Development and validation of an approach to produce large-scale quantities of CpG-methylated plasmid DNA.
The prokaryotic CpG-specific DNA methylase from Spiroplasma, SssI methylase, has been extensively used to methylate plasmid DNA in vitro to investigate the effects of methylation in vertebrate systems. Currently available methods to produce CpG-methylated plasmid DNA have certain limitations and cannot generate large quantities of methylated DNA without cost or problems of purity. Here we describe an approach in which the SssI methylase gene has been introduced into the Escherichia coli bacterial genome under the control of an inducible promoter. Plasmid DNA propagated in this bacterium under conditions which induce the methylase gene result in significant (>90%) CpG methylation. Methylated DNA produced by this approach behaves similarly to methylated DNA produced in vitro using the purified methylase. The approach is scalable allowing for the production of milligram quantities of methylated plasmid DNA.
Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 32610, USA. bsf@pharmacology.ufl.edu
Bradley S
BS
Fletcher
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D015257:DNA-Cytosine Methylases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D013149:Spiroplasma / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases
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