Isolation of plasmid by alkaline lysis
Plasmids are circular molecules of DNA that lead an independent existence in the bacterial cell. Plasmids almost carry one or more genes responsible for a useful characteristic displayed by the host. Classification of the naturally occurring plasmid is based on the main characteristic encoded by the plasmid. The five main types of plasmids are:
- Fertility or F-plasmids – these plasmids carry ‘tra’ genes which promote conjugal transfer of the plasmid.
- Resistance or R-plasmid – these plasmids carry genes that confer on the host, some antibiotic resistance.
- Col plasmids – these plasmids code for colicins (proteins that kill other bacteria)
- Degradative plasmids – these plasmids allow the host molecule to metabolise unusual molecules such as toluene and salicylic acid.
- Virulence plasmid – these plasmids confer pathogenicity on the host bacterium.
- Isolation of plasmids involves the use of three solutions for the extraction and purification of plasmid. These are:
• Solution I – contains EDTA which lyses the cells and chelates metal ions, thus weakening the cell wall and inactivating enzymes that digest DNA.
• Solution II – contains SDS and NaOH. SDS is an anionis detergent, which removes the lipid molecules and hence disrupts the cell membrane. It also helps denature the bacterial proteins. Addition of NaOH increases the pH of the solution (hence the name alkaline lysis) thereby denaturing bacterial chromosomal and plasmid DNA.
• Solution III – contains potassium acetate which reduces pH and therefore the plasmid DNA renatures due to its small size.
- The chromosomal DNA strands and bacterial proteins form a precipitate along with SDS which is removed by centrifugation. The renatured plasmid present in the supernatant solution is concentrated by the addition of isopropanol.