Gene technologyEdexcel A-Level Biology B: Revision notes

Recombinant DNA is DNA that contains genes from two different sources. It is made in four steps:

1. A restriction endonuclease cuts the DNA at a specific recognition sequence, isolating the gene. Many enzymes cut unevenly to leave single-stranded sticky ends.
2. The same enzyme cuts the vector (for example a plasmid), so its sticky ends are complementary to those of the gene.
3. The gene and vector are mixed and the sticky ends anneal by complementary base pairing.
4. DNA ligase forms phosphodiester bonds to join the sugar-phosphate backbones.

The recombinant plasmid can then be inserted into bacteria, which make the protein, for example human insulin.

A vector carries the recombinant DNA into a host cell.

• Plasmids: small circular DNA from bacteria, taken up by bacteria.
• Viruses: modified so that they cannot cause disease but still insert their DNA, with the new gene, into host cells (for example in gene therapy).
• Gene guns: tiny particles of gold or tungsten coated with the DNA are fired into plant cells, where the DNA can enter the nucleus and be incorporated into a chromosome.

Only a small proportion of the cells take up the DNA, so the cells that have done so must be identified.

A plasmid can carry two antibiotic resistance genes, for example for ampicillin and tetracycline. The human gene is inserted into the middle of the tetracycline resistance gene, which is then inactivated.

1. Bacteria are spread on agar containing ampicillin (the master plate). Only bacteria that took up a plasmid, recombinant or not, grow.
2. A velvet pad copies the colonies, in the same positions, on to agar containing tetracycline (replica plating).
3. Colonies that grow on ampicillin but not on tetracycline contain recombinant plasmids. Colonies that grow on both have non-recombinant plasmids.
4. The recombinant colonies are picked from the master plate.

A knockout mouse has had a gene inactivated (knocked out) in all its cells. Comparing its phenotype with a normal (wild type) mouse shows what the gene normally does. Mice are used as an animal model because they are mammals with genes similar to humans, breed quickly and are small.

Limitations: mice are not humans, so the gene may act differently; other genes may compensate; a gene may have several functions; and the knockout may be lethal before birth. There are also ethical issues about animal suffering and the use of animals, which is why such work is regulated.

Soya beans have been genetically modified for two main reasons.

• Improving production: a bacterial gene for herbicide tolerance is inserted, often with a gene gun. The crop survives spraying but weeds are killed, so there is less competition and a higher yield.
• Improving quality: natural soya oil has many polyunsaturated fatty acids, which are easily oxidised and make products go rancid. The gene for the enzyme converting oleic acid to polyunsaturated acids is silenced, giving a higher proportion of the monounsaturated oleic acid, which is more stable, so the oil lasts longer.

Widespread use of genetic modification in major crops and other transgenic organisms is debated.

Advantages: higher yields to feed a growing population, less use of pesticides or fewer cultivations, longer shelf life, improved nutrition and use of marginal land.

Disadvantages: gene flow to wild relatives and weeds producing herbicide-resistant weeds, effects on non-target organisms and biodiversity, expensive patented seed that makes farmers dependent on companies, and concern about eating GM food.

Examiners expect balanced, reasoned points and a conclusion supported by them.