Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna, 1090 Vienna, Austria.
Department of Medicine I, Institute of Cancer Research, Medical University of Vienna, 1090 Vienna, Austria.
Int J Mol Sci. 2021 Nov 20;22(22):12527. doi: 10.3390/ijms222212527.
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 () 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.
由于其成本效益高，高分辨率熔解（HRM）分析在候选单核苷酸多态性（SNP）的基因分型中起着重要作用。研究表明，HRM 分析不仅适用于单个 SNP 的基因分型，还允许在同一个扩增子中对多个 SNP 进行基因分型，尽管其鉴别能力有限。通过针对位于 O6-甲基鸟嘌呤-DNA 甲基转移酶（MGMT）基因启动子内的三个 C>T SNP rs527559815、rs547832288 和 rs16906252 进行靶向分析，这些 SNP 之间的距离为 45bp，我们研究了通过将 HRM 分析与焦磷酸测序（PSQ）相结合，是否可以提高鉴别能力。在优化聚合酶链反应（PCR）条件后，直接对进行 HRM 分析的 PCR 产物进行 PSQ 分析。通过分析寡脱氧核苷酸对照物，代表二倍体人类细胞的 36 种理论上可能的变体组合（8 种三重纯合子、12 种双重纯合子、12 种双重杂合子和 4 种三重杂合子组合），与 HRM 分析可明确鉴定 22 种变体组合和 PSQ 可明确鉴定 16 种变体组合相比，通过 HRM 和 PSQ 数据的联合分析，可明确鉴定 34 种变体组合。我们的方法成功地应用于不同来源的稳定细胞系、来自胶质母细胞瘤患者的原代人肿瘤细胞系和乳腺组织样本的基因分型。
