Basic steps involved in all DNA extraction methods
The basic criteria that any method of DNA isolation from any sample type should meet include: (1)
efficient extraction of DNA from the sample, (2) production of a sufficient amount of DNA for use in
downstream processes, (3) successful removal of contaminants, (4) isolation of high quality and high
purity DNA.
Assays to assess sample purity and quality control
Ultraviolet absorbance can be used to assess the purity of the extracted DNA. For a pure DNA sample,
the ratio of absorbance at 260 nm and absorbance at 280 nm (A260/A280) is 1.8. A ratio of < 1.8
indicates the sample is contaminated with protein or an organic solvent such as phenol, often used
during extraction processes. The quantification of double-stranded DNA can also be assessed by the
Qubit assay, which relies on the principle of DNA-selective fluorescent dyes although it may
underestimate in DNA extracted after RNA extraction with Trizol. DNA quality can be assessed by
visualization on agarose gels.
Common DNA extraction methods
Different extraction methods result in different yields and purity of DNA. Some of the extraction
methods have been systematically evaluated for specific applications such as soil and sediment samples,
human microbiome, and fecal samples.
Organic Extraction
In this conventional, widely used method, cells are lysed and cell debris is usually removed by
centrifugation. Then, proteins are denatured/digested using a protease, and precipitated with organic
solvents such as phenol, or 1:1 mixture of phenol and chloroform. The protein precipitate is removed
following separation by centrifugation. Purified DNA is usually recovered by precipitation using ethanol
or isopropanol. At some point in the process, RNAs are degraded through incubation with RNase. In the
presence of monovalent cations such as Na +, and at -20°C, absolute ethanol efficiently precipitates
polymeric nucleic acids and leaves behind short-chain and monomeric nucleic acid components,
including the ribonucleotides from RNase treatment in solution. This method uses hazardous organic
solvents, is relatively time-consuming, and residual phenol or chloroform may affect downstream
applications such as PCR. An example of a commercially available kit that relies on this chemistry is the
Easy-DNA® Kit from Thermo Fisher.
Silica-based technology
Silica-based technologies are widely employed in current kits. DNA adsorbs specifically to silica
membranes/beads/particles in the presence of certain salts and at a defined pH [10]. The cellular
contaminants are removed by wash steps. DNA is eluted in a low salt buffer or elution buffer. Chaotropic
salts are included in the kit buffers to aid in protein denaturation and extraction of DNA. This method
can be incorporated in spin columns and microchips, is cost-effective, has a simpler and faster procedure
than the organic extraction, and is suitable for automation. Kits based on this method include Purelink
Genomic DNA extraction kit from Thermo Fisher and DNeasy Blood and Tissue Kit from QIAGEN.
Magnetic separation
Magnetic separation is based on DNA reversibly binding to a magnetic solid surface/bead/particles that
have been coated with a DNA binding antibody, or a functional group that interacts specifically with
DNA. After DNA binding, beads are separated from other contaminating cellular components, washed,
and the purified DNA is eluted using ethanol extraction [12]. This method is rapid, simple to perform and
can be automated. However, it can be more costly than other methodologies. Examples of commercially
available kits include the Agencourt DNAdvance Kit from Beckman Coulter) and Magnetic Beads
Genomic DNA Extraction Kit from Geneaid.
Anion exchange technology
DNA extraction by anion exchange chromatography is based on the specific interaction between
negatively charged phosphates of the nucleic acid and positively charged surface molecules on the
substrate. DNA binds specifically to the substrate in the presence of low salt, contaminants are removed
by wash steps using a low or medium salt buffer, and purified DNA is eluted using a high salt buffer. This
technology is most commonly employed in plasmid isolation kits such as PureLink® HiPure Plasmid DNA
Purification Kits from Thermo Fisher, QIAGEN plasmid mini/midi kits and Genomic-tip, and NucleoBond®
PC kits from Macherey Nagel.
Others
Other methods of DNA extraction include salting out, cesium chloride density gradients, and chelex 100
resin. DNA isolation methods are often modified and optimized for different cell types or sample
sources. For example, cetyltrimethylammonium bromide (CTAB) and guanidium thiocyanate (GITC) are
often included in protocols for DNA extraction from plant materials.
DNA isolation from microbes
Bacterial cells are cultured in liquid media until they reach a maximum density of 2-3x109 cells/ml, and
then harvested. The collected cells are lysed, often done chemically, using reagents such as lysozyme,
EDTA, lysozyme and EDTA and other detergents, etc. Cellular components are then removed using one
of the above listed technologies, for example organic extraction or silica-based technologies. The final
step involves DNA precipitation to obtain pure DNA at a high concentration. This procedure can be
applied to a wide variety of microbes and other unicellular organisms such as yeast.
DNA extraction from animal cells and tissues
The basic steps involved in extracting DNA from animal cells and tissues is the same as discussed for
microbes. However, there are certain modifications to take into account the special features of animal
cells. Culturing and preparing of animal cells is often very different from that of microbial cells. Animal
cells do not have a cell wall like microbial cells, and consequently, are easier to lyse. Thus, they can be
lysed using only detergents. However, when cells are part of intact animal tissue, the tissue needs to
first be mechanically homogenized or treated with enzymes for lysis. Cell lysis is followed by the
isolation and purification of DNA from other cellular components.
DNA extraction from plant tissue and cells
The basic steps used for DNA isolation require adaptations to make them suitable for the different
characteristics of the plant cells and tissue. Chemicals or enzymes used to lyse microbial and mammalian
cells may not be equally effective on plant cells. For example, lysozyme is used to lyse bacterial cells but
has no effect on plant cells due to the presence of the cell wall. Furthermore, the biochemical content of
plant cells is very different from microorganisms and animal cells. Many plant species have a high
content of polysaccharides and polyphenols which are not removed by phenol extraction (unlike
microbes). Therefore, different methods and reagents need to be included in commercially available kits
to address the special features of plant cells.
One method is to utilize a detergent called cetyltrimethylammonium bromide (CTAB) which forms an
insoluble complex with nucleic acid and selectively precipitates DNA, leaving behind carbohydrates,
proteins and other contaminating components. The DNA-containing precipitate can be decomplexed by
dissolving it in NaCl. CTAB can be included in any step of the extraction process. To remove polyphenols
higher concentration of CTAB with polyvinylpyrrolidone (PVP) or polyvinylpolypyrrolidone (PVPP) can be
employed.
Another method is to use guanidium thiocyanate (GITC), which assists DNA purification from plant
materials in two ways. Firstly, it denatures and dissolves proteins, disintegrates cellular structures, and
dissociates nucleoproteins from the nucleic acid. Due to this property, GITC can be used to release DNA
from almost any type of tissue. Secondly, DNA binds strongly to silica particles in the presence of GITC.
This property can be utilized to separate DNA from the denatured proteins and other biochemical or
cellular components. Commonly, silica particles are packed in chromatography columns and a DNA
extract treated with GITC is applied. DNA binds selectively to the column and can be eluted in the last
step after washing away the cellular contaminants.
In some DNA extraction procedures, ascorbic acid, diethyldithiocarbamic acid and 2-mercaptoethanol
might be included to protect DNA against oxidation and degradation. RNA can be removed by using
RNase. The quality of the DNA isolated is largely dependent on the physiological condition of the plant
material,
