title: Rapid mutation detection in complex genes by heteroduplex analysis with capillary array electrophoresis.
abstract: 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.
authors.affiliation: Laboratorio de Genética del Cáncer, Instituto de Biología y Genética Molecular, Facultad de Medicina, Universidad de Valladolid, Valladolid, Spain. evelasco@ped.uva.es
authors.forename: Eladio
authors.initials: E
authors.lastname: Velasco
authors.forename: Mar
authors.initials: M
authors.lastname: Infante
authors.forename: Mercedes
authors.initials: M
authors.lastname: Durán
authors.forename: Eva
authors.initials: E
authors.lastname: Esteban-Cardeñosa
authors.forename: Enrique
authors.initials: E
authors.lastname: Lastra
authors.forename: Carlos
authors.initials: C
authors.lastname: García-Girón
authors.forename: Cristina
authors.initials: C
authors.lastname: Miner
mesh_terms: D000328:Adult; D019313:BRCA1 Protein / Q000235:genetics*; D024682:BRCA2 Protein / Q000235:genetics*; D001943:Breast Neoplasms / Q000235:genetics*; D018567:Breast Neoplasms, Male / Q000235:genetics; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods; D019075:Electrophoresis, Capillary / Q000379:methods*; D005260:Female; D052138:Genes, Neoplasm / Q000235:genetics*; D020180:Heteroduplex Analysis / Q000191:economics / Q000379:methods*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D010051:Ovarian Neoplasms / Q000235:genetics; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D019313:BRCA1 Protein; D024682:BRCA2 Protein