Detection of mutations in human genes by a new rapid method: cleavage fragment length polymorphism analysis (CFLPA).
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.
Rossetti
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S
Institute of Biology and Genetics, University of Verona, School of Medicine, Italy.
Englisch
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S
Bresin
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Pignatti
P F
PF
Turco
A E
AE
D000130:Achondroplasia / Q000235:genetics; D003094:Collagen / Q000235:genetics; D003550:Cystic Fibrosis / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D004262:DNA Restriction Enzymes; D004277:DNA, Single-Stranded / Q000378:metabolism; D005346:Fibroblast Growth Factors / Q000235:genetics; D006801:Humans; D009394:Nephritis, Hereditary / Q000235:genetics; D016891:Polycystic Kidney, Autosomal Dominant / Q000235:genetics; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D011505:Protein-Tyrosine Kinases; D011506:Proteins / Q000235:genetics; D051498:Receptor, Fibroblast Growth Factor, Type 3; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics; D016622:Silver Staining; D050396:TRPP Cation Channels
D003094:Collagen; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004262:DNA Restriction Enzymes; D004277:DNA, Single-Stranded; D005346:Fibroblast Growth Factors; D011505:Protein-Tyrosine Kinases; D011506:Proteins; D051498:Receptor, Fibroblast Growth Factor, Type 3; D017468:Receptors, Fibroblast Growth Factor; D050396:TRPP Cation Channels; C505032:CFTR protein, human; C496365:FGFR3 protein, human