Modified Allele-Specific qPCR (ASQ) Genotyping

Aigul Amangeldiyeva, Akmaral Baidyussen, Marzhan Kuzbakova, Raushan Yerzhebayeva, Satyvaldy Jatayev, Yuri Shavrukov

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review


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.

Original languageEnglish
Title of host publicationPlant Genotyping
Subtitle of host publicationMethods and Protocols
EditorsYuri Shavrukov
Place of PublicationNew York, NY.
Number of pages17
ISBN (Electronic)978-1-0716-3024-2
ISBN (Print)978-1-0716-3023-5
Publication statusPublished - 2023

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


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


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