![]() Kompetitive Allele Specific PCR (KASP) (LGC Biosearch Technologies, Teddington, UK) and PCR Allele Competitive Extension (PACE) (Integrated DNA Technologies, Inc.) genotyping represent developments to the original AS-PCR approach. Further improvements for AS-PCR include using fluorescence to detect the amplification of a specific allele. Single-plex PCRs for SNP analysis, such as TaqMan (Life Technologies, USA) and SimpleProbe (Roche Applied Science, USA) have been described as valuable additions for marker-assisted selection in plant breeding ( Makhoul et al., 2020 Della Coletta et al., 2021). Another method is allele-specific PCR with SNP targeting ( Didenko, 2001 Kaur et al., 2020). FRET (Fluorescence Resonance Energy Transfer) is one such method and is based on dual fluorescence that is quantified during ligation. There currently exists an enormous number and variety of established methods applicable for SNP analysis and genotyping based on distinctly different platforms and approaches ( Kim and Misra, 2007). In principle, AS-PCR assays can be developed to analyse almost any allelic variation ( Sommer et al., 1989 Ye et al., 1992). All these methods use specifically designed PCR-primer sets containing allele-specific primers (ASP) in which the allele specificity is determined by the base at or near the 3′ end. PCR can be adapted for rapid detection of single-base changes in genomic DNA by using a family of closely related methods, such as allele-specific PCR (AS-PCR) ( Ugozzoli and Wallace, 1991 Bottema and Sommer, 1993 Spierings et al., 2006), PCR-amplification of specific alleles (PASA) ( Sommer et al., 1989 Sarkar et al., 1990), allele-specific amplification (ASA), and amplification refractory mutation system (ARMS) ( Newton et al., 1989 Nichols et al., 1989 Wu et al., 1989). A variety of SNP genotyping methods are available, and new methods appear regularly with the aim of reducing cost and increasing throughput. In this regard, SNPs are very effective markers. The primary consideration for selecting a marker type for genotyping is the information content of the polymorphisms and the informative capacity of the test. The most frequently encountered sequence variations in any genome are single-nucleotide polymorphisms (SNP) ( Clevenger et al., 2015). High-throughput technologies for nucleotide sequence analysis and detection of sequence variation have been increasingly used for plant and animal genotyping, forensics, genetic medicine, and other fields of genetic testing. 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. FastPCR is a user-friendly and powerful Java-based software that is freely available ( ). The tool allows design of custom FRET cassette reporter systems for fluorescence-based assays. 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. In addition, the tool allows discrimination of up to four-allelic variants at a single SNP site. Probe sequences are designed in both directions, and both single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels) may be targeted. We developed a bioinformatic tool for designing probe sequences for PCR-based genotyping assays. 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. 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. 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. 4National Center for Biotechnology, Nur-Sultan, Kazakhstan.3National Laboratory Astana, Nazarbayev University, Nur-Sultan, Kazakhstan.1Institute of Biotechnology HiLIFE, University of Helsinki, Helsinki, Finland.Shustov 4 † Ilyas Akhmetollayev 4 † Ulykbek Kairov 3 † ![]()
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