Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis
Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis
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DOI：
10.1373/clinchem.2003.029751
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发表时间：
2004-10-01
影响因子：
9.3
通讯作者：
Wittwer, CT
中科院分区：
文献类型：
--
作者：
Reed, GH;Wittwer, CT
背景资料：PCR产物中杂合序列变化的筛查，又称“突变扫描”，是基因研究和临床应用的重要工具。传统的方法需要一个separationstep.Methods：我们评估了灵敏度和特异性的均相扫描，使用饱和的DNA染料和高分辨率的熔化。在GC含量为40%、50%和60%的三种不同序列背景下研究了杂合单核苷酸多态性（SNP）检测。在LCGreen（TM）I存在下产生50-1000 bp的PCR产物。结果：对于300 bp及以下的PCR产物，280例杂合子和296例野生型PCR产物均正确检出，无错误。在672例400 ~ 1000 bp之间的突变集中的病例中，敏感性和特异性分别为96.1%和99.4%。当使用具有最大错误率的序列背景和产物大小时，偏离中心SNPs（384例）的敏感性为95.6%，特异性为99.4%。大多数假阴性发生与SNPs相比，A或T野生型sequence.Conclusions：高分辨率熔解分析与染料LCGreen我确定杂合单碱基的变化，PCR产物的灵敏度和特异性可比或上级非同质技术。扫描的错误率取决于PCR产物的大小和碱基变化的类型，但与SNP的位置无关。该技术仅需要PCR试剂、染料LCGreen I和1-2分钟的闭管PCR后分析。(C)2004年美国临床化学协会。
Background: Screening for heterozygous sequence changes in PCR products, also known as "mutation scanning", is an important tool for genetic research and clinical applications. Conventional methods require a separation step.Methods: We evaluated the sensitivity and specificity of homogeneous scanning, using a saturating DNA dye and high-resolution melting. Heterozygous single-nucleotide polymorphism (SNP) detection was studied in three different sequence backgrounds of 40%, 50%, and 60% GC content. PCR products of 50-1000 bp were generated in the presence of LCGreen(TM) I. After fluorescence normalization and temperature overlay, melting curve shape was used to judge the presence or absence of heterozygotes among 1632 cases.Results: For PCR products of 300 bp or less, all 280 heterozygous and 296 wild-type cases were correctly called without error. In 672 cases between 400 and 1000 bp with the mutation centered, the sensitivity and specificity were 96.1% and 99.4%, respectively. When the sequence background and product size with the greatest error rate were used, the sensitivity of off-center SNPs (384 cases) was 95.6% with a specificity of 99.4%. Most false negatives occurred with SNPs that were compared with an A or T wild type sequence.Conclusions: High-resolution melting analysis with the dye LCGreen I identifies heterozygous single-base changes in PCR products with a sensitivity and specificity comparable or superior to nonhomogeneous techniques. The error rate of scanning depends on the PCR product size and the type of base change, but not on the position of the SNP. The technique requires only PCR reagents, the dye LCGreen I, and 1-2 min of closed-tube, post-PCR analysis. (C) 2004 American Association for Clinical Chemistry.