High-throughput mutation detection method to scan BRCA1 and BRCA2 based on heteroduplex analysis by capillary array electrophoresis.
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.
Esteban-Cardeñosa
Eva
E
Instituto de Biología y Genética Molecular, Facultad de Medicina, Universidad de Valladolid, Valladolid, Spain.
Duran
Mercedes
M
Infante
Mar
M
Velasco
Eladio
E
Miner
Cristina
C
D001322:Autoanalysis; D019313:BRCA1 Protein / Q000235:genetics*; D024682:BRCA2 Protein / Q000235:genetics*; D001943:Breast Neoplasms / Q000175:diagnosis; D019075:Electrophoresis, Capillary; D005260:Female; D020180:Heteroduplex Analysis; D006801:Humans; D009154:Mutation; D018807:Polymorphism, Single-Stranded Conformational
D019313:BRCA1 Protein; D024682:BRCA2 Protein