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initiated far more frequently than chromosomal replication. Hence a large number

of plasmid molecules will be produced per cell.

• Two genes coding for resistance to antibiotics have been introduced. One of these
  allows the selection of cells which contain plasmid: if cells are plated on medium
  containing an appropriate antibiotic, only those that contain plasmid will grow to
  form colonies. The other resistance gene can be used, as described below, for
  detection of those plasmids that contain inserted DNA.


• There are single recognition sites for a number of restriction enzymes at various
  points around the plasmid, which can be used to open or linearise the circular
  plasmid. Linearising a plasmid allows a fragment of DNA to be inserted and the
  circle closed. The variety of sites not only makes it easier to find a restriction
  enzyme that is suitable for both the vector and the foreign DNA to be inserted, but,
  since some of the sites are placed within an antibiotic resistance gene, the presence
  of an insert can be detected by loss of resistance to that antibiotic. This is termed
  insertional inactivation.

Insertional inactivation is a useful selection method for identifying recombinant

vectors with inserts. For example, a fragment of chromosomal DNA digested with

BamH1 would be isolated and purified. The plasmid pBR322 would also be digested at

a single site, usingBamH1, and both samples would then be deproteinised to inacti-

vate the restriction enzyme.BamH1 cleaves to give sticky ends, and so it is possible to

obtain ligation between the plasmid and digested DNA fragments in the presence of T4

DNA ligase. The products of this ligation will include plasmid containing a single

fragment of the DNA as an insert, but there will also be unwanted products, such as

BamHI

SphI

SalI

HindIII

EcoRI

SspI

ScaI

PstI

Ampicillin

resistance gene Tetracycline

resistance gene

Origin of

replication NdeI PuvII

pBR322

4.36 kb

ORI

ApR TcR

Fig. 6.11Map and important features of pBR322.

208 Recombinant DNA and genetic analysis