Plasmid DNA Isolation

By Gunisha Aggarwal | 31 August 2024

Isolation of DNA

Friedrich Miescher, a Swiss physician, isolated DNA for the first time. Isolation of DNA is the most crucial method of molecular biology. Without it, downstream molecular analysis is not possible. It is the starting point of any experiment.

DNA can be isolated from any biological material such as cells, tissues and viruses. Isolated DNA should have good quantity and quality as it should be pure means free of contamination like that of RNA and proteins. DNA isolation can be done by using organic, non-organic and adsorption methods.

Isolation of Plasmid DNA

Plasmids are small circular extrachromosomal DNA molecules present mostly in bacteria. These are present inside the cell but are physically separated from chromosomal DNA, and can replicate independently. While chromosomal or genomic DNA is large and mainly codes for all the necessary genetic information, plasmids are smaller in size and contain certain genes that confer various properties to the host like antibiotic resistance.

Plasmid isolation is a very important molecular process for gene cloning, gene expression analysis, sequencing and mutagenesis. Isolation and purification of plasmids consists of four steps:

  1. Growth of bacterial culture: Plasmids are isolated from bacterial cultures. These are inoculated with bacterial colonies picked from a cultured agar plate. The culture is grown and aliquots of these are used for plasmid DNA isolation.
  2. Harvesting of cells: It can be achieved by techniques like filtration or centrifugation.
  3. Lysis of cells: Harvested cells are lysed by using certain reagents like non-ionic or ionic detergents, organic solvents, alkali and heat.
  4. Purification of Plasmid DNA: After lysis plasmid DNA obtained is always contaminated with RNA and chromosomal DNA. So, it should be in purified form.

An alkaline lysis using sodium dodecyl sulphate (SDS), a strong anionic detergent at high pH opens the cell wall, denatures chromosomal DNA and proteins. So, it releases plasmid DNA in the solution. This alkaline solution breaks the base pairing. The question arises that then plasmid DNA should also get disrupted in such a case. It happens that the strands of plasmid DNA that are closed circular in shape are unable to separate from each other as they are topologically intertwined, due to which plasmid DNA does not get disrupted.

References:

  1. Sambrook J, Russel D. Molecular Cloning: A Laboratory Manual. 3rd edition. Vol. 1. New York, NY, USA: Cold Spring Harbor Laboratory Press; 2001.
  2. Brown, TA. (2010). Gene Cloning and DNA Analysis, An Introduction. 6th edition. (6th ed.) John Wiley &; Sons Ltd.
  3. Tan, S. C., & Yiap, B. C. (2009). DNA, RNA, and protein extraction: the past and the present. BioMed Research International, 2009(1), 574398.

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