Methods and Concepts in the Life Sciences/Isolation of Nucleic Acids
Plasmid preparation
[edit | edit source]The isolation of plasmid DNA from bacteria involves three steps:
- Growth of the bacterial culture
- Harvesting and lysis of the bacteria
- Purification of plasmid DNA
Bacteria containing the plasmid of interest are usually cultured overnight in a medium with an antibiotic that is suitable for the plasmid. Then a sample is centrifuged in order to concentrate cellular material (including DNA) into a pellet at the bottom of the containing vessel. The supernatant is discarded, and the pellet is then suspended in an EDTA-containing physiological buffer. The purpose of the EDTA is to chelate divalent metal cations such as Mg2+ and Ca2+, which are required for the function of DNA degrading enzymes (DNAses) and also serve to stabilize the DNA phosphate backbone.
Subsequently, a strong alkaline solution (pH 12.0-12.5) consisting of the detergent sodium dodecyl sulfate (SDS) and a strong base such as sodium hydroxide (NaOH) is added. The detergent disrupts the cells' membranes and the alkali denatures both the chromosomal and the plasmid DNA.
Finally, potassium acetate is added. This acidifies the solution and allows the renaturing of plasmid DNA. The larger and less supercoiled chromosomal DNA however precipitates along with proteins and the detergent. A final centrifugation is carried out, and this time the pellet contains only debris and can be discarded. The plasmid-containing supernatant on the other hand can be purified in further steps. This lysis method is referred to as alkaline lysis.
The traditional method for nucleic acid purification is phenol-chloroform extraction. In brief, aqueous samples are mixed with equal volumes of a phenol:chloroform mixture. After mixing, the mixture is centrifuged and two distinct phases are formed, because the phenol:chloroform mixture is immiscible with water. The aqueous phase is on top because it is less dense than the organic phase (phenol:chloroform). The proteins will partition into the lower organic phase while the nucleic acids (as well as other contaminants such as salts, sugars, etc.) remain in the upper aqueous phase. The upper aqueous phase is pipetted off and care is taken to avoid pipetting any of the organic phase or material at the interface. This procedure is often performed multiple times to increase the purity of the DNA.
If the mixture is acidic, DNA will precipitate into the organic phase while RNA remains in the aqueous phase since DNA is more readily neutralized than RNA.
Today, kits are commonly used to isolate plasmid DNA. These kits are named by the size of the bacterial culture and the corresponding plasmid yield. In increasing order, these are the miniprep, midiprep, maxiprep, megaprep, and gigaprep.
Miniprep kits combine alkaline lysis with plasmid purification via silica-based spin columns. Under high salt concentrations, positively charged ions can form salt bridges between the negatively charged DNA and the negatively charged silica matrix. Small nucleic acids (up to 70 bp) and proteins can be washed out with an ethanol solution containing a high salt concentration. Afterwards, the plasmid DNA is eluted with water or TE buffer.