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molecules, termedreannealing. All these pairings are transient, owing to the weak-

ness of hydrogen bonding between the few bases in the sticky ends, but they can be

stabilised by use of an enzyme termedDNA ligasein a process termedligation. This

enzyme, usually isolated from bacteriophage T4 and termedT4 DNA ligase, forms a

covalent bond between the 5^0 phosphate at the end of one strand and the 3^0 hydroxyl

of the adjacent strand (Fig. 6.1). The reaction, which is ATP dependent, is often carried

out at 10C to lower the kinetic energy of molecules, and so reduce the chances of

base-paired sticky ends parting before they have been stabilised by ligation. However,

long reaction times are needed to compensate for the low activity of DNA ligase in the

cold. It is also possible to join blunt ends of DNA molecules, although the efficiency of

this reaction is much lower than sticky-ended ligations.

Since ligation reconstructs the site of cleavage, recombinant molecules produced by

ligation of sticky ends can be cleaved again at the ‘joins’, using the same restriction

enzyme that was used to generate the fragments initially. In order to propagate

digested DNA from an organism it is necessary to join or ligate that DNA with

Table 6.1Numbers of clones required for representation of DNA

in a genome library

No. of clones required

Species Genome size (kb) 17 kb fragments 35 kb fragments

Bacteria (E. coli) 4 000 700 340

Yeast 20 000 3 500 1 700

Fruit fly 165 000 29 000 14 500

Man 3 000 000 535 000 258 250

Maize 15 000 000 2 700 000 1 350 000

Fragments produced by cleavage with BamHI

5 pGATCC

3  G

G

CCTAGp

pGATCC

G

G 3 

CCTAGp 5

GGATCC

CCTAGG

5  pGATCC

3  G

G 3 

CCTAGp 5

DNA ligase + ATP

Fig. 6.1Ligation molecules with cohesive ends. Complementary cohesive ends base-pair, forming a temporary

link between two DNA fragments. This association of fragments is stabilised by the formation of 3’ to 5’

phosphodiester linkages between cohesive ends, a reaction catalysed by DNA ligase.

197 6.2 Constructing gene libraries