Over the past 20 years, genetic engineering has been revolutionised by a new technique known as recombinant DNA with which scientists can directly alter genetic material. In recombinant DNA, the DNA of one organism is joined to that of a second organism to produce a recombinant DNA. When this recombinant DNA is introduced into another organism, it permanently changes the genetic makeup of that organism and all its descendants. The required fragment of DNA, cut from the chromosome of one organism using an enzyme (endonuclease) must be pasted to a 'cloning vehicle', another piece of DNA which will carry it into the host cell, using another enzyme (ligase). This is where plasmids come into play.
Plasmids are small circular pieces of DNA found in certain bacteria. They are separate from the bulk of the DNA and are automomous, i.e. can replicate independently from the rest of the DNA. They confer properties such as antibiotic resistance and sugar fermentation. They are used as cloning vehicles in the pharmaceutical industry:
Cloning vehicles
To act as a cloning vehicle, a DNA molecule must be capable of entering the host and once inside, replicate to produce multiple copies of itself. Two naturally occurring vehicles are plasmids and virus chromosomes. In practice, genes to be inserted into bacteria are first recombined into a plasmid. Because plasmid DNA is so much smaller than even highly fragmented chromosomal DNA, it is easily separable, and easily purified plasmid DNA is easily obtained. In the laboratory, when plasmid DNA is added to plasmid-free bacteria, in the presence of Ca2+, the DNA is taken up to yield bacteria that will soon contain many copies of the plasmid. In general, a given bacterial cell usually harbours only one form of the plasmid. The number of copies of a plasmid in a host cell depends on the genetic constitution of the plasmid and cell. So-called relaxed-control plasmids may multiply until each cell has on average 10 to 200 copies of the plasmid. In contrast, stringent-control plasmids replicate at about the same rate as the cell's main chromosome and are present in only one or a few copies per cell. Therefore, to maximise yield, relaxed plasmids are the ones used in recombinant DNA technology. A large number of daughter cells are cultured and their gene products extracted for human use. In certain cases, it is even possible to make the bacteria secrete the products in the culture medium. This greatly simplifies the extraction and purification process.
