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 vi sualization (CFLPA-SS) was used for the detection of mutations previou sly detected by single strand conformation (SSCA) or heteroduplex anal yses (HA), in order to assess this new method for mutation screening. The analysed mutations include single nucleotide transitions, transver sions, a deletion and a duplication in the following genes: CFTR (cyst ic fibrosis transmembrane conductance regulator), COL4A5 (collagen typ e 4 alpha 5 chain), PKD1 (polycystic kidney disease 1), and FGFR3 (fib roblast growth factor receptor 3). Peripheral blood leukocyte genomic DNA was isolated, amplified by polymerase chain reaction (PCR), and th en cleaved by Cleavase I enzyme at different temperatures. Electrophor esis of the fragments on denaturing polyacrylamide gel was followed by silver staining for 1 min. All 13 mutations investigated were reprodu cibly detected. CFLPA-SS proved to be a reliable method for mutation d etection and more rapid than SSCA and HA. (C) 1997 Academic Press Limi ted.