Detection of mutations in GC-rich DNA by bisulphite denaturing gradient gel electrophoresis.
Denaturing gradient gel electrophoresis (DGGE) in combination with PCR and 'GC-clamping' has proven highly efficient as a method for detection of DNA sequence differences. Due to strand dissociation phenomena, however, its use has been limited to the analysis of sequences with a relatively low content of GC pairs. This paper describes how treatment of template DNA with sodium bisulphite drastically lowers the melting temperature of very GC-rich sequences and renders them amenable to DGGE analysis. We demonstrate the use of bisulphite DGGE for rapid and efficient detection of mutations in the p16(INK4/CDKN2) tumour suppressor gene.
Guldberg
P
P
Department of Tumour Cell Biology, Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark. perg@bio.cancer.dk
Grønbak
K
K
Aggerholm
A
A
Platz
A
A
thor Straten
P
P
Ahrenkiel
V
V
Hokland
P
P
Zeuthen
J
J
D001482:Base Composition; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004586:Electrophoresis / Q000379:methods*; D005069:Evaluation Studies as Topic; D019942:Genes, p16; D006801:Humans; D009154:Mutation; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D013447:Sulfites
D017931:DNA Primers; D013447:Sulfites; D004247:DNA; C009279:sodium bisulfite