Detection of the most frequent mutations in BRCA1 gene on polyacrylamide gels containing Spreadex Polymer NAB.
Heterozygous carriers of germ-line mutations in the BRCA1 gene are at high risk for the development of breast and ovarian cancer. Inactivating mutations have been identified in the whole coding region of the gene, however, repeatedly occuring mutations can explain a large proportion of gene alterations detected in certain ethnic groups. In Czech patients, the 5382insC and 185delAG mutations may account for approximately 50% of all BRCA1 abnormalities (unpublished data). In the present study, a rapid and simple method to identify these short insertions and deletions that alter the size of the polymerase chain reaction (PCR) product is described. The analysis involves the separation of fragments amplified with primers that flank altered sites in the BRCA1 gene on non-denaturing polyacrylamide gels containing Spreadex Polymer NAB. The increased resolving power of Spreadex gels enables full separation of two DNA fragments that differ by 1-bp on gels that are 5 cm long. The method gave interpretable results with the genetic material obtained from all tested mutation carriers and control persons. Defective alleles were also detected in DNA samples from carriers of the 1135delA mutation in BRCA1 and the 4206ins4 mutation in BRCA2. These results suggest that electrophoresis on Spreadex gels can be used universally for detection of the most frequent frameshift mutations in BRCA genes. The method is suitable even for rapid screening of frequent germ-line mutations in BRCA genes in breast and ovarian cancer patients not selected for family history of cancer or age at diagnosis.
Janatová
M
M
Institute of Biochemistry and Experimental Oncology; Charles University in Prague, First Faculty of Medicine, Praha 2, 128 53 Czech Republic. mjana@lf1.cuni.cz
Pohlreich
P
P
Matous
B
B
D000180:Acrylic Resins; D001943:Breast Neoplasms / Q000175:diagnosis / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000032:analysis; D005260:Female; D019398:Genes, BRCA1; D006801:Humans; D008403:Mass Screening / Q000379:methods; D010051:Ovarian Neoplasms / Q000175:diagnosis / Q000235:genetics; D016133:Polymerase Chain Reaction; D044767:Ubiquitin-Protein Ligases
D000180:Acrylic Resins; D002352:Carrier Proteins; D017931:DNA Primers; D004273:DNA, Neoplasm; C016680:polyacrylamide gels; C111164:BRAP protein, human; D044767:Ubiquitin-Protein Ligases