A novel technique for detecting single nucleotide polymorphisms by analyzing consumed allele-specific primers

We present a simple and rapid polymerase chain reaction (PCR)-based techniq ue, termed consumed allele-specific primer analysis (CASPA), as a new strat egy for single nucleotide polymorphism (SNP) analysis. The method involves the use of labeled allele-specific primers, differing in length, with sever al noncomplementary nucleotides added in the 5'-terminal region. After PCR amplification, the amounts of the remaining primers not incorporated into t he PCR products are determined. Thus, nucleotide substitutions are identifi ed by measuring the consumption of primers. In this study, the CASPA method was successfully applied to ABO genotyping. In the present;method, the all ele-specific primer only anneals with the target polymorphic site on the DN A, so it is not necessary to analyze the PCR products. Therefore, this meth od is only little affected by modification of the PCR products. The CASPA m ethod is expected to be a useful tool for typing of SNPs.