Comparison of heteroduplex and single-strand conformation analyses, followed by ethidium fluorescence visualization, for the detection of mutations in four 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 naturally occurring mutations in 15 different DNA samples. The mutations included single nucleotide transitions, transversions and deletions, in CFTR (cystic fibrosis transmembrane conductance regulator), COL4A5 (collagen type IV alpha 5 chain), HEXB (hexosaminidase B), and COL1A2 (collagen type 1 alpha 2 chain) genes, responsible for diseases of medical interest. Genomic DNA from peripheral blood leukocytes or cDNA from reverse-transcribed fibroblast mRNA were amplified by polymerase chain reaction (PCR), and then analysed by two SSCAE and one HAE protocol. Fourteen 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 use of a different primer, located farther from the mutation was then identified with the use of a different primer, located farther from the mutation site, thus increasing the combined efficiency of the two methods to 100%. We believe that combined use of SSCAE and HAE is a good, cheap and safe approach for mutation screening in a human gene.