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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