Smith W M, Van Orsouw N J, Fox E A, Kolodner R D, Vijg J, Eng C
Charles A. Dana Human Cancer Genetics Unit, Dana-Farber Cancer Institute, Boston, MA 02115-6084, USA.
Genet Test. 1998;2(1):43-53. doi: 10.1089/gte.1998.2.43.
The human genome and related projects have resulted in the isolation of a rapidly growing number of genes that cause susceptibility to human cancer. With rare exception, these genes are large and have disease-associated mutations scattered along the length of the genes. Therefore, the development of accurate and cost-efficient mutation detection tests that can scan entire genes singly or in combination is warranted. hMLH1, encoding a mismatch repair enzyme, is a susceptibility gene for hereditary nonpolyposis colon cancer syndrome. This gene comprises 19 exons; mutations are scattered, typical of many susceptibility genes. Here, we present a strategy that combines extensive PCR multiplexing and two-dimensional DNA electrophoresis (Two-Dimensional Gene Scanning, TDGS) to scan accurately for mutations that lie within the exons and splice junctions of hMLH1. All target fragments, designed to have optimal melting characteristics, were prepared in a two-stage PCR--a four-plex long-distance PCR followed by short PCR in two multiplex groups of 10 and 11 amplicons. The mixture of amplicons was subjected to two-dimensional electrophoresis: separation by size in the first dimension and by melting characteristics in the second. Using this design, 41 samples containing known hMLH1 sequence variants or no alterations were blindly subjected to TDGS. All mutations were detected; there were no genuine false-positive or false-negative results. These results confirm that TDGS is a generally applicable, rapid, accurate, and reproducible mutation detection technology that would serve large-scale molecular epidemiologic studies as well as clinical molecular diagnostic purposes.
人类基因组计划及相关项目已分离出越来越多的、使人易患癌症的基因。除极少数例外，这些基因很大，且疾病相关突变分散在基因全长范围内。因此，开发能够单独或组合扫描整个基因的准确且经济高效的突变检测试验很有必要。hMLH1基因编码一种错配修复酶，是遗传性非息肉病性结直肠癌综合征的易感基因。该基因由19个外显子组成；突变分散，这是许多易感基因的典型特征。在此，我们提出一种策略，该策略结合了广泛的多重PCR和二维DNA电泳（二维基因扫描，TDGS），以准确扫描hMLH1外显子和剪接连接区内的突变。所有设计具有最佳解链特性的目标片段，通过两步PCR制备——先进行四重长距离PCR，然后在两个分别包含10个和11个扩增子的多重组中进行短PCR。扩增子混合物进行二维电泳：在第一维按大小分离，在第二维按解链特性分离。采用这种设计，对41个含有已知hMLH1序列变异或无改变的样本进行了TDGS盲测。所有突变均被检测到；无真正的假阳性或假阴性结果。这些结果证实，TDGS是一种普遍适用、快速、准确且可重复的突变检测技术，可用于大规模分子流行病学研究以及临床分子诊断。
