DNA Methyltransferases 209
BamHl(a) BamHl(b)
//~ ~ Mspl Mtase
CUT(a) ~BamHI ~ Plasmldlb) DNA (s~~
lal
Two DNA
fragments
Llnearlsed
PlssmidFig. 3. Inactivation of restriction sites using DNA Mtase of overlapping specificity.clearly would not methylate the full complement of CpG dinucleotides in
a given sequence of DNA. Unfortunately, no commercial preparations of
a CpG Mtase are currently available. However, a number of purification
schedules have been published for such enzymes from a range of mam-
malian sources.Apreparation (11) obtained from mouse erythroleukemia
cells has been well characterized and is probably the enzyme of choice for
this type of work.
As before, the DNA fragment or oligonucleotide duplex to be modified
is incubated with the Mtase preparation as described earlier. In order to
determine the extent and sequence-specific nature of the methylation,
the DNA can be subjected to strategic restriction analysis (if the DNA
sequence is known). A more thorough analysis involves the comparative
determination of the sequences of modified and unmodified DNA by the
Maxam Gilbert method (12). Methylated cytosines do not react with
hydrazine and, therefore, do not give bands in the C-track of the sequenc-
ing gel. Following successful methylation, the DNA can be used in com-
parative binding studies with transcription factors or can be used to transfect
cells, and so forth.