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Nucleic Acids Research 2005 33(14):e124; doi:10.1093/nar/gni121
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Published online 9 August 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Direct detection of methylation in genomic DNA

A. Bart*, M. W. J. van Passel, K. van Amsterdam and A. van der Ende

Department of Medical Microbiology, Academic Medical Center Amsterdam, The Netherlands

*To whom correspondence should be addressed. Tel: +31 20 5664863; Fax: +31 20 6979271; Email: a.bart{at}amc.uva.nl

Received June 10, 2005. Revised July 14, 2005. Accepted July 14, 2005.

The identification of methylated sites on bacterial genomic DNA would be a useful tool to study the major roles of DNA methylation in prokaryotes: distinction of self and nonself DNA, direction of post-replicative mismatch repair, control of DNA replication and cell cycle, and regulation of gene expression. Three types of methylated nucleobases are known: N6-methyladenine, 5-methylcytosine and N4-methylcytosine. The aim of this study was to develop a method to detect all three types of DNA methylation in complete genomic DNA. It was previously shown that N6-methyladenine and 5-methylcytosine in plasmid and viral DNA can be detected by intersequence trace comparison of methylated and unmethylated DNA. We extended this method to include N4-methylcytosine detection in both in vitro and in vivo methylated DNA. Furthermore, application of intersequence trace comparison was extended to bacterial genomic DNA. Finally, we present evidence that intrasequence comparison suffices to detect methylated sites in genomic DNA. In conclusion, we present a method to detect all three natural types of DNA methylation in bacterial genomic DNA. This provides the possibility to define the complete methylome of any prokaryote.


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