Rapid detection of deletion, insertion, and substitution mutations via heteroduplex analysis using capillary- and microchip-based electrophoresis
Rapid detection of deletion, insertion, and substitution mutations via heteroduplex analysis using capillary- and microchip-based electrophoresis
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DOI：
10.1101/gr.132700
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发表时间：
2000-09-01
期刊：
影响因子：
7
通讯作者：
Landers, JP
中科院分区：
文献类型：
--
作者：
Tian, HJ;Brody, LC;Landers, JP
在本报告中，我们探讨了毛细管和微芯片电泳在基于异源双链分析（HDA）的突变检测中的潜力。荧光染料标记的引物（6-FAM 标记）用于扩增 130 至 400 bp 的 DNA 片段。使用 BRCA1 和 BRCA1 中的六种杂合突变：185delAG、E1250X(3867GT)、R1443G (4446CG)、5382insC、5677insA，评估 DNA 片段长度、基质添加剂、pH 和盐对基于毛细管电泳 (CE) 和/或微芯片电泳的 HDA 的影响。 BRCA2 中的 6174delT。对于该系统，基于CE的HDA的有效片段大小在200-300 bp范围内，然而，对于基于微芯片的HDA，有效片段大小为150-260 bp。敏感性研究表明，基于 CE 的 HDA 只能检测到仅占总 DNA 1%-10% 的突变 DNA。使用基于 CE 的 HDA 可以区分 BRCA1 和 BRCA2 中的野生型和缺失或插入突变
In this report, we explore the potential of capillary and microchip electrophoresis for heteroduplex analysis-(HDA) based mutation detection. Fluorescent dye-labeled primers (6-FAM-tagged) were used to amplify the DNA fragments ranging from 130 to 400 bp. The effects of DNA fragment length, matrix additives, pH, and salt were evaluated for capillary electrophoresis- (CE) and/or microchip electrophoresis-based HDA, using six heterozygous mutations, 185delAG, E1250X(3867GT), R1443G (4446CG), 5382insC, 5677insA in BRCA1 and 6174delT in BRCA2. For this system, the effective Fragment size for CE-based HDA was found in the range of 200-300 bp, however, the effective range was 150-260 bp for microchip-based HDA. Sensitivity studies show CE-based HDA could detect a mutated DNA present at only 1%-10% of the total DNA. Discrimination between wild-type and deletion or insertion mutations in BRCA1 and BRCA2 with CE-based HDA could be achieved in