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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 and Kompetit...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8784781/ https://www.ncbi.nlm.nih.gov/pubmed/35082803 http://dx.doi.org/10.3389/fpls.2021.747886 |
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author | Kalendar, Ruslan Baidyussen, Akmaral Serikbay, Dauren Zotova, Lyudmila Khassanova, Gulmira Kuzbakova, Marzhan Jatayev, Satyvaldy Hu, Yin-Gang Schramm, Carly Anderson, Peter A. Jenkins, Colin L. D. Soole, Kathleen L. Shavrukov, Yuri |
author_facet | Kalendar, Ruslan Baidyussen, Akmaral Serikbay, Dauren Zotova, Lyudmila Khassanova, Gulmira Kuzbakova, Marzhan Jatayev, Satyvaldy Hu, Yin-Gang Schramm, Carly Anderson, Peter A. Jenkins, Colin L. D. Soole, Kathleen L. Shavrukov, Yuri |
author_sort | Kalendar, Ruslan |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-8784781 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-87847812022-01-25 Modified “Allele-Specific qPCR” Method for SNP Genotyping Based on FRET Kalendar, Ruslan Baidyussen, Akmaral Serikbay, Dauren Zotova, Lyudmila Khassanova, Gulmira Kuzbakova, Marzhan Jatayev, Satyvaldy Hu, Yin-Gang Schramm, Carly Anderson, Peter A. Jenkins, Colin L. D. Soole, Kathleen L. Shavrukov, Yuri Front Plant Sci Plant Science 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. Frontiers Media S.A. 2022-01-10 /pmc/articles/PMC8784781/ /pubmed/35082803 http://dx.doi.org/10.3389/fpls.2021.747886 Text en Copyright © 2022 Kalendar, Baidyussen, Serikbay, Zotova, Khassanova, Kuzbakova, Jatayev, Hu, Schramm, Anderson, Jenkins, Soole and Shavrukov. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Plant Science Kalendar, Ruslan Baidyussen, Akmaral Serikbay, Dauren Zotova, Lyudmila Khassanova, Gulmira Kuzbakova, Marzhan Jatayev, Satyvaldy Hu, Yin-Gang Schramm, Carly Anderson, Peter A. Jenkins, Colin L. D. Soole, Kathleen L. Shavrukov, Yuri Modified “Allele-Specific qPCR” Method for SNP Genotyping Based on FRET |
title | Modified “Allele-Specific qPCR” Method for SNP Genotyping Based on FRET |
title_full | Modified “Allele-Specific qPCR” Method for SNP Genotyping Based on FRET |
title_fullStr | Modified “Allele-Specific qPCR” Method for SNP Genotyping Based on FRET |
title_full_unstemmed | Modified “Allele-Specific qPCR” Method for SNP Genotyping Based on FRET |
title_short | Modified “Allele-Specific qPCR” Method for SNP Genotyping Based on FRET |
title_sort | modified “allele-specific qpcr” method for snp genotyping based on fret |
topic | Plant Science |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8784781/ https://www.ncbi.nlm.nih.gov/pubmed/35082803 http://dx.doi.org/10.3389/fpls.2021.747886 |
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