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

Autor: Katja Zappe, Christine Pirker, Heidi Miedl, Martin Schreiber, Petra Heffeter, Georg Pfeiler, Stefan Hacker, Werner Haslik, Sabine Spiegl-Kreinecker, Margit Cichna-Markl
Jazyk: angličtina
Rok vydání: 2021
Předmět:
Zdroj: International Journal of Molecular Sciences, Vol 22, Iss 22, p 12527 (2021)
Druh dokumentu: article
ISSN: 1422-0067
1661-6596
DOI: 10.3390/ijms222212527
Popis: 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 (MGMT) 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.
Databáze: Directory of Open Access Journals
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