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. 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. 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. The SNP site is positioned preferably at a penultimate base in each allele-specific primer, which increases the reaction specificity and allele discrimination. 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). In the proposed ASQ method, the fluorophores and quencher are located in separate complementary oligonucleotides. 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. 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). 5College of Science and Engineering, Biological Sciences, Flinders University, Adelaide, SA, Australia.4State Key Laboratory of Crop Stress Biology for Arid Areas, College of Agronomy, Northwest A&F University, Yangling, China.Seifullin Kazakh AgroTechnical University, Nur-Sultan, Kazakhstan 2Institute of Biotechnology HiLIFE, University of Helsinki, Helsinki, Finland.1National Laboratory Astana, Nazarbayev University, Nur-Sultan, Kazakhstan.Ruslan Kalendar 1,2 * †, Akmaral Baidyussen 3 †, Dauren Serikbay 3,4, Lyudmila Zotova 3, Gulmira Khassanova 3, Marzhan Kuzbakova 3, Satyvaldy Jatayev 3, Yin-Gang Hu 4, Carly Schramm 5, Peter A.
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