Tools of recombinant DNA technology

The essentials

Restriction enzymes belong to the class of nucleases. Exonucleases remove nucleotides from the ends of DNA, while endonucleases cut at specific positions within DNA. The first restriction endonuclease was Hind II, which cuts DNA at a particular sequence of six base pairs. Each restriction endonuclease recognises a specific palindromic nucleotide sequence: a sequence that reads the same on the two strands when read in the same direction (for example 5′-GAATTC-3′ and 3′-CTTAAG-5′ for EcoRI). Many enzymes cut slightly away from the centre of the palindrome, leaving single-stranded overhangs called sticky ends, which form hydrogen bonds with complementary cut ends. DNA ligase then seals the joints.

Naming rule for EcoRI: E = genus Escherichia, co = species coli, R = strain RY13, I = the first enzyme isolated from that strain.

Cut DNA fragments are separated by agarose gel electrophoresis. DNA is negatively charged, so it moves towards the anode; smaller fragments move farther. Separated DNA is stained with ethidium bromide and seen as orange bands under UV light. Bands are cut out and the DNA extracted (elution).

Cloning vectors such as plasmids and bacteriophages carry foreign DNA. Essential features:

• Ori: controls replication and copy number.
• Selectable marker: helps pick transformants; for example genes for resistance to ampicillin, chloramphenicol, tetracycline or kanamycin.
• Cloning (recognition) sites: preferably a single site for the chosen enzyme. In pBR322, inserting DNA at the BamH I site inactivates the tetracycline-resistance gene, so recombinants grow on ampicillin but not on tetracycline (insertional inactivation). An alternative is blue–white selection: insertion into the gene for β-galactosidase gives colourless colonies on a chromogenic substrate, while non-recombinants are blue.
• Plant and animal vectors: the Ti plasmid of Agrobacterium tumefaciens (disarmed so it no longer causes tumours) and disarmed retroviruses.

Competent hosts: DNA is hydrophilic and cannot cross membranes easily. Bacterial cells are made competent by treating them with a divalent cation such as calcium, then incubating on ice, giving a brief heat shock at 42 °C and returning to ice. Other methods: microinjection into an animal nucleus, the gene gun (biolistics) for plants, and disarmed pathogen vectors.