Modified Allele-Specific qPCR (ASQ) Genotyping

Abstract

The allele-specific qPCR (ASQ) method for SNP (single nucleotide polymorphism) detection is based on the FRET (fluorescence resonance energy transfer) system, a system using position-dependent fluorescent dyes and quenches. The modified ASQ method requires two separate components: (1) the allele-specific part, two AS primers targeting the SNP with identity in the penultimate positions at the 3'-end and specific tags in the 5'-end, and (2) the universal part, two universal probes (UPs) with corresponding tags and different fluorescent dyes in the 5'-end and a single common universal probe with a quencher in the 3'-ends (Uni-Q), complementary to all UP tags. There are two major variations of the ASQ method, with either short 4-bp tags (variant A) or longer 6-bp tags (variant B), both of which have been successfully used for SNP genotyping in plants. The modified ASQ method is much cheaper compared to other similar FRET-based methods because the most expensive parts, the universal probes, have a short and linear structure, where fluorophores and quenchers are located in the ends but not incorporated inside of the sequences.