The Good, The Bad & The Ugly: DNA Extraction Techniques
Getting great results with your DNA-based experiments is dependent on getting great quality from your samples — without spending too much time on extraction. In this article, we review the DNA extraction choices currently available so you can learn about the various pros and cons and identify the best choice for your lab.
Phenol–chloroform extraction – Cheap, but lots of preparation time
Phenol–chloroform extraction is one of the first widely used methods for extracting DNA and is still used by many labs. Cells are first disrupted in the presence of phenol and chloroform along with alcohol, salts, EDTA and SDS to improve the extraction and stabilize the DNA. Subsequently the samples are centrifuged to separate the mixture into two phases: a DNA-containing aqueous phase on the top and an organic phase on the bottom, with a layer of cellular debris in between. After transferring the aqueous phase to a new tube, DNA is further cleaned with ethanol washing and precipitation.
Advantages
Acceptable yield and purity of extracted DNA
Inexpensive
Challenges
Very time consuming
Potential contact with harmful chemicals
High risk of cross-contamination
Requires a high level of skill avoid transferring contaminants from the solid phase
Potential for carry-over phenol, which denatures proteins
Salting out DNA – reliable and easy, but lower yields and purity
Precipitation methods with salts, such as sodium chloride, potassium acetate and ammonium acetate, separate hydrophobic proteins and other components in the sample from hydrophilic nucleic acid molecules. These techniques might include cetyl trimethylammonium bromide (CTAB) to break up hard cell walls from plants, fungi and bacteria and to help to separate polysaccharides and pigments from the DNA. Polyvinylpyrrolidone (PVP) is a buffer component commonly added to prevent coprecipitation of polyphenols with nucleic acids. For applications that are sensitive to contaminants, such as PCR, extra steps for chloroform–isoamyl alcohol extraction and ethanol precipitation is generally needed.
Advantages
Softer than the phenol–chloroform method
Prevents hydrolysis of the bases for higher
DNA integrity Moderately easy with acceptable
DNA purity Generally low cost
Challenges
Variable yields
Time-consuming preparation of chemicals
Potential contamination with enzyme inhibitors
Reduced yields from loss in alcohol precipitations
Solid-phase DNA extraction kits – relatively quick and easy, but higher costs and waste
Column-based commercial kits are quicker and easier to use than the manual methods described above. In many of these, the negatively charged DNA binds to the positive charge of the stationary phase of the column due to the presence of chaotropic salts and alkaline conditions. Contaminants and high-concentration salts are removed with centrifugation and subsequent washing steps. The DNA is eluted from the column with nuclease-free water, often containing a buffer, such as Tris-EDTA.
Advantages
Very efficient and selective binding of DNA
Good quality and yield of nucleic acid
Reduced variability of yields compared to manual techniques
Reduced contamination with inhibitors in the sample
Quicker than manual methods
Challenges
DNA length affected by shearing forces during centrifugation
Losses in yield from each column wash
Loss of DNA molecules that are too small to stick to the column
Substantial increase in plastic waste
Magnetic beads – elegant, but hard to handle
Another method available as commercial kits is based on magnetic beads. Nucleic acids bind to various coatings applied to small particles containing magnetic iron oxide. Reversible binding of the nucleic acids is achieved with specific salt concentrations. After the nucleic acids bind to the beads, the beads are held in place with a magnet, allowing the supernatant to be removed. The nucleic acids are subsequently released into a new buffer.
Advantages
Fast and easy method
High-quality DNA
Option to select DNA based on size
No sheer forces from centrifugation for greater DNA integrity
Challenges
Time-consuming tube transfers
Carry-over contamination reduces DNA quality
Requires precisely correct salt concentrations
Beads carry-over interferes with downstream applications
Single-step purification — an advantageous new method
A relatively new technique for DNA purification offers the quickest way to get excellent DNA quality. This technique provides a unique combination of a component that protects genomic DNA as well as active enzymes that greatly improve lysis efficiency. In this process, the DNA flows through and everything else binds to the column, allowing highly pure and intact genomic DNA to be obtained with only one centrifugation step.
Advantages
Improve performance with better purity, DNA integrity and yield
Simplify your workflow with single-step purification after lysis
Reduce time to result
Keep samples under physiological, DNA-protecting conditions
Improve lysis efficiency for great yields
Eliminate mechanical disruption and overnight lysis for non-plant samples
Avoid the risk of DNA damage from repeated centrifugation
Eliminate separate disposal of harmful chemicals
Reduce plastic waste by 70% compared to silica kits
If you like to learn more about this DNA extraction technique, find more details here.