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 beta-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 of IVS2 (IVS2-666) of the human beta-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.
Takahashi
N
N
Department of Genetics, Radiation Effects Research Foundation, Hiroshima, Japan.
Hiyama
K
K
Kodaira
M
M
Satoh
C
C
D004247:DNA / Q000235:genetics*; D004586:Electrophoresis / Q000379:methods; D005260:Female; D005787:Gene Frequency; D005796:Genes; D005821:Genetic Techniques; D014644:Genetic Variation; D005838:Genotype; D005914:Globins / Q000235:genetics*; D006801:Humans; D008297:Male; D009691:Nucleic Acid Denaturation; D010957:Plasmids; D015347:RNA Probes; D015183:Restriction Mapping
D015347:RNA Probes; D005914:Globins; D004247:DNA