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probes and primers are usually designed with bioinformatics software using sequence

information from nucleic acid databases. Alternatively, gene family related sequences

as indicated in Section 5.11 may also be successfully employed. However, there are

many gene probes that have traditionally been derived from cDNA or from genomic

sequences and which have been cloned into plasmid and phage vectors. These require

manipulation before they may be labelled and used in hybridisation experiments.

Gene probes may vary in length from 100 bp to a number of kilobases, although this is

dependent on their origin. Many are short enough to be cloned into plasmid vectors

and are useful in that they may be manipulated easily and are relatively stable both in

transit and in the laboratory. The DNA sequences representing the gene probe are

usually excised from the cloning vector by digestion with restriction enzymes and

purified. In this way vector sequences which may hybridise non-specifically and cause

high background signals in hybridisation experiments are removed. There are various

ways of labelling DNA probes and these are described in Section 5.9.4.

6.4.2 RNA gene probes

It is also possible to prepare cRNA probes or riboprobes byin vitrotranscription of gene

probes cloned into a suitable vector. A good example of such a vector is the phagemid

pBluescript SK since at each end of the multiple cloning site where the cloned DNA

fragment resides are promoters for T3 or T7 RNA polymerase (Section 6.3.3). The vector is

then made linear with a restriction enzyme and T3 or T7 RNA polymerase is used to

transcribe the cloned DNA fragment. Provided a labelled NTP is added in the reaction a

riboprobe labelled to a high specific activity will be produced (Fig. 6.26). One advantage

of riboprobes is that they are single stranded and their sensitivity is generally regarded as

Vector containing gene probe

f1 ori

Ti

T7

T3

phagemid

Resistance marker

colE1

Labelled cRNA probes

(riboprobes) synthesised

Vector containing gene probe is

linearised by restriction digestion

T3 RNA polymerase added and

transcribes gene probe sequence

Labelled dNTP

Unlabelled dNTPs

Fig. 6.26Production of cRNA (riboprobes) using T3 RNA polymerase and phagemid vectors.

224 Recombinant DNA and genetic analysis