甲基化对脉冲场琼脂糖凝胶中染色体DNA电泳迁移率的影响。

Effect of methylation on the electrophoretic mobility of chromosomal DNA in pulsed-field agarose gels.

作者信息

Xydas S, Lange C S, Phil D, Neimark H C

机构信息

Department of Radiation Oncology, State University of New York, Health Science Center at Brooklyn, NY 11203, USA.

出版信息

Appl Theor Electrophor. 1996;6(1):43-7.

PMID:9072080

Abstract

Factors other than molecular weight are known to affect DNA electrophoretic mobility. DNA methylation has been found to affect the curvature of DNA, causing anomalous mobility in polyacrylamide gels; the effect of methylation on the mobility of large DNA molecules in agarose gels was unknown. Chromosomal DNA from Mycoplasma capricolum, a wall-less prokaryote which has a low intrinsic methylation rate, was methylated in agarose blocks by SssI methylase, a de novo methylase with a CpG recognition sequence. (A surprising finding was that SssI methylase altered the structure of InCert, but not SeaKem Gold, agarose.) Restriction enzyme analysis was used to estimate the extent of CpG methylation. DNA methylation was found to have no effect on the electrophoretic mobility of full-length chromosomal DNA (1,120 kbp) in agarose gels. Therefore, methylation is not a source of error in PFGE-based size estimation for chromosomal DNA molecules less than 1.12 Mbp in agarose gels.

摘要

已知除分子量外的其他因素会影响DNA的电泳迁移率。已发现DNA甲基化会影响DNA的曲率，导致其在聚丙烯酰胺凝胶中出现异常迁移；而甲基化对大DNA分子在琼脂糖凝胶中迁移率的影响尚不清楚。来自无壁原核生物山羊支原体的染色体DNA，其固有甲基化率较低，在琼脂糖块中被具有CpG识别序列的从头甲基化酶SssI甲基化。（一个惊人的发现是，SssI甲基化酶改变了InCert琼脂糖的结构，但未改变SeaKem Gold琼脂糖的结构。）使用限制酶分析来估计CpG甲基化的程度。结果发现，DNA甲基化对全长染色体DNA（1,120 kbp）在琼脂糖凝胶中的电泳迁移率没有影响。因此，对于琼脂糖凝胶中小于1.12 Mbp的染色体DNA分子，甲基化不是基于脉冲场凝胶电泳（PFGE）的大小估计误差来源。