Discrimination between 34 of 36 Possible Combinations of Three C>T SNP Genotypes in the <i>MGMT</i> Promoter by High Resolution Melting Analysis Coupled with Pyrosequencing Using A Single Primer Set

Due to its cost-efficiency, high resolution melting (HRM) analysis plays an important role in genotyping of candidate single nucleotide polymorphisms (SNPs). Studies indicate that HRM analysis is not only suitable for genotyping individual SNPs, but also allows genotyping of multiple SNPs in one and...

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Autores principales:	Katja Zappe, Christine Pirker, Heidi Miedl, Martin Schreiber, Petra Heffeter, Georg Pfeiler, Stefan Hacker, Werner Haslik, Sabine Spiegl-Kreinecker, Margit Cichna-Markl
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	genotyping multiple SNPs <i>MGMT</i> high resolution melting pyrosequencing Biology (General) QH301-705.5 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/99f9b8069d9140af89247b2e53ecf2fe
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spelling	oai:doaj.org-article:99f9b8069d9140af89247b2e53ecf2fe2021-11-25T17:57:30ZDiscrimination between 34 of 36 Possible Combinations of Three C>T SNP Genotypes in the <i>MGMT</i> Promoter by High Resolution Melting Analysis Coupled with Pyrosequencing Using A Single Primer Set10.3390/ijms2222125271422-00671661-6596https://doaj.org/article/99f9b8069d9140af89247b2e53ecf2fe2021-11-01T00:00:00Zhttps://www.mdpi.com/1422-0067/22/22/12527https://doaj.org/toc/1661-6596https://doaj.org/toc/1422-0067Due to its cost-efficiency, high resolution melting (HRM) analysis plays an important role in genotyping of candidate single nucleotide polymorphisms (SNPs). Studies indicate that HRM analysis is not only suitable for genotyping individual SNPs, but also allows genotyping of multiple SNPs in one and the same amplicon, although with limited discrimination power. By targeting the three C>T SNPs rs527559815, rs547832288, and rs16906252, located in the promoter of the O6-methylguanine-DNA methyltransferase (<i>MGMT</i>) gene within a distance of 45 bp, we investigated whether the discrimination power can be increased by coupling HRM analysis with pyrosequencing (PSQ). After optimizing polymerase chain reaction (PCR) conditions, PCR products subjected to HRM analysis could directly be used for PSQ. By analyzing oligodeoxynucleotide controls, representing the 36 theoretically possible variant combinations for diploid human cells (8 triple-homozygous, 12 double-homozygous, 12 double-heterozygous and 4 triple-heterozygous combinations), 34 out of the 36 variant combinations could be genotyped unambiguously by combined analysis of HRM and PSQ data, compared to 22 variant combinations by HRM analysis and 16 variant combinations by PSQ. Our approach was successfully applied to genotype stable cell lines of different origin, primary human tumor cell lines from glioma patients, and breast tissue samples.Katja ZappeChristine PirkerHeidi MiedlMartin SchreiberPetra HeffeterGeorg PfeilerStefan HackerWerner HaslikSabine Spiegl-KreineckerMargit Cichna-MarklMDPI AGarticlegenotypingmultiple SNPs<i>MGMT</i>high resolution meltingpyrosequencingBiology (General)QH301-705.5ChemistryQD1-999ENInternational Journal of Molecular Sciences, Vol 22, Iss 12527, p 12527 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	genotyping multiple SNPs <i>MGMT</i> high resolution melting pyrosequencing Biology (General) QH301-705.5 Chemistry QD1-999
spellingShingle	genotyping multiple SNPs <i>MGMT</i> high resolution melting pyrosequencing Biology (General) QH301-705.5 Chemistry QD1-999 Katja Zappe Christine Pirker Heidi Miedl Martin Schreiber Petra Heffeter Georg Pfeiler Stefan Hacker Werner Haslik Sabine Spiegl-Kreinecker Margit Cichna-Markl Discrimination between 34 of 36 Possible Combinations of Three C>T SNP Genotypes in the <i>MGMT</i> Promoter by High Resolution Melting Analysis Coupled with Pyrosequencing Using A Single Primer Set
description	Due to its cost-efficiency, high resolution melting (HRM) analysis plays an important role in genotyping of candidate single nucleotide polymorphisms (SNPs). Studies indicate that HRM analysis is not only suitable for genotyping individual SNPs, but also allows genotyping of multiple SNPs in one and the same amplicon, although with limited discrimination power. By targeting the three C>T SNPs rs527559815, rs547832288, and rs16906252, located in the promoter of the O6-methylguanine-DNA methyltransferase (<i>MGMT</i>) gene within a distance of 45 bp, we investigated whether the discrimination power can be increased by coupling HRM analysis with pyrosequencing (PSQ). After optimizing polymerase chain reaction (PCR) conditions, PCR products subjected to HRM analysis could directly be used for PSQ. By analyzing oligodeoxynucleotide controls, representing the 36 theoretically possible variant combinations for diploid human cells (8 triple-homozygous, 12 double-homozygous, 12 double-heterozygous and 4 triple-heterozygous combinations), 34 out of the 36 variant combinations could be genotyped unambiguously by combined analysis of HRM and PSQ data, compared to 22 variant combinations by HRM analysis and 16 variant combinations by PSQ. Our approach was successfully applied to genotype stable cell lines of different origin, primary human tumor cell lines from glioma patients, and breast tissue samples.
format	article
author	Katja Zappe Christine Pirker Heidi Miedl Martin Schreiber Petra Heffeter Georg Pfeiler Stefan Hacker Werner Haslik Sabine Spiegl-Kreinecker Margit Cichna-Markl
author_facet	Katja Zappe Christine Pirker Heidi Miedl Martin Schreiber Petra Heffeter Georg Pfeiler Stefan Hacker Werner Haslik Sabine Spiegl-Kreinecker Margit Cichna-Markl
author_sort	Katja Zappe
title	Discrimination between 34 of 36 Possible Combinations of Three C>T SNP Genotypes in the <i>MGMT</i> Promoter by High Resolution Melting Analysis Coupled with Pyrosequencing Using A Single Primer Set
title_short	Discrimination between 34 of 36 Possible Combinations of Three C>T SNP Genotypes in the <i>MGMT</i> Promoter by High Resolution Melting Analysis Coupled with Pyrosequencing Using A Single Primer Set
title_full	Discrimination between 34 of 36 Possible Combinations of Three C>T SNP Genotypes in the <i>MGMT</i> Promoter by High Resolution Melting Analysis Coupled with Pyrosequencing Using A Single Primer Set
title_fullStr	Discrimination between 34 of 36 Possible Combinations of Three C>T SNP Genotypes in the <i>MGMT</i> Promoter by High Resolution Melting Analysis Coupled with Pyrosequencing Using A Single Primer Set
title_full_unstemmed	Discrimination between 34 of 36 Possible Combinations of Three C>T SNP Genotypes in the <i>MGMT</i> Promoter by High Resolution Melting Analysis Coupled with Pyrosequencing Using A Single Primer Set
title_sort	discrimination between 34 of 36 possible combinations of three c>t snp genotypes in the <i>mgmt</i> promoter by high resolution melting analysis coupled with pyrosequencing using a single primer set
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/99f9b8069d9140af89247b2e53ecf2fe
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Discrimination between 34 of 36 Possible Combinations of Three C>T SNP Genotypes in the <i>MGMT</i> Promoter by High Resolution Melting Analysis Coupled with Pyrosequencing Using A Single Primer Set

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