Restriction enzymes

Recombinant DNA technology needs these key tools: restriction enzymes, polymerase enzymes, ligases, vectors and a host organism.

In 1963 two enzymes were isolated that restrict the growth of bacteriophage in E. coli. One adds methyl groups to DNA and the other cuts DNA; the cutting one was named restriction endonuclease.

Five years later Hind II was isolated. It was the first restriction endonuclease whose action depended on a specific nucleotide sequence. It always cuts at a particular point, recognising a specific six-base-pair recognition sequence.

More than 900 restriction enzymes are now known, isolated from over 230 bacterial strains, and each recognises a different sequence.

Naming: the first letter comes from the genus and the next two from the species of the source prokaryote. EcoRI comes from Escherichia coli RY 13: 'R' is from the strain name, and the Roman numeral gives the order in which enzymes were isolated from that strain.

Restriction enzymes are nucleases. Exonucleases remove nucleotides from the ends of DNA; endonucleases cut at specific positions within it.

An endonuclease scans the DNA for its recognition sequence, binds there, and cuts both strands at specific points in their sugar-phosphate backbones. Each recognises a specific palindromic sequence.

A DNA palindrome reads the same on both strands when both are read in the same orientation. For example, 5'-GAATTC-3' pairs with 3'-CTTAAG-5'.

The cut falls a little away from the centre of the palindrome, between the same two bases on each strand, leaving single-stranded overhangs called sticky ends. They hydrogen-bond with complementary cut ends, which helps DNA ligase join them.

Source DNA and vector must be cut with the same restriction enzyme. The fragments then carry matching sticky ends that ligase can join end to end, forming recombinant DNA from different genomes.