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BRCA1 mutation screening using restriction endonuclase fingerprinting‐single‐strand conformation polymorphism in an automated capillary electrophoresis system
Author(s) -
Kringen Pedro,
Egedal Sidsel,
Pedersen Jan C.,
Harbitz Thorstein B.,
Tveit Kjell M.,
Berg Kåre,
BørresenDale AnneLise,
Andersen Tone I.
Publication year - 2002
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.200290025
Subject(s) - single strand conformation polymorphism , capillary electrophoresis , biology , microbiology and biotechnology , restriction enzyme , genetics , polymerase chain reaction , exon , genomic dna , endonuclease , coding region , gene
Efficient mutation scanning techniques are needed for the rapid detection of novel disease‐associated mutations and rare‐sequence variants of putative importance. The large size of the breast cancer 1 gene ( BRCA1 ) and the many mutations found throughout its entire coding sequence make screening for mutations in this gene particularly challenging. We have developed a method for screening exon 11 of the BRCA1 gene based on restriction enzyme digestion of fluorescence‐labeled polymerase chain reaction (PCR) products followed by single‐strand conformation polymorphism (SSCP) using an automated capillary electrophoresis system, denoted capillary restriction endonuclease fingerprinting (REF)‐SSCP electrophoresis. Using this strategy on a control set of samples, we were able to detect 17 of 18 known sequence alterations. The method was then applied to screen 73 Norwegian females with family histories of breast and/or ovarian cancer. A total of 172 sequence alterations were detected, including substitutions, insertions, and deletions. One novel substitution of unknown function was identified. Sequencing of all samples negative in the capillary REF‐SSCP system gave no additional mutations confirming the high sensitivity of the described methodology. Capillary REF‐SSCP electrophoresis appeared as a technically convenient technique, requiring amplification of fewer PCR fragments than traditional SSCP. The novel strategy allows high‐throughput mutation scanning without radioactive labeling and polyacrylamide gel electrophoresis (PAGE).