Tian H, Brody L C, Landers J P
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Genome Res. 2000 Sep;10(9):1403-13. doi: 10.1101/gr.132700.
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 <8 min, while the substitution mutations required 14 min of analysis time. For each mutation region, 15 samples were run to confirm the accuracy and reproducibility of the method. Using the method described, two previously reported mutations, E1038G (3232AG, missense) and 4427 C/T (4427CT, polymorphism), were detected in the tested samples and confirmed by DNA sequencing. Translation of the CE-based methodology to the microchip format allowed the analysis time for each mutation to be decreased to 130 sec. Based on the results obtained with this model system, it is possible that CE-based HDA methodologies can be developed and used effectively in genetic testing. The fast separation time and automated operation afforded with CE instrumentation provide a powerful system for screening mutations that include small deletions, insertions, and point mutations. Translation to the microchip platform, especially to a multichannel microchip system, would allow for screening mutations with high throughput.
在本报告中，我们探讨了毛细管电泳和微芯片电泳用于基于异源双链分析（HDA）的突变检测的潜力。使用荧光染料标记的引物（6-羧基荧光素标记）扩增130至400 bp的DNA片段。利用BRCA1基因中的六个杂合突变185delAG、E1250X（3867G>T）、R1443G（4446C>G）、5382insC、5677insA以及BRCA2基因中的6174delT，评估了DNA片段长度、基质添加剂、pH值和盐对基于毛细管电泳（CE）和/或微芯片电泳的HDA的影响。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，基于微芯片的HDA的有效范围是150 - 260 bp。灵敏度研究表明，基于CE的HDA能够检测到仅占总DNA 1% - 10%的突变DNA。基于CE的HDA在<8分钟内即可实现BRCA1和BRCA2中野生型与缺失或插入突变之间的区分，而替换突变则需要14分钟的分析时间。针对每个突变区域，运行15个样本以确认该方法的准确性和可重复性。使用所描述的方法，在测试样本中检测到两个先前报道的突变E1038G（3232A>G，错义突变）和4427 C/T（4427C>T，多态性），并通过DNA测序进行了确认。将基于CE的方法转换为微芯片形式可使每个突变的分析时间缩短至130秒。基于该模型系统获得的结果，有可能开发并有效利用基于CE的HDA方法进行基因检测。CE仪器所提供的快速分离时间和自动化操作，为筛选包括小缺失、插入和点突变在内的突变提供了一个强大的系统。转换到微芯片平台，特别是多通道微芯片系统，将能够实现高通量的突变筛选。
