Single-strand conformation polymorphism analysis by capillary and microchip electrophoresis: A fast, simple method for detection of common mutations in BRCA1 and BRCA2
Single-strand conformation polymorphism analysis by capillary and microchip electrophoresis: A fast, simple method for detection of common mutations in BRCA1 and BRCA2
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DOI：
10.1006/geno.1999.6067
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发表时间：
2000-01-01
期刊：
影响因子：
4.4
通讯作者：
Landers, JP
中科院分区：
文献类型：
--
作者：
Tian, HJ;Jaquins-Gerstl, A;Landers, JP
作为对遗传性乳腺癌和卵巢癌的深入研究的结果，已经确定了两种乳腺癌易感基因BRCA 1和BRCA 2。在每个基因中，在某些种族人群中以相对高的频率发现了少量特定突变，BRCA 1中的185 delAG和5382 insC以及BRCA 2中的6174 delT突变已被确定为德系犹太人群体中的常见突变，组合频率为2.0至2.5%，具有上述三种常见突变之一的女性患乳腺癌或卵巢癌的风险很高。因此，这三种突变的准确和具有成本效益的检测可能对易感女性和男性的风险评估具有重要意义。本文介绍了一种快速、简便的基于毛细管电泳（CE）的聚合物网络检测BRCA 1和BRCA 2基因3种常见突变的方法。用荧光染料标记的引物（B-FAM-taged）扩增3个258、296和201 bp的DNA片段，分别检测185 delAG、5382 insC和6174 delT突变。PCR产物变性后，在聚合物网络中通过CE可以在不到10 min的时间内获得每个突变的单链构象多态性（SSCP）图谱。我们证明了通过将该测定转化为微芯片形式所提供的潜力，其中SSCP分析在120 s内完成，仅代表微芯片技术可以实现的分析时间减少的一小部分。用于检测这些突变的SSCP方法的速度和简单性使其在临床诊断实验室中具有吸引力。(C)北京大学出版社.
As a result of intensive studies on hereditary breast and ovarian cancers, two breast cancer susceptibility genes, BRCA1 and BRCA2, have been identified. In each gene, a small number of specific mutations have been found at relatively high frequency in certain ethnic populations, The mutations, 185delAG and 5382insC in BRCA1 and 6174delT in BRCA2, have been identified as common mutations in the Ashkenazi Jewish population, with a combined frequency of 2.0 to 2.5%, Women who have one of the above three common mutations are at a high risk of developing breast or ovarian cancer. Consequently, accurate and cost-effective detection of these three mutations may have important implications for risk assessment in susceptible women and men. In this report, we describe a fast and simple capillary electrophoresis (CE)-based method using a polymer network for screening the three common mutations in BRCA1 and BRCA2, Fluorescent dye-labeled primers (B-FAM-tagged) were used to amplify three DNA fragments of 258, 296, and 201 bp for detection of the 185delAG, 5382insC, and 6174delT mutations, respectively. After the PCR products were denatured, a single-strand conformation polymorphism (SSCP) profile could be obtained for each mutation in less than 10 min by CE in a polymer network. We demonstrate the potential provided by translating this assay to the microchip format where the SSCP analysis is complete in 120 s, representing only a fraction of the reduction ill analysis time that can be achieved with microchip technology. The speed and simplicity of the SSCP methodology for detection of these mutations make it attractive for use in the clinical diagnostic laboratory. (C) 2000 Academic Press.