Enzymes Used in Molecular Biology

The discovery and characterisation of a number of key enzymes have permitted the development of various techniques for the analysis and manipulation of DNA. In particular, the enzymes termed type II restriction endonucleases have come to play a key role in all aspects of molecular biology. These enzymes recognise specific DNA sequences, usually 4–6 base pairs (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 (Figure 1).
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 (Table 1). 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. This is widely exploited in molecular biology to allow the construction of recombinant DNA, i.e. the joining of DNA fragments from different sources.

Figure 1. Examples of digestion of DNA by restriction endonucleases. The upper panel indicates the result of a restriction digestion forming blunt fragments with the enzyme HindIII. The bottom panel indicates the cohesive fragments produced by digestion with the enzyme EcoR1.

Approximately 500 restriction enzymes have been characterised that recognise over 100 different target sequences. A number of these, termed isoschizomers, recognise different target sequences but produce the same staggered ends or overhangs. A number of other enzymes have proved to be of value in the manipulation of DNA, as summarised in Table 2, and are indicated at appropriate points within the text.

Table 1. Examples of restriction endonucleases that recognise different target
sequences and the resulting fragments following digestion.

Table 2. Comparison of the various labelling methods for DNA.