GENE CLONING / CLONING

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Cloning is a molecular biology technique which is used to make millions of copies of a particular gene of interest or a piece of DNA molecule. It generally involves taking a piece of DNA molecule (containing specific gene sequence of interest) from a donor organism where it naturally exists and putting same into a cloning host cell (e.g. Escherichia coli) through the help of a vehicle such as a vector or plasmid which carries the gene of interest into the recipient host.At the end of a gene cloning process, many identical copies of the cloned DNA or gene known as clones will be generated for further molecular studies. The cloned gene of interest also expresses or produces the proteins which it actually encodes within its host cell. A clone is a population of identical cells or DNA molecules.

Vectors in gene cloning technique are pieces of circular DNA molecule that are competent enough to transport DNA between hosts and are also capable of independent replication within the cells of their host organisms. A vector is a vehicle for foreign or passenger DNA molecules. Most vectors used for gene cloning techniques are either plasmids or phages. Cloning vectors therefore are DNA molecules that are used to transport cloned sequences of a gene or DNA between biological hosts and the test tubes. Typical examples of cloning vectors used for gene cloning techniques and other recombinant DNA technological applications include plasmids, yeast artificial chromosomes (YAC), reporter gene vectors, bacteriophage lambda or phage, cosmid vectors and shuttle vectors. However, the type of cloning vector used for the gene cloning technique is usually dependent on the expertise of the researcher and the type of experimentation to be undertaken because each of these vectors varies in their ability to transport the gene of interest from one host or test tube to another.

Plasmids are mostly used in gene cloning techniques because they have small sizes and are easy to manipulate. They are extra-chromosomal DNA molecules that are capable of independent (autonomous) replication. Their circular structure makes them to be more stable than other vectors. Plasmids have biomarkers such as antibiotic resistance markers which aid their easy detection and recovery from a cloning process. The millions of copies of the cloned DNA or gene generated after a given gene cloning technique have a wide variety of medical and biomedical as well as industrial applications. For example, copies of the cloned DNA can be used for sequencing analysis, molecular probes and for paternity tests. Cloned DNA or gene can also be used to generate transgenic animals and crops with immense resistance to pests. Cloned gene can also be used to study the gene expression and function of a given protein molecule in a living system. Several biopharmaceutical products (e.g. insulin) and proteins that are of medical significance have also been generated in large amounts through gene cloning techniques. The significance of this important technique of molecular biology is enormous.

The term cloning encompasses gene cloning and molecular cloning. Gene cloning can be achieved in a variety of ways but gene cloning by PCR (in vitro technique) and cell-based gene cloning techniques (in vivo method) are usually the main methods involved in performing any cloning experiment in the molecular biology laboratory. While the cell-based gene cloning technique is mainly performed in a living system which may include a bacterial cell, yeast cell, mammalian cell or arthropods; the PCR gene cloning technique is carried out in a piece of test tube using the thermocycler. Cloning allows a copy of any specific part of a DNA or RNA sequence to be selected among many other copies and be reproduced in an unlimited amount in a host cell.

PROPERTIES OF PLASMIDS USED FOR GENE CLONING TECHNIQUES

  • Plasmids contain selectable marker such as antibiotic resistance gene which aid in their easy detection and recovery after the gene cloning experiment.
  • Plasmids used for gene cloning have relatively small sizes (e.g. 10 kb), and thus they can be easily purified and manipulated. However, they cannot be used for the cloning of large sized DNA molecules (especially eukaryotic nucleic acid) whose sizes are above 10 Kb (e.g. 100 Kb). Such large DNA molecules are cloned with other vectors such as the YAC vector.
  • Their circular shape or structure makes them more stable than other vectors used for gene cloning manipulations.
  • Plasmids can produce several copies of the DNA insert unlike other vectors which are limited in their generated clones.
  • They are capable of autonomous replication in vivo.

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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