The Concept of DNA Cloning

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Table of Contents

Introduction
Steps
Recombinant Fusion Protein
Chimeric mutation
Conclusion
References

Introduction

When two or more genes that have different kinds of gene code are combined they form Chimeric or fusion proteins. These genes are coded for separate proteins. Eventually a new polypeptide is formed with the functional properties of the proteins that were combined (Tierney, 2007).

DNA cloning can either be based on cells or achieved by using polymerase chain reaction(PCR).In the approach based on cells both the replicating molecule or the biological vehicle known as the vector and the foreign DNA fragment are cut using the same restriction enzyme(s ) to produce compatible cohesive (“sticky”) or blunt ends on the DNA molecules, then the foreign DNA fragment is permanently joined to the DNA of the vector using an enzyme known as DNA ligase which catalyzes the formation of a phosphodiester bond between the two DNA chains thus producing a chimera or a recombinant DNA molecule (Rychlik, 1990). The replicating molecule is meant to carry the foreign DNA fragment into the host cell (Chou & Bloch, 1992).

Fusion proteins can occur naturally by a process known as chimeric mutation. This process occurs when there is a high level of mutation. This creates a new coding system from the two genes.

Fusion proteins can be created in two ways. There is the artificial way and this is known as Recombinant fusion. This kind of fusion is got from the process known as recombinant DNA technology. This technology is used for research in biological fields.

Steps

DNA cloning involves the following steps;

DNA recombination-This involves the identification and isolation of the DNA fragment containing the gene of interest from the chromosomal DNA using restriction enzymes or by using the polymerase chain reaction(PCR),gel electrophoresis and sonication of DNA (Tierney, 2007). The fragment of DNA isolated must be joined to a replicating molecule or vector which acts as a vehicle that transports the DNA into the host cell. Both the isolated DNA fragment and the vector are cut using restriction enzymes at their restriction sites into sizeable fragments suitable for cloning. The desired DNA fragment is inserted into the cut ends of the vector and permanently linked using the enzyme DNA ligase thus forming a recombinant DNA molecule or chimera, but in some instances if processed under in vivo conditions, the enzyme terminal transferase may be added in order to avoid free sticky ends to rejoin instead of forming a chimera since it catalyzes the addition of “tails” of the nucleotide to the 3’ends of the DNA chains.
Transformation-This is whereby the recombinant DNA molecule enters the host cell (which is usually a bacterium) and proliferates. The recombinant plasmid molecule also contains color selection markers which show white/blue screening on a media of X-gal (Saiki & Arnheim, 1985).
Selective amplification-Within the host bacterium the vector multiplies producing numerous identical copies not only of itself but also of the gene that it carries. After a large number of cell divisions, a colony or a clone of identical host cells is produced.
Isolation of desired DNA clones-Culturing of transfected cells is done. The selectable antibiotic resistance markers are used as well as the color selection markers if present in the recombinant plasmid, though further confirmation.

This paper aims to discuss how the fusion proteins are formed from the two processes.

Recombinant Fusion Protein

The first step in this process is identification of the regions of the DNA that are going to be used in the synthesis. This is done by use of bioinformatics. When these regions are synthesized they form antigenic fragments. The region that binds to the antigen on an antibody is known as the fragment. It has a single constant and variable domain of the chain (Talpaz, 2003).

The fragments can be designed for a specific region if required (Perrone, 2005).

The second step is the design of polymerase chain reaction primers. The primers are used to amplify these regions. Primers are used as the starting point to synthesis of DNA. The polymerase chain reaction is used to magnify DNA strands through several orders of magnitude. This leads to the replication of the DNA. This process consists of a subsequent repetition of 20-40 temperature changes. These steps known cycles consist of 2-3 discrete temperature steps. This process happens in three stages:

Exponential amplification: This involves doubling the product in every cycle.
Leveling off stage: This involves slowing down of the reaction until the DNA polymerase loses activity.
Plateau: There is no more formation of products as the entire reagent and enzyme for the reaction is used up (McFarland, 2009).

