A simple and rapid modified--new method for SNP typing by fragment length discrepant allele specific PCR / 法医学杂志

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.