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5.6 The manipulation of nucleic acids – basic tools and techniques

5.6.1 Enzymes used in molecular biology

The discovery and characterisation of a number of key enzymes has enabled the

development of various techniques for the analysis and manipulation of DNA. In

particular the enzymes termed type IIrestriction endonucleaseshave come to play a

key role in all aspects of molecular biology. These enzymes recognise certain DNA

sequences, usually 4–6 bp in length, and cleave them in a defined manner. The

sequences recognised are palindromic or of an inverted repeat nature. That is they read

the same in both directions on each strand. When cleaved they leave a flush-ended or

staggered (also termed a cohesive-ended) fragment depending on the particular enzyme

used (Fig. 5.20). An important property of staggered ends is that those produced from

different molecules by the same enzyme are complementary (or ‘sticky’) and so will

anneal to each other. The annealed strands are held together only by hydrogen bonding

between complementary bases on opposite strands. Covalent joining of ends on each of

the two strands may be brought about by the enzyme DNA ligase (Section 6.2.2). This is

widely exploited in molecular biology to enable the construction of recombinant DNA,

i.e. the joining of DNA fragments from different sources. Approximately 500 restriction

(a)Enzyme Recognition sequence Products

(b)

HpaII

HaeIII

BamHI

HpaI

EcoR I

HindIII

PvuII

BamHI

5 –CCGG–3

3 –GGCC–5

5 –GGCC–3

3 –CCGG–5

5 –GGATCC–3

3 –CCTAGG–5

5 –GTTAAC–3

3 –CAATTG–5

GAATTC

AAGCTT

CAGCTG

GGATCC

5 –C

3 –GGC

5 –GG

3 –CC

5 –G

3 –CCTAG

5 –GTT

3 –CAA

CGG–3

C–5

CC–3

GG–5

GATCC–3

G–5

AAC–3

TTG–5

Fig. 5.20Recognition sequences of some restriction enzymes showing (a) full descriptions and (b) conventional

representations. Arrows indicate positions of cleavage. Note that all the information in (a) can be derived

from knowledge of a single strand of the DNA, whereas in (b) only one strand is shown, drawn 5’ to 3’; this

is the conventional way of representing restriction sites.

162 Molecular biology, bioinformatics and basic techniques