Gene Cloning

Key Tools for Gene Cloning 51
same place on both strands of the DNA, and hence give blunt or flush ends
(see SmaI in Figure 3.9). These blunt or flush ends are more difficult to join
together with ligase because there is no transient base pairing to hold the
molecules together. T4 DNA ligase is capable, however, of ligating bluntended
molecules if used at a higher concentration and there are a number
of situations in which these restriction enzymes are particularly useful.
Restriction enzymes, which cut to give blunt ends, are in fact very versatile.
Because there is no transient base pairing of compatible sticky ends
required for a successful ligation, you do not have to use the same restriction
enzymes to generate the fragments that you want to ligate together.
Thus a fragment generated by cutting with any restriction enzyme giving
blunt ends can be ligated to a fragment cut with any other blunt-ended cutter.
Also, as we will see later, it is possible to convert restriction fragments
from blunt- to sticky-ended by adding linkers (Box 4.3).
Q3.11. You have a restriction fragment generated by digestion with PvuII
which you want to clone into the multiple cloning site in pUC18. Which
restriction enzyme would you cut the vector with?
Bacteria sometimes produce restriction enzymes that recognize the
same sequence of bases as those from other bacteria; these enzymes are
called isoschizomers. Some pairs of isoschizomers, although they recognize
the same sequence, cut in different positions. SmaI and XmaI are
isoschizomers, which both recognize the sequence GGGCCC, but whereas
SmaI cuts to give blunt ends, XmaI cuts to give sticky ends. In some situations
it would not matter which of these enzymes you use but in others you
might choose one in preference to another. Another pair of isoschizomers
is Asp718 and KpnI, and whereas cutting with KpnI leaves a 3′ overhang
with a hydroxyl group at the end (Figure 3.11) Asp718 leaves a 5′ overhang
which, as discussed above, is preferable in gene cloning because it is a
better substrate for DNA ligase.
Some restriction enzymes cut DNA much less frequently than the ones
we have looked at so far; these are often referred to as “rare cutters”. Some
“rare cutters” cut infrequently because they have a long recognition site for
example NotI which recognizes the 8 bp sequence GC^GGCCGC. Others cut
rarely because they recognize a sequence which is not commonly found in
DNA. These restriction enzymes are useful when you want to cut whole
chromosomes or whole genomes up into a few large fragments for applications
like RFLP mapping (Box 6.3) and genome sequencing (Chapter 7).
3.8 Alkaline Phosphatase
In Section 3.4 we discussed the ways in which the conditions under which
the ligation reaction is carried out can increase the chances of forming a