166 Pingoud, Alves, and Geigerenzymes. Substrate impurities vary with the method used to isolate
and purify the DNA. In particular, "miniprep" methods developed for
rapid plasmid isolation tend to give DNA material containing impu-
rities that may interfere with the restriction digests: salt contained in
the lysis buffer, organic solvents remaining from the extraction with
phenol/chloroform or from the precipitation with alcohols, as well as
cellular proteins, carbohydrates, lipids, and other nucleic acids that
were not completely removed during the purification.
Salt and organic solvents can be removed by repeated washing of
the DNA after an ethanol precipitation with 70% ethanol at room
temperature prior to drying the pellet in a desiccator, or alternatively
by dialysis against 0.3M sodium acetate, pH 4.8, and precipitation by
ethanol. Contaminating proteins, especially nonspecific nucleases, must
be separated by phenol/chloroform extraction after incubation with
proteinase K. Contaminating cellular RNA can be destroyed by the
addition of DNase-free RNase to the lysis buffer (0.1 mg/mL). Chro-
mosomal DNA fragments must be separated by chromatographic pro-
cedures or density gradient centrifugation.
- Specific Inhibition of Restriction Enzymes
by Methylation of the Substrate
As discussed in Section 2.4.1., the activity and specificity of restric-
tion enzymes are influenced by the methylation state of the DNA
substrate (for a review, cf [128]). Site-specific methylation of DNA is
owing to the following methyltransferase activities: modification
methyltransferases as part of bacterial restriction/modification sys-
tems, modification methyltransferases as part of bacterial DNA-repair
systems, and eukaryotic cytosine-specific methyltransferases. Of inter-
est, in particular during cloning work, is DNA methylation by the dam
and dcm methyltransferases, which are present in the majority of the
commonly used E. coli strains. The dam methyltransferase transfers a
methyl group from S-adenosylmethionine to the N 6 position of adenine
within the sequence GATC. The dcm methyltransferase specifically
methylates the internal cytosine at C 5 within the sequence CCWGG.
Methylation by dam or dcm methyltransferases inhibits DNA cleav-
age by certain restriction enzymes whose recognition sites overlap or
are identical with the dam or dcm modification sites (e.g., TaqI, MboI).
Other restriction enzymes are not influenced by dam or dcm methyla-
- Specific Inhibition of Restriction Enzymes