Recombinant DNA technology is the process of joining DNA molecules from two different species and inserting them into a host organism to create new genetic combinations that are valuable in science, medicine, agriculture, and industry.
Recombinant DNA (rDNA) is the general term for a piece of DNA that has been created by combining at least two different DNA strands.
These DNA molecules are formed using laboratory methods of genetic recombination, such as molecular cloning, which allow genetic material from multiple biological sources to be brought together.
The resulting DNA sequences produced through this process are not naturally found together in the genome and represent novel genetic arrangements.
The first successful creation of recombinant DNA in a living organism was achieved in 1973.
This work was carried out by Herbert Boyer at the University of California, San Francisco, and Stanley Cohen at Stanford University.
They used restriction enzymes derived from Escherichia coli (E. coli) to cut DNA and insert foreign DNA fragments into plasmid vectors, enabling the formation of recombinant DNA.
Isolation of Genetic Material
The first step in recombinant DNA (rDNA) technology is the isolation of the desired DNA in its pure form, meaning it must be free from other cellular macromolecules.
DNA is located inside the cell along with other substances such as RNA, polysaccharides, proteins, and lipids, which makes separation and purification essential.
The cell wall and cell membrane are broken down using specific enzymes such as lysozyme, cellulase, and chitinase, depending on the type of organism.
Ribonuclease (RNase) is used to remove contaminating RNA from the sample.
Proteases are used to digest and remove proteins associated with DNA.
Other macromolecules are removed using additional enzymatic or chemical treatments to ensure high purity of the DNA.
Finally, ethanol is added to the solution, causing the DNA to precipitate out as fine, visible threads.
These DNA threads are then carefully spooled out to obtain highly purified DNA.
Restriction Enzyme Digestion
Restriction enzymes act as molecular scissors that cut DNA at specific nucleotide sequences and precise locations.
These cutting reactions are known as restriction enzyme digestions.
The process involves incubating purified DNA with a selected restriction enzyme under optimal conditions specific to that enzyme, including proper temperature, pH, buffer composition, and ionic strength.
The progress and success of the restriction enzyme digestion are checked using a technique called agarose gel electrophoresis.
In this technique, DNA samples are loaded into wells of an agarose gel and an electric current is applied across the gel.
Because DNA is negatively charged, it moves towards the positive electrode (anode) during electrophoresis.
DNA fragments are separated based on their size, with smaller fragments moving faster and farther through the gel than larger ones.
This separation allows visualization and identification of the digested DNA fragments.
The desired DNA fragments can then be carefully cut out from the gel for further use.
The vector DNA (such as plasmid DNA) is processed using the same restriction enzyme digestion and agarose gel electrophoresis procedure to prepare it for ligation.
Amplification Using PCR
Polymerase Chain Reaction (PCR) is a laboratory technique used to make multiple copies of a specific DNA sequence using the enzyme DNA polymerase under in vitro conditions.
PCR allows the amplification of a single DNA copy or a few copies into thousands to millions of copies within a short time.
PCR reactions are carried out in a machine called a thermal cycler, which precisely controls temperature changes required for the reaction.
The main components required for a PCR reaction include the template DNA, which contains the sequence to be amplified.
Primers are short, chemically synthesized oligonucleotides that are complementary to specific regions of the target DNA and provide a starting point for DNA synthesis.
The enzyme DNA polymerase is used to synthesize new DNA strands by extending the primers.
Nucleotides (dNTPs) are the building blocks that are incorporated into the new DNA strands during the extension process.
The DNA fragments that have been cut using restriction enzymes can be amplified using PCR.
After amplification, these DNA fragments can be ligated with the similarly cut vector DNA for further cloning and recombinant DNA formation.
Ligation of DNA Molecules
The purified DNA fragment of interest and the vector DNA are cut using the same restriction enzyme to generate compatible ends.
This process produces a cut DNA fragment and a cut (opened) vector, making both ready for joining.
The joining of these two DNA pieces is carried out by the enzyme DNA ligase, and this process is known as ligation.
DNA ligase forms phosphodiester bonds between the DNA fragments, permanently linking the insert DNA to the vector DNA.
The resulting DNA molecule is a hybrid composed of two different DNA molecules: the gene of interest and the vector.
In genetic terminology, the intermixing of DNA strands from different sources is called recombination.
Therefore, the newly formed hybrid DNA molecule is called recombinant DNA, and the overall method is referred to as recombinant DNA technology.
Insertion of Recombinant DNA Into Host
In this step, the recombinant DNA molecule is introduced into a recipient host cell, most commonly a bacterial cell.
