Methods of Genetic Modification
The double-helix structure of DNA allows modification of plasmids to take place.
Genetic modification involves genetic engineering, also known as gene splicing, a technique to splice together DNA fragments from more than one organism and thus preparing a «recombinant» DNA molecule in a test tube, producing a single piece of genetic material containing the original information from multiple fragments which can then be inserted into another organism. This is achieved by cutting up DNA molecules with restriction enzymes and splicing these fragments together using DNA ligase. A transgenic organism that contains such DNA sequences from н foreign organism integrated into its own genome, the term «transgenic» literally means across gene. A mouse or fish engineered to express the green fluorescence protein, for example, would be considered a transgenic organism, since the gene coding for the protein originated from a species of jellyfish.
With current technology, transgenic organisms can be produced with only a very small proportion of extraneous DNA For example, the genome of most mammals contains three billion basepairs of DNA, while it becomes relatively difficult to insert more than 10,000 to 20,000 basepairs of foreign DNA More sophisticated techniques using yeast artificial chromosomes and bacterial artificial chromosomes allow insertions of up to 320,000 basepairs - approximately 0,01 % of the total genome. In concept, multiple rounds of transgenesis or interbreeding of transgenics could lead to organisms with a higher proportion of foreign DNA but cost and time соnsiderations prevent this.
In order to introduce new DNA into the receiving host, vectors are used. Vectors range from small circular pieces of DNA such as plasmids, to various viruses that can carry and transmit genetic information. Three processes are known by which the genetic composition of bacteria can be altered.
Transformation is a process by which some bacteria are naturally capable of taking up DNA to acquire new genetic traits. This phenomenon was discovered in cows by Frederick Griffith in 1928, although the fact that it was specifically DNA molecules that carried the genetic information was not proven until 1944. Bacteria that are competent to undergo transformation are frequently used in molecular biology. The foreign DNA uptake is facilitated by the presence of certain cations, such as Сa2+, or by the use of electric current (electroporation). Transformation does not normally integrate new DNA into the bacterial chromosome. Instead, it remains on a plasmid.
In conjugation, DNA is transferred from one bacterium to another via a temporary connecting tube of protein called a pilus (a process analogous to but biologically distinct from mating). A plasmid is transferred through the pilus. Conjugation is not widely used for the artificial genetic modification of bacteria, but happens often in nature.
Transduction refers to the introduction of new DNA into a bacterial cell by a bacteriophage, a virus that infects bacteria.
In order to gain knowledge about a particular gene’s function, researchers often use knock out organisms. These organisms have a specific gene that has been functionally destroyed or «knocked out.» They are used extensively in disease research with model organisms. For example, when investigating the cause of cystic fibrosis, researchers identified the CFTR gene as a likely candidate for the disease, found t lie mouse equivalent, bred a mouse with this gene «knocked out», and noted that the knockout mouse also had cystic fibrosis.