Designing allele-specific competitive-extension PCR-based assays for high-throughput genotyping

Description

PCR is simple, rapid, and is the method of choice for analysis of nucleotide polymorphisms and detection of gene variations for basic research, agriculture, and medicine. Variants of PCR, collectively known as allele-specific PCR (AS-PCR), use a competitive reaction in the presence of allele-specific primers to preferentially amplify only certain alleles. This method, originally named by its developers as Kompetitive Allele Specific PCR (KASP), is an AS-PCR variant adapted for fluorescence-based detection of amplification results. We created a bioinformatics tool and propose a new, more versatile allele-specific quantitative PCR (ASQ) method. The tool described in this work facilitates determining primers for KASP and ASQ that target single-nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms (InDels). The described tool allows for creation of assays for SNP typing with a high discriminatory power. For this goal, the program calculates primers such that in an allele-specific primer, the penultimate base before the primer’s 3' end base is positioned on the SNP site; this allows using proofreading polymerases in PCR, which increases specificity of KASP and ASQ. FastPCR is a user-friendly and powerful Java-based software that is freely available at http://primerdigital.com/tools/. Using the FastPCR environment and the tool for designing KASP and ASQ provides an unparalleled flexibility for developing genotyping assays and specific and sensitive diagnostic PCR-based tests, which translates into a greater likelihood of success in research.

Period	Oct 20 2022 → Oct 21 2022
Event title	MODERN PERSPECTIVES FOR BIOMEDICAL SCIENCES
Event type	Conference
Location	Astana, KazakhstanShow on map
Degree of Recognition	International