Genotyping of Single Nucleotide Polymorphism by Modified Tetra-primer PCR

Objectives: Genotyping of single nucleotide polymorphisms (SNPs) has often been utilized in genetic studies of diseases. We have examined SNP of the human b-defensin-2 gene, which encodes an antimicrobial peptide in oral tissues. In this study, we developed a modified tetra-primer PCR for simple and definitive determination of SNP. Methods: Tetra-primer PCR is used as a method to identify SNPs using four primers (two allele-specific primers and two outer primers). Basically, three PCR bands are amplified; the largest band is amplified by the two outer primers, the second band is from the SNP allele, and the third band is from the normal allele. Originally, allele-specific primers that had single or double mismatches at the 3'-ends were used. For each allele, a single mismatched primer was functional in the PCR but double mismatched primers were not functional in this system. Based on this principle, two and three PCR bands are detected for homozygotes and heterozygotes, respectively. However, we observed that the double-mismatched primer was sometimes functional in the PCR. Furthermore, we observed that the triple-mismatched primer never was functional. Consequently, we applied double- and triple-mismatched primers to a modified tetra-primer PCR. The human b-defensin-2 gene had a SNP at Ð475 in the 5'-flanking region. We determined the effectiveness of SNP detection by a modified tetra-primer PCR using 100 ng of genomic DNA from KB and SSC-9 cells. Results: In analyses of genomic DNA both from KB and SSC-9, the modified tetra-primer PCR was fully functional. Three and two PCR bands were observed in KB and SSC-9 cells, respectively. These results suggested that KB and SSC-9 were heterozygous and homozygous, respectively, at the position. Conclusion: This new modified tetra-primer PCR, using double and triple mismatched primers, appears to be able to detect any SNP in a simple, definitive, and low cost fashion.