High resolution melting analysis for gene scanning.
High resolution melting analysis for gene scanning.
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DOI：
10.1016/j.ymeth.2010.01.013
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发表时间：
2010-04
期刊：
影响因子：
4.8
通讯作者：
Wittwer, Carl T.
中科院分区：
文献类型：
--
作者：
Erali, Maria;Wittwer, Carl T.
高分辨率熔解是一种新的基因分型和变异扫描方法，可以无缝地附加到PCR扩增。通过扩增子解链进行基因分型的局限性可以通过未标记探针或快速恢复引物分析来解决，所有分析均在未标记探针的情况下进行。高分辨率解链也可用于扫描具有多个外显子的大基因中的罕见序列变异，这是本文的重点。通过在PCR之前简单地添加异源检测染料，在没有任何添加、处理或分离步骤的情况下进行高分辨率解链。通过与野生型纯合子相比不同形状的非典型熔解曲线来鉴定杂合变体。通过事先与野生型DNA混合来检测纯合或半合变体。介绍了高分辨率扫描测定的设计、优化和性能考虑因素，以实现基因扫描的快速周转。设计关注点包括引物选择和计算机模拟预测解链曲线。优化包括退火温度的温度梯度选择、常见变体的随机群体筛选以及用机器人沉积和干燥的引物对批量制备引物板。性能包括快速DNA制备、PCR和高分辨率熔解扫描，当不存在变体时，总共只需要3小时。当检测到变异时，可以通过快速循环测序和毛细管电泳在另外3小时内鉴定它们。对于方案中的每一步，提供了原则的一般概述，然后深入分析了一个例子，扫描CYBB，在X连锁慢性肉芽肿病中突变的基因。
High resolution melting is a new method of genotyping and variant scanning that can be seamlessly appended to PCR amplification. Limitations of genotyping by amplicon melting can be addressed by unlabeled probe or snapback primer analysis, all performed without labeled probes. High resolution melting can also be used to scan for rare sequence variants in large genes with multiple exons and is the focus of this article. With the simple addition of a heteroduplex-detecting dye before PCR, high resolution melting is performed without any additions, processing or separation steps. Heterozygous variants are identified by atypical melting curves of a different shape compared to wild type homozygotes. Homozygous or hemizygous variants are detected by prior mixing with wild type DNA. Design, optimization, and performance considerations for high resolution scanning assays are presented for rapid turnaround of gene scanning. Design concerns include primer selection and predicting melting profiles in silico. Optimization includes temperature gradient selection of the annealing temperature, random population screening for common variants, and batch preparation of primer plates with robotically deposited and dried primer pairs. Performance includes rapid DNA preparation, PCR, and scanning by high resolution melting that require, in total, only 3 hours when no variants are present. When variants are detected, they can be identified in an additional 3 hours by rapid cycle sequencing and capillary electrophoresis. For each step in the protocol, a general overview of principles is provided, followed by an in depth analysis of one example, scanning of CYBB, the gene that is mutated in X-linked chronic granulomatous disease.
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DOI：
10.1016/b978-0-12-374537-8.00015-8
发表时间：
2010-01-01
期刊：
MOLECULAR DIAGNOSTICS, 2ND EDITION
影响因子：
--
作者：
Farrar, Jared S.;Reed, Gudrun H.;Wittwer, Carl T.
通讯作者：
Wittwer, Carl T.
影响因子：
3.1
作者：
Pornprasert, Sakorn;Phusua, Arunee;Sanguansermsri, Torpong
通讯作者：
Sanguansermsri, Torpong
影响因子：
2.9
作者：
FIXMAN, M;FREIRE, JJ
通讯作者：
FREIRE, JJ
影响因子：
9.3
作者：
Herrmann, Mark G.;Durtschi, Jacob D.;Voelkerding, Karl V.
通讯作者：
Voelkerding, Karl V.
影响因子：
4.1
作者：
Lyon, Elaine;Wittwer, Carl T.
通讯作者：
Wittwer, Carl T.