Abstract: Objective To establish a new method of SNP typing. Methods Based on the principle of allele specific PCR and capillary electrophoresis technique, 11 diallelic SNP loci were selected and two forward primers with different length were designed for each SNP, with their 3′ends matched to the two alleles, respectively. An artificially mismatched base was also introduced into the third or fourth base in the 3′end area of the two forward primers in order to enhance the specificity of amplification. A common reverse primer was designed 100-300 bp away from the forward primers, and labeled with fluorescence. The PCR products were separated and analyzed by ABI PrismTM 310 Genetic Analyzer after all of the 11 SNPs were multiply amplified. Results A single product peak was observed while the SNP was homozygous, and two product peaks with different height were observed while the SNP was heterozygous. The length of PCR products was different with the different SNPs. According to the length of the products and the number of the product peaks, the genotypes of the 11 SNPs can simultaneously be analyzed, and the results were in accordance with the direct sequencing. Conclusion Fragment length discrepant allele specific fluorescence labeled multi-PCR (FLDASFLM-PCR) is a simple, rapid and efficient new method for SNP typing.

Key words: forensic biology, single nucleotide, polymorphism, allele specific PCR, multiplex amplification