This process of introducing foreign DNA into a host cell is known as transformation.
Bacterial cells do not naturally take up foreign DNA easily, so they must be specially treated to become competent cells that can accept recombinant DNA.
One common method used is heat (thermal) shock, which creates temporary pores in the bacterial cell membrane to allow DNA entry.
Calcium ion (Ca²⁺) treatment is also used to increase the permeability of the bacterial cell wall and membrane.
Electroporation is another technique in which a brief electrical pulse is applied to create temporary pores in the cell membrane, enabling the recombinant DNA to enter the bacterial cell.
Once inside the host cell, the recombinant DNA can replicate and be expressed.
Isolation of Recombinant Cells
The transformation process produces a mixed population of host cells, consisting of both transformed cells (containing recombinant DNA) and non-transformed cells (without foreign DNA).
A selection process is used to separate and isolate only the transformed host cells from the non-transformed ones.
For the isolation of recombinant cells from non-recombinant cells, marker genes present on the plasmid vector are used.
These marker genes usually confer easily detectable traits, most commonly antibiotic resistance, which helps in identifying successful transformants.
For example, the pBR322 plasmid vector contains two different marker genes: an ampicillin resistance gene (ampR) and a tetracycline resistance gene (tetR).
When the restriction enzyme PstI (PstI RE) is used, it cuts within and knocks out the ampicillin resistance gene in the plasmid.
As a result, recombinant cells that have successfully taken up the plasmid with the inserted DNA become sensitive to ampicillin because the resistance gene is disrupted.
Non-recombinant cells, which still carry an intact plasmid without an insert, remain resistant to ampicillin.
This difference in antibiotic sensitivity allows scientists to selectively identify and isolate recombinant cells from non-recombinant cells.
Application of Recombinant DNA technology
Recombinant DNA technology is widely used in biotechnology, medicine, and scientific research.
One of the most common applications is in basic biological and biomedical research, where it plays a central role in modern experimental work.
Recombinant DNA is used to identify, map, and sequence genes, and to determine their biological functions.
Recombinant proteins are widely used as laboratory reagents and for producing antibody probes that help study protein synthesis in cells and whole organisms.
Numerous practical applications exist in industry, food production, human and veterinary medicine, agriculture, and bioengineering.
DNA technology is used for the detection of HIV in infected individuals.
In agriculture, recombinant DNA technology is used to produce Bt-cotton, which provides protection against bollworms (ball worms) and improves crop resistance to pests.
In medicine, a classic application is the production of human insulin using recombinant DNA technology.
Gene therapy uses recombinant DNA methods as an attempt to correct defective genes responsible for hereditary diseases.
In clinical diagnosis, techniques such as ELISA (Enzyme-Linked Immunosorbent Assay) are examples where recombinant DNA technology has practical applications.
Limitations of Recombinant DNA technology
Introduction of genetically modified species into an environment can lead to the destruction of native species and disruption of natural ecosystems.
Genetically engineered resilient plants can potentially give rise to super weeds that are difficult to control and manage.
There is a risk of cross-contamination and migration of proprietary or modified DNA between different organisms.
Recombinant organisms may escape and contaminate natural environments, leading to unintended ecological consequences.
Recombinant organisms are often genetically uniform populations (clones) and can be vulnerable in exactly the same ways.
A single disease or pest could potentially wipe out an entire cloned population very quickly.
The creation of “superbugs” (highly resistant microorganisms) is a major theoretical concern associated with this technology.
There are significant ethical concerns, with some people believing that humans are “playing God” by interfering with nature’s natural processes of selection.
Fear of the unknown long-term effects of recombinant DNA technology increases concerns about its potential impact on civilization and ecosystems.
There is a concern that genetic technologies could lead to genetic information being stolen or misused without proper consent.
Many people worry about the safety of genetically modified foods and medicines produced using recombinant DNA technology.
References
Verma, P. S., & Agrawal, V. K. (2006). Cell Biology, Genetics, Molecular Biology, Evolution and Ecology (1st ed.). S. Chand & Company Ltd.
Klug, W. S., & Cummings, M. R. (2003). Concepts of Genetics. Prentice Hall, Upper Saddle River, New Jersey.
Educational resource on Recombinant DNA Technology, Byju’s Biology.
General overview of Recombinant DNA, Wikipedia.
Encyclopaedia Britannica article on Recombinant DNA Technology.
Online discussion resource on the advantages and disadvantages of Recombinant DNA technology, Quora.
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