Variable fragment length allele-specific polymerase chain reaction (VFLASP), a method for simple and reliable genotyping

Abstract

Single-nucleotide polymorphism (SNP) is a substitution of a single nucleotide at a specific position in the genome. Until now, 585 million SNPs have been identified in the human genome, and therefore, a widely applicable method is desirable to detect a specific SNP. Herein we report a simple and reliable genotyping assay, which seems to be suitable for medium and small size laboratories, as well, to easily genotype most of the SNPs. In our study, all of the possible base variations (A-T, A-G, A-C, T-G, T-C, G-C) were tested to prove the general feasibility of our technique. The basis of the assay is a fluorescent PCR, in which both allele-specific primers, differing only at the 3′ end according to the sequence of the SNP, were present, and the length of one of them was modified with 3 bp by adding an adapter sequence to the 5’ end of that primer. The competitive presence of both allele-specific primers excludes the false amplification of the absent allele (which can happen in simple allele-specific PCR (AS-PCR)) and ensures the amplification of the proper allele(s). Unlike other complicated genotyping methods that use of manipulation of fluorescent dyes for genotyping, we apply an approach based on the length of amplicons from different alleles to differentiate between them. In our experiment (named variable fragment length allele-specific polymerase chain reaction (VFLASP)), the investigated six SNPs, containing the six available base variations, gave clear and reliable results after detecting the amplicons by capillary electrophoresis.