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, Zn2+-Phos-tag), which selectively captures the T-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. (c) 2008 Elsevier Inc. All rights reserved.