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.
Highlights
The method we propose here is suitable for uniplex application and includes elements previously used in various approaches for single nucleotide polymorphism (SNP) genotyping
The aim of this report was to demonstrate the functionality of the proposed Allele-specific q-PCR” (ASQ) method, the development of all components and conditions, and to show examples of their application for SNP genotyping in barley with comparisons to Amplifluor and Kompetitive allele specific PCR (KASP) methods
Melting temperature was calculated for oligonucleotides at a concentration of 100-500 nM, depending on the specific oligonucleotide, and for standard PCR reaction buffer with 50 mM K+ and 3 mM Mg2+, or without magnesium for allele-specific primers (ASPs) sequences calculated without a tag
Summary
Many SNP genotyping platforms exist (reviewed in Kim and Misra, 2007; Schramm et al, 2019). Several use fluorescence resonance energy transfer (Didenko, 2001; Kaur et al, 2020a) as the basis for SNP visualization (Table 1). Like Scorpions (Thelwell et al, 2000; Solinas et al, 2001) TaqMan (Schena et al, 2006; Jawhari et al, 2015) and Molecular Beacons (Hardinge and Murray, 2019), these two parts are combined in a single molecular probe
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