Process of DNA Recombinant Technology Steps & Application

When a genetically modified vector is introduced and incorporated into the genome of an entity (host), the process of recombinant DNA technology modifies the phenotypic of the entity (host). As a result, the procedure comprises inserting a foreign DNA fragment into the genome that contains the target gene.

Recombinant DNA Technology is also known as Genetic Engineering. In this article, you will learn about the fundamentals of the artificial approach – recombinant DNA technology – as well as the methods required.

What Recombinant DNA Technology?

Recombinant DNA Technology is defined as the method of joining together molecules of DNA from two different species, which is then inserted into a host organism to produce GMOs used in science, medicine, agriculture and industry.

What is the Process of Recombinant DNA Technology?

The process of Recombinant DNA technology involves the following steps in a specific sequence:

1. Isolation of genetic material (DNA)
2. Cutting or fragmentation of DNA at specific locations by restriction endonuclease
3. Isolation and amplification of the desired DNA fragment
4. Formation of Recombinant DNA (rDNA)
5. Insertion or transfer of rDNA into the host cell
6. Obtaining the foreign gene product
7. Downstream Processing

1. Isolation of the Genetic Material (DNA)

a) Isolation of DNA is the foremost step for rDNA technology. DNA needs to be pure form, i.e., from any other molecule.
b) Many enzymes are needed to isolate the DNA like Lysozyme– to break open bacterial cells, Cellulase– to break plant cells, and Chitinase– to break fungal cells.
c) Various macromolecules like RNA and proteins are removed by treatment with specific enzymes. For example, ribonucleases help remove RNA, and proteases help remove proteins.
d) DNA released from cells is intertwined with proteins such as histones which are also removed for getting pure DNA.
e) Purified DNA is finally precipitated after adding chilled ethanol, which can be removed by spooling.

2. Cutting or Fragmentation of DNA at Specific Locations by Restriction Endonucleases

a) A restriction endonuclease enzyme is used to cleave the source DNA into fragments at specific locations.
b) Agarose gel electrophoresis is used to check the progress of the restriction endonuclease cleavage.
c) This process is also repeated with the vector DNA.

3. Isolation and Amplification of the Desired DNA Fragment

After the identification of the particular gene of interest or the desired DNA fragment, it is taken out from the gel and is known as elution. The final need is to make multiple copies of it which can be made in two ways:

i) By inserting the DNA fragment into a bacterium and after repeated cell divisions of the bacterium, millions of cells containing the copies of this DNA fragment can be obtained.

ii) Another quicker and far more direct method is to use DNA polymerase enzymes to copy the identified gene sequence through the polymerase chain reaction (PCR).

Kary Mullis developed the polymerase chain reaction in (1983), in which multiple copies of the desired DNA fragment are synthesised in vitro by the following three steps:

a) Denaturation: The desired DNA fragment is mixed with the two sets of primers and heated to about 95 ℃ for 30 seconds, dissociating the double-stranded DNA fragment into single strands.
b) Annealing primers: In this step, the resultant solution is allowed to cool at about 55-60℃ for about 1 minute. The complementary primers get attached to the base pairs flanking the DNA fragment, making it double-stranded and leaving the rest of the DNA single-stranded. The temperature for annealing depends on the melting temperature.
c) Primer extension or synthesis: In this step, the heat-stable DNA polymerase (Taq polymerase) enzyme, extends the primer by adding complementary nucleotides (dNTPs). The synthesis is carried out at 72℃ for about 2 minutes.

The above three steps are repeated again and again and more copies of the desired DNA fragment are produced.

Fig: Polymerase Chain Reaction (PCR)

4. Formation of Recombinant DNA (rDNA)

In this step, the two molecules of DNA, i.e. the gene of interest and the vector DNA, are cut by the same restriction enzyme to produce sticky ends and then joined together with the help of the DNA ligase enzyme. The resultant DNA formed is known as recombinant DNA or hybrid DNA, or chimeric DNA.

