Recombinant DNA technology

	Recombinant DNA Technology

 

The term recombinant DNA means the joining or recombining of two pieces of DNA from two different sources. Genetic modification using recombinant DNA allows us to move genes whose function are known. By making manipulations more precise and outcomes more certain, the risk of producing organisms with unexpected traits are decreased.

·        Steps involved in recombinant DNA technique (or gene cloning): -

·        The basic 7 steps involved in gene cloning are:

Isolation of DNA [gene of interest] fragments to be cloned.

Insertion of isolated DNA into a suitable vector to form recombinant DNA.

Introduction of recombinant DNA into a suitable organism known as host.

Selection of transformed host cells and identification of the clone containing the gene of interest.

Multiplication/Expression of the introduced Gene in the host.

Isolation of multiple gene copies/Protein expressed by the gene.

Purification of the isolated gene copy/protein

                                  Photo courtesy: - Pearson

·        Tools for recombinant DNA technology

·        RESTRICTION ENZYMES (the molecular scissors)

The foundations of rDNA technology were laid by the discovery of restriction enzymes. These enzymes exist in many bacteria where they function as a part of a defense mechanism called the Restriction-Modification System. This System consists of two components:

1. A restriction enzyme that selectively recognizes a specific DNA sequence and digests any

DNA fragment containing that sequence. The term restriction is derived from the ability of

these enzymes to restrict the propagation of foreign DNA (e.g. Viral/phage DNA) in a

bacterium.

2. A modification enzyme that adds a methyl group to one or two bases within the sequence

recognized by the enzyme. Once a base is modified by methylation, the sequence cannot be

digested. It is thus obvious that the Restriction-Modification enzyme system within a given

bacterium protects its DNA from digestion by methylation but can digest foreign DNA which is not protected by similar methylation.

Different species of bacteria contain their own sets of restriction endonucleases and corresponding methylases. Three main classes of restriction endonucleases- type I, type II and type III are present, of which, only type II restriction enzymes are used in r-DNA technology. As they recognize and cut DNA within a specific sequence typically consisting of 4-8 bp.

                               Photo courtesy: -NCERT Biotechnology Class12th

In addition to restriction enzymes, there are several other enzymes that play an important role in rDNA technology. Two of these are DNA ligase and alkaline phosphatase.

·        DNA ligase: This enzyme forms phosphodiester bonds between adjacent nucleotides and

covalently links two fragments of DNA. The reaction requires one of the fragments to have a 5' phosphate residue and the other a 3' hydroxyl group. In a previous section it was indicated how two fragments cut with Eco RI could stick together; DNA ligase seals this by forming a covalent bond. DNA ligase isolated from the bacteriophage T4 is frequently used to ligate different DNA fragments in order to generate rDNA molecules.

·        Alkaline phosphatase: Ligation requires the presence of a 5'phosphate group. If some of the fragments are treated with alkaline phosphatase to remove their phosphate groups then these cannot ligate within themselves and are forced to ligate with other fragments containing 5'phosphate groups. Hence this is a useful strategy to prevent self-ligation which would otherwise lead to wasteful ligation of fragments treated with restriction enzymes. An insert is ligated to the vector in generating rDNA as the vector is prevented from self-ligation by treating it with alkaline phosphatase. Alkaline phosphatase used for this purpose is purified from bacteria or calf intestines.

Cloning Vectors: - Another major component of a gene cloning experiment is a vector such as a plasmid. A vector serves as a vehicle to carry a foreign DNA sequence into a host cell.

                            Photo courtesy- NCERT Biotechnology.(class 12th)

APPLICATIONS: -

Recombinant DNA is widely used in biotechnology, medicine and research.

The most common application of recombinant DNA is in basic research, in which the technology is important to most current work in the biological and biomedical sciences. Recombinant DNA is used to identify, map and sequence genes, and to determine their function. rDNA probes are employed in analyzing gene expression within individual cells, and throughout the tissues of whole organisms. Recombinant proteins are widely used as reagents in laboratory experiments and to generate antibody probes for examining protein synthesis within cells and organisms.

Recombinant chymosin

Found in rennet, chymosin is an enzyme required to manufacture cheese. It was the first genetically engineered food additive used commercially. Traditionally, processors obtained chymosin from rennet, a preparation derived from the fourth stomach of milk-fed calves. Scientists engineered a non-pathogenic strain (K-12) 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. Today about 60% of U.S. hard cheese is made with genetically engineered chymosin. In 1990, FDA granted chymosin "generally recognized as safe" (GRAS) status based on data showing that the enzyme was safe.

Recombinant human insulin

Almost completely replaced insulin obtained from animal sources (e.g. pigs and cattle) for the treatment of insulin-dependent diabetes. A variety of different recombinant insulin preparations are in widespread use. Recombinant insulin is synthesized by inserting the human insulin gene into E. coli, or yeast (Saccharomyces cerevisiae) which then produces insulin for human use.

Recombinant human growth hormone (HGH, somatotropin)

Administered to patients whose pituitary glands generate insufficient quantities to support normal growth and development. Before recombinant HGH became available, HGH for therapeutic use was obtained from pituitary glands of cadavers. This unsafe practice led to some patients developing Creutzfeldt–Jakob disease. Recombinant HGH eliminated this problem, and is now used therapeutically. It has also been misused as a performance-enhancing drug by athletes and others.

Recombinant blood clotting factor VIII

A blood-clotting protein that is administered to patients with forms of the bleeding disorder hemophilia, who are unable to produce factor VIII in quantities sufficient to support 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.

Recombinant hepatitis B vaccine

Hepatitis B infection is controlled through the use of a recombinant hepatitis B vaccine, which contains a form of the hepatitis B virus surface antigen that is produced in yeast cells. The development of the recombinant subunit vaccine was an important and necessary development because hepatitis B virus, unlike other common viruses such as polio virus, cannot be grown in vitro. Vaccine information from Hepatitis B Foundation.

Diagnosis of infection with HIV

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. The DNA test looks for the presence of HIV genetic material using reverse transcription polymerase chain reaction (RT-PCR). Development of the RT-PCR test was made possible by the molecular cloning and sequence analysis of HIV genomes. HIV testing page from US Centers for Disease Control (CDC)

Golden rice

A recombinant variety of rice that has been engineered to express the enzymes responsible for β-carotene biosynthesis. This variety of rice holds substantial promise for reducing the incidence of vitamin A deficiency in the world's population. Golden rice is not currently in use, pending the resolution of regulatory and intellectual property issues.

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. These crops are in common commercial use in several countries.

Insect-resistant crops

Bacillus thuringeiensis is a bacterium that naturally produces a protein (Bt toxin) with insecticidal properties. The bacterium has been applied to crops as an insect-control strategy for many years, and this practice has been widely adopted in agriculture and gardening. Recently, plants have been developed that express a recombinant form of the bacterial protein, which may effectively control some insect predators. Environmental issues associated with the use of these transgenic crops have not been fully resolved.

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