Multiplex primer prediction software for divergent targets

Gardner, Shea N.; Hiddessen, Amy L.; Williams, Peter L.; Hara, Christine; Wagner, Mark C.; Colston, Bill W.

For comparison, we considered other software options for designing primers for these heterogeneous viruses. FastPCR (www.biocenter.helsinki.fi/bi/programs/fastpcr .htm) and GeneUp (25) were the only programs that did not require an MSA as input. The FastPCR algorithm for group-specific PCR (i.e. universal amplification) designs PCR primer pairs individually for each target sequence without regard for primer conservation among targets, and then compares each primer pair to the other targets. This is a brute force strategy that is only suitable for small target sets and short target sequences (appropriate for gene lengths, but not for viral genome-length sequences). We ran the FastPCR software on our internal servers, but it did not complete 'group-specific PCR' for the smallest data set, Norwalk virus, after running for 18 h, and for 'multiplex PCR' gave the error message 'No compatible combination of pair primers for multiplex PCR found'.