[priyasaravanan_1406]

Being first coined by a molecular biologist in 1952 plasmids, the small, circular, extra chromosomal DNA mainly found in bacteria made its entry into the field of molecular biology as vectors in 1970s. Plasmids are able to self replicate without any dependence on the host chromosomal DNA, carry genes useful for the host organism (for example, antibiotic resistance gene) and also possess the ability to transfer among different bacterial species through horizontal gene transfer. These characteristic makes plasmids a useful vector tool in molecular biology in developing recombinant molecules. It is not that a single bacterial cell contains only one plasmid as there is a possibility of different plasmids existing in a single bacterial cell. Based on their ability to transfer between species plasmids are classified as conjugative and non-conjugative plasmids and another classification of plasmids made based on the function and the type of genes they carry include fertility plasmid, resistance plasmid, virulence plasmid, col plasmid and degradative plasmid.

With its wide application in molecular biology in the preparation of recombinants, plasmids need to be extracted or isolated from its host organism and thus isolated plasmids need to be purified. The initial step required in the extraction or isolation of the plasmid from the host bacteria is the preparation of the host bacterial culture which is called as minipreparation or miniprep from which the plasmid is extracted. As the name indicates miniprep involves preparation of few ml of the culture by inoculating the host organism containing the desired plasmid in the organism specific broth and allowing to grow overnight to attain the stationary phase. At this stage the culture is subjected to centrifugation, as a result the bacterial cells settle down forming a pellet which is then harvested and exposed to various processes like alkaline lysis, phenol extraction or cesium chloride gradient and ethanol precipitation to obtain pure plasmids.

In alkaline lysis, the harvested cell pellet is treated with buffer solution followed by the addition of special solution which initiates lysis of the cell and hence called as cell lysis solution. This solution is a combination of sodium dodecyl sulfate and sodium hydroxide, whereas sodium dodecyl sulfate acts on the cell membrane, lyses the membrane and also acts on the proteins by denaturing them and the alkaline environment created by the addition of the sodium hydroxide enables the denaturation and hydrolysis of the DNA and RNA respectively. The solution is then neutralized by the addition of potassium acetate which enables the precipitation of the proteins (denatured) and DNA (chromosomal) which upon centrifugation settles down leaving the plasmid DNA, RNA and fewer proteins in the supernatant.

The supernatant containing the plasmid, RNA and less protein is subjected to either phenol extraction method or cesium chloride gradient method to extract the plasmid alone from the mixed supernatant. The cesium chloride gradient is effective in obtaining pure plasmids and is adopted for plasmid production in large scale. In phenol extraction method, the addition of phenol to the supernatant followed by vigorous mixing denatures the proteins present in the supernatant which is finally seen as a separate precipitated layer below the plasmid - RNA layer. The plasmid - RNA layer is removed and processed with ethanol (ethanol precipitation) which enables the pelleting of the plasmid alone which is then treated with Tris hydrochloride and EDTA solution whereas the former is added to create buffer environment and the later takes care of any Magnesium ions present in the solution by chelating the ions and thus protects the plasmid DNA. The fear of RNA presence is cured by adding Ribonuclease A to the solution which takes care of the RNA and thus pure plasmid can be obtained.

In cesium chloride gradient method, as already said used for large scale plasmid production, the supernatant is treated with the solution of cesium chloride and ethidium bromide which upon centrifugation forms different fractions in the tube based on the density. The fractionation in the tube is seen as protein layer on top of all, followed by layer of chromosomal DNA, below which is the layer of super coiled plasmid and finally RNA is seen as pellets at the bottom of the tube. The plasmid layer is then recovered and subjected to ethanol precipitation method to obtain the pure plasmid.

Thus the plasmids with unique characteristics can be isolated and purified to be used as vectors in molecular biology.