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

Mol Cell Probes. 1997 Apr;11(2):155-60. doi: 10.1006/mcpr.1996.0085.

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 FGFR3 (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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Achondroplasia / genetics
  • Collagen / genetics
  • Cystic Fibrosis / genetics
  • Cystic Fibrosis Transmembrane Conductance Regulator / genetics
  • DNA Mutational Analysis / methods*
  • DNA Restriction Enzymes
  • DNA, Single-Stranded* / metabolism
  • Fibroblast Growth Factors / genetics
  • Humans
  • Nephritis, Hereditary / genetics
  • Polycystic Kidney, Autosomal Dominant / genetics
  • Polymerase Chain Reaction
  • Polymorphism, Restriction Fragment Length*
  • Protein-Tyrosine Kinases*
  • Proteins / genetics
  • Receptor, Fibroblast Growth Factor, Type 3
  • Receptors, Fibroblast Growth Factor / genetics
  • Silver Staining
  • TRPP Cation Channels

Substances

  • CFTR protein, human
  • DNA, Single-Stranded
  • Proteins
  • Receptors, Fibroblast Growth Factor
  • TRPP Cation Channels
  • polycystic kidney disease 1 protein
  • Cystic Fibrosis Transmembrane Conductance Regulator
  • Fibroblast Growth Factors
  • Collagen
  • FGFR3 protein, human
  • Protein-Tyrosine Kinases
  • Receptor, Fibroblast Growth Factor, Type 3
  • DNA Restriction Enzymes

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