Multiplex primer prediction software for divergent targets

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

GreeneSCPrimer (20) did generate a number of degenerate primer candidates from the MSAs for Norwalk and FMDV, requiring the user to manually select a combination of forward and reverse groups from a set of options. We ran GreeneSCPrimer using length, T m , etc. settings mirroring or more lenient than those we used for Table 3 (T m = 55-65 C, GC% = 20-80%, length 17-25 bp, 100% coverage, product size 80-620 bp, allowed T m difference 10 C, others left as defaults). HYDEN (22) also generated degenerate candidates from a MSA, although it does not check T m and the length is limited to a single value rather than a range. For our tests using HYDEN, we used a length of 18 rather than 17 because of the lack of T m control, and allowed 0 mismatches. The GreeneSCPrimer option requiring the fewest total primers for the Norwalk set required 18 primers, four of which had either 2-fold or 4-fold degeneracy so the actual number of priming sequences would be 26, compared to a total of 20 non-degenerate primers predicted by MPP (Supplementary Data). HYDEN generated four degenerate primers covering only 34 of 41 sequences, each with 3-or 4-fold degeneracy for Norwalk, which translates to 15 priming sequences in the reaction. One would need to find primers to amplify the remaining seven sequences. Small degenerate priming sets (e.g. four primers in this case) are less expensive to purchase, but because of dilution effects from the many sequence combinations actually present (15 priming sequences in the PCR), sensitivity may be reduced compared to nondegenerate priming. However, using a smaller set of degenerate signatures such as those from HYDEN may be preferable, and is a capability that could improve MPP in a future version.