A mobility shift detection method for DNA methylation analysis using phosphate affinity polyacrylamide gel electrophoresis.
A mobility shift detection method for DNA methylation analysis using phosphate affinity polyacrylamide gel electrophoresis.
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DOI：
10.1016/j.ab.2008.03.021
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发表时间：
2008-07
影响因子：
2.9
通讯作者：
Emiko Kinoshita-Kikuta;E. Kinoshita;T. Koike
中科院分区：
文献类型：
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作者：
Emiko Kinoshita-Kikuta;E. Kinoshita;T. Koike
我们描述了一种DNA甲基化分析方法，使用亚硫酸氢盐介导的胞嘧啶到尿嘧啶的目标DNA转化，随后是甲基化特异性聚合酶链式反应(MSP)和磷酸亲和聚丙烯酰胺凝胶电泳(PAGE)。用1：1的5‘-磷酸化甲基化特异性和5’-羟基非甲基化特异性的引物进行MSP。使用固定的磷酸盐结合标签分子(即，聚丙烯酰胺结合的双锌(II)络合物，锌离子-磷酸标签)选择性地捕获5‘-磷酸化DNA片段的PAGE，使甲基化特异产物的迁移率漂移检测为较慢的迁移带。使用这一新的程序，我们证明了在pUC19质粒中检测到甲基化的胞嘧啶碱基。
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 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.