Modified Allele-Specific qPCR (ASQ) Genotyping

Amangeldiyeva A. Baidyussen A. Kuzbakova M. Yerzhebayeva R. Jatayev S. Shavrukov Y.
2023 NLM (Medline)

Methods in molecular biology (Clifton, N.J.)
2023 #2638 231 - 247 pp.

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.

Allele-specific primers , Fluorescent dyes and quenches , Genotyping , Single nucleotide polymorphism (SNP) , Universal molecular probes

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Kazakh Research Institute of Agriculture and Plant Growing, Almaty, Kazakhstan
S.Seifullin Kazakh AgroTechnical University, Astana, Kazakhstan
College of Science and Engineering, Biological Sciences, Flinders University, Adelaide, SA, Australia

Kazakh Research Institute of Agriculture and Plant Growing
S.Seifullin Kazakh AgroTechnical University
College of Science and Engineering

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