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screening may be undertaken in 3–4 h whereas it may be several days before detection

by hybridisation is achieved. The PCR screening technique gives an indication of the

size of the cloned insert rather than the sequence of the insert; however, PCR primers

that are specific for a foreign DNA insert may also be used. This allows a more

rigorous characterisation of clones from cDNA and genomic libraries.

6.5.3 Hybrid select/arrest translation

The difficulty of characterising clones and detecting a desired DNA fragment from a

mixed cDNA library may be made simpler by two useful techniques termedhybrid

select (release) translationorhybrid arrest translation. Following the preparation

of a cDNA library in a plasmid vector the plasmid is extracted from part of each

colony, and each preparation is then denatured and immobilised on a nylon mem-

brane (Fig. 6.29). The membranes are soaked in total cellular mRNA, under stringent

conditions (usually a temperature only a few degrees belowTm) in which hybridisa-

tion will occur only between complementary strands of nucleic acid. Hence each

membrane will bind just one species of mRNA, since it has only one type of cDNA

immobilised on it. Unbound mRNA is washed off the membranes, and then the bound

mRNA is eluted and used to directin vitrotranslation (Section 6.7). Byimmunopre-

cipitation or electrophoresis of the protein, the mRNA coding for a particular

protein can be detected, and the clone containing its corresponding cDNA isolated.

This technique is known as hybrid release translation. In a related method called

Forward

primer

Non-recombinant

M13 multiple cloning site

Reverse

primer Agarose gel electrophoresis

225 bp

Recombinant 125 bp
Forward
primer

M13 multiple cloning site

Reverse

primer

100 bp DNA insert

Fig. 6.28PCR screening of recombinant vectors. In this figure, the M13 non-recombinant has no insert and

so the PCR undertaken with forward and reverse sequencing primers gives rise to a product 125 bp in length.

The M13 recombinant with an insert of 100 bp will give rise to a PCR product of 125 bpþ100 bp¼225 bp

and thus may be distinguished from the non-recombinant by analysis on agarose gel electrophoresis.

227 6.5 Screening gene libraries