5. Insertion or Transfer of Recombinant DNA Into the Host Cell

a) DNA is a hydrophilic molecule that cannot pass through the host’s cell membrane. So, the host cells are made competent to accept the new DNA because they do not easily accept foreign DNA.

b) Various methods like the indirect method involves the transfer of recombinant DNA through the vectors or carriers or the direct method that includes electroporation, microinjection and biolistic (or gene particle gun) are used by the recombinant DNA can be introduced into the recipient or host cell. This method is also known as transformation.

6. Obtaining the Foreign Gene Product

a) After introduction of the recombinant DNA into the host cell, it gets multiplied, and the foreign gene expresses itself under appropriate conditions in the form of the desired protein product.
b) For the best benefit of human mankind, the desired gene product needs to be produced on a large scale, and for this purpose, large vessels called bioreactors are used.
c) In the bioreactors, the cells with the desired gene are multiplied in a continuous culture system. The used medium is drained from one side while the fresh medium is added from the other side to maintain the cells in their physiologically active exponential phase.

7. Downstream Processing

a) This step includes the processes involved with separating and purifying the bioproducts formed before it is released for marketing as a finished product.
b) The product is subjected to quality control testing and kept in suitable preservatives. If drugs are to be manufactured, such formulation must undergo clinical trials.
c) Proper quality control testing for each product is also needed. Downstream processing and quality control tests differ from product to product.

Fig: Process of Recombinant DNA Technology

Applications of Recombinant DNA Technology

Following are the applications of Recombinant DNA Technology:

1. It is widely used in biotechnology, medicine and research.
2. It is used for the production of vaccines and recombinant therapeutics such as human insulin, interferon and human growth hormone.
3. It is used in agriculture for the development of insect-resistant crops.
4. It is used in the development of gene therapy.
5. It is used to detect the presence of HIV in a person and for hepatitis diagnostic tests.
6. It is used in the creation of transgenic animals.
7. It is used in tests for determining hereditary disease and paternity.
8. It is used for the production of clotting factors for treating haemophilia.

Summary

Recombinant DNA Technology is defined as the method of joining together molecules of DNA from two different species, which is then inserted into a host organism to produce GMOs used in science, medicine, agriculture and industry. It is also known as Genetic Engineering. The recombinant DNA technology process includes isolating and cutting the DNA by the restriction endonuclease enzyme. Then amplification of the desired DNA fragment and then insertion of the recombinant DNA into the host cell, multiplying further at an exponential rate for producing a large amount of the desired gene product.

With the help of recombinant DNA techniques, bacteria have been created capable of synthesising human insulin, human growth hormone, alpha interferon, hepatitis B vaccine and other medically useful substances. It is also used in industry, food production, human and veterinary medicine, agriculture and bioengineering.

FAQs

Q.1. What is a recombinant DNA process?
Ans: Recombinant DNA Technology is defined as the method of joining together molecules of DNA from two different species, which is then inserted into a host organism to produce GMOs that are used in science, medicine, agriculture and industry.

Q.2. What are the steps in recombinant DNA technology?
Ans: 1. Isolation of genetic material (DNA)
2. Cutting or fragmentation of DNA at specific locations by restriction endonuclease
3. Isolation and amplification of the desired DNA fragment
4. Formation of Recombinant DNA (rDNA)
5. Insertion or transfer of rDNA into the host cell
6. Obtaining the foreign gene product
7. Downstream Processing

Q.3. What is recombinant DNA technology used for?
Ans: 1. It is widely used in biotechnology, medicine and research.
2. It is used for the production of vaccines and protein therapies such as human insulin, interferon and human growth hormone.
3. It is used to identify, map and sequence genes and to determine their function.
4. It is used in agriculture for the development of insect-resistant crops.
5. It is used in gene therapy.

Q.4. What is the first step in Recombinant DNA technology?
Ans: The first step in the Recombinant DNA technology is the isolation of DNA in its pure form, i.e., from any other macromolecules like RNA and proteins.

Q.5. Why is recombinant DNA technology necessary nowadays?
Ans: Recombinant DNA technology is necessary nowadays because it has made human life easier. It has developed advanced strategies for biomedical applications such as cancer treatment, genetic diseases, diabetes, etc.

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