Nucleic Acids in Biotechnology 173
Figure 5.4 Transposon tagging
5.2.1.6 Chromosome Walking. If an overlapping series of clones can be isolated, it is possible to use
one clone to isolate the next in line and thus ‘walk’along the DNA to the required sequence. This is a very
time-consuming process, but nevertheless has been used frequently.
5.2.1.7 Chromosome Jumping. This is an extension of chromosome walking that proceeds by larger
steps and ignores the large DNA stretches in the middle of each step (hence the word ‘jumping’).
5.2.2 The Polymerase Chain Reaction
A DNA or gene of interest is best isolated now directly from the total DNA of the organism in question by
use of PCR.^8 Since the complete DNA sequences of many organisms (human, mouse, etc.) are now known,
it is easy to design and synthesise chemically a pair of oligonucleotide primers of 20–30 residues flanking the
region to be amplified, with each complementary to a different DNA strand (Figure 5.5). If it is required
to clone the DNA into a vector, the oligonucleotide primers each can contain a restriction site at the 5-ends,
whereas the 3-ends are complementary to the ends of the sequence to be cloned.
The target duplex DNA is denatured by heat and annealed to the primers, which are in vast excess to
prevent the target DNA strands renaturing with each other. The two primers are next elongated on the sep-
arated target DNA template strands by use of a thermostable DNA polymerase, for example, Taq DNA
polymerase from the thermophilic bacterium Thermus aquaticus, to give a twofold amplification. Since
the primers are derived from different DNA strands, each newly synthesised strand now contains a bind-
ing site for the primer used for copying of the otherstrand. A second round of denaturation by heat,
annealing and primer extension results in a fourfold amplification. After 20 rounds of amplification, 2^20
copies of the original target DNA are formed. Such a powerful technique can produce as much DNA as
can be made by classical cloning methods. For cloning, the DNA is either treated with both restriction
enzymes to create sticky ends, and the product joined to a similarly treated vector with T4 DNA ligase, just
as in classical cloning (Section 5.2.1), or cloned without restriction digestion. In the latter case, the PCR
products are often cloned into a special cleaved vector with single 3-T overhanging bases at the cleavage
sites, because PCR products usually contain a single A base overhang at both 3-ends, which is added in a
template-independent manner. But of course, it is not essential to clone the DNA, since the PCR reaction can
be repeated again if further amounts of DNA are required.