Introduction and History
Recombinant DNA molecules are developed in the laboratory by bringing together genetic materials from multiple organisms to produce new genetic combinations that are not previously found in biological organisms. These new combinations are made to benefit science, medicine, agriculture, and industry (Khan, S et al., 2016).
Stanford researcher Paul Berg, in 1972, ligated DNA fragments of two viruses for the first time. Later in 1973, the first organism containing recombinant DNA molecules was engineered by Herb Boyer (UCSF) and Stanley Cohen (Stanford University). Boyer and Cohen introduced antibacterial resistance recombinant DNA molecules they created in E.coli bacteria.
Paul Berg was awarded the 1980 Nobel Prize in Chemistry due to his significant contributions to deciphering nucleic acids biochemistry with regard to recombinant DNA technology.
The development of recombinant DNA technology was not only a major scientific breakthrough, but it was the fundamental technique that served as a foundation of the entire genetic engineering industry.
In 1976, Gentech, the first biotechnology company, was launched by Herb Boyer. In 1982, Gentech produced synthetic forms of human insulin from bacteria, which were engineered with the insulin gene.
Since then, there has been no stop. Recombinant DNA technology is used extensively in various research labs exploring new genetic combinations that could be of benefit to mankind. Many therapeutic drugs and vaccines, which are the products of recombinant DNA technology, have revolutionized human medicine.
Unlike traditional approaches, rDNA technology uses modern approaches and tools to overcome health, agricultural, and environmental challenges yielding more reliable products in less time.
In this review, the steps of rRNA technology and techniques used in recombinant DNA technology are discussed.
Steps of Recombinant DNA Technology
Selection and isolation of desired DNA
The first step of recombinant DNA technology is the selection of desired DNA molecules and its isolation in pure form (without any contamination from proteins, RNA, lipids, etc.). Different enzymes are used for isolation and purification of DNA like proteases, nucleases, cellulases, etc. After the enzyme treatment, ethanol is used to precipitate DNA.
Selection of cloning vector
The next step in genetic recombination is to select a suitable vector. A vector is a self-replicating molecule that acts as a vehicle to transfer genetic material from one cell to another. A vector containing a foreign DNA material is termed as ‘Recombinant DNA.’
The most commonly used vectors are plasmids and bacteriophages.
Restriction enzyme digestion
Restriction enzymes, also known as molecular scissors, cut DNA at specific locations. Isolated DNA is incubated with suitable restriction enzymes, for digestion, at optimal conditions.
Agarose gel electrophoresis of the digested sample is performed. The technique separates DNA fragments based on the size of the application of electric current. Resolved DNA fragments are then cut from the gel.
Vector DNA is also treated using the same protocol.
Amplification of DNA
The polymerase chain reaction (PCR) is a method to make multiple copies of DNA using a DNA polymerase enzyme. Millions of copies of single DNA fragments can be made by doing a PCR reaction.
Steps of PCR and reagents required for the reaction are mentioned below:
PCR reaction mixture contains:
- Template DNA: DNA that has to be amplified.
- Primers: These are short oligonucleotide sequences that serve as a starting point for DNA synthesis.
- DNA polymerase enzyme
- Nucleotides and co-factors like (Mgcl2).
Steps of PCR
- Denaturation: Two strands of double-stranded DNA are separated by heating at 94-98°C.
- Annealing: Forward and reverse primers bind with each strand at a lower temperature (50-56 oC)
- Extension: DNA polymerase adds nucleotides to the new strand using the original strand as a template. This cycle is usually repeated 20-30 times to generate multiple copies of DNA.
Once the cut DNA fragment is amplified using PCR, now it can be ligated with the vector.
Insertion of desired DNA into a vector to form recombinant DNA
As purified DNA and vectors were cut with the same restriction enzyme. Both DNA and vectors contain cuts that can be easily joined. The process of ligating DNA fragments of interest with the vector is called DNA ligation and is performed by the ligase enzyme. The resultant DNA molecule formed is the recombinant DNA molecule, and this technique is referred to as recombinant DNA technology
Insertion of recombinant DNA in host cells
The next step is the insertion of recombinant DNA molecules in the host cell. Mostly the host cell used is a bacterial cell. Yeast and fungi can also be used as hosts. The process of transferring recombinant DNA into the host cell is called transformation.
For transformation, bacterial cells are first made competent by thermal treatment, electroporation, etc. to accept foreign DNA molecules.
Selection of positive clones
It is important to separate positive cells (transformed cells containing foreign DNA) from non-transformed cells. For this purpose, the marker gene present in the plasmid is used. Different vectors contain different marker genes, for example, antibiotic resistance genes like ampicillin resistance gene, so cells with the plasmid when grown in media containing ampicillin continue to live. However, cells without plasmid die.
It is checked that foreign DNA inserted in the host is expressing the desired characteristic in the host. Transformed hosts are then multiplied to obtain sufficient copies of genes.
Applications of Recombinant DNA Technology
Recombinant DNA technology has a wide range of applications, from crop agriculture, gene therapy, and pharmaceuticals to bioremediation.
- In the agricultural sector, genetic engineering improves both yield and resistance to pests and herbicides. The first genetically engineered crop licensed was ‘Flavr Savr’ tomato, which has increased shelf life feature in it through recombinant DNA technology.
- Recombinant DNA technology is used in gene therapy to correct genes involved in hereditary diseases.
- This technology is widely used in bioremediation, for example, the use of oil-eating bacteria (bacteria with improved ability to digest oil) to clear up oil spillage.
- Recombinant DNA technology has a wide range of benefits to the medical industry. One of the classic examples is the production of human insulin.
- Proteins synthesized using recombinant DNA technology can be used as antibody probes to monitor protein synthesis.
- It can be used to detect, map, and sequence genes.
There are many ethical issues to be considered regarding GMOs, and these are highly argued upon. Some of the issues are mentioned below.
- The introduction of new genetically modified species in the environment can disturb the natural ecosystem; for example, it is being argued that resistant plants may give rise to resistant weed, which can be difficult to control.
- Cross-contamination and horizontal gene transfer between organisms is another argument of the anti-GMO community.
- Recombinant organisms are clones, and they lack genetic variability. Therefore, one disease or pest can wipe out the whole GMO population.
- Many people have safety concerns about the use of GMO food and medicines (Robinson AW, T, 2016).
- Reid, N. (2018, February). Statistical Science in the World of Big Data. Journal of Statistics and Probability, 138, 42-45.
- Khan, S., Ullah, M. W., Siddique, R., Nabi, G., Manan, S., Yousaf, M., & Hou, H. (2016). Role of Recombinant DNA Technology to Improve Life. International journal of genomics, 2016, 2405954. https://doi.org/10.1155/2016/2405954
- Robinson AW, T. (2016). Application of Recombinant DNA Technology (Genetically Modified Organisms) to the Advancement of Agriculture, Medicine, Bioremediation and Biotechnology Industries. Journal Of Applied Biotechnology & Bioengineering, 1(3). doi: 10.15406/jabb.2016.01.00013