Adaptation of conformation-sensitive gel electrophoresis to an ALFexpress DNA sequencer to screen BRCA1 mutations.
Conformation-sensitive gel electrophoresis (CSGE) has been introduced as the most reliable method for the screening of large and multi-exon genes because of its simplicity, sensitivity and specificity. Based on heteroduplex formation and with the use of mildly denaturing solvents, it allows detection of single-base mutations with accuracy. This is important in genes such as BRCA1 and BRCA2, in which alterations span the entire gene. We have adapted the CSGE assay to a fluorescent platform--a DNA sequencer one-color technology--that reduces the time involved and enhances resolving power for the complete scanning of the BRCA genes. Electrophoresis has high sensitivity and is performed in less than three hours, and the gel does not require staining with ethidium bromide. Eighteen single-base and six frameshift mutations in the BRCA1 gene were analyzed. We compared the manual and fluorescent CSGE methods, and all mutations were detected with accuracy.
Blesa
J R
JR
Instituto de Investigaciones Citológicas, Valencia, Spain.
Hernández-Yago
J
J
D019313:BRCA1 Protein / Q000235:genetics*; D001943:Breast Neoplasms / Q000175:diagnosis / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005260:Female; D005456:Fluorescent Dyes; D016368:Frameshift Mutation; D006580:Genetic Carrier Screening; D005820:Genetic Testing / Q000379:methods; D020180:Heteroduplex Analysis / Q000379:methods; D006720:Homozygote; D006801:Humans; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D019313:BRCA1 Protein; D017931:DNA Primers; D004273:DNA, Neoplasm; D005456:Fluorescent Dyes