Primer design for PCR and sequencing in high-throughput analysis of SNPs

To achieve high-throughput analysis of allele frequencies in human SNPs; we have developed automated methods for designing PCR and DNA sequencing primers. We found we could run the PCR assays at quite stringent, uniform conditions. The design process used freely available databases, including dbSNP SNPper, and TSC, and publicly available software including RepeatMasker and Primer3. We describe parameters for the software and other considerations that increase experimental success. As anticipated, some assays failed at the design stage due primarily to the genomic locations of repetitive sequences, extreme GC content regions, or lack of sufficient sequence. However, over 23 000 assays, including 96% of those recently analyzed, have,been experimentally successful. Similar design methods could be used for PCR assays in any organism with substantial available sequence.