Primer design of allele-specific (AS) primers. Two AS primers, one for each allele of a SNP are designed. The AS primers contain one of two polymorphic nucleotides at the primer 3' end. Two sets of primers, either forward or reverse primers can be designed. If a common reverse or forward primer is used in a PCR reaction, the reaction is called allele-specific PCR (AS-PCR). Generally, two PCR reactions are needed for detection of both alleles of a SNP. A variant of AS-PCR is to use only one AS primer and two outer SNP-flanking primers in a single PCR reaction, i.e., three-primer nested system . A mismatch (represented by *) may be deliberately introduced at the third position from the 3' end of each of AS primers to increase allelic specificity (See Table 1). The SNP R (G/A) is illustrated as an example, and the other types of SNPs can be applied in the same way.