Detection of mutations in human genes by a new rapid method: Cleavage fragment length polymorphism analysis (CFLPA)

Sandro Rossetti, Sabine Englisch, Elena Bresin, Pier Franco Pignatti, Alberto E. Turco

Research output: Contribution to journalArticle

Abstract

Cleavage fragment length polymorphism analysis with silver staining visualization (CFLPA-SS) was used for the detection of mutations previously detected by single strand conformation (SSCA) or heteroduplex analyses (HA), in order to assess this new method for mutation screening. The analysed mutations include single nucleotide transitions, transversions, a deletion and a duplication in the following genes: CFTR (cystic fibrosis transmembrane conductance regulator), COL4A5 (collagen type 4 alpha 5 chain), PKD1 (polycystic kidney disease 1), and FCFR3 (fibroblast growth factor receptor 3). Peripheral blood leukocyte genomic DNA was isolated, amplified by polymerase chain reaction (PCR), and then cleaved by Cleavase I enzyme at different temperatures. Electrophoresis of the fragments on denaturing polyacrylamide gel was followed by silver staining for 1 min. All 13 mutations investigated were reproducibly detected. CFLPA-SS proved to be a reliable method for mutation detection and more rapid than SSCA and HA.

Original languageEnglish
Pages (from-to)155-160
Number of pages6
JournalMolecular and Cellular Probes
Volume11
Issue number2
DOIs
Publication statusPublished - Apr 1997

Keywords

  • Cleavage fragment length polymorphism analysis (CFLPA)
  • Mutation detection
  • Mutation screening
  • Polymerase chain reaction (PCR)
  • Silver staining

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology

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