Since DNA was first extracted by Johannes Friedrich Miescher in 1869, several methods and commercial kits for DNA extraction have been developed. Such methods range from the simple use of crude lysates to the more sophisticated use of columns and kits, tailored specifically to almost any sample.
Crude lysates can be prepared using any of the lysis methods, such as detergents and ultrasonication, followed by heating at high temperatures or performing a proteinase K digestion, to denature or digest the proteins, respectively.
By far the most simplistic method of extracting DNA, the use of crude lysates is limited to very few molecular applications. Indeed, the lack of a purification step results in high levels of contamination, which can lead to DNA degradation.
Another simple and inexpensive method, the salting-out procedure begins with a crude lysate and involves the addition of salt, such as potassium or ammonium acetate, to precipitate out proteins and other contaminating components. The precipitate is subsequently removed via centrifugation, and the DNA is retrieved via alcohol precipitation.
As with the use of crude lysates, the salting out method generally results in higher contamination than other methods, and the extracted DNA often requires further processing before it is suitable for molecular testing.
Phenol-Chloroform (Organic Extraction)
Effective at extracting large amounts of high-molecular weight genomic DNA, the organic extraction method has been used for several years.
Using this multi-step method, cells are lysed and a mixture of phenol, chloroform and isoamyl alcohol is employed to remove lipids, carbohydrates, proteins and cell debris from the lysate. Next, the DNA is precipitated using a mixture of alcohol and salt. The precipitated DNA is then purified by centrifugation and washed in ethanol, prior to re-suspension.
This technique can be employed to isolate DNA from a variety of samples; however, compared with the more modern techniques, it is very time-consuming and labour-intensive.
Alkaline Extraction Method (for Plasmid DNA Isolation)
First developed in 1979, this simple DNA extraction method enables the isolation of purified plasmid DNA that may be used immediately, for example, to transform other bacterial cells.
Also referred to as “alkaline lysis”, this method usually involves re-suspending a pellet of bacterial cells in a buffer solution, prior to lysing the cells in an alkaline sodium hydroxide solution with the detergent sodium dodecyl sulphate (SDS). This reagent combination disrupts the cell membrane and denatures the proteins and DNA (i.e. the double strands come apart). Following a neutralisation step with potassium acetate at pH 5, the chromosomal and plasmid DNA reanneal (i.e. the double strands reform). During this step, the (covalently closed) plasmid DNA strands recombine correctly and remain in solution. The genomic DNA, however, reanneals randomly; thereby forming a precipitate with the denatured proteins and most of the detergent. To complete the process, the unwanted material is removed by centrifugation and the plasmid DNA is recovered via alcohol precipitation.
This method of plasmid DNA extraction is far safer and less expensive than the caesium chloride (CsCl) density gradient method, which requires the use of an ultracentrifuge and ethidium bromide.
Caesium Chloride (CsCl) Density Gradient Centrifugation
This method is suitable for the extraction of RNA, genomic DNA and the large-scale extraction of highly purified (supercoiled) plasmid DNA. It delivers high-quality nucleic acids, but is extremely laborious and time-consuming.
The DNA extraction procedure typically involves the lysis of intact cells in alkaline conditions with a protease (e.g. proteinase K), a detergent (e.g. SDS) and RNase, followed by alcohol precipitation. The re-suspended DNA is subsequently centrifuged on a CsCl density gradient, in the presence of ethidium bromide. Covalently closed DNA binds less ethidium bromide than linear DNA; hence, plasmid DNA accumulates at lower densities in the CsCl gradient. The solution containing the desired DNA band is then collected, the ethidium bromide is removed using isopropanol and the DNA is recovered via alcohol precipitation.
This technique can be employed to isolate various nucleic acids for sensitive downstream applications, including sequencing and transfection; however, its usefulness is limited by the requirement of specialist equipment, long centrifugation times and the use of toxic materials.
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