STEPS INVOLVED IN GENE CLONING

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Gene cloning (both cell-based and in vitro technique) involves some series of procedures and these shall be highlighted in this unit. Gene cloning in general terms involves the reproduction or making of several copies of the gene of an individual organism through molecular biology techniques (e.g. PCR). The gene of interest is cut with a specific restriction enzyme (endonuclease) and it is inserted into a vector (cut with the same endonuclease). The vector acts as a vehicle and transports the gene of interest into the host cell (e.g. a bacterial cell). But before then, the cut gene of interest or DNA is joined with the vector (e.g. plasmid) using DNA ligase; and this forms a recombinant DNA (rDNA) molecule. The rDNA molecule is inserted into the host cell where it is further replicated as the chromosomal DNA of the host organism is being replicated as well. Specific protein molecule which the gene of interest encodes will be produced and expressed in the host cell. Note: The gene cloning process can be visualized by the gel electrophoresis technique; and this enables the researcher to monitor the reactions going on in the test tube where most of the reaction is taking place.

  • Isolation of plasmid DNA and chromosomal DNA molecule: Plasmid DNA isolation as well as the isolation of chromosomal DNA molecule is usually the first step in any gene cloning experiment. Several techniques are available for the isolation of plasmid DNA and chromosomal DNA from microbial cells. To isolate plasmid DNA molecule from a microbial cell, the bacteria containing the plasmid is first grown overnight in nutrient agar plates and incubated at the correct temperature and growth conditions. The growth medium is incorporated with the antibiotic that corresponds to the antibiotic resistance marker on the plasmid DNA to be isolated; and this allows only the bacteria that contain the plasmid to grow. Several methods exist for the isolation and purification of plasmid DNA molecule. Kits are commercially available for the selective isolation and purification of plasmid DNA molecule and chromosomal DNA molecule from samples.

In the simplest method, the bacterial cells are lysed in test tube by incorporating lysozymes, RNase enzyme and a chelating agent (e.g. EDTA) to suspensions of the organism. The RNase enzyme degrades RNA molecules while the EDTA chelates divalent ions such as Mg++. Lysozyme is a cell wall degrading enzyme, and it attacks the peptidoglycan layer of the bacterial cell wall. The mixture is centrifuged in order to pellet large molecules. The plasmid DNA molecule is usually contained in the supernatant which also contain other soluble cell components. The supernatant in this case is known as the cell lysate. The lysate is treated with NaOH to denature all DNA molecules; and a column resin technique which normally binds DNA molecules is used to purify the plasmid DNA molecule. In the column resin method, small amounts of the resin are mixed with the supernatant containing the plasmid DNA molecule. Plasmid DNA binds to the resin. Plasmid-bound resin is collected in a small column; and the plasmid DNA is eluted from the resin while the remaining cell components are washed away. Several rapid techniques are available for the isolation of plasmid DNA molecule from cells. The isolation of chromosomal DNA molecule from cells is similar to the procedure used for the isolation of plasmid DNA molecule. However, chromosomal DNA molecule unlike the plasmid DNA is precipitated; and the cell lysate is extracted with phenol. Chromosomal DNA is much fragile than plasmid DNA and thus, the former cannot be purified using columns as is the case for plasmids. Centrifugation technique is used obtain the precipitated chromosomal DNA molecule.          

  • Cutting of DNA or gene of interest and vector: The gene of interest or DNA is cut or nicked with specific restriction enzyme or endonuclease in vitro. The same restriction enzyme used in cutting the DNA of interest is also used to cut the plasmid vector that will carry the gene of interest or DNA into the host cell. The restriction enzyme specifically cut the plasmid vector at one of its restriction sites in order to allow for the insertion of the foreign or exogenous DNA. The reason for using the same restriction enzyme to cut both the DNA and plasmid vector is to ensure specificity of nicking so that the foreign DNA can be properly inserted into the plasmid. Complementary sequences on the DNA fragment and the plasmid vector will be generated after cutting (Figure 1).
Figure 1.Illustration of gene cloning technique. Photo courtesy: https://www.microbiologyclass.com
  • Ligation: After nicking the DNA molecule and plasmid vector respectively, both are mixed in a test tube where DNA ligase enzyme is added for ligation. Ligation is the molecular biology technique of joining two DNA molecules together with the help of DNA ligase enzyme. After ligation, a recombinant DNA molecule carrying the gene of interest to be cloned will be formed (Figure 6.3). The restriction enzyme used for cutting should be inactivated before the addition of DNA ligase enzyme for ligation activity in order to prevent the endonuclease from cutting the DNA molecule every time it is joined by the DNA ligase enzyme.    
  • Transformation: Transformation is the process of transferring exogenous DNA molecules into host cells.  
  • Isolation and selection: Plasmids used for gene cloning techniques normally contain selectable biomarkers such as antibiotic resistance genes. And by isolating and plating them onto agar plate containing the antibiotic to which the organism is resistant to, only successfully transformed bacterial cells which have taken up the plasmid will be able to grow. The bacterial cells are resistant to the antibiotic in the agar medium because they harbour plasmid that expresses a particular antibiotic resistance gene.
  • Expression: The inserted and cloned gene of interest is transcribed and translated into proteins. Thus, the gene of interest is therefore expressed within the host cell with the gene product being a specific protein molecule. After cloning a gene, it is important to have the cloned gene express the product it encodes. The cloned gene can also be sequenced to know the exact sequence of bases in the DNA molecule.

Further reading

Cooper G.M and Hausman R.E (2004). The cell: A Molecular Approach. Third edition. ASM Press.

Das H.K (2010). Textbook of Biotechnology. Fourth edition. Wiley edition. Wiley India Pvt, Ltd, New Delhi, India.

Davis J.M (2002). Basic Cell Culture, A Practical Approach. Oxford University Press, Oxford, UK. 

Mather J and Barnes D (1998). Animal cell culture methods, Methods in cell biology. 2rd eds, Academic press, San Diego.

Noguchi P (2003).  Risks and benefits of gene therapy.  N  Engl J Med, 348:193-194.

Sambrook, J., Russell, D.W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York.

Tamarin Robert H (2002). Principles of Genetics. Seventh edition. Tata McGraw-Hill Publishing Co Ltd, Delhi.     

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