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Rapid mutation detection in complex genes by heteroduplex analysis with capillary array electrophoresis
Author(s) -
Velasco Eladio,
Infante Mar,
Durán Mercedes,
EstebanCardeñosa Eva,
Lastra Enrique,
GarcíaGirón Carlos,
Miner Cristina
Publication year - 2005
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200410425
Subject(s) - heteroduplex , dna sequencer , microbiology and biotechnology , multiplex , genetics , biology , capillary electrophoresis , multiplex polymerase chain reaction , gene , mutation , mlh1 , polymerase chain reaction , dna , msh2 , dna mismatch repair , dna repair
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.