Recombinant DNA technology / Genetic engineering

Basic steps involved in rec DNA technology (or genetic engineering) are given below :

(i) Selection and isolation of DNA insert: First step in rec DNA technology is the selection of a DNA segment of interest which is to be cloned. This desired DNA segment is then isolated enzymatically. This DNA segment of interest is termed as DNA insert or foreign DNA or target DNA or cloned DNA.
(ii) Selection of suitable cloning vector: A cloning vector is a self-replicating DNA molecule, into which the DNA insert is to be integrated. A suitable cloning vector is selected in the next step of rec DNA technology. Most commonly used vectors are plasmids and bacteriophages.
(iii) Introduction of DNA-insert into vector to form recDNA molecule: The target DNA or the DNA insert which has been extracted and cleaved enzymatically by the selective restriction endonuclease enzymes [in step (i)] are now ligated (joined) by the enzyme ligase to vector DNA to form a rec DNA molecule which is often called as cloning-vector-insert DNA construct.
(iv) rec DNA molecule is introduced into a suitable host: Suitable host cells are selected and the rec DNA molecule so formed [in step (iii)] is introduced into these host cells. This process of entry of rec DNA into the host cell is called transformation. Usually selected hosts are bacterial cells like E. coli, however yeast, fungi may also be utilized.
(v) Selection of transformed host cells: Transformed cells (or recombinant cells) are those host cells which have taken up the recDNA molecule. In this step the transformed cells are separated from the non-transformed cells by using various methods making use of marker genes.
(vi) Expression and Multiplication of DNA insert in the host: Finally, it is to be ensured that the foreign DNA inserted into the vector DNA is expressing the desired character in the host cells. Also, the transformed host cells are multiplied to obtain sufficient number of copies. If needed, such genes may also be transferred and expressed into another organism.

Tools for Recombinant DNA Technology:

(a) Enzymes:
- a. Restriction Endonucleases: to cut DNA molecules at specific sites
- b. Exonucleases: removes nucleotides from the ends of a nucleic acid molecule
- c. DNA ligases : to join two fragments of DNA
- d. DNA polymerase : synthesize a new complementary DNA strand of an existing DNA or RNA template
(b) Cloning Vectors: It is another important natural tool which geneticists use in rec DNA technology. The cloning vector is the DNA molecule capable of replication in a host organism, into which the target DNA is introduced producing the rec DNA molecule. A cloning vector may also be termed as a cloning vehicle or earner DNA or simply as a vector or a vehicle a great variety of cloning vectors are present for use with E. coli is the host organism. However under certain circumstances it becomes desirable to use different host for cloning experiments. So, various cloning vectors have been developed based on other bacteria like Bacillus, Pseudomonas, Agrobacterium, etc. and on different eukaryotic organisms like yeast and other fungi. The cloning vector which has only a single site for cutting by a particular restriction endormclease is Considered as a good cloning vector. Different types of DNA molecules may be used as cloning vehicles such as they may be plasmids, bacteriophages, cosmids, phasmids or artificial chromosomes.
(c) Host Organism: A good host organism is an essential tool tor genetic engineering. Most widely used host for rec DNA technology is the bacterium E. coli. because cloning and isolation of DNA inserts is very easy in this host. A good host organism is the one winch easy to transform and in which the replication of rec DNA is easier. There should not be any interfering element against the replication of rec DNA in the host cells
(d) DNA Insert Or Foreign DNA: The desired DNA segment which is to be cloned is called as DNA insert or foreign DNA or target DNA. The selection of a suitable target DNA is the very first step of rec DNA technology. The target DNA (gene) may be of viral, plant, animal or bacterial origin.

Following points must be kept in mind while selecting the foreign DNA:

1. CD It can be easily extracted from source.
2. It can be easily introduced into the vector.
3. The genes should be beneficial for commercial or research point of view.

A number of foreign genes are being cloned for benefit of human beings. Some of these DNA inserts are the genes responsible for the production of insulin, interferon’s, lymphotoxins various growth factors, interleukins, etc.

Applications of Recombinant DNA Technology:

Genetic engineering or rec DNA technology has enormous and wide-spread applications in all the fields of biological sciences.

