Recombinant DNA Technology

It is possible to get the foreign gene transcribed and translated and produce the protein coded by the gene

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Dr. Arshed Iqbal Dar Srinagar, Publish Date: Oct 29 2017 11:25PM | Updated Date: Oct 29 2017 11:25PM

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Recombinant DNA technology is a set of techniques that enables the DNA from different sources to be identified, isolated are recombined so that new characteristics can be introduced into an organism. The farmost technique in recombinant DNA technology is the transfer of a sequence of DNA (gene) from one organism to other organism. Hence, it is also called as Genetic engineering, molecular cloning,or gene cloning. Recombinant DNA technology refers to joining of two (or more) DNAs of different origin to create a new (or novel)DNA molecule. This DNA is transferred to an organism, usually bacteria, where it can multiply using the host machinery.The process of inserting a foreign DNA in a host is also referred as Gene cloning.The cloned gene has to undergo replication, usually independent of the replication of the host genome to make multiple copies. If necessary, regulatory elements are also provided along with the foreign gene. Thus, it is possible to get the foreign gene transcribed and translated and produce the protein coded by the gene. This process is known as expression of cloned gene. A suitable carrier is required to transport the foreign gene to the host cell. This carrier, which is usually an autonomously replicating small DNA molecule, is known as cloning vector. A number of DNA modifying enzymes are needed for the construction of a cassette suitable for gene cloning. These enzymes include the restriction enzymes ligases, polymerases, nucleases etc. If a gene from one species is introduced into another species, that species should produce the same product from that gene. In other words, it should be possible to place a human gene into bacteria and have those bacteria produce the human protein encoded by that gene.infact there are many human genes that produce human proteins in bacteria, and these proteins can be used to treat serious medical problems.

Birth of rDNA technology:

To many of the biologists who had played a part in that birth, it seemed that molecular genetics was entering a period of consolidation. They felt there would be further growth, but that this would be incremental; new conceptual advances seemed unlikely as the fine details of the old problems were worked out and biochemists dissected in ever more minute detail the pathway involved. At that time, in the 1960s, molecular genetics was almost entirely based on the study of bacteria and phages. New advances would come in working out the functioning of genes in higher organisms, but the experimental tools were lacking and it seemed as far in the future as putting a man on the moon. So to avoid possibly marking time, several distinguished contributors to our primary knowledge on the storage of genetic information in DNA left molecular genetics to start up new carrers in neurobiology. What they could not have forseen was the very rapid development over the next decade of the enzymological and chemical techniques that gave rise to recombinant DNA, which by now has come to include any technique for manipulating DNA or RNA.

The birth of rDNA technology took place due to some major discoveries viz.

 The first major breakthrough was the discovery of endonucleases to cut the DNA. On the basis of rDNA technology, researchers made tissue plasminogen activators (rt-PA) such as Hirudin, superoxide dismutase, urokinase/prokinase. These medicines brought paradigmatic shift in the therapy of myocardial infarction (heart attack). If the injection of prokinase/urokinase is given to the patients suffering with myocardial infarction within 6 to 12 hours, there is no salvage of myocardium. Apart from prokinase, Insulin, Human growth hormone, Interleukin-2 and Taxol were also unique medicines.

During 1970, it was found that bacteria contained nucleases that would recognize short nucleotide sequences within DNA duplex and cleave the DNA backbone at specific sites on both strands of the duplex. These enzymes are called typeII restriction endonucleases or simply restriction enzymes. They were given this name because they function in bacteria to destroy viral DNAs that might enter the cell, thereby restricting the growth of the viruses.The bacterium protects its own DNA from nucleolytic attack by methylating the bases at susceptible sites, a chemical modification that blocks the action of enzyme.

 Interferons:- Interferons are the glycoprotins which have an inhibitory action upon the multiplication of viruses in cells more or less adjacent to the affected ones. They are being used for the treatment of several diseases including a rare form of cancer called hairy cell leukemia. Interferons are being used for the treatment of several diseases. 

 The advent of Human erythropoitin (EPO) manage to combat patients with renal diseases, enabling us to minimize the risk associated with blood transfusion.

