Genetics Engineering Genetic engineering is an umbrella term that can cover a wide range of ways of changing the genetic material — the DNA code — in a living organism. This code contains all the information, stored in a long chain chemical molecule, which determines the nature of the organism. Apart from identical twins, genetic make-up is unique to each individual. Individual genes are particular sections of this chain, spaced out along it, which determine the characteristics and functions of our body. Defects of individual genes can cause a malfunction in the metabolism of the body, and are the roots of many “genetic” diseases.
In a sense, man has been using genetic engineering for thousands of years. We weren’t changing DNA molecules directly, but we were guiding the selection of genes. For example the domestication of plants and animals. Recombinant DNA technology is the newest form of genetic engineering, which involves the manipulation of DNA on the molecular level. This is a totally new process based on the science of molecular biology, a relatively new science only forty years old. It represents a major increase in our ability to improve life.
But a negative aspect is that it changes the forms of life we know of, possibly damaging our environment It has been known for some time that genetic information can be transferred between micro-organisms. This is process it done via plasmids (small circular rings of DNA) or phages (bacterial viruses). Both of these are termed vectors, this is because of their ability to move genetic material. In general this is limited to simpler species of bacteria. nevertheless, this can restriction can be overcome with the use of genetic engineering because it allows the introduction of any gene. While genetic engineering is beginning to be used to produce enzymes, the technology itself also depends on the harnessing of enzymes, which are available in nature.
In the early 1970s Herbert Boyer, working at the University of California Health Science Centre in San Francisco, and Stanley Cohen at Stanford University found that it was possible to insert into bacteria genes they had removed from other bacteria. First they learned the trick of breaking down the DNA of a donor organism into manageable fragments. Second, they discovered how to place such genes into a vector, which they used to ferry the fragments of DNA into recipient bacteria. Once inside its new host, a transported gene divided as the cell divided, leading to a clone of cells, each containing exact copies of the gene. This technique became known as gene cloning, and was followed by the selection of recipient cells containing the desired gene.
The enzymes used for cleaving out the DNA pieces act in a highly specific way. Genes can, therefore, be removed and transferred from one organism to another with extraordinary precision. Such manoeuvres contrast sharply with the much less predictable gene transfers that occur in nature. By mobilising pieces of DNA in this way (including copies of human genes), genetic engineers are now fabricating genetically modified microbes for a wide range of applications in industry, medicine and agriculture. The underlying idea of transferring genes between cells is quickly explained. However the actual practice is an extremely complicated process.
The scale of the problem can be gauged from the astronomical numbers involved: the DNA of even the simplest bacterium contains 4,800,00 pairs of bases. But there is only one copy of each gene in each cell. First, restriction enzymes are used to snip the DNA into smaller pieces, each containing one or just a few genes. These enzymes cut DNA in very precise ways. They recognise particular stretches of bases (termed recognition sequences) and snip each strand of the double helix at a particular place. Whenever the recognition sequence appears in the long DNA chain, the enzyme makes a cut. Whenever the same enzymes are used to break up a certain piece of DNA, they always produce the same set of fragments.
The cuts produce pieces of double helix with short stretches of single stranded DNA at each end. These are know as sticky ends. If the enzyme is allowed to act for a limited time, it may not have a chance to attack all the recognition sequences in the chain. This will result in longer fragments. As in natural DNA replication, bases have an inherent propensity to join up with their partners A with T, for example, and G with C.
So too with sticky ends. For example, the sequence TTAA will tend to re-associate with AATT. Genetic engineers use another type of enzyme, DNA ligase, to make the union permanent. This is the key principle of genetic engineering the use of two types of enzyme to cut out one piece of DNA and then to attach it to another piece. The genetic engineer’s toolkit now contains several hundred different restriction enzymes.
Each is a precision instrument for fragmenting DNA in a particular way. Some recognise different base sequences; others recognise the same sequence but snip at a different point within or next to the sequence. Ferrying DNA to a new home Once a piece of DNA has been broken up into a mixture of fragments, these can be separated into different sized pieces. The next stage is to insert a particular DNA fragment into a vector. Often this is a plasmid, a selfreplicating circular piece of DNA that can become incorporated in the bacterial nucleus and later become detached, carrying genes with it. Plasmids seem to have evolved as a natural mechanism for moving genes around among bacteria. To insert a DNA fragment into a vector, the genetic engineer first splits open the plasmid by adding the same restriction enzyme that was used to release the DNA fragment from the DNA of the donor organism. This creates sticky ends complementary to those on the fragment to be transplanted.
The fragment thus fits neatly into the gap in the vector DNA, where it is firmly annealed by DNA ligase. Next, the plasmid is allowed to infect a bacterium, in which it can replicate. Once inside, the vector and thus the foreign gene replicates every time the cell divides. As bacteria divide about once every 20 minutes, gene cloning can lead to a billionfold increase in the number of copies of a particular gene within 10 hours or so. The bacterium simply treats it and replicates it as part of its own DNA.
Not all the countless cells in a culture of bacteria become infected when a vector is added. One method of distinguishing those that do contain the vector is to incorporate into it a gene that confers resistance to a particular antibiotic. When the bacteria are cultured later, that antibiotic is included in the nutrient medium to inhibit any non resistant organisms. Only bacteria that have taken up the vector (and thus the resistance gene) are able to grow. A similar trick distinguishes bacteria carrying the vector plus a new gene from unwanted ones containing the unaltered vector. By using a variety of restriction enzymes to cut up DNA into manageable pieces, and then cloning these sequences, it is possible to create a DNA library a collection of sequences carrying all the genetic information in a particular organism. But much of this information is not expressed at any particular moment.
Genetic engineers are usually interested only in the genes that are actually functioning at any one time for example, one responsible for producing a specific enzyme. The DNA that codes for hereditary messages specifying current activities of this sort is much smaller in quantity than the total DNA in a cell. This information is to be found in messenger RNA. An enzyme called reverse transcriptase allows its messages to be translated into DNA. This copy DNA (cDNA) is then cloned into bacteria, giving a library, much smaller than that of a cell’s total DNA, that will certainly contain the desired gene. But this still leaves the final challenge of locating the specific bacteria containing the spliced gene.
One method is to spread the bacteria infected with the vector onto a nutrient medium, on which each individual cell can spawn millions of progeny and thus appear as a visible colony. The genetic engineer also needs to know the amino acid sequence of the protein coded by the gene. By following the genetic code, a corresponding stretch of RNA can now be synthesised chemically. During the synthesis, radioactive atoms are incorporated into the RNA, making a gene probe. The next step is to make, on special filter paper, a replica of the plate with the colonies of the cloned bacteria. Treated with caustic soda, the bacteria burst open and release their DNA, which is also broken into single strands that stick to the filter. The gene probe is now added. If the correct sequence is present, the probe will pair tenaciously with it.
The filter is now washed to remove the u …