Tetra-primer amplification refractory mutation system PCR, or ARMS-PCR, employs two pairs of primers to amplify two alleles in one PCR reaction. The primers are designed such that the two primer pairs overlap at a SNP location but each match perfectly to only one of the possible SNPs. The basis of the invention is that unexpectedly, oligonucleotides with a mismatched 3'-residue will not function as primers in the PCR under appropriate conditions. As a result, if a given allele is present in the PCR reaction, the primer pair specific to that allele will produce product but not to the alternative allele with a different SNP. The two primer pairs are also designed such that their PCR products are of a significantly different length allowing for easily distinguishable bands by gel electrophoresis or melt temperature analysis. In examining the results, if a genomic sample is homozygous, then the PCR products that result will be from the primer that matches the SNP location and the outer opposite-strand primer, as well from the two outer primers. If the genomic sample is heterozygous, then products will result from the primer of each allele and their respective outer primer counterparts as well as the outer primers.
An alternative strategy is to run multiple qPCR reactions with different primer sets that target each allele separately. Well-designed primers will amplify their target SNP at a much earlier cycle than the other SNPs. This allows more than two alleles to be distinguished, although an individual qPCR reaction is required for each SNP. To achieve high enough specificity, the primer sequence may require placement of an artificial mismatch near its 3'-end, which is an approach generally known as Taq-MAMA.