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a requirement for the cloning of PCR-amplified DNA. For example certain techniques

such asin vitroprotein synthesis are best achieved with the DNA fragment inserted

into an appropriate plasmid or phage cloning vector (Section 6.7.1). Cloning methods

for PCR follow closely the cloning of DNA fragments derived from the conventional

manipulation of DNA. The techniques with which this may be achieved are through

one of two ways, blunt-ended or cohesive-ended cloning. Certain thermostable

DNA polymerases such asTaqDNA polymerase andTthDNA polymerase give rise

to PCR products having a 3^0 overhanging A residue. It is possible to clone the PCR

product into dT vectors termed dA : dT cloning. This makes use of the fact that the

terminal additions of A residues may be successfully ligated to vectors prepared

with T residue overhangs to allow efficient ligation of the PCR product (Fig. 6.9).

The reaction is catalysed by DNA ligase as in conventional ligation reactions

(Section 6.2.2).

It is also possible to carry out cohesive ended cloning with PCR products. In this

caseoligonucleotide primers are designed with a restriction endonuclease site

incorporated into them. Since the complementarity of the primers needs to be

absolute at the 3^0 end the 5^0 end of the primer is usually the region for the location

of the restriction site. This needs to be designed with care since the efficiency of

digestion with certain restriction endonuclease decreases if extra nucleotides, not

involved in recognition, are absent at the 5^0 end. In this case the digestion and

ligation reactions are the same as those undertaken for conventional reactions

(Section 6.2.1).

A

A

PCR product amplified with Taq DNA polymerase

T

T

Ligation T4 DNA ligase

Vector (dT) + PCR insert

Vector (dT)

Fig. 6.9Cloning of PCR products using dA : dT cloning.

205 6.2 Constructing gene libraries