Restriction enzymes and gels

Restriction enzymes are the 'molecular scissors'. Bacteria make them to defend themselves: they cut up the DNA of attacking viruses. The first one characterised was Hind II, which always cuts DNA at a particular site with a sequence of six base pairs: its recognition sequence. Today more than 900 restriction enzymes are known, from over 230 strains of bacteria.

They belong to a group called nucleases. Exonucleases remove nucleotides from the ends of DNA; endonucleases cut at specific positions within the DNA. Restriction enzymes are restriction endonucleases.

Naming follows a fixed rule. In EcoRI: E is the genus, Escherichia; co is the species, coli; R is the strain, RY 13; and I (Roman one) means it was the first enzyme isolated from that strain.

Each enzyme recognises a palindrome: a stretch of DNA that reads the same on both strands when read in the same direction (5'→3'). A word such as MALAYALAM reads the same forwards and backwards; for DNA, EcoRI's site reads 5'-GAATTC-3' on one strand and 5'-GAATTC-3' on the other (written 3'-CTTAAG-5' under it).

Many restriction enzymes cut a little away from the centre of the palindrome, between the same two bases on each strand. This leaves short single-stranded overhangs at each end, called sticky ends, because they can form hydrogen bonds with matching sticky ends.

This is the key to joining DNA: if the source DNA and the vector are cut with the same enzyme, their sticky ends match, and DNA ligase can join them into one recombinant DNA.

Gel electrophoresis separates and shows DNA fragments. DNA is negatively charged, so in an electric field it moves towards the anode (positive end) through a gel of agarose, a natural polymer extracted from seaweed. The gel acts like a sieve: smaller fragments move farther. The DNA is then stained with ethidium bromide and exposed to UV light, which makes it show up as bright orange bands. A wanted band can be cut out and the DNA extracted from the gel: this is elution.