COMPARISON OF HETERODUPLEX AND SINGLE-STRAND CONFORMATION ANALYSES, FOLLOWED BY ETHIDIUM FLUORESCENCE VISUALIZATION, FOR THE DETECTION OF MUTATIONS IN 4 HUMAN GENES

Non-isotopic DNA single-strand conformation analysis and heteroduplex analysis by ethidium bromide fluorescence visualization (SSCAE and HAE , respectively) were compared for the detection of 15 different natura lly occurring mutations in 15 different DNA samples. The mutations inc luded single nucleotide transitions, transversions and deletions, in C FTR (cystic fibrosis transmembrane conductance regulator), COL4A5 (col lagen type IV alpha 5 chain), HEXB (hexosaminidase B), and COL1A2 (col lagen type 1 alpha 2 chain) genes, responsible for diseases of medical interest. Genomic DNA from peripheral blood leukocytes or cDNA from r everse-transcribed fibroblast mRNA were amplified by polymerase chain reaction (PCR), and then analysed by two SSCAE and one HAE protocol. F ourteen out of 15 mutations (93%) were detected with one or the other method. HAE was more sensitive than SSCAE for the larger products (257 -426 bp). The only undetected mutation was then identified with the us e of a different primer, located farther from the mutation site, thus increasing the combined efficiency of the two methods to 100%. We beli eve that combined use of SSCAE and HAE is a good, cheap and safe appro ach for mutation screening in a human gene.