Modified “Allele-Specific qPCR” Method for SNP Genotyping Based on FRET

Abstract

The proposed method is a modified and improved version of the existing “Allele-specific q-PCR” (ASQ) method for genotyping of single nucleotide polymorphism (SNP) based on fluorescence resonance energy transfer (FRET). This method is similar to frequently used techniques like Amplifluor and Kompetitive allele specific PCR (KASP), as well as others employing common universal probes (UPs) for SNP analyses. In the proposed ASQ method, the fluorophores and quencher are located in separate complementary oligonucleotides. The ASQ method is based on the simultaneous presence in PCR of the following two components: an allele-specific mixture (allele-specific and common primers) and a template-independent detector mixture that contains two or more (up to four) universal probes (UP-1 to 4) and a single universal quencher oligonucleotide (Uni-Q). The SNP site is positioned preferably at a penultimate base in each allele-specific primer, which increases the reaction specificity and allele discrimination. The proposed ASQ method is advanced in providing a very clear and effective measurement of the fluorescence emitted, with very low signal background-noise, and simple procedures convenient for customized modifications and adjustments. Importantly, this ASQ method is estimated as two- to ten-fold cheaper than Amplifluor and KASP, and much cheaper than all those methods that rely on dual-labeled probes without universal components, like TaqMan and Molecular Beacons. Results for SNP genotyping in the barley genes HvSAP16 and HvSAP8, in which stress-associated proteins are controlled, are presented as proven and validated examples. This method is suitable for bi-allelic uniplex reactions but it can potentially be used for 3- or 4-allelic variants or different SNPs in a multiplex format in a range of applications including medical, forensic, or others involving SNP genotyping.

Zinc finger knuckle genes are associated with tolerance to drought and dehydration in chickpea (Cicer arietinum L.)

“…The allele discrimination and genotyping results are clear using this ASQ method with medium throughput working effectively in 96-or 384-well microplates and qPCR instruments. Successful results for the ASQ method were recorded earlier in the genotyping of barley (Kalendar et al, 2022), sugar beet, and tomato (Amangeldiyeva et al, 2023).…”

“…In general, it is much faster to work with medium throughput methods, for example, based on fluorescence (Förster) resonance energy transfer (FRET). Recently, allele-specific qPCR (ASQ) methods for plant genotyping have been developed that are also suitable for chickpea (Kalendar et al, 2022;Amangeldiyeva et al, 2023). Microarray technology employing thousands of simultaneous reactions on a microchip represents the next generation of plant genotyping (Varshney et al, 2012), and diversity array technology (DArT) is also very powerful and popular for the identification of SNP and haplotypes (a group of closely located SNPs) in genetic regions of entire genomes (James et al, 2008;Deres and Feyissa, 2022), including chickpea (Roorkiwal et al, 2014;Thudi et al, 2014).…”

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

“…These results for SNP for genotyping of humans validated our conclusion that the proposed ASQ method is very accurate and effective for SNP genotyping; we expect similar results in other animal species. In addition, we developed ASQ sets and applied SNP genotyping in the barley genes HvSAP16 and HvSAP8 (controlling stress-associated proteins); these are validated examples and have been accepted for publication ( Baidyussen et al, 2021 ; Kalendar et al, 2022 ). Our tool and the ASQ method are suitable for two-, three-, or four-allelic uniplex reactions but can potentially be used for different SNPs in a multiplex format in a range of applications, including medical or forensic studies or other studies involving SNP genotyping.…”

The power of retrotransposons in high-throughput genotyping and sequencing

“…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.…”