AN IMPROVED METHOD FOR THE DETECTION OF GENETIC VARIATIONS IN DNA WITH DENATURING GRADIENT GEL-ELECTROPHORESIS

We have examined the feasibility of denaturing gradient gel electrophoresis (DGGE) of RNA : DNA duplexes to detect variations in genomic and cloned DNAs. The result has demonstrated that employment of RNA : DNA duplexes makes DGGE much more practical for screening a large number of samples than that of DNA : DNA heteroduplexes originally developed by Lerman et al. (1986), because preparation of RNA probes is easier than that of DNA probes. Three different 32P-labeled RNA probes were produced. Genomic or cloned DNAs were digested with restriction enzymes and hybridized to labeled RNA probes, and resulting RNA : DNA duplexes were examined by DGGE. The presence of mismatch(es) was detected as a difference in mobility of bands on the gel. The experimental conditions were determined using DNA segments from cloned normal and 3 thalassemic human β-globin genes. The results of the experiments on the cloned DNAs suggest that DGGE of RNA : DNA duplexes will detect nucleotide substitutions and deletions in DNA. In the course of these studies, a polymorphism due to a single-base substitution at position 666 IVS2 of (IVS2-666) of the human β-globin gene was directly identified using genomic DNA samples. A study of 59 unrelated Japanese from Hiroshima was made in which the frequency of the allele with C at IVS2-666 was 0.48 and that of the allele with T was 0.52. This approach was found to be very effective for the detection of heritable variation and should be a powerful tool for the detection of fresh mutations in DNA, which occur outside the known restriction sites. © 1990.