DNA Methyltransferases 2073.1. Construction of Gene Libraries
A partial restriction digest of genomic DNA from any organism can
form the starting material for the construction of a genomic library. In
order to take advantage of the versatile bacteriophage ~, vectors, which
contain single unique restriction sites (e.g., ~,gtl 1, ~,gtl0, and so on),
DNA must be either digested with EcoR1 (compatible with the
unique cloning site of ~,gtl0 and ~,gtl 1) or with any enzyme followed
by the addition of EcoRI linkers to the ends of the DNA fragments.
The "activation" of the linkers requires an incubation of the frag-
ments with an excess of EcoRI. As a consequence, DNA that has been
digested with, say, SaulIIA and therefore probably contains one or
more internal EcoRI sites will be further reduced in size. In order to
prevent this secondary fragmentation of the DNA, the genomic digest
is incubated with EcoRI Mtase in the presence of SAM prior to the
addition of the linkers. In this way, only the tandem linker sites are
cleaved, and the DNA fragment remains intact. This procedure
can of course be applied to any restriction enzyme, providing a com-
patible Mtase is available. Moreover, the principle of protection of
specific restriction sites can be extended to other related DNA
manipulations as required. This strategy is particularly suitable for
the construction of cDNA libraries (see Chapters 17 and 19 in vol. 4
of this series).3.2. Modifying Restriction Sites
Methylation of a DNA sequence that is the target for a restriction
enzyme with partially degenerate specificity by a DNA Mtase of over-
lapping specificity can reduce the number of such sites that are cleaved
by the cognate restriction enzyme. For example, as shown in Fig. 2, the
enzyme HinclI recognizes and cleaves the sequence GTPyPuAC. The
two sequences GTCAAC and GTCGAC, which are found in the plas-
mid pBR322, are both sites for HinclI. However, the sequence
GTCGAC, but not the sequence GTCAAC, is a target for the TCGA-
specific TaqI Mtase. Therefore, methylation ofpBR322 by TaqI Mtases
produces a plasmid with effectively a single HinclI site. This approach
has been promoted by New England Biolabs (10), who offer an exten-
sive range of DNA Mtases for such purposes.