Velasco Eladio, Infante Mar, Durán Mercedes, Pérez-Cabornero Lucía, Sanz David J, Esteban-Cardeñosa Eva, Miner Cristina
Laboratorio de Genética del Cáncer, Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid, Valladolid, Spain.
Nat Protoc. 2007;2(1):237-46. doi: 10.1038/nprot.2006.482.
Heteroduplex analysis (HA) has proven to be a robust tool for mutation detection. HA by capillary array electrophoresis (HA-CAE) was developed to increase throughput and allow the scanning of large multiexon genes in multicapillary DNA sequencers. HA-CAE is a straightforward and high-throughput technique to detect both known and novel DNA variants with a high level of sensitivity and specificity. It consists of only three steps: multiplex-PCR using fluorescently labeled primers, heteroduplex formation and electrophoresis in a multicapillary DNA sequencer. It allows, e.g., the complete coding and flanking intronic sequences of BRCA1 and BRCA2 genes from two patients (approximately 25 kb each) to be scanned in a single run of a 16-capillary sequencer, and has enabled us to detect 150 different mutations to date (both single nucleotide substitutions, or SNSs, and small insertions/deletions). Here, we describe the protocol developed in our laboratory to scan BRCA1, BRCA2, MLH1, MSH2 and MSH6 genes using an ABI3130XL sequencer. This protocol could be adapted to other instruments or to the study of other large multiexon genes and can be completed in 7-8 h.
异源双链分析（HA）已被证明是一种用于突变检测的强大工具。通过毛细管阵列电泳进行的异源双链分析（HA-CAE）得以开发，以提高通量并允许在多毛细管DNA测序仪中扫描大型多外显子基因。HA-CAE是一种直接且高通量的技术，能够以高灵敏度和特异性检测已知和新型DNA变体。它仅由三个步骤组成：使用荧光标记引物进行多重PCR、形成异源双链以及在多毛细管DNA测序仪中进行电泳。例如，在16毛细管测序仪的单次运行中，它可以扫描两名患者的BRCA1和BRCA2基因的完整编码及侧翼内含子序列（每个约25 kb），并且截至目前已使我们能够检测到150种不同的突变（包括单核苷酸替换，即SNSs，以及小的插入/缺失）。在此，我们描述了在我们实验室开发的使用ABI3130XL测序仪扫描BRCA1、BRCA2、MLH1、MSH2和MSH6基因的方案。该方案可适用于其他仪器或其他大型多外显子基因的研究，并且可以在7 - 8小时内完成。
