DNA
isolation is an essential technique in molecular biology. Isolation of
high-molecular weight DNA has become very important with the increasing demand
for DNA fingerprinting, restriction fragment length polymorphism (RFLP),
construction of genomic or sequencing libraries and PCR analysis in research
laboratories and industry. DNA isolation is the first step in the study of
specific DNA sequences within a complex DNA population, and in the analysis of
genome structure and gene expression. The quantity, quality and integrity of
DNA will directly affect these results. DNA constitutes a small percentage of
the cell material and is usually localized in a defined part of the cell. In
prokaryotic cells, DNA is localized in the nucleoid that is not separated from
the rest of the cell sap by a membrane. In eukaryotic cells, the bulk of DNA is
localized in the nucleus, an organelle that is separated from the cytoplasm by
a membrane. The nucleus contains about 90% of the total cellular DNA, the
remaining DNA is in other organelles like mitochondria, chloroplasts or
kinetochores. In viruses and bacteriophages, the DNA is encapsulated by a
protein coat, and constitutes between 30 and 50 percent of the total mass of
the virion. In prokaryotic and eukaryotic cells, DNA constitutes only about 1%
of the total mass of the cell.
There
are five basic steps of DNA extraction: 1) disruption of the cellular structure
to create a lysate, 2) separation of the soluble DNA from cell debris and other
insoluble material, 3) binding the DNA of interest to a purification matrix, 4)
washing proteins and other contaminants away from the matrix and 5) elution of
the DNA.
1. Creation of Lysate
The
first step in any nucleic acid purification reaction is releasing the DNA into
solution. The goal of lysis is to rapidly and completely disrupt cells in a
sample to release nucleic acid into the lysate. There are four general
techniques for lysing materials: physical methods, enzymatic methods, chemical
methods and combinations of the three.
Physical Methods
Physical
methods typically involve some type of sample grinding or crushing to disrupt
the cell walls or tough tissue. A common method of physical disruption is
freezing and grinding samples with a mortar and pestle under liquid nitrogen to
provide a powdered material that is then exposed to chemical or enzymatic lysis
conditions. Physical methods are often used with more structured input
materials, such as tissues or plants. Other devices use bead beating or shaking
in the presence of metallic or ceramic beads to disrupt cells or tissues, or
sonication to disrupt tissues and lyse cells.
Chemical Methods
Chemical
methods can be used alone with easy-to-lyse materials, such as tissue culture
cells or in combination with other methods. Cellular disruption is accomplished
with a variety of agents that disrupt cell membranes and denatures proteins.
Chemicals commonly used include detergents (e.g., SDS) and chaotropes (e.g.,
guanidine salts and alkaline solutions).
Enzymatic Methods
Enzymatic
methods are often used with more structured starting materials in combination
with other methods with tissues, plant materials, bacteria and yeast. The
enzymes utilized help to disrupt tissues and tough cell walls. Depending on the
starting material, typical enzymatic treatments can include: lysozyme, zymolase
and liticase, proteinase K, collagenase and lipase, among others. Enzymatic
treatments can be amenable to high throughput processing, but may have a higher
per sample cost compared to other disruption methods.
2. Clearing of Lysate
Depending
on the starting material, cellular lysates may need to have cellular debris
removed prior to nucleic acid purification to reduce the carryover of unwanted
materials (proteins, lipids and saccharides from cellular structures) into the
purification reaction, which can clog membranes or interfere with downstream
applications. Usually clearing is accomplished by centrifugation, filtration or
bead-based methods. Once a cleared lysate is generated, the DNA can then be
purified by many different chemistries, such as silica, ion exchange, cellulose
or precipitation-based methods.
3. Binding to the Purification Matrix
Regardless
of the method used to create a cleared lysate, the DNA of interest can be
isolated using a variety of different methods. The most commonly used method is
genomic DNA isolation systems based on sample lysis by detergents, and
purification by binding to matrices (silica, cellulose and ion exchange). Bind
capacity is an indication of how much nucleic acid an isolation chemistry can
bind before it reaches the capacity of the system and no longer isolates more
of that nucleic acid.
Solution-Based Chemistry
This
type of chemistry does not rely on a binding matrix, but rather on alcohol
precipitation. Following the creation of lysate, the cell debris and proteins
are precipitated using a high-concentration salt solution. The high
concentration of salt causes the proteins to fall out of solution, and then
centrifugation separates the soluble nucleic acid from the cell debris and
precipitated protein. The DNA is then precipitated by adding isopropanol to the
high-concentration salt solution. This forces the large genomic DNA molecules
out of solution, while the smaller RNA fragments remain soluble. The insoluble
DNA is then pelleted and separated from salt, isopropanol and RNA fragments via
centrifugation. Additional washing of the pellet with ethanol removes the
remaining salt and enhances evaporation. Lastly, the DNA pellet is resuspended
in an aqueous buffer like Tris-EDTA or nuclease-free water and, once dissolved,
is ready for use in downstream applications.
