Abstract: Objective To establish a new method for single nucleotide polymorphism (SNP) typing based on allele specific PCR: fragment length discrepant allele specific PCR (FLDAS-PCR), and study the influence on specific extension by introducing a mismatch at the third or fourth 3′-terminal base of allele specific primers. Methods For SNP loci rs759117 and rs760887, two allele specific forward primers, with different length and a mismatch introduced at the third or fourth 3′-terminal base, and a public reverse primer were designed for SNP typing. The genotyping of SNP was determined by the two allele specific fragments different in size after polyacrylamide gel and silver staining. Results The different homozygote genotypes comprised a single band with different size respectively, and the heterozygote genotypes comprised two bands. Typing results were completely consistent with those by direct sequencing. Non-specific primer extension was decreased remarkably after introducing a mismatch at the third or fourth 3′-terminal base of allele specific primers, and the stringency of PCR reaction was cut down. Conclusion FLDAS-PCR is a simple, rapid and efficient new method for SNP typing. During FLDAS-PCR, specific primers with a mismatch at the third or fourth 3′-terminal base have more power to identify two alleles.

Key words: single nucleotide polymorphism, fragment length discrepant allele specific PCR, base mismatch