One of the most common tasks in molecular biology is cleaning up DNA from aqueous solutions to remove buffer salts, enzymes or other substances that could affect downstream applications. Examples include cleaning up PCR reactions, digests or other enzymatic treatments and cleaning up genomic or plasmid DNA contaminated with cellular proteins/debris. There are several ways to approach DNA clean-up, here are five of them.
1. Phenol-Chloroform Extraction
Phenol chloroform extraction (see Kirby, 1957), normally followed by ethanol precipitation, is the traditional method to remove protein from a DNA sample. In this procedure, the DNA solution is mixed with phenol and chloroform. The water-soluble DNA partitions into the aqueous phase, while the proteins are denatured by the organic solvents and stay in the organic phase. The aqueous phase containing the protein-free DNA can then be collected.
Advantages: A cheap and effective way to remove proteins from DNA solutions.
Disadvantages: Slow compared to most modern methods, there is a risk of phenol/chloroform carry-over into the final sample (which could inhibit downstream enzymatic reactions), chloroform and phenol are both hazardous chemicals.
2. Ethanol Precipitation
Ethanol precipitation is a tried and tested method for de-salting and concentrating DNA. 0.1 to 0.5 M monovalent cations (normally in the form of the acetate salt of sodium) is added to the DNA, along with ethanol to a final concentration of 70%. Ethanol changes the DNA structure so that the DNA molecules aggregate and precipitate from solution (see Eickbush and Moudrianakis, 1978). Since most salts and small organic molecules are soluble in 70% ethanol they stay in solution and the precipitated DNA can be separated from them by centrifugation.
Advantages: A cheap and effective way to de-salt and concentrate DNA.
Disadvantages: Time consuming and risk of ethanol carry-over into the final sample
3. Silica Column-based Kits
Column-based kits offer a convenient approach to DNA cleanup. The principle is that chaotrophic salts are added to the sample to denature the DNA by disrupting it’s hydrogen bonding. Under these conditions, the DNA will selectively bind to the silica resin in the column, allowing it to be separated from the rest of the sample. After washing the DNA is eluted from the column with a low salt solution which allows the re-naturing of the DNA, causing it to lose affinity for the silica. A good example of this technology is Qiagen’s Qiaquick series, which has several kits for agarose gel extraction, enzymatic reaction, nucleotide and PCR clean-up.
Advantages: Convenient, relatively fast and the user can process large number of samples using the vacuum manifold option.
Disadvantages: Fairly expensive, and in my experience low yields (as low as 25%) and chaotrophic salt carry-over are common.
Note: Zymo’s DNA clean-up and concentrator kit offers an alternative, based on the same silica resin technology, where the sample can be eluted in a very small volume to give high DNA concentrations.
4. Strataclean Resin
Stratagene’s StrataClean approach uses a slurry of hydroxylated silica, which (almost magically it seems) binds protein with a high affinity, while having a low affinity of DNA at near neutral pH. The slurry is added directly to the DNA sample, which is then mixed and centrifuged and the supernatant containing the protein-free DNA is collected. The protocol takes just a few minutes, although 2 or 3 clean-ups may be required to certain stubborn enzymes (details are available in the kit’s protocol).
Advantages: Very fast and cheap, no chaotrophic salts or organic washing solutions
Disadvantages: Only removes proteins. Removal of salts requires a traditional ethanol precipitation step.
5. Magnetic Beads
This approach uses magnetic beads that conditionally bind DNA and can be immobilized on a magnetic to separate the DNA from the rest of the sample and allow washing etc. Invitrogen’s ChargeSwitch technology is the best example of this I have seen. It uses magnetic beads that are positively charged, and will therefore bind to DNA, at low pH but at high pH they are negatively charged and release the DNA.
Advantages: Fast, no chaotrophic salts or organic washing solutions, very good yields and spectophotmetric purity in my experience
Disadvantages: The inital outlay for the magnets is reasonably high and the procedure is a bit tricky when handling multiple samples.
I think that covers it! If you have any more suggestions, or indeed any questions, please feel free to add a comment.