Detection of germline BRCA1 mutations by Multiple-Dye Cleavase Fragment Length Polymorphism (MD-CFLP) method

S. Casadei, L. Cortesi, V. Pensotti, P. Radice, M. Pierotti, D. Amadori, D. Calistri

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
Pages (from-to)845-849
Number of pages5
JournalBritish Journal of Cancer
Volume85
Issue number6
DOIs
Publication statusPublished - Sep 14 2001

Keywords

  • BRCA1
  • CFLP
  • Hereditary breast cancer
  • Mutational analysis

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

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