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Recombinant DNA Technology Process
Gene transfer from one species to another is possible artificially with the introduction of Recombinant DNA Technology in medical science. The rDNA technology is not only become beneficial in the medical sector, but also other sectors like agriculture, animal husbandry, and industrial sectors. The gene modification process is done through several steps.
What is Recombinant DNA Technology?
Recombinant DNA technology is a step-by-step process of altering the phenotype of a host or entity with the introduction and incorporation of a genetically modified vector into the host’s genome. In rDNA technology, a foreign DNA fragment with the desired gene is introduced into the genome.
Figure 1: Recombinant DNA Technology
A recombinant gene is a particular gene introduced for modification and the complete process is called recombinant DNA technology. Embedding interest gene to host’s genome is not a simple process. The development process of recombinant DNA occurs in some interconnected processes.
Process of Recombinant DNA Technology
The rDNA technology is processed through seven steps. The steps are discussed below.
Step 1 - Genetic Material Isolation
Nucleic acid is the genetic material of living organisms. RNA is the genetic material of some living organisms whereas DNA is the most common genetic substance. In the rDNA technology, the initial step is to take the desired DNA to purely isolate it and keep it macromolecule-free. In human cells, the DNA is located within the cell membrane along with macromolecules. Several enzymes are used to free the DNA from these cell substances. They are Lysozyme, Chitinase, Cellulase, Protease, and Ribonuclease. DNA appears as precipitate due to the addition of ethanol and finally it becomes purified.
Step 2 - Restriction Enzyme Digestion
In restriction enzyme digestion the DNA is cut at specific locations by restriction enzymes. Purified DNAs are run out on agarose gel by the Agarose Gel Electrophoresis technique. The DNAs are charged negatively by applying current and travel to the positive electrode to spread out.
Step 3 - Amplification Using PCR
PCR or Polymerase Chain Reaction is the DNA sequence replication method using the DNA polymerase enzyme. In PCR reactions, the components are primers, enzymes, nucleotides, and templates. Template refers to the DNA for simplification and DNA polymerase is the involved enzyme. Primers are small chemically synthesized oligonucleotides of the DNA region. The nucleotides are required for primer extension by enzymes.
Figure 2: Amplification using PCR
Step 4 - DNA Molecules Ligation
The vector of interest and purified DNA are cut with a similar restriction enzyme. Cut vectors and DNA fragment is open now. DNA ligase joins these pieces to make recombinant DNA in the ligation process.
Step 5 - Inserting Recombinant DNA Into the Host
Transformation occurs with the introduction of recombinant DNA to a recipient host cell. Bacterial cells are treated to become competent to accept new DNA. Recombinant DNA is shifted to recipient E.coli cells if it has the gene for ampicillin resistance and these cells become ampicillin-resistant.
Step 6 - Securing Foreign Gene Product
Recombinant protein is made under optimal conditions in the host by expressing recombinant DNA as protein. If the protein amount is large, cell culture will not yield a small volume. Bioreactors are used for large-scale production for human benefit.
Bioreactors collect mediums from one side and return the used medium from another side. They can convert raw substances into enzymes or proteins. Stirred-tank bioreactor is the most commonly known bioreactor.
Step 7 - The Downstream Process
The protein involves some downstream processes at the final stage. These include - purification and separation, formulation with suitable preservatives, tests for quality control, and clinical trials.
Objectives of Recombinant DNA Technolog
The most common goals of this technology are
Synthesizing new genes artificially
Gene characterization and isolation
Modifying the genome of living organisms
Explanation of hereditary diseases and related treatments
Modifying isolated genes as per desire
Human genome enhancement
Application of Recombinant DNA Technolog
The rDNA technology has wide usage in industries, medical sciences, agriculture, and animal husbandry. The applications of this technology are mentioned below.
The technology is used for the production of insulin for humans.
Recombinant HB vaccines are produced in rDNA technology.
In agriculture, Recombinant DNA Technology is used for producing better crops.
Figure 3: Recombinant DNA Technology used to make a plant with a new trait
Growth hormones are required for the treatment of dwarfism. This technology helps in such hormone production.
Recombinant DNA Technology is especially used to obtain DNA fingerprinting.
Gene therapy can be highly emphasized by using this technology.
It provides aid in the diagnosis of several disease types.
Recombinant DNA Technology is used to introduce foreign DNA fragments to the host genome. It is an artificial gene modification process by which the desired gene can be processed. The process starts with isolating genetic substances and ends with obtaining foreign genes. The downstream process could occur as an additional step in some cases. The rDNA technology is used widely in medical science, agricultural sectors, industries, and animal husbandries.
Q1. What is the key process behind the insertion of recombinant DNA into a host cell?
Ans. The interest gene carried by foreign DNA and vector DNA is cut by the same restriction enzyme. Two DNAs are joined with the formation of sugar-phosphate bonds by the ligase enzyme.
Q2. Is there any kind of risk attached to the processes of recombinant DNA technology?
Ans. Gene pollution occurring in rDNA technology is a safety issue that results in antibiotic-resistant microbes named super weeds. Allergenicity, health effects, adverse immune reactions, and other issues can occur due to the recombinant DNA technology process.
Q3. Which enzymes play a crucial role in the production of recombinant DNA when performing the technology?
Ans. In rDNA technology, hybrid genes are prepared from two more genes. The two enzymes that play a crucial role in this process are ligase and restriction endonucleases.
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