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asymmetric they are similar to restriction sites and allow the phage DNA to be

circularised. Phage may be replicated very efficiently in this way, the result of which

are concatemers of many phage genomes which are cleaved at the cos sites and

inserted into newly formed phage protein heads.

Much use of phagelhas been made in the production of gene libraries mainly

because of its efficient entry into theE. colicell and the fact that larger fragments of

DNA may be stably integrated. For the cloning of long DNA fragments, up to

approximately 25 kb, much of the non-essentiallDNA that codes for the lysogenic

life cycle is removed and replaced by the foreign DNA insert. The recombinant phage

is then assembled into pre-formed viral protein particles, a process termedin vitro

packaging. These newly formed phage are used to infect bacterial cells that have been

plated out on agar (Fig. 6.16).

Once inside the host cells, the recombinant viral DNA is replicated. All the genes

needed for normal lytic growth are still present in the phage DNA, and so multipli-

cation of the virus takes place by cycles of cell lysis and infection of surrounding cells,

giving rise to plaques of lysed cells on a background, orlawn, of bacterial cells. The

viral DNA including the cloned foreign DNA can be recovered from the viruses from

these plaques and analysed further by restriction mapping (Section 5.9.1) and agarose

gel electrophoresis (Section 5.7.4).

In general two types oflphage vectors have been developed,linsertion vectors

andlreplacement vectors(Fig. 6.17). Thelinsertion vectors accept less DNA than

the replacement type since the foreign DNA is merely inserted into a region of the

phage genome with appropriate restriction sites; common examples arelgt10 and

lcharon16A. With a replacement vector a central region of DNA not essential for lytic

growth is removed (a stuffer fragment) by a double digestion with for exampleEcoRI

andBamHI. This leaves two DNA fragments termed right and left arms. The central

stuffer fragment is replaced by inserting foreign DNA between the arms to form a

functional recombinantlphage. The most notable examples oflreplacement vectors

arelEMBL andlZap.

In Vitro Packaging

Single Strain Mix Double Strain Mix

λ virus has defective cos sites

therefore lost ability for

viral packaging

λ virus strain produces incomplete

capsid protein E, the other produces

defective capsid protein D

Capsid protein isolated Capsid proteins isolated

Fig. 6.16Two strategies for producingin vitropackaging extracts for bacteriophagel.

213 6.3 Cloning vectors