Velasco Eladio, Infante Mar, Durán Mercedes, Esteban-Cardeñosa Eva, Lastra Enrique, García-Girón Carlos, Miner Cristina
Laboratorio de Genética del Cáncer, Instituto de Biología y Genética Molecular, Facultad de Medicina, Universidad de Valladolid, Valladolid, Spain.
Electrophoresis. 2005 Jun;26(13):2539-52. doi: 10.1002/elps.200410425.
Mutational analysis of large multiexon genes without prevalent mutations is a laborious undertaking that requires the use of a high-throughput scanning technique. The Human Genome Project has enabled the development of powerful techniques for mutation detection in large multiexon genes. We have transferred heteroduplex analysis (HA) by conformation-sensitive gel electrophoresis of the two major breast cancer (BC) predisposing genes, BRCA1 and BRCA2, to a multicapillary DNA sequencer in order to increase the throughput of this technique. This new method that we have called heteroduplex analysis by capillary array electrophoresis (HA-CAE) is based on the use of multiplex-polymerase chain reaction (PCR), different fluorescent labels and HA in a 16-capillary DNA sequencer. To date, a total of 114 different DNA sequence variants (19 insertions/deletions and 95 single-nucleotide substitutions - SNS) of BRCA1 and BRCA2 from 431 unrelated BC families have been successfully detected by HA-CAE. In addition, we have optimized the multiplex-PCR conditions for the colorectal cancer genes MLH1 and MSH2 in order to analyze them by HA-CAE. Both genes have been amplified in 13 multiplex groups, which contain the 35 exons, and their corresponding flanking intronic sequences. MLH1 and MSH2 have been analyzed in nine hereditary nonpolyposis colorectal cancer patients, and we have found six different DNA changes: one complex deletion/insertion mutation in MLH1 exon 19 and another five SNS. Only the complex mutation and one SNS may be classified as cancer-prone mutations. Our experience has revealed that HA-CAE is a simple, fast, reproducible and sensitive method to scan the sequences of complex genes.
对没有常见突变的大型多外显子基因进行突变分析是一项艰巨的任务，需要使用高通量扫描技术。人类基因组计划推动了用于大型多外显子基因突变检测的强大技术的发展。我们已将通过构象敏感凝胶电泳对两个主要的乳腺癌（BC）易感基因BRCA1和BRCA2进行的异源双链分析（HA）转移至多毛细管DNA测序仪，以提高该技术的通量。我们将这种新方法称为毛细管阵列电泳异源双链分析（HA-CAE），它基于在16毛细管DNA测序仪中使用多重聚合酶链反应（PCR）、不同的荧光标记和HA。迄今为止，通过HA-CAE已成功检测到来自431个无关BC家族的BRCA1和BRCA2的总共114种不同的DNA序列变异（19个插入/缺失和95个单核苷酸替换 - SNS）。此外，我们优化了用于结直肠癌基因MLH1和MSH2的多重PCR条件，以便通过HA-CAE对其进行分析。这两个基因已在包含35个外显子及其相应侧翼内含子序列的13个多重组中进行了扩增。已对9名遗传性非息肉病性结直肠癌患者的MLH1和MSH2进行了分析，我们发现了6种不同的DNA变化：MLH1外显子19中的一个复杂缺失/插入突变和另外5个SNS。只有复杂突变和一个SNS可被归类为易癌突变。我们的经验表明，HA-CAE是一种简单、快速、可重复且灵敏的方法，用于扫描复杂基因的序列。
