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fragment of DNA, a non-functional enzyme results which is unable to carry out

hydrolysis of X-gal. Thus, a recombinant pUC plasmid may be easily detected since

it is white or colourless in the presence of X-gal, whereas an intact non-recombinant

pUC plasmid will be blue since its gene is fully functional and not disrupted. This

elegant system, termedblue/white selection, allows the initial identification of

recombinants to be undertaken very quickly and has been included in a number of

subsequent vector systems. This selection method and insertional inactivation of anti-

biotic resistance genes do not, however, provide any information on the character of the

DNA insert, just the status of the vector. To screen gene libraries for a desired insert

hybridisation to gene probes is required and this is explained in Section 6.5.

6.3.2 Virus-based vectors

A useful feature of any cloning vector is the amount of DNA it may accept or have

inserted before it becomes unviable. Inserts greater than 5 kb increase plasmid size to

the point at which efficient transformation of bacterial cells decreases markedly, and

so bacteriophages (bacterial viruses) have been adapted as vectors in order to propa-

gate larger fragments of DNA in bacterial cells. Cloning vectors derived froml

bacteriophageare commonly used since they offer an approximately 16-fold advan-

tage in cloning efficiency in comparison with the most efficient plasmid cloning vectors.

Phagelis a linear double-stranded phage approximately 49 kb in length (Fig. 6.15).

It infectsE. coliwith great efficiency by injecting its DNA through the cell membrane.

In the wild-type phagelthe DNA follows one of two possible modes of replication.

Firstly the DNA may either become stably integrated into theE. colichromosome

where it lies dormant until a signal triggers its excision. This is termed thelysogenic

life cycle. Alternatively, it may follow alytic life cyclewhere the DNA is replicated

Non-recombinant vector (no insert)

Induce with IPTG

MCS

Recombinant vector (insert within MCS)

β-Galactosidase gene

X-gal hydrolysed

(white to blue)

BLUE plaque

DNA inserted in MCS

β-galactosidase gene

Induce with IPTG

X-gal NOT hydrolysed

(white)

WHITE plaque

Fig. 6.14Principle of blue/white selection for the detection of recombinant vectors.

211 6.3 Cloning vectors