Modified "Allele-Specific qPCR" Method for SNP Genotyping Based on FRET
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 an...
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Published in | Frontiers in plant science Vol. 12; p. 747886 |
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Main Authors | , , , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
Switzerland
Frontiers Media S.A
10.01.2022
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Subjects | |
Online Access | Get full text |
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Summary: | 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
and
, 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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Reviewed by: Alexandr Muterko, Institute of Cytology and Genetics, Russian Academy of Sciences (RAS), Russia; Mark Owen Winfield, University of Bristol, United Kingdom Edited by: Roger Deal, Emory University, United States These authors have contributed equally to this work and share first authorship This article was submitted to Technical Advances in Plant Science, a section of the journal Frontiers in Plant Science |
ISSN: | 1664-462X 1664-462X |
DOI: | 10.3389/fpls.2021.747886 |