Biomedical Engineering Reference
In-Depth Information
CHAPTER 3
Cutting and joining DNA
molecules
more simply. That is, the enzyme recognizes a par-
ticular target sequence in a duplex DNA molecule
and breaks the polynucleotide chain within that
sequence to give rise to discrete fragments of defined
length and sequence.
The presence of restriction and modification
systems is a double-edged sword. On the one hand,
they provide a rich source of useful enzymes for
DNA manipulation. On the other, these systems can
significantly affect the recovery of recombinant DNA
in cloning hosts. For this reason, some knowledge of
restriction and modification is essential.
Cutting DNA molecules
Before 1970 there was no method of cleaving DNA
at discrete points. All the available methods for
fragmenting DNA were non-specific. The available
endonucleases had little site specificity and chemical
methods produced very small fragments of DNA.
The only method where any degree of control could
be exercised was the use of mechanical shearing.
The long, thin threads which constitute duplex
DNA molecules are sufficiently rigid to be very easily
broken by shear forces in solution. Intense sonica-
tion with ultrasound can reduce the length to about
300 nucleotide pairs. More controlled shearing
can be achieved by high-speed stirring in a blender.
Typically, high-molecular-weight DNA is sheared
to a population of molecules with a mean size of
about 8 kb by stirring at 1500 rev/min for 30 min
(Wensink
et al.
1974). Breakage occurs essentially
at random with respect to DNA sequence. The ter-
mini consist of short, single-stranded regions which
may have to be taken into account in subsequent
joining procedures.
During the 1960s, phage biologists elucidated the
biochemical basis of the phenomenon of host restric-
tion and modification. The culmination of this work
was the purification of the restriction endonuclease
of
Escherichia coli
K12 by Meselson and Yuan (1968).
Since this endonuclease cuts unmodified DNA into
large discrete fragments, it was reasoned that it must
recognize a target sequence. This in turn raised the
prospect of controlled manipulation of DNA. Unfor-
tunately, the K12 endonuclease turned out to be
perverse in its properties. While the enzyme does
bind to a defined recognition sequence, cleavage
occurs at a 'random' site several kilobases away
(Yuan
et al.
1980). The much sought-after break-
through finally came in 1970 with the discovery
in
Haemophilus influenzae
(Kelly & Smith 1970,
Smith & Wilcox 1970) of an enzyme that behaves
Host-controlled restriction
and modification
Restriction systems allow bacteria to monitor the
origin of incoming DNA and to destroy it if it is
recognized as foreign. Restriction endonucleases
recognize specific sequences in the incoming DNA
and cleave the DNA into fragments, either at specific
sites or more randomly. When the incoming DNA is
a bacteriophage genome, the effect is to reduce the
efficiency of plating, i.e. to reduce the number of
plaques formed in plating tests. The phenomena of
restriction and modification were well illustrated
and studied by the behaviour of phage
λ
on two
E. coli
host strains.
If a stock preparation of phage
, for example, is
made by growth upon
E. coli
strain C and this stock is
then titred upon
E. coli
C and
E. coli
K, the titres
observed on these two strains will differ by several
orders of magnitude, the titre on
E. coli
K being the
lower. The phage are said to be
restricted
by the sec-
ond host strain (
E. coli
K). When those phage that do
result from the infection of
E. coli
K are now replated
on
E. coli
K they are no longer restricted; but if they
are first cycled through
E. coli
C they are once again
restricted when plated upon
E. coli
K (Fig. 3.1). Thus
the efficiency with which phage
λ
λ
plates upon a
