Detection of germline BRCA1 mutations by Multiple-Dye Cleavase Fragment Length Polymorphism (MD-CFLP) method.
We describe the Multiple-Dye Cleavase Fragment Length Polymorphism (MD-CFLP) method set up for a sensitive and preliminary rapid screening of BRCA1 mutations. We analysed exons 11 and 16, which are known to cover slightly more than 70% of the whole coding region of the gene, subdivided into 4 amplicons and labelled with different fluorescent dUTPs. MD-CFLP was first utilised on a panel of 30 DNA samples in which the presence of single-base substitutions or small deletions/insertions had been previously identified by direct sequencing as gold standard, in order to define the optimal conditions in terms of PCR amplification and temperature of digestion. In a second step, we blindly analysed 21 DNA samples by MD-CFLP to verify its reliability. The sensitivity and specificity of MD-CFLP were both 100% in the first study, and 80% and 94%, respectively, in the blind sample assay. Our results demonstrate the capability of the MD-CFLP method to detect DNA sequence alterations in fragments of more than 1 kb. We conclude that CFLP is a powerful tool in mutational analysis, offering reliable results in a shorter time and at a lower cost than conventional methods, and its potential can be enhanced when internal fluorescent labelling and laser detection are used.
Casadei
S
S
Istituto Oncologico Romagnolo, 47100 Forlì, Italy.
Cortesi
L
L
Pensotti
V
V
Radice
P
P
Pierotti
M
M
Amadori
D
D
Calistri
D
D
D019313:BRCA1 Protein / Q000235:genetics*; D001943:Breast Neoplasms / Q000175:diagnosis / Q000453:epidemiology / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000032:analysis; D005091:Exons; D005260:Female; D005456:Fluorescent Dyes; D020022:Genetic Predisposition to Disease; D018095:Germ-Line Mutation; D006801:Humans; D009690:Nucleic Acid Conformation; D010051:Ovarian Neoplasms / Q000175:diagnosis / Q000453:epidemiology / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D019313:BRCA1 Protein; D017931:DNA Primers; D004273:DNA, Neoplasm; D005456:Fluorescent Dyes