Vectors Used in Biotechnology

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1. Plasmids
Plasmids are closed circular, double-stranded, extrachromosomal DNA molecules which occur naturally in bacteria, yeast, and some higher eukaryotic cells, and exist in a parasitic or symbiotic relationship with their host cell (Lodish et al., 2000) The main application of plasmids is as cloning vectors in gene cloning. In gene cloning, a fragment of DNA, containing the gene to be cloned is inserted into a circular molecule called the “vector” to produce recombinant DNA molecule. Plasmids are one of the most commonly used “vectors” for this purpose. They transport the gene into a host cell, such as a bacterium, which is said to be transformed with the recombinant molecule. Here, these plasmid vectors multiply, producing numerous identical copies of itself and the gene that it carries. Like the host-cell chromosomal DNA, plasmid DNA is duplicated before every cell division; they replicate independent of the chromosomal DNA. During cell division, at least one copy of the plasmid DNA is segregated to each daughter cell, assuring continued propagation of the plasmid through successive generations of the host cell. Thus after a number of successive cell divisions, various identical host cells are produced, thus, the gene carried by the plasmid is “cloned” (Brown, 2006; Lodish et al., 2000). Furthermore, the transformed bacterial host cells may have the ability to express the gene, thus producing proteins encoded by the gene included in the recombinant plasmid. This application can be used for producing proteins in large quantities which can be purified further, as is done for the production of recombinant insulin. Also, plasmids could be used in gene therapy, for delivery of therapeutic gene of interest into human host cells, without causing cell injury, oncogenic mutations and an immune response (Daugherty, 2007; Lipps, 2008). Various features of plasmid that make it a suitable cloning vector are: 1. Plasmids contain an “Origin of replication” sequence (ori), which allows the plasmid to replicate/ multiply inside the cell independent of the bacterial chromosome, and thus maintains the plasmid DNA in the cell population as the host organism grows and divides, allowing a large number of copies of the plasmid to be made (Brown, 2006). 2. Plasmids have antibiotic resistance genes which allow the plasmid to survive in normally toxic concentrations of antibiotics such as ampicillin, and this feature can be used as a selectable marker to conveniently select for “transformed” bacterial cells containing a particular plasmid from a culture (Brown, 2006). 3. Plasmids have a region that contains several unique restriction endonuclease sites in a short stretch of the DNA, known as a “polylinker” region or Multiple Cloning Site (MCS). This provides a variety of choices of sites available for insertion of DNA fragments during recombinant DNA production, because it provides a greater choice for restriction enzymes that can be used in cloning (Brown, 2006; Nicholl, 2008). 4. Plasmids have a packaging capacity (that is they can take foreign DNA) upto 10 kb in length which is usually very suitable for cloning. Moreover, a cloning vector should be ideally less than 10kb in size, as large molecules tend to break down during purification. Plasmids usually range from about 1.0kb to over 250 kb for the largest plasmids. Thus, plasmids, especially small ones, are very relevant and useful for cloning purposes, as far as size is concerned (Brown, 2006; Lipps, 2008). 5. Plasmids are inherited stably, without integration and they have reduced toxicity, features that make plasmids more advantageous than viral vectors in gene therapy (Stoll & Calos, 2002). They can be transferred into many cell types, thus increasing their usefulness (Brown, 2006). 6. Also it is easy to find plasmids with a high copy number, which is useful because multiple copies of the cloning vector need to be present in a cell so that large quantities of the recombinant DNA...
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