Application of High-Resolution Melting to Large-Scale, High-Throughput SNP Genotyping: A Comparison with the TaqMan® Method
Application of High-Resolution Melting to Large-Scale, High-Throughput SNP Genotyping: A Comparison with the TaqMan® Method
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DOI：
10.1177/1087057110365900
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发表时间：
2010-07-01
影响因子：
--
通讯作者：
Rossi, Anna Maria
中科院分区：
文献类型：
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作者：
Martino, Alessandro;Mancuso, Tommaso;Rossi, Anna Maria
由于单核苷酸多态性（SNPs）作为遗传变异标记的广泛应用，在过去的几十年中，已经开发并应用了几种高通量基因分型方法。高分辨率熔解（High-resolution melting，HRM）是一种非常有吸引力的、先进的、快速的、成本效益高的SNP基因分型技术，其基于对PCR产物熔解曲线的分析，使用嵌入荧光染料来监测从未熔解的DNA到熔解的DNA的转变。作者使用HRM对215份人类DNA样本进行ABCB 1、NQO 1和SLC 19 A1基因中SNP的基因分型，并对96份样本进行IL 1A和IL 12 B基因中SNP的基因分型，目的是评估HRM与TaqMan（R）检测相比的灵敏度和准确性，以便进行大规模、高通量SNP分型应用。使用对于这些因素来说是异质的扩增子探索了PCR产物大小、T（M）、GC含量和SNP位置对HRM性能的潜在影响。区分能力范围为91.4%至98.4%，只有当稀有纯合子的数量下降到1个或几个单位时才显着降低。除了HRM实验条件的更好标准化之外，特异性和经验证的测定的可用性可以大大减少大规模基因分型研究的时间和成本，失败或错误分类的风险可以忽略不计。（Journal of Biomolecular Screening 2010：623-629）
Because of the wide use of single-nucleotide polymorphisms (SNPs) as markers of genetic variation, several high-throughput genotyping methods have been developed and applied during the past decades. High-resolution melting (HRM) is a very attractive, advanced, fast, and cost-effective SNP genotyping technology based on the analysis of the melting profile of PCR products, using intercalating fluorescent dyes to monitor the transition from unmelted to melted DNA. The authors used HRM for genotyping 215 human DNA samples for SNPs in the ABCB1, NQO1, and SLC19A1 genes and 96 samples for SNPs in the IL1A and IL12B genes with the aim of assessing HRM sensitivity and accuracy in comparisons with the TaqMan (R) assay in view of large-scale, high-throughput SNP-typing applications. The potential effect of PCR product size, T(M), GC content, and SNP position on HRM performances was explored with amplicons that were heterogeneous for these factors. Discrimination power ranged from 91.4% to 98.4%, being significantly lower only when the number of rare homozygotes dropped to 1 or few units. The availability of specific and validated assays, in addition to a better standardization of HRM experimental conditions, can considerably reduce time and costs of large-scale genotyping studies with a negligible risk of failure or misclassification. (Journal of Biomolecular Screening 2010: 623-629)