Genetic Engineering

 

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4   RECOMBINANT DNA TECHNOLOGY

 

        Escherichia coli (E. coli), has a) a well-defined genetics, b) reproduces itself once every twenty minutes, c) is easy to grow in the laboratory, and d) has many non-pathogenic strains. Consequently, E. coli has been a natural choice for experiments in molecular genetics and genetic engineering. The first object of this work was to insert DNA from one E. coli strain into another E. coli strain (gene cloning). The second object was to get this ‘cloned’ DNA to produce a functional protein. The next step was to repeat the process using eukaryotic DNA. Since the mechanisms of gene expression in prokaryotes and eukaryotes differed very considerably and a cell may not tolerate a foreign protein, the objectives may not be met for every gene.

        The actual process of gene cloning is comparatively straightforward. In E. coli (and in bacteria in general), a circular DNA molecule occurs, in addition to its regular genome, which is called the ‘plasmid’, and which is usually used as the `vector’, whereby the foreign DNA is introduced. The DNA being cloned is first cut up into small pieces using enzymes which break the DNA only at specific base sequences (restriction endonucleases). If the plasmid is also cut with the same enzyme at one site, its ring form is opened up into a linear configuration. If the plasmid is then mixed with the chopped foreign DNA in the presence of suitable enzymes to reconnect the DNA strands (ligases), the plasmid circle reforms incorporating a new piece. This process of recombining selected DNA pieces leads to `recombinant DNA’. When these plasmids which contain the cloned DNA are used to infect E. coli cells, they can then replicate themselves. A cell may contain many plasmids of various types and sometimes many copies of one type. The plasmids can also integrate into their host’s DNA which, in prokaryotes, is all in one long chromosome.

       E. coli are quite easy to work with in the laboratory but their cells do not secrete any of their proteins into the culture medium. Thus even if by clever manipulation one has cloned a gene and achieved expression of the protein intracellularly, the bacterial cells will have to be broken open before the product can be recovered, with the cloned protein having to be purified from all the other cellular proteins. For this reason, many genetic engineers use other organisms, such as Bacillus subtilis and yeasts which secrete some compounds. Now Agrobacterium tumefaciens is the much favoured organism in genetic manipulations. Animal and plant cell systems are also being developed using viruses as vectors to insert the cloned DNA.

       The purpose of most genetic engineering research has been to design bacteria that make biological products which are very rare and expensive as it is very difficult to extract them from eukaryotic cells. The best known examples are, a) insulin, formerly recovered from pig pancreas, b) human growth hormones, which used to be made from pituitary glands taken from cadavers, and c) rennin, a milk clotting enzyme used in cheesemaking but extracted from the stomachs of calves.

        It is now possible to use genetic engineering to correct some defects which arise from a single DNA mutation (such as thalassaemia) and to introduce new characteristics, such as drought and salt resistance, in plants. Another possibility is that certain specific proteins, perhaps useful enzymes, could be redesigned to make them more efficient or more thermostable. However, in spite of commendable progress, there are formidable technical hurdles to such work, which may sound simple but, in practice it is not. In addition, the moral, ethical and safety issues associated with genetic manipulation of whole organism are complex and will still require many years before they are resolved.

 

4 POLYMERASE CHAIN REACTION

 

        In order to study genetic structure and function, and to manipulate genes, adequate quantities of the DNA of interest must be available. Earlier, this was done using micro-organisms to generate multiple copies of specific gene sequences. Now this is done much more easily, using the cell-free technique called Polymerase Chain Reaction (PCR). PCR procedures are designed after the natural process of DNA synthesis by cells. PCR technique amplifies microgram quantities of DNA a million-fold in a day, through repeated cycles of the same process, directed by a primer DNA sample, using deoxyribonucleotide triphosphates in solution, in a thermal cycler, that maintains the high temperatures required to denature the DNA sample, before it can be amplified. The enzyme DNA polymerase is essential for DNA synthesis. DNA polymerase from normal sources cannot withstand the high temperatures of the PCR process, and so is denatured by the end of each cycle, when it had to be replenished. The discovery that the DNA polymerase from thermophilic organisms (that thrive only at high temperatures), particularly the hot spring bacteria, can withstand temperatures over 90 o C, has made the PCR technique immensely practicable. Taq-polymerase, from the bacterium Thermus aquaticus was the earlier favourite. But now two other thermophilic bacteria, Thermococcus littoralis and Pyrococcus furiosus, provide DNA polymerase that is even more thermostable than Taq-polymerase, and so are preferred.

        There are several variants and modifications on the basic PCR procedure, to suit different specific requirements. The PCR technique is now central to a very large number of procedures of molecular biology and genetic engineering, as is the use of restriction endonucleases.

        PCR procedures are impressively simple and efficient. But what is more amazing is the fact that this process occurs in vivo at ambient temperatures or the body temperature of the organism, while in vitro it requires temperatures closer to the boiling point of water.