Comparison of DGGE and CDGE in detection of single base changes in the hamster hprt and human N-ras genes.
Denaturing gradient gel electrophoresis (DGGE) and constant denaturant gel electrophoresis (CDGE) are methods based on sequence-determined melting characteristics of DNA and thus detect different types of single base changes in the amplified fragments. We have studied detection of 19 mutations in the human N-ras oncogene and 10 mutations in exon 3 of the Chinese hamster hypoxanthine-guanine phosphoribosyltransferase (hprt) gene using GC-clamped DGGE and CDGE. After allowing formation of heteroduplexes with the corresponding wild type sequence, all the mutations separated from the wild type in at least one concentration of the denaturants used in CDGE but two of the mutations in hprt exon 3 did not show separation in any of the DGGE runs. Melting behavior of the mutant fragments was dependent, as expected, on both the type and the location of a mutation. We describe conditions allowing separation of the mutations in the fewest possible DGGE and CDGE runs.
Ridanpää
M
M
Finnish Institute of Occupational Health, Helsinki.
Burvall
K
K
Zhang
L H
LH
Husgafvel-Pursiainen
K
K
Onfelt
A
A
D000818:Animals; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005091:Exons / Q000235:genetics; D011905:Genes, ras / Q000235:genetics*; D005821:Genetic Techniques; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D009691:Nucleic Acid Denaturation; D017354:Point Mutation; D016133:Polymerase Chain Reaction
D007041:Hypoxanthine Phosphoribosyltransferase