An improved high throughput heteroduplex mutation detection system for screening BRCA2 mutations-fluorescent mutation detection (F-MD).
We describe an improved, fast, automated method for screening large genes such as BRCA2 for germline genomic mutations. The method is based on heteroduplex analysis, and has been adapted for a high throughput application by combining the fluorescent technology of automated sequencers and robotic sample handling. This novel approach allows the entire BRCA2 gene to be screened with appropriate overlaps in four lanes of an ABI 377 gel. The method will detect all types of mutations, especially point mutations, more reliably and robustly than other commonly used conformational sensitive methods (e.g. CSGE). In addition we show that this approach, which relies on band shift detection, is able to detect single base substitutions that have hitherto only been detectable by direct sequencing methods.
Cancer Genetics Team, Section of Cancer Genetics, Institute of Cancer Research, Sutton, Surrey. UK. edwards@icr.ac.ukros@icr.ac.uk
S M
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Edwards
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Kote-Jarai
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R
Hamoudi
R A
RA
Eeles
D024682:BRCA2 Protein; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D004586:Electrophoresis / Q000379:methods; D005453:Fluorescence; D005782:Gels; D005820:Genetic Testing; D006801:Humans; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics*; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*
D024682:BRCA2 Protein; D005782:Gels; D009363:Neoplasm Proteins; D009692:Nucleic Acid Heteroduplexes; D014157:Transcription Factors; D004247:DNA