Received on July 7, 2003
Accepted on November 24, 2003

Molecular Diagnostics and Genetics

High-Throughput Mutation Detection Method to Scan BRCA1 and BRCA2 Based on Heteroduplex Analysis by Capillary Array Electrophoresis

¹ Laboratorio de Genética del Cáncer, Unidad de Diagnóstico Genético y Perinatal, Instituto de Biologia y Genética Molecular (IBGM), Facultad de Medicina, Universidad de Valladolid, Valladolid, Spain

^* To whom correspondence should be addressed. E-mail: evelasco{at}ped.uva.es.

Background: Scanning for mutations in BRCA1 and BRCA2 in a large number of samples is hampered by the large sizes of these genes and the scattering of mutations throughout their coding sequences. Automated capillary electrophoresis has been shown to be a powerful system to detect mutations by either single-strand conformation polymorphism or heteroduplex analysis (HA).

Methods: We investigated the adaptation of gel-based HA of BRCA1 and BRCA2 to a fluorescent multicapillary platform to increase the throughput of this technique. We combined multiplex PCR, three different fluorescent labels, and HA in a 16-capillary DNA sequencer and tested 57 DNA sequence variants (11 insertions/deletions and 46 single-nucleotide changes) of BRCA1 and BRCA2.

Results: We detected all 57 DNA changes in a blinded assay, and 2 additional single-nucleotide substitutions (1186 A>G of BRCA1 and 3624 A>G of BRCA2), previously unresolved by conformation-sensitive gel electrophoresis. Furthermore, different DNA changes in the same PCR fragment could be distinguished by their peak patterns.

Conclusions: Capillary-based HA is a fast, efficient, and sensitive method that considerably reduces the amount of "hands-on" time for each sample. By this approach, the entire coding regions of BRCA1 and BRCA2 from two breast cancer patients can be scanned in a single run of 90 min.