Methods of DNA extraction
DNA extraction is a procedure used for purifying DNA from the nucleus of cells to apply it for further investigations, like PCR or sequencing.
Generally, four necessary steps are required for successful DNA purification:
1. Cell disruption or cell lysis of the cellular membranes, breaking the cell walls open by sonicating or heating the sample with a lysis buffer.
2. Protein denaturation; dehydration and precipitation of the cellular proteins.
3. Removing the insoluble cell debris; cellular proteins and other cellular components are separated from the nucleic acid.
4. Precipitation and dissolving the DNA; leave the cell extract as a clear supernatant via centrifuging.
DNA extraction methods are comprised of four district methodology based on the sample’s source and size to pure the DNA extract.
1. Chemical-based DNA extraction method
Chemical DNA extraction that also called solution-based is subdivided into organic solvent-based DNA extraction and inorganic solvent-based DNA extraction.
1.1 Organic solvent-based DNA extraction
DNA extraction procedures using organic solvents were initially derived from several related RNA extraction methods. The routine protocol is including four distinct steps as follows:
1. Cell lysis by adding lysis buffer that contains Tris, EDTA, MgCl2, NaCl, and SDS. The components of the lysis buffer assist in the lysing of the cell membrane and the nuclear envelope.
2. Inactivation of DNases and RNases usually occurs through using organic solvents such as phenol and chloroform, denaturing the protein portion of the cell denatured.
3. Purification of DNA and removal of RNA, lipids, and proteins by adding isoamyl alcohol.
4. Resuspension of extracted nucleic acids.
After centrifugation, the phenol settles in the bottom of the tube and DNA in the aqueous phase while the denatured protein remains between both layers as a whitish cloud. Therefore, care must be taken to collect nucleic acid from this method. The collected nucleic acid is precipitated with the help of chilled alcohol (isoamyl alcohol). Finally, the DNA is dissolved in TE buffer. Because of the use of the organic solvents, this method is also named as an organic solvent-based DNA extraction method.
Disadvantages: Although the yield and quality (800 to 900 ng) of DNA obtained from this process are very demanding, the phenol and chloroform’s harmful nature restricts it. Furthermore, the amount of sample required is high. Therefore, it is not valid to apply this method for samples like hair and nail.
Role of chemicals:
| Tris | preserving the pH of the solution, Facilitating the cell membrane lysis by interacting the membrane lipopolysaccharides |
| EDTA | blocks DNase activity |
| SDS | helps in breaking the cell membrane and nuclear envelope open |
| NaCl | Neutralizes the negative charge of DNA, protects DNA from denaturation |
| MgCl2 | protects DNA in the lysis cell |
| Phenol | precipitates the protein impurities |
1.2 Inorganic DNA extraction
The inorganic DNA extraction also called the salting-out technique, is a non-toxic and straightforward DNA extraction technique comprised of two main methods: proteinase K and salt use.
In the salting-out method, firstly, the lysis buffer is used to break the cell membrane, and then proteinase K and RNase are added. Next, high concentrations of saturated salts (usually 6 M NaCl) are used to precipitation the proteins to the bottom, leaving DNA as a clear supernatant. Finally, the DNA is precipitated in the supernatant via washing with ethanol or isopropanol in the same manner as described for organic solvent methods.
Advantages: This technique is rapid, cost-effective, and not toxic for use in laboratory settings. Furthermore, since the detergents and NaCl solution are used instead of incubation with enzyme for protein precipitation, the higher DNA yield with more correction is produced.
Disadvantage: if not maintained well in a cold chain, the proteinase K cannot be utilized longer.
2. Solid-phase DNA extraction method using silica matrices
Most solid-phase techniques use a spin column to bind nucleic acid under centrifugal force. Spin columns are made of silica matrices generally needed buffer solutions, like sodium cations, at a specific pH to turn them into positively charged surfaces to absorb DNA. Accordingly, sodium cations bind tightly to the negatively charged oxygen in the phosphate backbone of DNA, usually conducted on a microchip coated in silica channels. Some proteins and other biochemical compounds that may also bind to the column are removed via tandem washing steps. DNA is finally eluted under low ionic strength using TE buffer or sterile distilled water.
Advantages: Less risk of pipetting error, reduced number of sample transfers, and less needed sample volume and time.
Disadvantages: The method is expensive due to the high cost of some of the available equipment choices.
3. DNA extraction using anion-exchange resins
Anion exchange resins are positively charged chemical substances that bind to negatively charged molecules like DNA or enzymes like nucleases. The yield and biological activity of DNA purified by this method compared to purification via two rounds of CsCl gradient. This method provides the condition to remove salts and low molecular weight contaminants from DNA in solution without binding or extracting the DNA of interest. The strength of bound DNA, RNA, and other impurities to the column can be modified by changing the salt concentrations and pH conditions of buffers. Accordingly, protein and RNA can be washed from the DNA-containing column via lower-concentration salt buffers.
The procedure starts by lysing the samples via using proteinase K or incubation at high temperatures. Then, contaminants are removed by adding resin, precipitating them at the bottom. The remaining DNA suspended in the supernatant can be directly used or transferred to a new tube for long-term storage.
Advantages: Allowing rapid separation of DNA from contaminating RNA, proteins, carbohydrates, and metabolites.
4. DNA extraction methods using magnetic-particle beads
Positively charged magnetic beads are the alternative techniques that attract the negatively charged DNA. Magnetic particle comprises one or multiple magnetic cores covered by a matrix of polymers, silica, or hydroxyapatite with terminal functionalized groups.
In this technique, the whole blood sample is mixed with an SDS solution just by inverting the tube for two or three times, and then incubate it at room temperature. After that, magnetic nanoparticles are added to this mixture, followed by the addition of binding buffer (the mix of sodium chloride and polyethylene glycol). The solution is again mixed via inversion and allowed to rest at room temperature. Then an external magnet is used for fixing the magnetic pellet at the bottom to discard the supernatant. Finally, the magnetic pellet is washed ethanol and dried. The magnetic pellet then resuspends in TE buffer, where the bounding magnetic particles bound to DNA are eluted by incubation at 65°C with continuous agitation.
Advantages: Magnetic beads are useful separation methods since they protect the DNA molecules towards either physical or chemical conditions. Moreover, the use of advanced devices like centrifuge is not necessary.
