Recombinant DNA Tech: Invented by Berg, Cohen & Boyer for Gene Cloning - Essay Sample

Introduction

Recombinant DNA technology which forms the basis of gene cloning was invented largely through the works of scientists Paul Berg, Stanley N Cohen, and Herbert W Boyer. Gene cloning is a technique in molecular biology that involves the introduction of the foreign gene into another vector or organism. The recombinant DNA is replicated within host organisms and introduced into the cloning vector. The knowledge behind gene cloning is the manipulation of organisms' nucleic acids (Brown, 2013). A known DNA sequence is cut from a particular organism and introduced into another organism hence creating a genetically modified organism (GMO). Manipulating the nucleic acid of a given organism alters its genotype as well as its phenotype. In this regard, gene cloning has been used for various purposes such as creating new and improved varieties of vegetables and fruits, creating new strains of crops, and producing human insulin for diabetics (Brown, 2013). The gene cloning technique has also applied in the transfer of nitrogen-fixing bacterial genes into food crops with the aim of boosting yield without using expensive fertilizers. The technology of introducing an organism's genetic material into another organism's genome has proved to create a significant impact in the development of genetically modified organisms.

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Methods

The process for gene cloning involves the use of bacteria. The bacteria such as E. coli are commonly used within the laboratory setting as cloning organisms due to their ease of use and high rate of reproduction. Moreover, the bacteria comprise of circular DNA molecules called plasmids. The plasmids are separate from chromosomal DNA. Restrictions enzymes such as EcoRI, HindIII, or BamHI are used to treat plasmids (Brown, 2013). After the treatment the plasma DNA is cleaved in unique way along a stand of nucleotides. The same restriction enzyme must also be used in the treatment of foreign DNA to be inserted. This helps in creating compatible DNA bonding areas. It is necessary to introduce DNA ligase to enhance the attachment of recombinant DNA to the plasmid. DNA ligase helps in the creation of phosphodiester bonds between nucleotides. After the process of introducing DNA ligase, the DNA mixture becomes ready to be reintroduced to the host organism. Provided with sufficient nutrients, host organisms can uptake the foreign DNA and undergo transformation.

Results

A clone is an exact copy of a macromolecule, organelle, single cell, organ, or organism. Gene cloning therefore is the technique in which copies of a single gene are made. Gene cloning gives rise to a pure sample of an individual gene. After identifying a specific gene, clones can be used in various fields of industrial and biomedical research. In this regard, genetic engineering has been used to alter genetic sequence with the aim of changing the protein product or give rise to new organisms. Hence, gene cloning has many advantages such as cure for diseases, enhancement of organ replacement and helping infertile couples to bear kids (Brown, 2013). However, gene cloning has some notable disadvantages such as in-breeding, decreases in gene diversity, disruption of family and parenting life and is likely to bring about new diseases. (Brown, 2013).Notably, critical ethical concerns regarding gene cloning have been raised by the anticipated harvest of organs from clones. According to the advocates of human therapeutic cloning, the practice could give rise to genetically identical for tissue transplant and regenerative medicine.

Discussion

Gene cloning involves four major steps. These steps include isolation, ligation, transformation, and screening. Basically, various techniques are involved in these steps. For instance, gene regulation, arrangement and expression, is studied. The genes are then modified to acquire a changed protein product. Gene expression is also modified either to suppress or enhance a given product. Multiple copies of a particular segment of nucleic acid are artificially made. The genes are then introduced from the host organism to the cloning vector resulting in a transgenic organism. This process is basically aimed at creating an organism with altered or desirable characteristics.