Silica-Binding Chemistry
The
technology for these genomic DNA purification systems is based on binding of
the DNA to silica under high-salt conditions. The key to isolating any nucleic
acid with silica is the presence of a chaotropic salt like guanidine
hydrochloride. Chaotropic salts present in high quantities are able to disrupt
cells, deactivate nucleases and allow nucleic acid to bind to silica. Once the
genomic DNA is bound to the silica membrane, the nucleic acid is washed with a
salt/ethanol solution. These washes remove contaminating proteins,
lipopolysaccharides and small RNAs to increase purity while keeping the DNA
bound to the silica membrane column. Once the washes are finished, the genomic
DNA is eluted under low-salt conditions using either nuclease-free water or TE
buffer.
Cellulose-binding chemistry
Nucleic
acid binds to cellulose in the presence of high salt and alcohols. Generally
speaking, the binding capacity of cellulose-based methods is very high.
Conditions can be adjusted to preferentially bind different species and sizes
of nucleic acid. As a result of the combination of binding capacity and
relatively small elution volume, we can get high concentration eluates for
nucleic acids.
Ion
Exchange Chemistry
Ion
exchange chemistry is based on the interaction that occurs between
positively-charged particles and the negatively-charged phosphates that are
present in DNA. The DNA binds under low salt conditions, and contaminating
proteins and RNA can then be washed away with higher salt solutions. The DNA is
eluted under high salt conditions, and then recovered by ethanol precipitation.
4. Washing
Wash
buffers generally contain alcohols and can be used to remove proteins, salts
and other contaminants from the sample or the upstream binding buffers.
Alcohols additionally help associate nucleic acid with the matrix.
5. Elution
DNA
is soluble in low-ionic-strength solution such as TE buffer or nuclease-free
water. When such an aqueous buffer is applied to a silica membrane, the DNA is
released from the silica, and the eluate is collected. The purified,
high-quality DNA is then ready to use in a wide variety of demanding downstream
applications, such as multiplex PCR, coupled in vitro transcription/translation
systems, transfection and sequencing reactions.
RNA Isolation and Purification
Obtaining
pure RNA is an essential step in the analysis of patterns of gene expression
and understanding the mechanism of gene expression. Thus, isolation of pure,
intact RNA is one of the central technique in molecular biology and represents
an important step in Northern analysis, nuclease protection assays, RNA
mapping, RT-PCR, eDNA library construction and in vitro translation
experiments. RNA isolation generally consists of several steps: (1)
cell lysis and homogenization, (2) quenching of biochemical processes, (3)
nucleic acid partitioning, (4) RNA retrieval and crude purification, and (5)
assessing the quality of the extracted RNA.
Step 1: Cell lysis and homogenization
The
first step requires effective cell lysis following homogenization for the complete
release of nucleic acid. Several methods include chemical treatments such as
TRIzol or detergents that disrupt cells to release cellular contents. In
addition to chemical methods, enzymatic means may be employed, such as
treatment with lysozymes or enzymatic spheroplasting to weaken the cell walls
for homogenization. For cells that are refractory to these treatments,
mechanical disruption such as reciprocal bead-beating or mechanical shearing
with a French pressure cell may be employed.
Step 2: Quenching of biochemical processes
During
cell lysis, previously compartmentalized biomolecules are released and
subjected to the milieu of enzymatic activities that may compromise RNA
integrity. Therefore, solvents that solubilize cell contents should be denaturing
or contain chaotropic agents such as guanidinium thiocyanate or urea. In most
instances, homogenization and quenching of biochemical processes are performed
in the same step.
Step 3: Nucleic acid partitioning
Many
commercial preparations use phenol-chloroform-based extractions to isolate
nucleic acids. Phase separation is usually achieved when their constituents are
centrifuged to separate aqueous and organic phases. In nucleic acid isolation,
the partitioning of DNA from RNA depends on pH. At an alkaline pH, DNA and RNA
are retained in the aqueous phase. However, as pH decreases, DNA increasingly
migrates from the aqueous phase to the organic phase and interphase. Therefore,
phenol equilibrated to a pH of ~ 8 is used for the extraction of DNA while
acid phenol of pH 4.8 is used to isolate RNA.
