Techniques of modern gene technology
Gene technology involves considerable expertise, expensive laboratory equipment and some specialised scientific techniques, some of which are briefly outlined below.
Polymerase chain reaction (PCR) produces large amounts of a specific DNA fragment, providing the supply of DNA for insertion into another organism. An original piece of DNA is used as a template to make many copies. DNA polymerase is the enzyme responsible for making the copies of DNA, and the technique involves a chain reaction to produce a large amount of the copied DNA sequence.
Gel electrophoresis is a technique used to separate large biological molecules, including proteins or fragments of DNA. It helps scientists identify genes, or proteins, based on their size. The molecules move through an electric field in a gel, much like dessert jelly, at different speeds according to their size. Smaller molecules will move faster than large ones. This allows DNA segments of different sizes to be separated from each other. This technique is also used to produce the DNA ‘fingerprints’ that are often used in forensic science.
Blotting is a technique for isolating and identifying individual DNA molecules. Once DNA molecules have been separated by gel electrophoresis, a special absorbent material is placed on top of the gel where it picks up DNA molecules in the same way as blotting paper soaks up ink. Once the DNA molecules are on the blot, it is possible to probe them using labelled DNA to identify a specific gene or sub-set of DNA molecules.
Restriction enzymes and ligases are naturally-occurring enzymes used to cut and join pieces of DNA respectively. There is a whole family of restriction enzymes, which can be thought of as ‘DNA scissors’. Each one cuts DNA at a specific, known place. DNA ligase is used to rejoin the DNA after cutting. DNA ligase can be thought of as ‘DNA glue’. By cutting and rejoining the DNA, a specific gene can be transferred into an existing DNA sequence.
Gene insertion involves the insertion of new genes into the cell’s existing genetic material. Different methods are used to transfer genes into different living things.
In animals, the desired gene can be inserted by injecting the gene into a single-celled embryo. This embryo is then allowed to develop into an adult animal. This technique is called microinjection.
In plants, the gene of interest can be coated onto tiny metal particles which are then shot into the cell using a special gun. A second method uses bacteria, usually one from the Agrobacterium family as they have a natural ability to infect plant cells and incorporate the bacterial DNA into the plant cell. Scientists can add the desired gene to the DNA of the bacteria, which then enter the cells of the plant, transporting the gene in the process. The gene integrates into the DNA of the plant cell. This added or foreign gene is called a transgene. If you would like to know brief information about membrane protein isolation you could choose here.
With both techniques, the place where the transgene inserts and the number of insertion events are impossible to predict. The unpredictable nature of the transgene’s insertion can be a cause for concern. Although an inserted gene may successfully function, its random insertion may have disrupted an existing complex of genes. Insertion, and the methods used to achieve it, can delete sections of existing DNA, or cause the addition of superfluous DNA. Thus effects on the plant other than that intended by the addition of the transgene may occur. These effects are called pleiotropic effects.
Plants have the interesting ability – under the right conditions – to develop from a single cell taken from an adult plant. Growing plants in this way is called regeneration and requires techniques known as tissue culture. Plant cells containing an added transgene that is stable and functioning are grown using tissue culture until they develop into a whole plant. This plant will then produce seed containing the added gene, and the seeds can be used just like conventional seeds, to produce more transgenic plants.
For both animals and plants, the chance of new DNA becoming permanently fixed into the organism’s existing DNA is relatively low. For this reason, scientists expose many cells to the gene and then select those that have successfully taken up the new gene. Knowing which cells contain the inserted gene is made easier by the use of ‘markers’, explained below.
Marker genes/proteins are used to keep track of inserted genes. As gene insertion is rather ‘hit or miss’, scientists usually insert a marker gene along with the gene being transferred. A marker gene is chosen to produce an easily detectable product or effect, but one which is neutral in terms of the organism’s function. By detecting the effects of the marker gene or the protein it produces, scientists know when the inserted DNA has been incorporated.
Marker genes therefore enable researchers to select among cells for the ones which have successfully received the new gene. Commonly used marker genes are those which give the cell the ability to withstand treatment with a chemical, such as an antibiotic or a herbicide. Scientists will treat all the experimental cells with the antibiotic or herbicide; only those that have received the gene for resistance will survive. Other marker genes may make the cell turn a particular colour or glow when exposed to ultra-violet light.
There have been serious concerns about the use of antibiotic resistance genes or herbicide resistance genes as markers, because of the risk of such genes spreading. If disease-causing bacteria acquire these genes, then the relevant antibiotic will not kill them. The same applies to plant weeds acquiring herbicide tolerance. Other marker genes include genes which enable a cell to use specific food sources that it would not normally be able to digest, or that allow cells to survive in the absence of particular growth additives.
Marker genes are tools that are designed for use only in the research stages of development of a genetically modified organism. New techniques are also being used which can ensure markers are removed once their job is complete.