Screening for BRCA1 gene mutations by capillary electrophoretic REF-SSCP

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The identification of novel mutations in large genes requires efficient mutation-scanning techniques. Except for DNA-sequencing and PTT (protein truncation test), SSCP (single strand conformation polymorphism) is the technique that has been the most extensively applied to scan for mutations in the BRCA1-gene. In the present study a capillary electrophoretic (CE) restriction endonuclease fingerprinting (REF) modification of the SSCP technique was established for BRCA1 exon 11.

Samples containing a total of 16 known nucleotide changes in BRCA1 exon 11 were examined. Exon 11 was amplified in four overlapping PCR-fragments. Aliquots of labelled PCR products were submitted to digestion with three or four fragment-specific restriction enzymes and submitted to electrophoresis. Each sample was analysed using (1) radioactive labelling and polyacrylamide gel electrophoresis (PAGE) and (2) fluorochrome labelling and capillary electrophoresis in an ABI 310 sequencer. Samples giving abnormal electropherograms were reamplified and sequenced to identify the exact nature of the nucleotide change. All of the 16 known nucleotide changes could be detected by method 1. The aberrant band indicating the presence of one of the mutations (G484X) was, however, difficult to reproduce. All nucleotide changes but G484X were detected by fluorochrome-labelled CE-REF-SSCP (method 2). This method appeared as the faster and technically more convenient of the two.

CE-REF-SSCP was chosen as the mutation-scanning technique in our further BRCA1 studies. A series consisting of 75 affected members of Norwegian breast cancer families was first screened for a set of Norwegian BRCA1 mutations using restriction enzyme-based tests. The samples were then screened for novel mutations in exon 11 by CE-REF-SSCP. The results of this mutation screening will be presented.