Gene Cloning: A Valuable Tool in Biotechnology

Although in the biosciences world, gene cloning is a standard procedure conducted by the majority of labs daily, the word “clone” still concerns a great number of people. The truth is that in the last 20 years, gene cloning has opened the way for new scientific findings, facilitating the access to large numbers of DNA molecules. Moreover, the technology of dozens of pharmaceutical products, biological tests and medical devices is based on gene cloning techniques. Gene cloning is defined as the procedure in which a certain gene is isolated from an organism’s genome and amplified in millions or even billions of copies (National Institute of Health, 2023). The laboratory procedures that include DNA isolation and manipulation are known under the term “Recombinant DNA Technology”. This article thoroughly analyses the basic steps for the creation of gene clones and refers to some of the most popular application of them in current biotechnology.

Step-by-step Guide to Gene Cloning

Although gene manipulation techniques are constantly evolving, the basic core of gene cloning remains the same. The first step towards creating a gene clone is the selection of the gene. Online databases, nowadays give the opportunity to observe the sequence of a gene, its location within the DNA molecule and its size.

Next step is the isolation of the desired gene from a DNA molecule. The traditional technique requires the use of a restriction nuclease. Restriction nucleases are enzymes found bacteria, that recognize specific nucleic acid sequences and cleave the DNA molecule at those specific sites. These enzymes act as a defensive mechanism of the bacterium against bacteriophages. Each restriction nuclease recognizes different DNA sequences and cleaves fragments of different sizes. The nuclease cleaves the DNA molecule, isolating the desired gene (Celie, Parret, & Perrakis, 2016).

Figure 1: Restriction Enzyme (University of Waikato, 2007)

Before the start of the amplification process, the gene fragment has to be inserted into a cloning vector. A cloning vector is a small piece of DNA into which foreign genomic material can be incorporated. The most well-used vectors in biotechnology are plasmids and bacteriophages. Before the insertion of the gene fragment, the vector is being cleaved by a restriction nuclease, usually the same as the one that was utilized to cleave the DNA of interest. The gene fragment is then incorporated into the DNA of the vector. A new recombinant DNA molecule containing the vector’s DNA, gene fragment and often many more sequences is created (Carter, Essner, Goldstein, Iyer, & Carter, 2022).

In order for the gene fragment to amplify and create clones, the recombinant molecule is transferred into a host cell, which is usually a bacterium. The process by which foreign genetic material enters a bacterium is called “transformation”. Once the recombinant DNA molecule is inside the host cell, the replication machinery of the bacterium allows it to amplify. The host cell is then grown in culture media. It undergoes cell division and the recombinant molecule keeps amplifying, creating multiple cloned molecules (Wilson & Hunt, 2002).

It is also important to mention the presence of marker genes in the vector DNA. Marker genes are DNA sequences that are also incorporated into the vectors to allow the detection of the desirable clone in the cell culture. There are two types of marker genes known as selectable markers and screenable markers. Selectable markers are usually genes that express a protein which protects the cell from agents that would normally be toxic to it. One of the most popular selectable markers is the ampicillin resistance gene. If ampicillin is added into the bacterial culture, only the cells that present ampicillin resistance will be kept alive. Screenable markers, on the other hand, allow the visual detection of clones (Simões et al., 2016). These marker genes express proteins that offer a distinctive visual characteristic to the transformed cell, such as green fluorescence or another color (Sakanyan et al., 1993).

The amplification process can be accelerated with the use of the Polymerase Chain Reaction (PCR). PCR is a very common laboratory technique that allows for the rapid production of millions or even billions of copies of a specific DNA molecule. This technique does not require the use of bacteria and it is automatically conducted in PCR machines (Roberts, 2019).

Closing this chapter, it is important to mention genomic libraries. These libraries are collections of cloned DNA pieces that constitute the entirety of the genome. A genomic library is created exactly as described above. The whole genomic DNA of a cell is cleaved by restriction enzymes, each fragment of DNA is incorporated in a separate vector and finally, each recombinant vector transforms a bacterium. Thousands of clones are then generated after the culture of the bacterium (Godiska, Wu, & Mead, 2013).

Figure 2: Creation of Genomic Library (Encyclopaedia Britannica)

Gene Cloning Applications

DNA clones are widely used in biosciences research and drug development. The transformed host bacteria can be used as protein factories to produce large quantities of a specific protein. Most gene therapy techniques also majorly rely on gene cloning. The healthy genes that are incorporated into diseased cells are usually products of gene cloning, which have been developed in the lab. In research, gene clones are also very often used for genetic analysis. After sequencing, cloned genes can be used to study the nucleic acid sequence, the molecular function of a protein or even the genetic basis of a disease. Finally, gene cloning has a leading role in the biopharmaceutical research and drug development. As mentioned above, transformed bacteria act as protein factories that produce huge amounts of a desired protein. As such, proteins associated with human disease, like insulin or human growth hormone, can be mass-produced and purified from DNA libraries. Interestingly, gene cloning is also used in agriculture for the production of more natural, less toxic crops.

Figure 3: Genetic Revolution (D'Urbino, 2022)

Conclusion

Gene cloning is one of the most powerful tools of biotechnology at the moment. This technique is behind the majority of lab-based academic research, drug development and modern agriculture. Innovation in lab technology and use of more and more molecular tools is perfecting the gene cloning procedures, giving the opportunity to produce more clones in small amounts of time.

Bibliographical References

Carter, M., Essner, R., Goldstein, N., Iyer, M., & Carter, M. (2022). Guide to research techniques in Neuroscience. Amsterdam: Academic Press.

https://doi.org/10.1016/C2018-0-04205-X

Celie, P. H., Parret, A. H., & Perrakis, A. (2016). Recombinant cloning strategies for protein expression. Current Opinion in Structural Biology, 38, 145–154. doi:10.1016/j.sbi.2016.06.010

https://doi.org/10.1016/j.sbi.2016.06.010

Godiska, R., Wu, C.-C., & Mead, D. A. (2013). Genomic libraries. Brenner’s Encyclopedia of Genetics, 306–309. doi:10.1016/b978-0-12-374984-0.00641-0

https://doi.org/10.1016/B978-0-12-374984-0.00641-0

Biesecker, L. G. (2023). Cloning. National Human Genome Research Institute https://www.genome.gov/genetics-glossary/Cloning

Roberts, M. A. (2019). Recombinant DNA technology and DNA sequencing. Essays in Biochemistry, 63(4), 457–468. doi:10.1042/ebc20180039

https://doi.org/10.1042/EBC20180039

Sakanyan, V., Desmarez, L., Legrain, C., Charlier, D., Mett, I., Kochikyan, A., … Pierard, A. (1993). Gene cloning, sequence analysis, purification, and characterization of a thermostable aminoacylase from bacillus stearothermophilus. Applied and Environmental Microbiology, 59(11), 3878–3888. doi:

https://doi.org/10.1128/aem.59.11.3878-3888.1993

Simões, G. A. R., Xavier, M. A. S., Oliveira, D. A., Menezes, E. V., Magalhães, S. S. G., Gandra, J. A. C. D., & Xavier, A. R. E. O. (2016). Genetic markers for detection of escherichia coli K-12 harboring ampicillin-resistance plasmid from an industrial wastewater treatment effluent pond. Genetics and Molecular Research, 15(2). doi:10.4238/gmr.15028528

http://dx.doi.org/10.4238/gmr.15028528

Wilson, J. H., & Hunt, T. (2002). Molecular biology of the cell, 4th edition: A problems approach. New York: Garland Science.

https://www.ncbi.nlm.nih.gov/books/NBK26837/

Visual Sources