Cutting and joining DNA molecules - Principles of Gene Manipulation - page 34

mcr C mcr B hsd S hsd M hsd R mrr

Fig. 3.4 The immigration control region of
E. coli strain K12.

14 kb

Whereas the Eco KI restriction system, encoded by
the hsd RMS genes, cleaves DNA that is not protected
by methylation at the target site, the Mcr A, Mcr BC
and Mrr endonucleases cut DNA that is methylated
at specific positions. All three endonucleases restrict
DNA modified by CpC methylase (M. Sss I) and the
Mrr endonuclease will attack DNA with methylade-
nine in specific sequences. The significance of these
restriction enzymes is that DNA from many bacteria,
and from all plants and higher animals, is extensively
methylated and its recovery in cloning experiments
will be greatly reduced if the restriction activity is
not eliminated. There is no problem with DNA from
Saccharomyces cerevisiae or Drosophila melanogaster
since there is little methylation of their DNA.
All the restriction systems in E. coli are clustered
together in an 'immigration control region' about
14 kb in length (Fig. 3.4). Some strains carry muta-
tions in one of the genes. For example, strains DH1
and DH5 have a mutation in the hsd R gene and so
are defective for the Eco KI endonuclease but still
mediate the Eco KI modification of DNA. Strain DP50
has a mutation in the hsd S gene and so lacks both the
Eco KI restriction and modification activities. Other
strains, such as E. coli C and the widely used cloning
strain HB101, have a deletion of the entire mcr C-
mrr region and hence lack all restriction activities.

where subsequent ligation is achieved, the resulting
product may contain small deletions. The message is
clear: cheap restriction enzymes are in reality poor
value for money!
A typical QC procedure is as follows. DNA frag-
ments are produced by an excessive overdigestion of
substrate DNA with each restriction endonuclease.
These fragments are then ligated and recut with the
same restriction endonuclease. Ligation can occur
only if the 3

termini are left intact, and only
those molecules with a perfectly restored recogni-
tion site can be recleaved. A normal banding pattern
after cleavage indicates that both the 3

′ and 5 ′

termini
are intact and the enzyme preparation is free of de-
tectable exonucleases and phosphatases (Fig. 3.5).

′ and 5 ′

The importance of enzyme quality

Restriction enzymes are available from many differ-
ent commercial sources. In choosing a source of
enzyme, it is important to consider the quality of the
enzyme supplied. High-quality enzymes are purified
extensively to remove contaminating exonucleases
and endonucleases and tests for the absence of such
contaminants form part of routine quality control
(QC) on the finished product (see below). The absence
of exonucleases is particularly important. If they are
present, they can nibble away the overhangs of
cohesive ends, thereby eliminating or reducing the
production of subsequent recombinants. Contamin-
ating phosphatases can remove the terminal phos-
phate residues, thereby preventing ligation. Even

Fig. 3.5 Quality control of the enzyme Pst I. DNA was
overdigested with the endonuclease and the fragments were
ligated together and then recut. Note that the two digests give
an identical banding pattern upon agarose gel electrophoresis.