Some important applications of rec DNA technology are enlisted below:

(1) Production of Transgenic Plants: By utilizing the tools and techniques of genetic engineering it is possible to produce transgenic plants or the genetically modified plants. Many transgenic plants have been developed with better qualities like resistance to herbicides, insects or viruses or with expression of male sterility, etc. Also they allow the production of commercially important biochemical, pharmaceutical compounds, etc. Genetic engineering is capable of introducing the improved post-harvest characteristics in plants also. Transgenic plants also aid in the study of the functions of genes in plant species.
(2) Production of Transgenic Animals: By the use of rec DNA technology, desired genes can be inserted into the animal so as to produce the transgenic animal. The method of rec DNA technology aids the animal breeders to increase the speed and range of selective breeding in case of animals. It helps for the production of better farm animals so as to ensure more commercial benefits. Another commercially important use of transgenic animals is the production of certain proteins and pharmaceutical compounds. Transgenic animals also contribute for studying the gene functions in different animal species. Biotechnologists have successfully produced transgenic pigs, sheep, rats and cattle.
(3) Production of Hormones: By the advent of techniques of rec DNA technology, bacterial cells like E.coli are utilized for the production of different fine chemicals like insulin, somatostatin, somatotropin and p-endorphin. Human Insulin Hormone i.e., Humulin is the first therapeutic product which was produced by the application of rec DNA technology. The genes of interest are incorporated into the bacterial cells which are then cloned. Such clones are capable of producing a fair amount of hormones like insulin which have great commercial importance.
(4) Production of Vaccines: Vaccines are the chemical preparations containing a pathogen in attenuated (or weakened) or inactive state that may be given to human beings or animals to confer immunity to infection. A number of vaccines have been synthesized biologically through rec DNA technology. These vaccines are effective against numerous serious diseases caused by bacteria, viruses or protozoa. These include vaccines for polio, malaria, cholera, hepatitis, rabies, smallpox, etc. The generation of DNA vaccines has revolutionized the approach of treatment of infectious diseases. DNA-vaccine is the preparation that contains a gene encoding an immunogenic protein from the concerned pathogen.
(5) Biosynthesis of Interferon: Interferon’s are the glycoproteins which are produced in very minute amounts by the virus-infected cells. Interferon’s have antiviral and even anti-cancerous properties. By recDNA technology method, the gene of human fibroblasts (which produce interferon’s in human beings) is inserted into the bacterial plasmid. These genetically engineered bacteria are cloned and cultured so that the gene is expressed and the interferon’s are produced in fairly high quantities. This interferon, so produced, is then extracted and purified.
(6) Production of Antibiotics: Antibiotics produced by microorganisms are very effective against different viral, bacterial or protozoan diseases. Some important antibiotics are tetracyclin, penicillin, streptomycin, novobiocin, bacitracin, etc. recDNA technology helps in increasing the production of antibiotics by improving the microbial strains through modification of genetic characteristics.
(7) Production of Commercially Important Chemicals: Various commercially important chemicals can be produced more efficiently by utilizing the methods of rec DNA technology. A few of them are the alcohols and alcoholic beverages obtained through fermentation; organic acids like citric acid, acetic acid, etc. and vitamins produced by microorganisms.
(8) Application in Enzyme Engineering: As we know that the enzymes are encoded by genes, so if there are changes in a gene then definitely the enzyme structure also changes. Enzyme engineering utilizes the same fact and can be explained as the modification of an enzyme structure by inducing alterations in the genes which encode for that particular enzyme.
(9) Prevention and Diagnosis of Diseases: Genetic engineering methods and techniques have greatly solved the problem of conventional methods for diagnosis of diseases. It also provides methods for the. prevention of a number of diseases like AIDS, cholera, etc. Monoclonal antibodies are useful tools for disease diagnosis. Monoclonal antibodies are produced by using the technique called hybridoma technology. The monoclonal antibodies bear specificity against a specific antigen. These are used in the diagnosis of diseases due to their specificity. Genetic engineering allows the production of hybridoma which is a cell obtained by the fusion of a lymphocyte cell capable of producing antibodies and a single myeloma cell (tumour cell).
(10) Gene Therapy: Gene therapy is undoubtedly the most beneficial area of genetic engineering for human beings. It involves delivery of specific genes into human body to correct the diseases. Thus it is the treatment of diseases by transfer and expression of a gene into the patients’ cells so as to ensure the restoration of a normal cellular activity. On the basis of types of cells into which the functional genes are introduced, the gene therapy may be classified as somatic gene therapy and germ line gene therapy. Gene therapy is done either by using in vivo strategy (also called as patient therapy) or by using the ex vivo strategy.
(11) Applications in forensic science: The applications of rec DNA technology (or genetic engineering) in forensic sciences largely depend on the technique called DNA profiling or DNA fingerprinting. It enables us to identify any person by analysing his hair roots Wood stains, serum, etc. DNA fingerprinting also helps to solve the problems of parentage and to identify the criminals.