Designing Allele-Specific Competitive-Extension PCR-Based Assays for High-Throughput Genotyping and Gene Characterization

Kalendar R. Shustov A.V. Akhmetollayev I. Kairov U.
1 March 2022 Frontiers Media S.A.

Frontiers in Molecular Biosciences
2022 #9

Polymerase chain reaction (PCR) is a simple and rapid method that can detect nucleotide polymorphisms and sequence variation in basic research applications, 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 developed a bioinformatic tool for designing probe sequences for PCR-based genotyping assays. Probe sequences are designed in both directions, and both single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels) may be targeted. In addition, the tool allows discrimination of up to four-allelic variants at a single SNP site. To increase both the reaction specificity and the discriminative power of SNP genotyping, each allele-specific primer is designed such that the penultimate base before the primer’s 3′ end base is positioned at the SNP site. The tool allows design of custom FRET cassette reporter systems for fluorescence-based assays. FastPCR is a user-friendly and powerful Java-based software that is freely available (http://primerdigital.com/tools/). Using the FastPCR environment and the tool for designing AS-PCR provides unparalleled flexibility for developing genotyping assays and specific and sensitive diagnostic PCR-based tests, which translates into a greater likelihood of research success. Copyright

diagnostic system , genotyping assay design software , genotyping system , insertion-deletion polymorphism , polymerase chain reaction-based markers , single nucleotide polymorphism

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Institute of Biotechnology HiLIFE, University of Helsinki, Helsinki, Finland
PrimerDigital Ltd, Helsinki, Finland
National Laboratory Astana, Nazarbayev University, Nur-Sultan, Kazakhstan
National Center for Biotechnology, Nur-Sultan, Kazakhstan

Institute of Biotechnology HiLIFE
PrimerDigital Ltd
National Laboratory Astana
National Center for Biotechnology

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