Genetic engineering is considered as a kind of biotechnology. This is a process in which the alteration of the genetic make-up of cells is done by deliberate and artificial means.
This process involves transfer or replacement of genes to create recombinant DNA. This can be done by cutting DNA molecules at specific sites to get fragments containing desirable and useful genes from one type of cell.
Thereafter, these genes can be inserted into a suitable carrier or vector. Now, these recombinant DNA can be put into completely different cell of a bacterium or plant or animal cell. By this technique they acquire useful characteristics, such as disease resistance or to make useful enzymes, hormones, vaccines, etc.
Tools and Techniques of Genetic Engineering:
Genetic engineering involves cutting and pasting fragments of DNA may be cut and pasted in the vector, a plasmid or a virus whose DNA then becomes the recombinant DNA.
The three tools for recombinant DNA technology are as follows:
1. Cell culture containing cells:
Cell culture containing cells with the desired DNA segment (gene).
2. DNA modifying enzymes such as:
(a) Restriction endonuclease from various bacteria. It cleaves double stranded DNA but only at a limited number of sites
(b) DNA ligase from Escherichia coli which joins DNA molecules,
(c) DNA polymerase from E. coli which synthesises a new strand of DNA complementary to an existing DNA or RNA template.
DNA segment can be excised by “molecular scissors” or ‘chemical scalpels’ which are known as restriction enzymes in biotechnological language.
Restriction enzymes, synthesized by microbes as a defense mechanism, are specific endonucleases, which can cleave
However, they can do this only at limited number of sites depending on the number of recognition sequences in DNA.
With the use of restriction enzymes it is possible to cut a DNA sequence. For example, a restriction enzyme EcoRI will cut DNA only if sequence GAATC/CTTAG is present in the double stranded DNA. Hundreds of such restriction enzymes with different sequence specificities have been isolated from several bacteria and are commercially available.
These enzymes are known as restriction enzymes because they restrict infection by bacteria by certain bacteriophages, by degrading the viral DNA without affecting the bacterial DNA. Thus, such enzymes are helpful in breaking DNA molecule at a specific point. In recombinant DNA technology, they are used to cut DNA into specific fragments that contain specific genes.
DNA Synthesising Enzymes:
DNA synthesising enzymes, such as DNA polymerase or reverse transcriptase to make DNA complementary to an existing DNA or RNA template respectively called c DNA (complementary or copy DNA) are also required.
Because of the presence of an universal genetic code, the DNA polymerases (synthesising enzymes) can accurately transcribe a DNA sequence from one organism to another organism, and finally molecular sutures, the DNA ligase enzymes to join DNA fragments. This happens so by the formation of phosphodiester bond, again.
Any pair of fragments produced by the same restriction enzyme, from any DNA source (e.g., fruit fly, any other animal or human), can be joined together.
3. Cloning Vectors:
The desired gene is inserted in the vectors or gene carrier to form DNA. Vectors carrying recombinant DNA (r-DNA) divide and thus help in producing several copies of the r-DNA.
There are five cloning vectors which are used in experiments with E. coli animals/humans are:
(a) Plasmids
(b) Bacteriophages (viruses that infect bacteria)
(c) Plant/Animal Viruses
(d) Transposons (Jumping Genes), and
(e) Artificial Chromosomes of Bacteria, Yeast and Mammals.
The most commonly used vectors are plasmids and viruses.
