Recombinant DNA technology

Introduction
DNA (Deoxyribonucleic acid) is the hereditary material in humans and almost all other living organisms. The information stored in the DNA is in the form of four nitrogen bases adenine (A), thymine (T), guanine (G), and cytosine (C). Each base is also attached to a sugar molecule and a phosphate molecule. The DNA bases pair up with each other, i.e. A with T, and G with C. these four bases are the same for all organisms. The sequence and number of these bases determines the information available for building and maintaining an organism and this is what creates diversity. The DNA is transcribed into mRNA and mRNA is translated into protein, and the protein then forms the organism. By changing the DNA sequence, the way in which the protein is formed changes. This leads to either a different protein, or an inactive protein. The term recombinant DNA (sometimes called chimeric DNA) is generally used to describe the process of taking a piece of one DNA, and combining it with another strand of DNA. The DNA sequences used in the construction of recombinant DNA molecules can originate from any species. For example, plant DNA may be joined to bacterial DNA, or human DNA to fungal DNA. DNA sequences that do not occur anywhere in nature may be created by the chemical synthesis of DNA, and incorporated into recombinant molecules. Proteins that result from the expression of recombinant DNA within living cells are termed recombinant proteins. Expression of foreign proteins requires the use of specialized expression vectors and often necessitates significant restructuring of the foreign coding sequence.

Methods for obtaining recombinant DNA

Formation of recombinant DNA requires a cloning vector, a DNA molecule that replicates within a living cell. Vectors are generally derived from plasmids or viruses, and represent relatively small segments of DNA that contain necessary genetic signals for replication. The choice of vector depends on the choice of host organism, the size of the DNA to be cloned, and whether and how the foreign DNA is to be expressed. The DNA segments can be combined by using a variety of methods, such as restriction enzyme/ligase cloning etc. The cloning of any DNA fragment essentially involves seven steps: 1. 2. 3. 4. Choice of host organism and cloning vector, Preparation of vector DNA Preparation of DNA to be cloned Creation of recombinant DNA

5. Introduction of recombinant DNA into the host organism 6. Selection of organisms containing recombinant DNA 7. Screening for clones with desired DNA inserts and biological properties Depending on the choice of host organism (and hence the choice of vector), there may be three methods by which recombinant DNA may be made. In all cases, the first step is to select a piece of DNA to be inserted into a vector. The second step is to cut that piece of DNA with a restriction enzyme and then ligate the DNA insert into the vector with DNA Ligase. The insert contains a selectable marker which allows for identification of recombinant molecules. The vector is then inserted into a host cell. The host cells must be specially prepared to take up the foreign DNA. Transformation: The host in this case is a bacterial cell such as E. Coli. Often an antibiotic marker is used in this case, so that a host cell without a vector dies when exposed to a certain antibiotic, and the host with the vector will live because it is resistant. Non-bacterial transformation: The only difference from the above process is that a bacterial cell is not used as a host. In microinjection, the DNA is injected directly into the nucleus of the cell being transformed. In biolistics, the host cells are bombarded with high velocity micro projectiles, such as particles of gold or tungsten that have been coated with DNA. Phage introduction: Phage introduction is a process which is equivalent to transformation, except a phage is used instead of bacteria.

Expression of recombinant DNA (rDNA)

Once the foreign DNA id transplanted into the host, there is a possibility that the DNA might simply be replicated without being expressed i.e. recombinant protein may not be formed. Expression of the foreign gene require restructuring to include sequences necessary for producing mRNA molecule that can be used by the hosts translational (i.e. DNA to protein) apparatus. In addition, changes may be needed to the coding sequence as well to make the protein soluble, direct the recombinant protein to the proper cellular or extracellular location, and stabilize the protein from degradation. Production of recombinant proteins in eukaryotic systems generally takes place in yeast and filamentous fungi. The use of animal cells is difficult due to the fact that they many need a solid support surface, unlike bacteria, and have complex growth needs. However, some proteins are too complex to be produced in bacterium, so eukaryotic cells must be used.

Applications of rDNA technology

Recombinant chymosin: Traditionally, processors obtained chymosin (enzyme necessary to manufacture cheese) from rennet, a preparation derived from the fourth stomach of milk-fed calves. Scientists engineered a non-pathogenic strain (K12) of E. coli bacteria for large-scale laboratory production of the enzyme. This microbiologically produced recombinant enzyme, identical structurally to the calf derived enzyme, costs less and is produced in abundant quantities.

Recombinant human insulin : Almost completely replaced insulin obtained from animal sources (e.g. pigs and cattle) for the treatment of insulin-dependent diabetes. Recombinant insulin is synthesized by inserting the human insulin gene into E. coli, which then produces insulin for human use.

Recombinant human growth hormone (HGH): Before recombinant HGH became available, HGH for therapeutic use (for patients whose pituitary glands generate quantities insufficient to support normal growth and development) was obtained from pituitary glands of cadavers. This unsafe practice led to some patients developing Creutzfeldt-Jacob disease.

Recombinant blood clotting factor VIII: A blood-clotting protein that is administered to patients with forms of the bleeding disorder haemophilia (incapable of normal blood coagulation). Before the development of recombinant factor VIII, the protein was obtained by processing large quantities of human blood from multiple donors, which carried a very high risk of transmission of blood borne infectious diseases, for example HIV and hepatitis B.

Diagnosis of HIV infection: each of the three widely used methods for diagnosing HIV infection has been developed using recombinant DNA. The antibody test (ELISA or western blot) uses a recombinant HIV protein to test for the presence of antibodies that the body has produced in response to an HIV infection (made possible by the molecular cloning and sequence analysis of HIV genomes).

Herbicide-resistant crops: commercial varieties of important agricultural crops (including soy, maize/corn, sorghum, canola, alfalfa and cotton) have been developed that incorporate a recombinant gene that results in resistance to the

herbicide glyphosate (trade name Roundup), and simplifies weed control by glyphosate application.

Insect-resistant crops: Bacillus thuringeiensis is a bacterium that naturally produces a protein (BT toxin) with insecticidal properties. Application of this bacterium to crops has been widely adopted in agriculture and gardening as an insect-control strategy. Recently, plants have been developed that express a recombinant form of the bacterial protein, which may effectively control some insect predators.

Future Scope
Recombinant DNA technology will play an increasingly significant role in the diagnosis, prevention and treatment of human diseases, by such methods as detecting carriers of recessive gene defects, production of antigens for vaccine preparation and of specific human proteins, identification of microbial pathogens, producing targeted medicines, and providing patients with less toxic pharmaceuticals. It will also impact agriculture and livestock as researchers find ways to optimize the genetic codes of plants and animals to resist disease.

Concerns with rDNA

Concerns regarding the ethical, legal and environmental impact of recombinant DNA technologies have increased mostly due to the rapid pace of advancement in the field as well as the increased use of genetically modified plants and animals for our daily necessities.