Synthesis of cytosine methylated DNAs in vitro --J. Colasanti and V. Sundaresan In our studies of the effects of transposon activity on DNA methylation it was necessary to devise a simple method to synthesize cytosine methylated DNA. We have found that this can be done by PCR in which 5-methyl-dCTP is substituted for dCTP (Colasanti and Sundaresan, 1990, submitted). The reaction conditions are much the same as for regular PCR except that the extension time had to be increased to 5' for a 1 kb fragment as the enzyme operates less efficiently with the methylated nucleotide, and the yields decrease significantly for amplification of larger fragments. Using this method we could demonstrate that cytosine-methylated DNA is completely resistant to the restriction enzyme Hinf1 at the concentrations normally used to cut genomic DNA, i.e. 10-100 fold enzyme excess. This would account for the observed resistance to Hinf1 digestion of the Mu1 elements in inactive-Mu lines (Chandler and Walbot, PNAS 83:1761771, 1986). We find that Hinf1 at 15000 fold excess will completely digest cytosine methylated DNA, but use of such vast excess of enzyme is impractical in most genomic digests.

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