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.