 Mitogen activated protein was discovered:- The studies suggested that the functional role of (MAP) Kinase is signal transduction pathway which plays a critical role in many aspects of cardiovascular responses.

 In somatic gene therapy:- The recipients genome is cahnged, but the change isn’t passed along to the next generation.

 In germline gene therapy:- the patients eggs and sperm cells are changed with the goal of passing on the changes to their offsprings.

 DNA vaccines:- The immune system generates antibodies in response to the recognition of proteins and other large molecules carried by pathogens. The functional component of the vaccine introduced into the host is the protein responsible for the elicitation of the immune response. The introduction of DNA into animals doesnot generate an immune response against the DNA molecule, but, if that DNA is expressed to yield a protein, that protein can stimulate the immune system. This is the basis of DNA vaccination. DNA vaccines have several advantages over conventional vaccines. It can stimulate both cell-mediated and humoral immunity. rDNA technology can be used to clone the gene for the protective antigen protein. A number of vaccines against virus have been developed using this technology. These vaccines are- Hepatitis B, Influenza, HIV (AIDS), Herpes, Foot and mouth diseases, etc.

 Formation of rDNA:-

DNA molecules from two different sources are treated with restriction enzyme that make staggered cuts in the DNA duplex. Staggered cuts leave short single stranded tails/ends that act as sticky ends or cohesive end, because they form base pairs with the cohesive ends of any molecule cut with the same enzyme.One of the DNA fragments that will make up the recombinant molecule is a bacterial plasmid.The other DNA fragment is obtained from human cells, following treatment with the same restriction enzyme used to open the plasmid. When the Human DNA fragments and treated plasmid are incubated together in the presence of DNA-ligase, the two types of DNA become hydrogen bonded to one another by their sticky ends and then ligated to form circular DNA recombinants. The first recombinant DNA molecules were formed by this method in 1972 by Paul Berg, Boyer, Chang and Cohen (University of California) marking the birth of modern genetic engineering.

Now, suppose you were interested in isolating a single gene from human genome e.g a gene that codes for insulin because your goal is to obtain a purified preparation of one type of recombinant DNA that contain the insulin coding fragment, you must separate this one fragment from all of the others. This is done by a process called DNA cloning.

 Applications of rDNA technology:-

 Hemophilia:- Lack of a particular protein that helps the blood to clot when blood vessels are broken in a cut or bruise. Without treatment, a person with hemophilia can bleed to death from a small cut. For many years, people with hemophilia recieved injection of the clotting proteins that was taken from donated blood. When AIDS epidemics begin, one of the groups at highest risk was people with hemophilia because many of the injections of clotting proteins were contaminated with HIV, a virus that causes AIDS. In the mid 1990s, genetic engineers placed the DNA of human gene that encodes the clotting factor into bacteria. The bacteria now produces clotting factors that is identical to the natural human clotting factors. It is not contaminated with HIV or any other virus that infect humans.

 Gene therapy:- 

(A) Germline gene therapy:- Insertion of a corrected gene into a person’s germline cells so that the person then transmits the corrected version of the gene to his or her offsprings. It is contraversial and banned in humans.

(B) Somatic cell gene theraphy:- Scientists have focused their effort on somatic cell gene theraphy. Insertion of corrected gene into the tissue where the gene is normally expressed. Genetic disorder corrected but doesn’t pass the corrected gene to his or her offsprings. 

First successful somatic cell gene therapy was correction of the genetic disorder. ADA (Adenosine deaminase) enzyme deficiency. The result is complete failure of the immune sysytem. Children with this disorder can quickly die from a simple cold or case of the flu,because their bodies can’t fight against infection.

