Genotyping by allele-specific PCR

SNPs are the basis for many polymorphisms that are detected using systems such as restriction fragment length polymorphisms (RFLPs), randomly amplified polymorphic DNAs (RAPDs) and amplified fragment length polymorphisms (AFLPs). Cleaved amplified polymorphic site (CAPS or PCRRFLP) markers have been adopted by a number of groups to enable mapping of SNP markers identified in ESTs. Because this system relies on each SNP being associated with a restriction enzyme site, only a proportion of SNPs are amenable to CAPS. In addition, the enzyme digest step is both time-consuming and often unreliable. An alternative method is allele-specific PCR (AS-PCR). The reliability of AS-PCR can be increased by the addition of destabilizing mismatches within the allele-specific primer (ASP), reducing the false positive rate, and a parallel positive control PCR reducing false negatives. The positive control PCR takes place in the same tube as the diagnostic PCR, competing with it for access to polymerase and nucleotides and this too may contribute to a reduction in the false positive rate. The outcomes of these experiments allowed the formation of guidelines which were successfully used to design assays for three additional SNPs. Briefly, the guidelines were as follows: (i) the melting temperature (Tm) of the positive control primers should be 20-25°C lower than that of the 300-1000 bp PCR product itself; (ii) the Tm of the inner PCR products should be - 35°C lower than the positive control PCR product; and (iii) the annealing temperature should be 20°C below the Tm of the positive control PCR product. The recommended optimization pathway involved alterations to both positive control and ASP concentrations