The third step involves cloning of the DNA using different expression systems. The regions of the DNA that were amplified are first purified using a gel in preparation for the cloning stage. They are then isolated and after that screened for expression performance (Perrone, 2005).

Sequencing and Alignment Analysis is the fourth step in this process. The DNA is arranged in such a way that the regions will be similar in function and structure.

The fifth step is to identify the clones that are relevant for the synthesis. This is can be done in two ways depending on the expression yields required (Kitcher, 2007).

The two are Higher-plex techniques and Low-to-mid-plex techniques.

After that the conditions and additives are optimised so as to improve on the yield of the clones.( Othman& Hart, 1988).

Lastly purification is done to the clones so as to get the required yield. The isolated DNA is purified and placed on electrophoretic gel. Purification is done to produce a yield of more than 94% in purity (McLaren, 2000). Purification of the DNA fragments is important to ensure the integrity of the results. Consequently, the presence of these fragments may affect the clarity and the precision of the results.

Chimeric mutation

The other way that fusion proteins can be formed is through chimeric mutation. The process in which this mutation occurs is known as chromosome translocation. This is a naturally occurring process caused by non homologous chromosomes reconfiguring themselves (Khan & Park, 2008). A fusion is then formed when the two or more genes combine. This is most profound in cancer. This occurrence can be detected by cytogenetic of the cells affected. Translocations can either be balanced or unbalanced. When balanced there is exchange that is in consequential to the gene code but for unbalanced it leads to the missing of some genes thus a chromosome imbalance (Nekton, 1989).

There are two types of translocations (Liu, 1995).

Reciprocal (non Robertsonian) this translocation occurs when there is exchange of materials between chromosomes that are non homologous. This kind of translocation is not harmful. This is normally found in prenatal diagnosis. At times carries of balanced translocations react with those unbalanced translocations. This may lead to miscarriages or abnormality in newborns (Kitcher, 2007).

Robertsonian translocation involves the combining of two or more chromosomes. This happens at the centromere region which leads to the loss of a short arm. The chromosomes are acrocentric. The rearrangement results in a karyotype. Newborns have a have translocations. They occur at chromosomes 13 and 14 (Vicente, 2004).

Translocations are known to cause several diseases in human beings:

Cancer
Infertility
Down syndrome

Conclusion

This paper has so far analyzed the making of fusion tags and their future use in our day to day lives. Fusion tags can be used in different applications, thus enhancing and benefiting the way bodily processes are carried out (Higuchi, 1988).

References

Chou, M. & W. Bloch (1992). Prevention of pre- PCR mis-priming and primer dimerization improves low-copy-number amplifications. Japan: Hanoi publishers.

Higuchi, R. (1988). Primer-directed enzymatic amplification of DNA with a Thermo stable DNA polymerase. Japan: Longhorn publishers.

Khan, Z. & Park, D. (2008). Analytical Biochemistry. India: Longhorn publishers.

Kitcher, P. (2007). There will never be another you. Chicago: University of Illinois Press.

Liu, Y. (1995). Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Madrid: Lion publishers.

McFarland, D. (2009). Preparation of Fusion Tags.London: Oxford Publishers.

McLaren, A. (2000). Fusion Tags in the 21^st Century. Perth: Wiley Publishers.

Nektons, R. (1989). Analysis of any point mutation in DNA. London: Oxford Publishers.

Othman, H. & Hart, D. (1988). Genetic applications of an inverse polymerase chain reaction, London: Oxford Publishers.

Perrone, J. (2005). Government legislation designed to prevent cloning of human beings is on track. London: Oxford Publishers.

Rychlik, W. (1990). Optimization of the annealing temperature for DNA amplification in vitro.Perth: Wiley Publishers.

Saiki, R & Arnheim, N. (1985). Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Berkley: University of Berkley press.

Talpaz, M. (2003). Philadelphia chromosome-positive leukemias: from basic mechanisms to molecular therapeutics. Philadelphia: University of Philadelphia Press.

Tierney, J. (2007). Are Scientists Playing God? It Depends on Your Religion. New York: and CRC publishers.

Vicente, W. (2004). Helicase-dependent isothermal DNA amplification. London: Wiley Publishers.

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