Methylation of DNA by the host strain can have a great effect on DNA cleavage and/or transformation. Most laboratory strains of E. coli contain 3 DNA methylases that methylate distinct nucleotides in specific DNA sequences:
|Dam methylase||methylates adenine residues in the sequence GATC|
|Dcm methylase||methylates the internal cytocine residues in the sequence CCAGG and CCTGG|
|EcoK1 methylase||methylates adenine residues in the sequence AAC(N6)GTGC and GCAC(N6)GTT|
When the DNA is isolated from strains expressing one of these methylases, especially Dam or Dcm methylase because their sites are much more common than EcoK1 sites, some or all sites for a restriction enzyme may be resistant to cleavage. This occurs because DNA is protected from cleavage when a particular base in the recognition site of the restriction enzyme is methylated. For example, plasmid DNA isolated from Dam+ E. coli is completely resistant to cleavage by Mbo I, which cleaves GATC sites. Not all restriction enzymes are sensitive to methylation. For example, BamH I still cleaves GGATCC sites of plasmid DNA from Dam+ E. coli.
Further, methylation of plasmid DNA can affect the frequency of transformation in special situations. For example, the transformation efficiency is reduced when Dam-modified plasmid DNA is introduced into Dam- strains or when Dam- or Dcm-modified DNA is introduced into certain other bacterial species.
In some cases it will be necessary to use a Dam- and/or Dcm-negative E. coli strain, for instance GM48 and JM110.
For more information, see page 221 of the New England Biolabs 2000/01 catalogue.