Transferring the Gene
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Transferring the Gene
A vector is a carrier DNA molecule into which the desired gene can be inserted.
Most commonly, this vector is a plasmid. This is a small, extra-chromosomal, circular piece of DNA often found in bacteria in addition to their functional DNA.
The plasmids are modified so that they have two or more genes for resistance to antibiotics. They should also contain a sequence that can be recognised by the same restriction enzyme used to cut the fragments. The site that is cut should be in one of the genes for antibiotic resistance.
A Plasmid cut by Bam HI:
How the gene is introduced into the plasmid:
Cut the genome with a restriction enzyme (RE) and mix with the plasmid that has also been cut with the same R.E so that the sticky ends of the fragments and the plasmid are complementary. Hopefully, some fragments will insert into the plasmid DNA before either segment joins with itself.
The fragments are added to the plasmids with these possible outcomes:
- Plasmid rejoins, tetracycline gene now intact.
- Fragment joins with plasmid. Tetracycline resistance gene is interrupted; the fragment does not contain the desired gene.
- Fragment joins with plasmid. Tetracycline gene is interrupted; this time the fragment does contain the desired gene.
- The fragment joins with itself.
- How the recombinant cells are identified:
Some plasmids will now contain the recombinant DNA fragment.
Other plasmids, however will not contain a fragment. If the plasmids are recombinants then one of the antibiotic resistance genes (e.g, for tetracycline) will have been disrupted. However, the other gene for antibiotic resistance (e.g. for ampicillin) will still be intact.
- Add this mixture of recombinant and non-recombinant plasmids to bacteria. Some will take up the plasmids if they are in calcium chloride solution.
- The bacteria are transferred to a plate containing the antibiotic ampicillin. Those bacteria that have taken up any plasmid will be resistant to the antibiotic so will survive and form colonies. These bacteria will include recombinant and non-recombinant plasmids.
- These colonies are then replicated onto plates containing the antibiotic tetracycline. Those bacteria with recombinant plasmids will not survive because the fragment has disrupted the gene for resistance.
- The 2 plates are compared and those colonies resistant to ampicillin but not to tetracycline can be identified. All these colonies contain recombinant plasmids.
There is still one problem - how do you identify the recombinant plasmid with the desired gene in it?
You need to know the DNA base sequence of the gene for the desired protein so that a section of that base sequence can be radioactively labelled.
This section of DNA with the correct base sequence is called a probe.
The DNA in the bacteria is "unzipped" so that it becomes single stranded and a probe would anneal (attach) if there were complementary bases.
The probe is added and sticks to the correct complementary fragment. The correct fragment can now be identified, as it is radioactive.
All bacteria in a colony have been produced by the replication of one individual. They will all therefore have the same genes to produce the same proteins. This colony can then be isolated and multiplied so that the protein is synthesised and can be harvested for further use.
Bacteria can be used in this way to produce human insulin for diabetics who do not produce their own.