Cutting the gene out of the DNA

1. The first step is to find which chromosome carries the required gene and where along that chromosome it lies, so that the correct length of DNA is taken.
2. A restriction enzyme is then used to cut the DNA, and each restriction enzyme cuts only where it finds one specific sequence of bases called its recognition site.
3. Because the enzyme cuts at a fixed sequence, the same gene is cut out in the same place every time, which is what makes the process repeatable.
4. The cut is staggered, so the two strands of the double helix are cut a few bases apart rather than straight across.
5. That leaves a short length of unpaired bases on each end of the cut piece, and these are the sticky ends.
6. Sticky ends matter because unpaired bases can form hydrogen bonds with a complementary set of unpaired bases, so the gene can later be fitted into another piece of DNA.

Cutting open the vector

1. A vector is a piece of DNA used to carry a gene into a host cell, and the two vectors used are plasmids and viruses.
2. A plasmid is a small circular loop of DNA found in a bacterial cell, separate from the single large bacterial chromosome.
3. Plasmids are convenient vectors because they are small, they are easy to take out of and put back into bacteria, and they are copied by the bacterium along with its own DNA.
4. A virus can be used instead, because a virus naturally injects its own genetic material into a host cell, and the required gene is carried in with it.
5. The plasmid is cut open using the same restriction enzyme that was used to cut out the gene.
6. Using the same enzyme is essential, because it leaves sticky ends on the plasmid whose unpaired bases are complementary to those on the gene, so the two fit together and no other fragment does.

Joining the gene into the vector

1. The cut gene and the cut plasmid are mixed, and the sticky ends pair up by complementary base pairing, with adenine pairing to thymine and cytosine pairing to guanine.
2. Base pairing alone holds the pieces together only weakly, because the sugar-phosphate backbone of each strand is still broken.
3. DNA ligase is added, and this enzyme seals those breaks so the gene becomes a permanent part of the circular plasmid.
4. The result is recombinant DNA, a single molecule made from the DNA of two different organisms.

Transferring the plasmid into host bacteria

1. The recombinant plasmids are mixed with host bacteria in a solution containing calcium ions, which makes the bacterial cell membrane more permeable.
2. The mixture is given a brief heat shock, and some of the bacteria take a plasmid in through the membrane, which is called transformation.
3. Only a small proportion of the bacteria take up a plasmid, so the mixture contains transformed and untransformed cells together.
4. Bacteria are used as hosts because they reproduce roughly every twenty minutes, so a few transformed cells become millions within a day, each carrying a copy of the gene.

Selecting the bacteria that took up the gene

1. A marker gene is added to the plasmid alongside the required gene before the transfer, and the marker used is usually a gene for resistance to a named antibiotic.
2. Every bacterium that takes up the plasmid therefore takes up the resistance gene as well, so it can survive that antibiotic while untransformed bacteria cannot.
3. All of the bacteria are spread over agar that contains the antibiotic, and the plates are left in a warm incubator.
4. Untransformed bacteria are killed by the antibiotic, while transformed bacteria divide repeatedly and each one grows into a visible colony.
5. Every colony that appears is therefore a clone of cells carrying the required gene, and those colonies are the ones taken forward.
6. The selected bacteria are then grown in large numbers and the protein they make is collected and purified.

Transferring genes into plants and animals

1. For a plant, the gene is usually carried in by a bacterium that naturally inserts part of its plasmid into plant cells, or it is fired into the cells on tiny metal particles.
2. A single transformed plant cell is then grown in a nutrient medium into a whole plant, and every cell of that plant carries the gene.
3. For an animal, the gene is inserted into a fertilised egg cell at a very early stage, before the cells have begun to specialise.
4. Because the modified cell divides by mitosis to form the whole organism, every cell receives a copy of the gene and the characteristic appears throughout the body.
5. A gene inserted into an adult organism would only be present in the cells that received it, so the whole organism would not show the characteristic.