Description and Validation of High-Throughput Simultaneous Genotyping and Mutation Scanning by High-Resolution Melting Curve Analysis
Description and Validation of High-Throughput Simultaneous Genotyping and Mutation Scanning by High-Resolution Melting Curve Analysis
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DOI：
10.1002/humu.20949
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发表时间：
2009-06-01
期刊：
影响因子：
3.9
通讯作者：
Tavtigian, Sean V.
中科院分区：
文献类型：
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作者：
Nguyen-Dumont, Tu;Le Calvez-Kelm, Florence;Tavtigian, Sean V.
使用高分辨率熔解曲线分析 (HR-melt) 进行突变扫描是检测序列变异的有效且灵敏的方法。然而，突变扫描扩增子中常见 SNP 的存在可能会使结果的解释变得相当复杂，并通过干扰根据熔解曲线的样品聚类来增加标记用于测序的样品数量。已经报道了描述同时高分辨率基因扫描和基因分型的方案。在这里，我们证明它可以提高大规模病例对照突变筛查的灵敏度和效率。 ATM 的两个外显子均含有干扰标准突变扫描的 SNP，被选中用于对国际乳腺癌遗传学研究中的 1,356 名受试者进行筛查。不对称 PCR 在 SNP 特异性未标记探针存在的情况下进行。定制的 HR-melt 软件帮助根据探针目标熔化对样品进行分层。这种方法改进了对罕见的已知和未知变异的识别，同时大大减少了测序工作。它甚至允许使用单个探针对串联 SNP 进行基因分型。因此，HR-melt 是一种快速、高效且经济高效的工具，可用于研究的高通量突变筛选以及分子诊断和临床目的。 Hum Mutat 30, 884-890, 2009。(C) 2009 Wiley-Liss, Inc.
Mutation scanning using high-resolution melting curve analysis (HR-melt) is an effective and sensitive method to detect sequence variations. However, the presence of a common SNP within a mutation scanning amplicon may considerably complicate the interpretation of results and increase the number of samples flagged for sequencing by interfering with the clustering of samples according to melting profiles. A protocol describing simultaneous high-resolution gene scanning and genotyping has been reported. Here, we show that it can improve the sensitivity and the efficiency of large-scale case-control mutation screening. Two exons of ATM, both containing an SNP interfering with standard mutation scanning, were selected for screening of 1,356 subjects from an international breast cancer genetics study. Asymmetric PCR was performed in the presence of an SNP-specific unlabeled probe. Stratification of the samples according to their probe-target melting was aided by customized HR-melt software. This approach improved identification of rare known and unknown variants, while dramatically reducing the sequencing effort. It even allowed genotyping of tandem SNPs using a single probe. Hence, HR-melt is a rapid, efficient, and cost-effective tool that can be used for high-throughput mutation screening for research, as well as for molecular diagnostic and clinical purposes. Hum Mutat 30, 884-890, 2009. (C) 2009 Wiley-Liss, Inc.