A mobility-shift detection method for DNA methylation analysis using phosphate-affinity polyacrylamide gel electrophoresis

Kinoshita-Kikuta Emiko

Kinoshita Eiji

Koike Tohru

Analytical Biochemistry

Herein, we describe a procedure for DNA methylation analysis using the bisulfite-mediated C-to-U 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 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.

DNA methylation; Epigenetics

Methylation-specific PCR

Phosphate-affinity PAGE

Phos-tag

医学 [ 490 ]

英語

Academic Press Inc.

Elsevier Science

Copyright (c) 2008 Elsevier Inc.

[ISSN] 0003-2697

[DOI] 10.1016/j.ab.2008.03.021

[NCID] AA00524867

[DOI] http://dx.doi.org/10.1016/j.ab.2008.03.021