R DNA Technology
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Recombinant DNA technology is the process used for producing new genetic combinations by joining different genetic material (DNA) together and inserting them into host organisms from two different species or sources. These new combinations are of value to medicine, science, industry, and agriculture. The insertion of a gene into the host genome is not an easy task. Firstly, the desired gene needs to be selected for its administration into the host then, a suitable vector is selected to form recombinant DNA by integrating the gene with the vector. In living organisms, recombinant DNA was achieved by Herbert Boyer and Stanley Cohen who insert foreign DNA from plasmids by using E.Coli restriction enzymes.
Tools Used in Process of Recombinant DNA technology
1. Restriction Enzymes:- Its role is to identify the site where the desired gene is introduced into the vector genome
Endonucleases: Make cuts within the DNA strand
Exonucleases: Remove nucleotides from the DNA stand end
2. Enzyme Ligase: It is used to join two fragments. Sticky ends of the desired gene and the vector is attached with the help of ligase enzyme
3. Vector: It is generally a plastid plasmid used to carry and integrate the desired gene
4. Host: Competent host cell into which the Recombinant DNA is inserted.
Steps involved in DNA Technology
1. Isolation of Genetic Material
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Isolation of desired DNA in its pure form meaning free from other macromolecules.
In the normal cell, DNA exists along with other macromolecules like proteins, RNA, polysaccharides within the cell membrane.
It must be separated from other macromolecules and also purified by the help of enzymes which include cellulose, Lysozyme, Chitinase, proteases, ribonuclease.
Ultimately, DNA is precipitated out as a fine thread by the addition of ethanol and purified DNA is spoiled out which is called spooling.
2. Restriction Enzyme Digestion
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Restriction enzyme digestion is a reaction in which DNA is cut at a specific location by restriction enzymes that act as 'Molecular Scissors'.
Purified DNA is then incubated with the selected restriction enzyme at optimal conditions for a certain enzyme.
Agarose gel electrophoresis is the technique in which Agarose gel is used for running out the DNA. With the help of current, Being negatively charged, DNA travels to the positive electrode and on the basis of the size, they are separated out.
This allows digested DNA fragments to separate and cut out. Using the same procedure vector DNA can also be used.
3. Amplification using PCR
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PCR or polymerase chain reaction is the process in which multiple copies of DNA sequence can be made in vitro by using the enzyme DNA polymerase.
Millions of copies of DNA can be produced by amplifying single copy with the help of PCR
The following components are used to run PCR reactions on Thermal cyclers'.
Template: DNA to be amplified.
Primers: They are chemically synthesized and small oligonucleotides that are complementary to a specific DNA region.
Enzyme: DNA polymerase
Nucleotides: Needed for the extension of primers by the enzyme.
Using PCR, the DNA fragments that are cut can be amplified and then ligated with cut vectors.
4. Ligation of DNA Molecules
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The same restriction enzyme is used for cutting the purified DNA and selected vector
Both the cut DNA fragments and cut vector are given when the process opens.
Ligation is the method in which these two pieces are joined together using enzyme ligase.
The resulting DNA molecule is a desired DNA of interest and vector.
5. Insertion of DNA Recombinant Host
In this step transformation takes place. It is a process in which the Recombinant DNA is inserted into a recipient cell, mainly a bacterial cell which is first made competent, so they can accept new DNA.
The methods used for making a cell competent are Ca+2 ion treatment, thermal shock, electroporation, etc.
6. Obtaining/Culturing the Foreign Gene Product
A piece of alien DNA is inserted into a cloning vector and alien DNA gets multiplied by transferring it into a bacterial cell.
Production of desirable protein expression is the ultimate aim. Recombinant protein is the protein in the encoded gene which is expressed in the heterologous host.
Large volume or Cell culture is needed to produce a large amount of Recombinant protein that benefits humans. To accomplish, vessels used are known as bioreactors
Bioreactors are basically large containers and can process about 100-1000 liters of cell culture. A bioreactor offers optimum conditions like pH, temperature to convert raw material biologically into specific enzymes, proteins.
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7. Downstream Processing
This process involves the marketing of protein as a final product after going through a quality control test purification clinical test etc.
FAQs on Recombinant DNA Technology Process
1. What exactly is Recombinant DNA (rDNA) technology?
Recombinant DNA technology, also known as genetic engineering, is a process used to alter the genetic material of an organism. It involves taking a specific gene from one organism and inserting it into another, creating a new combination of DNA. This allows us to give the host organism a new, desirable trait, like the ability to produce a specific medicine.
2. What are the main steps involved in the recombinant DNA technology process?
The process generally follows several key steps to create a genetically modified organism. These are:
- Isolation of DNA: The genetic material (DNA) is extracted from the source organism.
- Cutting the DNA: Special enzymes are used to cut both the source DNA and a vector at specific sites.
- Amplification: The gene of interest is copied many times using a technique called PCR (Polymerase Chain Reaction).
- Joining DNA: The gene of interest is inserted into a carrier molecule, called a vector (like a plasmid), to form recombinant DNA.
- Transformation: This recombinant DNA is introduced into a suitable host cell, such as a bacterium.
- Obtaining the Product: The host cells multiply, and the desired product (like a protein) is extracted and purified for use.
3. Why are restriction enzymes often called 'molecular scissors' in this process?
Restriction enzymes are called 'molecular scissors' because of their ability to cut DNA molecules at very precise and specific locations. Each enzyme recognises a unique sequence of nucleotides, known as a recognition site, and makes a clean cut. This precision is crucial for cutting out the desired gene and opening the vector DNA so the new gene can be inserted correctly.
4. What is the role of a vector, like a plasmid, in rDNA technology?
In recombinant DNA technology, a vector acts as a delivery vehicle. Its main job is to carry the foreign gene of interest into the host cell. Plasmids, which are small circular DNA molecules found in bacteria, are commonly used as vectors. Once inside the host, the vector ensures that the new gene is replicated along with the host's own DNA, allowing the trait to be passed on to all daughter cells.
5. How is the desired gene actually introduced into a host organism?
Introducing the recombinant DNA into a host organism is a process called transformation. For this to happen, the host cell must be made 'competent' to accept foreign DNA. A common method for bacteria is the heat shock method, where cells are briefly exposed to a high temperature. This creates temporary pores in the cell membrane, allowing the recombinant plasmid to enter the cell.
6. What are some important real-world applications of rDNA technology?
This technology has revolutionised many fields. Some key applications include:
- Medicine: Mass production of human insulin for treating diabetes, manufacturing vaccines, and developing gene therapy to treat genetic disorders.
- Agriculture: Creating genetically modified crops (GM crops) like Bt-cotton, which is resistant to pests, and Golden Rice, which is enriched with Vitamin A.
- Research: Studying the function of genes and understanding diseases at a molecular level.
7. What happens during 'downstream processing' in biotechnology?
Downstream processing is the final and very important stage that occurs after the host cells have produced the desired product (like a protein or drug). It involves a series of steps to separate, purify, and preserve the product from the complex mixture in which it was made. This ensures the final product is pure, effective, and safe for use, for example, as a medicine.
