Restriction enzymes and methylation

Daniel Kim dkim at nmsu.edu
Thu Mar 2 15:38:45 EST 1995


Hello:

I was thinking of a way to clone PCR products into restriction sites using
a linker-primer.  In Stratagene's cDNA synthesis kit, the first strand
synthesis was done using a methyl-dCTP, ensuring that all internal
restriction sites for XhoI (I think) were methylated and thus protected. 
Then the linker-primer site could be digested with no danger of cutting
internal sites.

Could a similar system be used in a PCR reaction?  For instance, if the
primers were nonmethylated, but the reactions were done with methylated
dNTPs, then the primers would have hemimethylated sites while the internal
sites would be methylated on both strands.  Alternatively, a single cycle
at the end could be done with nonmethylated dNTPs overwhelming the
original methylated dNTPS, creating hemimethylated internal sites and one
nonmethylated primer site.

Does this make sense?  I sometimes don't think these things through and
make up schemes that are doomed to failure.  Please help.

Daniel Kim
dkim at verdi.nmsu.edu




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