The target gene or the DNA fragment to be cloned must be isolated. Isolation is done using a restriction enzyme which can be found in bacteria as well as many other prokaryotes. The restriction enzyme recognizes and binds to a particular sequence of DNA which is known as the restriction site. Notably, the recognition enzyme can recognize a single or multiple restriction sites. When the restriction enzyme finds its target DNA sequence, it cuts a double-stranded DNA at a given point, producing either blunt ends or sticky end. A number of restriction enzymes are fond of producing sticky ends which are characterized by single-stranded overhangs. Molecules with matching overhangs tend to pair each other due to DNA ligase that connects the two strands at the backbone of the DNA. Notably some restriction enzymes produce blunt ends as they cut across the middle the target DNA sequence hence not leaving any overhang.

After isolating the target gene, the next step that follows is ligation. This step involves inserting the amplified fragment into plasmid to facilitate in the production of the recombinant DNA molecule. During the process of ligation, the restriction enzymes used at the previous stage are used to digest the plasmid to open it up so that the target gene can be inserted. Since both the plasmid and the target gene hare a common restriction enzyme, they have complementary sticky ends. The DNA ligase is then used to stick the two DNAs together at the sticky ends. The DNA ligase has the capability of attaching together DNA strands that have double strand breaks. DNA ligase uses ATP as the source of energy to catalyze a reaction in which hydroxyl group sticking off the 3' end of one strand of DNA is linked to the phosphate group sticking off 5' end of the other strand. The reaction results in the production of a sugar-phosphate backbone which is very intact. Consequently, the production of a recombinant DNA molecule takes place. DNA ligase and restriction enzymes are in most cases, used for the insertion of genes as well as other DNA pieces into plasmids during the process of gene cloning.

The step that follows ligation is the transfection or transformation process. This step involves placing the recombinant DNA into the cloning vector or a host cell. The transfection process also involves the practice of culturing cells in an antibiotic mixture. The cells are also subjected to heavy shock such as high temperatures to enable them easily take up foreign DNA. Notably a significant number of these cultures are likely to lack a plasmid with the target DNA since the success rate of the transformation process cannot be predicated and it is often not guaranteed. In this regard, it is a must to appropriately select cultures with DNA of interest. Typically, a plasmid comprises of an antibiotic resistance gene which provides the survival media for the bacteria in a particular antibiotic (Ditta, 1985). Without a plasmid, the bacterial will die as the plasmid supports the bacteria supports the bacteria and enables the bacterial to reproduce in the mixture.

The last step of gene cloning is screening. This process involves selecting the bacteria that took up the plasmid from the nutrient mixture that has the antibiotic. Each bacterium that has a plasmid gives rise to a particular colony comprising of cluster of plasma-containing bacteria that are apparently identical. However, some colonies may not contain the right plasmid. The reason for this is that during the ligation process, fragments of DNA do not often get "pasted" in the way that was supposed to be (Ditta, 1985). It is therefore, necessary to obtain DNA from various colonies and find out whether each of the collected colonies contains the right plasmid. Checking the right plasmid requires the use of appropriate methods such as Polymerase Chain Reaction (PCR) and restriction enzyme digestion.

Conclusion

Gene Cloning is the technology of introducing an organism's genetic material into another organism's genome and has proved to create a significant impact in the development of genetically modified organisms. A colony that contains the right plasmid is grown in bulk to be used in the production of protein or plasmid. Once the colony with the right plasmid has been found, growing of a large culture of plasma-bearing bacterial becomes much easy. The bacterial are them given a chemic signal instructing them to produce the target protein. In this regard, the bacterial play a significant role of miniature "factories" facilitating the production of large volumes of protein. Taking an example of a plasmid that contains the human insulin gene, the role of the bacterial would be the transcription of the gene and translation of the mRNA to enhance the production of several molecules of human insulin protein.

References

Brown, T. A. (2013). Gene cloning and DNA analysis: An introduction. John Wiley & Sons.Ditta, G., Schmidhauser, T., Yakobson, E., Lu, P., Liang, X., Finlay, D. R., Guiney, D., & Helinski, D. R. (1985). Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid, 13(2), 149-153. https://doi.org/10.1016/0147-619x(85)90068-x