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capacity). Vectors have in general been developed from naturally occurring molecules

such as bacterial plasmids, bacteriophages or combinations of the elements that make

them up, such ascosmids(Section 6.3.4). For gene library constructions there is a choice

and trade-off between various vector types, usually related to ease of the manipulations

needed to construct the library and the maximum size of foreign DNA insert of the

vector (Table 6.2). Thus, vectors with the advantage of large insert capacities are usually

more difficult to manipulate, although there are many more factors to be considered,

which are indicated in the following treatment of vector systems.

6.3.1 Plasmids

Many bacteria contain an extrachromosomal element of DNA, termed aplasmid,

which is a relatively small, covalently closed circular molecule, carrying genes for

antibiotic resistance, conjugation or the metabolism of ‘unusual’ substrates. Some

plasmids are replicated at a high rate by bacteria such asE. coliand so are excellent

potential vectors. In the early 1970s a number of natural plasmids were artificially

modified and constructed as cloning vectors, by a complex series of digestion and

ligation reactions. One of the most notable plasmids, termed pBR322 after its devel-

opers Bolivar and Rodriguez (pBR), was widely adopted and illustrates the desirable

features of a cloning vector as indicated below (Fig. 6.11).

• The plasmid is much smaller than a natural plasmid, which makes it more resistant
  to damage by shearing, and increases the efficiency of uptake by bacteria, a process
  termedtransformation.


• A bacterial origin of DNA replication ensures that the plasmid will be replicated by
  the host cell. Some replication origins display stringent regulation of replication, in
  which rounds of replication are initiated at the same frequency as cell division.
  Most plasmids, including pBR322, have a relaxed origin of replication, whose
  activity is not tightly linked to cell division, and so plasmid replication will be

Table 6.2Comparison of vectors generally available for cloning

DNA fragments

Vector Host cell Vector structure Insert range (kb)

M13 E. coli Circular virus 1–4

Plasmid E. coli Circular plasmid 1–5

Phagel E. coli Linear virus 2–25

Cosmids E. coli Circular plasmid 35–45

BACs E. coli Circular plasmid 50–300

YACs S. cerevisiae Linear chromosome 100–2000

Notes:BAC, bacterial artificial chromosome; YAC, yeast artificial chromosome.

207 6.3 Cloning vectors