Heteroduplex analysis by capillary array electrophoresis for rapid mutation detection in large multiexon genes.
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.
Velasco
Eladio
E
Laboratorio de Genética del Cáncer, Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid, Valladolid, Spain. evelasco@ped.uva.es
Infante
Mar
M
Durán
Mercedes
M
Pérez-Cabornero
Lucía
L
Sanz
David J
DJ
Esteban-Cardeñosa
Eva
E
Miner
Cristina
C
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D005091:Exons / Q000235:genetics*; D005453:Fluorescence; D005796:Genes / Q000235:genetics*; D019398:Genes, BRCA1; D024522:Genes, BRCA2; D020180:Heteroduplex Analysis / Q000379:methods*; D006801:Humans; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction
D048868:Adaptor Proteins, Signal Transducing; D017931:DNA Primers; D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; C501486:MLH1 protein, human; D009687:Nuclear Proteins; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein