Mutation and propagation
Genetic change can be brought about by using wild type organisms with their default DNA and inducing mutation e.g. through physiochemical routes like exposure to UV radiation or mutagenic chemicals; selective breeding to bring out recessive characteristics or emphasise particular traits; and culture of recombinant DNA that introduces artificial elements to the existing DNA, e.g. new sequences taken from a different species, an inhibitory sequence that switches off an existing function, etc.
Bacterial species can readily exchange DNA between them, as well as assimilate it from their environment. Fungi can undergo sexual reproduction to produce offspring with potentially new genotypes and phenotypes.
Culturing newly made or found mutants isn’t necessarily easy, due to their genetic instability. If introduced to a continuous culture, they may revert back to their wild type.
Say we are interested in the gene for insulin. Sure, we could take it straight from people, but remember humans are eukaryotes and eukaryotes have non-coding sequences within their genes called introns. The mRNA following splicing, on the other hand, has no introns! How can we make DNA from mRNA?
Take this arbitrary bit of mRNA: UCCAUGCCAUUUGGG
If we had an enzyme which could reverse the transcription back into DNA, this time intron-free, that would be great. We do – it’s called reverse transcriptase and it produces DNA. This special case of DNA is called complementary DNA – cDNA.
cDNA via reverse transcriptase: AGGTACGGTAAACCC (remember that DNA unlike mRNA is double-stranded; not shown for simplicity)
If we wanted the portion after the second G above, is there a way we could cut the DNA? It appears so. Some microorganisms have actually evolved enzymes whose job it is to invade a host and chop its DNA up at specific sequences. These enzymes are called restriction endonucleases. Each has its own short sequence which it recognises. There is a restriction endonuclease called CviQI which has the recognition site GTAC and cuts between G and T. That fits our bill!
The other DNA strand will also have a GTAC site read in the opposite direction. Notice that the complementary sequence of GTAC backwards (starting from the C) is… GTAC! This is called a palindrome and all restriction endonucleases will have one simply by the virtue of DNA bases being complementary.
DNA samples can be amplified by PCR to make more of it, before further processing in experiments or applications. Larger fragments of DNA can be amplified directly in bacteria, as they grow quickly and are easy to handle.
In vivo (inside a living organism) gene cloning involves stimulating bacteria to take up target DNA by inserting it in a circular bit of DNA they normally carry beside their “main” DNA, called a plasmid. This is often transferred horizontally between bacteria, and always passed down through the generations. Target DNA may be inserted in plasmids via PCR when restriction endonuclease leave sticky ends which can be joined back with the aid of the enzyme DNA ligase which catalyses the reaction between the sugar-phosphate groups to form phosphodiester bonds.
Plasmids also contain an antibiotic resistance gene which, if taken up successfully by bacteria, will enable their growth on a medium containing that antibiotic. This allows the selection of only bacteria which have taken up the plasmid (vector), and with it our DNA of interest.
The host cells (bacteria) can now be grown on a large scale. They will express the new DNA in the plasmid, and pass it on to their offspring cells to do the same. Shortly, there will be a massive number of bacteria producing whatever the gene of interest codes for. This could be human insulin.
The product can then be isolated and used. It is in a pure form and is in fact being used worldwide to treat Type I diabetes. This breakthrough enabled better management of the condition which had previously been treated with non-human insulin which had side effects.
Many elements can be added to a plasmid, and a few of them are standard.
Restriction sites enable the easy addition of new genes as flanked by them. A promoter indicates the start of transcription for the sequence of DNA upstream of itself. The aforementioned antibiotic resistance gene enables selection of microorganisms with this plasmid by exposure to antibiotic and observing the ones that grew regardless, due to having the resistance gene contained on the plasmid.
The origin of replication indicates the point where the plasmid itself begins its own replication. The selectable marker can be used alternatively to the antibiotic resistance gene as a form of selection, and could be any sequence that the bacterium cannot survive without. It can be more effective than the antibiotic resistance gene in the event that antibiotic-based selection reaches a point where even bacteria without this plasmid are able to mount a resistant response, hence diluting the power of this selection method.
As a precaution against unforeseen effects of genetic modification, bacteria used in research contain genes that render them unable to survive in the outside world.
Since bacteria are prokaryotic and many genes introduced for them to process are of human sequence, processes that are exclusive to eukaryotes, e.g. post-translational modification of proteins, may not be successful in bacteria. This is why an alternative cultured microorganism for these purposes is the yeast Pichia pastoris, a eukaryote. This can also improve the folding of polypeptides.