Before gene therapy was available, these children had to live in plastic bubble in a hospital where they could be protected from all bacteria and viruses. Their food had to be sterilized and no one could touch them directly. Their parents couldn’t hug them. The first succesful case was with a four year old girl. Genetic engineers took a functional version of the gene and placed it in a virus that had been disabled so that it couldn’t produce a disease. They then removed some of the girl’s non-functioning immune system cells from her body and added the enginerred virus to them. The virus infected the cells and as it did it introduced the corrected gene into cells. Physicians then reintroduced the treated cells into the girl’s body, and these cells began to produce functional adenosine deaminase. Soon the girl’s immune sysytem was functioning normally and she was able to leave the bubble and live with her family. Many diseases which can be cured by using rDNA technology  are: Insulin, Interferon, Somatotropin, Somatostatin, endorphin, human blotting factor (VIIIC), Immunogenic proteins etc.

 rDNA technology in Environment:-

A vast majority  of applications of environmental biotechnolgy use naturally occurring microorganisms (bacteria, fungi, etc)to identify and filter manufacturing waste before it is introduced into the environment. Bioremediation programme involving the use of microorganisms are currently in progress to clean up contaminated air, tracks of land, lakes and waterways. Recombinant technology helps in improving the efficacy of these processes so that their basic biological processes are more efficient and can degrade more complex chemicals and higher volumes of waste materials. rDNA technology also is being used in development of bioindicators where bacteria have been genetically modified as bioluminescors that gave off light in response to several chemical pollutants. These are being used to measure the presence of some hazardous chemicals in the environment. Other genetic sensors that can be used to detect various chemical contaminants are also undergoing trials and include sensors that can be used to track how pollutants are naturally degrading in ground water. For example when gene such as the mercury resistance gene (mer) or toluene degradation gene (tol) gene is linked to genes that code for bioluminescence within living bacterial cells, the biosensor cells can signal extremely low levels of inorganic mercury or tolune that are present in contaminated waters and soils by emitting visible light, which can be measured with fiber-optic fluro meters.

 rDNA technology in crop improvement:-

Transgenic crops depend on the basic productivity of the germplasm into which the genes are introduced. There is a tremendous need to be able to handle the existing genes in crop plants and their sexually compatible relatives much more effectively than can be done by traditional breeding and back-crossing technology. The development and use of marker technology provides a way in which breedres can identify those regions of the chromosomes that contain important genes covering agronomic traits of interest. A wide range of different markers is now available and in use in enhancing covenetional breeding programmes in many of our major crops. They also provide a way in which favourable alleles at important loci, present in the wild relatives of crops, can be identified and transferred. In addition, they have been used to incorporate introduced transgenes rapidly into a wide range of adapted germplasm. A major problem in the production of crops is protecting them from the ravages of pests and diseases. This is achieved by agronomic practices, genetic resistance and crop protection chemicals. The use of the latter should be as careful as possible to ensure they donot affect areas outside the target, are only used when necessary and when they bring a cost benefit. Again, recombinant DNA technology can provide some help through its ability to recognise very precisely biological individuals. The increase in diagnostic power coming from the technology is being, and will increasingly be, used to aid agronomic decisions to enhance the use of crop protection chemicals. Finally, it is often assumed by critics that transgenic technology is only being used by commercial companies to generate commercial profits. This is not true. Transgenic technology is an important research tool to understand plant function and is being used in research-labs world wide for that aim. The performance of a crop in a given field is a function of its phenotype.

 Diagnosis of infectious diseases:- Modern medical practice depends on laboratory tests for the specific and correct diagnosis of many diseases. rDNA technology allows for the production of highly specific diagnosis tests. Some of the infectious diseases are cholera, small pox, measles, meningitis, hepatitis, etc. These diseases led the serious damage to the human health. Infectious diseases diagnosis mainly depend upon isolation and identification of pathogens, which may take several days. Development of diagnostic kits to identify pathogenic organisms by knowing the organism-specific DNA sequence has provided rapid, specific and correct diagnosis. In this way, advancement in biotechnology has made easy early, correct and quick diagnosis of infectious diseases. Various diagnostic kits have been developed for AIDS, CANCER, Foot and Mouth diseases, Tuberculosis, etc. Different biotechnological tools used in diagnosis of infectious diseases and prenatal diseses are ELISA, PCR based technique, RIA Essays, etc.

Dr. Arshed Iqbal Dar is Assistant Professor, Zoology at GDC Sumbal Sonawari.