primer Design Paradigm

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4.3.1 Modern sequence databases

Perhaps the most important new contribution to the field of PCR is the availability of modern 232 sequence databases – they are a treasure trove that can be used to improve the design of the PCR 233 assay to maximize coverage (i.e. the number of variants that are efficiently amplified by a given 234 PCR reaction) and also guide the testing of a PCR assay by identifying potential false positives 235 or false negatives (e.g., due to sequence variations at primer and probe sites). When designing a 236 PCR assay, it is helpful to first collect sets of sequences that represent the inclusivity, 237 exclusivity, and background panels ( see Glossary for definitions). In addition to the generalized 238

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