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

Abstract

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.

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“…Unfortunately, this version of the primers was not as robust and would sometimes fail to amplify the target (Figure 2). This is not altogether surprising, since mismatches close to the 3′ end of a primer can drastically lower PCR efficiency and are the basis of allele-specific PCR diagnostic assays [14,15].…”

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“…These advancements have increased efficiency and reduced costs ( Elbaidouri et al., 2013 ; Mir and Varshney, 2012 ). With such NGS-based high-throughput technological developments, low-throughput molecular markers, such as Kompetitive allele-specific PCR (KASP) ( Makhoul et al., 2020 ), nevertheless remain indispensable for tracking specific genomic regions in molecular breeding programs when analyzing large numbers of samples ( Kalendar et al., 2022a ; Kalendar et al., 2022c ). Therefore, single nucleotide polymorphism (SNP) markers continue to be the most preferred marker systems for development of high-throughput genotypic platforms for genome-wide marker scanning.…”

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“…The MAF detection limits of quantitative PCR ranges between 10 and 20%. However, variations in PCR techniques, like allele-specific amplification [ 54 ], allele-specific nonextendable primer blocker PCR [ 55 ], and peptide nucleic acid-locked nucleic acid PCR clamp [ 56 ], increase the sensitivity. Several genome-wide sequencing methods have been developed in the last decade.…”