A mobility shift detection method for DNA methylation analysis using phosphate affinity polyacrylamide gel electrophoresis.
We describe a procedure for DNA methylation analysis using the bisulfite-mediated cytosine-to-uracil conversion of a target DNA followed by methylation-specific polymerase chain reaction (MSP) and phosphate affinity polyacrylamide gel electrophoresis (PAGE). The MSP was performed using a 1:1 mixture of 5'-phosphorylated methylation-specific and 5'-OH non-methylation-specific primers. The PAGE using an immobilized phosphate-binding tag molecule (i.e., a polyacrylamide-bound dizinc(II) complex, Zn(2+)-Phos-tag), which selectively captures the 5'-phosphorylated DNA fragment, enabled the mobility shift detection of the methylation-specific product as a slower migration band. Using this novel procedure, we demonstrated the detection of a methylated cytosine base in a pUC19 plasmid.
Kinoshita-Kikuta
Emiko
E
Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8553, Japan.
Kinoshita
Eiji
E
Koike
Tohru
T
D004247:DNA / Q000032:analysis* / Q000235:genetics; D019175:DNA Methylation; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D015394:Molecular Structure; D010710:Phosphates
D010710:Phosphates; D004247:DNA