Overview of Recombinant DNA Technology

Introduction of Recombinant DNA Technology

Screening microorganisms for valuable products is the key to industrial production. Initially, the focus was on finding microbes from different natural sources. However, there are limitations to the best way to produce the desired product. Although the strain modification method is very successful, the process of strain production is tedious, time-consuming, expensive, and random. At the same time, it is impossible to predict the effects of mutations on other metabolic processes in an organism. In this case, recombinant DNA technology (RDT) was developed.

The concept of RDT was proposed by scientists Stanley Cohen from Stanford University and Herbert Boyer from the University of California during a conversation at a conference in 1973. Cohen had been looking for ways to introduce small circular DNA molecules into bacterial cells, while Boyer was trying to use enzymes to cut DNA on specific nucleotide sequences. In their conversation, they both realized the two technologies may integrate. As a result, the construction of biofunctional bacterial plasmids in vitro laid the foundation for the era of recombinant biotechnology.

Applications of Recombinant DNA Technology

The continued progress of RDT has broadened the horizons of the biotech industry. From the initial production of insulin in E. coli, the generation of various proteins in bacteria, and genetic manipulation spread to plants and animals, RDT creates better conditions for the invention and production of more beneficial products. It replaces traditional strategies and provides a new way to deal with a variety of problems, such as malnutrition, disease, environmental pollution, and agriculture-related problems. The onset of the genomic age shifts the focus of treatment from symptoms to causes, and the identification of correlations between disease phenotypes and patient genotypes opens several avenues for the development of new treatments. These include designing small molecule and biological drugs, developing animal models (disease models) with the same genetic defects as patients, using gene therapy to treat diseases, and xenotransplantation.

Applications of RDTFig 1. Applications of RDT1

RDT facilitates the development of novel vaccine development methods and the development of less pathogenic and more immunogenic vaccines, e.g., subunit vaccines, plant vaccines, DNA vaccines. Metabolic engineering is one of the applications of RDT, where wild-type strains are transformed into more productive ones by making necessary changes in sensible manners. Many of the products prepared by RDT have been approved for commercial use. The high cost and long turnaround time of biopharmaceutical production have forced biotech companies to look for alternatives like biosimilars. Biosimilars are "highly similar" to the original biologic drugs. There are some minor differences in the active ingredients but no significant changes in clinical properties. Recombinant proteins with better properties than existing drugs have also been synthesized, which are called biobetters. So far, trastuzumab biobetter and filgrastim biobetter have been approved by FDA.

AThe strategy used to make the first rDNA moleculeFig 2. The strategy used to make the first rDNA molecule1

λdvgal 120 is a variant of bacteriophage λ, which includes three galactose genes from E. coli. Circular SV40 and λdvgal 120 are linearized using a set of endonuclease and exonuclease. In the next step, the poly A tail is attached to the 3' end of SV40, while the poly T tail is attached to the 3' end of λdvgal 120, which allows hybridization between the DNA, and then covalently attached to the DNA using DNA ligase.

Due to the complexity and size of gene structure, gene modification in higher eukaryotic cells is a challenge. These obstacles have led scientists to develop alternative methods of gene editing. Therefore, comprehensive efforts and numerous experiments have contributed to the development of genome-editing techniques. For example:

  • Transcription Activator-Like Effector Nucleases (TALENs)

TALENs are artificial chimeras of transcription factors and Fok I restriction endonuclease. TF in the DNA-binding domain carries a repeat-variable di-residue (RVD), which is responsible for recognizing the specificity of one of the four nucleotides, while FokI cuts DNA. TALENs enjoy advantages like cost effectiveness, safety, high efficiency, and targeting specificity. And TALENs have been shown to have a high success rate in the animal embryo and plant genome editing.

  • CRISPR/Cas9

CRISPR/Cas9 system is a common platform in genome editing. It is the third generation of genome editing tools developed in 2013. CRISPR, in its natural form, gives organisms immunity by degrading foreign genetic material. Due to its recognition and gene editing capabilities, the CRISPR/Cas system is ideal for editing eukaryotic genomes in the lab, which allows a small RNA (sRNA) that serves as a guide to be cut into DNA to achieve modification. Previous studies have revealed that CRISPR can make changes, or mutations, in the genome more efficiently through these interventions than other gene-editing techniques such as TALEN. In December 2018, the FDA approved CRISPR trials for hereditary childhood blindness.

  • RNA interference (RNAi)

RNAi is one of the post-transcriptional gene silencing techniques that rely on mRNA breakdown. So far, RNAi has been an effective tool for understanding the mechanisms of gene regulation, gene targeting, or gene therapy. The first clinical use of RNAi was to cure blindness caused by age-related macular degeneration. RNAi-based therapies for viral infections, neurodegenerative diseases, and cancer are also being developed. The first RNAi-based drug, Patisiran, was approved by the FDA in 2018. However, safety and efficacy are major limitations to the development of RNAi-based therapies.

Advances in Recombinant DNA Technology

In the past decade, many biotech companies have begun to commercialize biotechnology. It is estimated that the future global biotechnology market value is expected to grow at a CAGR of 15.83%, with many industries contributing to the growth. As expected, the health applications sector accounted for the largest share of 48.64% in 2020, with innovative biopharmaceutical companies developing about 40% of drugs derived from biotechnology. Agricultural companies are also expected to continue this trend by bringing about sustainable productivity improvements through agricultural innovations. All of these innovations are aimed at increasing crop productivity, gaining access to pressure-resistant plants, and providing better diagnostics and affordable medicines.

The RDT marks a paradigm shift in the life sciences. It reships the way researchers approach problems. Building new rDNA and introducing it into microbes, plants, and laboratory animals is standard practice in research laboratories today. This technology helps scientific experts to treat challenging genetic diseases, increase agricultural productivity, biofortify crops, and bring about dramatic changes in forensic medicine, bio-remediation, and more. However, the ethical dilemmas surrounding the growth of rDNA biological commercialization have been the subject of concern and debate. But the accumulation of research and experience helps to understand the potential risks associated with rDNA organisms and predict the outcomes.


  1. Nayana Patil and Aruna Sivaram, A Complete Guide to Gene Cloning: From Basic to Advanced, Springer, 2022, Pages 1-14.

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