Step 4: RNA retrieval and crude purification
RNA
contained in the aqueous phase can be extracted using a variety of methods. One
of the commonly used methods is through precipitation with isopropanol.
However, as the final step involves drying the RNA pellet, caution must be
exercised as excessive drying may result in poor resolubilization of RNA, which
may affect subsequent purification and downstream analysis. As an alternative
to precipitation, silica-based microcentrifuge spin-columns provide rapid
purification of RNA. In general, ethanol is added to the aqueous phase and the
resulting mixture is applied to the columns, with the RNA-containing column
washed to remove contaminants and the RNA subsequently eluted.
Step 5: Assessing the quality of extracted RNA
The
most efficient approach for assessing both RNA quantity and integrity involves
the microfluidic chip-based platforms offered by Agilent Technologies
(Bioanalyzer) and BioRad (Experion), with options for RNA, DNA, and proteins
using specific chips. Using fluorescent dye-based electrophoresis, the 2100
Bioanalyzer detects and resolves up to picogram quantities of total RNA or
small RNA depending on the chip used. As a measure of RNA integrity, the
instrument software assigns a RNA integrity number from 1 to 10 based on the
electrophoretic trace of the sample. The Bioanalyzer tracings show the full
range of ncRNA species with excellent resolution. The chips and reagents for
RNA analysis are highly cost-effective for analysis of multiple samples,
relative to other rigorous techniques, though the specialized instrument for
processing and analyzing the chips can be expensive for small research
laboratories.
RNA
isolation is a flexible process that can be customized according to specific
laboratory requirements and costs. However, designing an RNA isolation strategy
should incorporate considerations such as the toughness of the cell, pH of the
extraction solvent, and an unbiased method of RNA retrieval—issues that are
fundamentally important to producing reliable and biologically meaningful
results.
Protein Isolation and Purification
Proteins
can be obtained from a wide variety of samples. For diagnostic purposes, they
may be obtained from a patient's cells or tissues, for experimental use the
proteins may originate from microorganisms or from cell lines derived from
insects, vertebrate animals, or plants. Protein isolation and purification
requires multiple steps.
Isolation of proteins
- Choosing
the best protein isolation method depends on the properties of the source
sample (e.g., whether the sample is liquid or solid). Supposing that a
solid sample contains a large number of cells, a process must be used for
homogenizing the tissues and lysing the cells. In the case of tissue
samples, mechanical homogenization methods are useful.
- Methods
for lysing cells range from physical methods, such as heat treatment and
sonication, to chemical methods, such as treatment with a detergent
solution. Detergents that increase the solubility of proteins can be used
most effectively by taking into consideration the conditions of the
experimental medium, especially the buffer. Using appropriate detergents,
proteins that are difficult to extract (membrane proteins or nuclear
proteins) can also be obtained in desirable amounts. In addition,
chaotropic reagents, such as urea and guanidine hydrochloride, can be used
to increase the efficiency of extraction because they break down the
structure of the protein and dissolve well in water. However, treatment
with chaotropic reagents usually requires high salt concentrations.
Therefore, if not removed using membrane dialysis, the high salt
concentration can cause problems in further steps of the experiment. The
salt removal process itself can lead to loss of the protein.
- In
the case of liquid samples, a decision should be made about whether to
obtain the dissolved protein or to extract it from the cells it is
contained in. In order to extract the protein from the cells where it is
present, it is necessary to isolate the cells by centrifugation. In
particular, centrifugation using media with different densities may be
useful to isolate proteins expressed in specific cells. For example, to
obtain only the immune cells from bodily fluids or to separate adipocytes
or keratinocytes from skin tissue, centrifuging the liquid containing the
cells in a high-density medium may precipitate the desired cells depending
on the density of each constituent cell. Density-gradient
ultracentrifugation is additionally applicable for eliminating undesired
cellular impurities or obtaining certain cell organelles.
- If a
soluble protein is obtained from bodily fluids, it is treated similarly to
a cell lysate from solid samples. Protein solutions are generally dilute
when they undergo analysis. Thus, it is necessary to perform an enrichment
process, such as concentration or precipitation. The traditional
techniques of salting out and heat denaturation have the advantage of
being very simple. In addition, the precipitated protein is very stable,
meaning that this process may be used as a means of increasing the shelf
life of a protein. Salting out is a method of lowering the solubility of
proteins through competing solubility in water using salts that are more
soluble in water, such as ammonium sulfate. Since proteins precipitate at
a specific concentration of salt, this procedure also has the advantage
that the desired proteins can be separated from other proteins and
precipitated.
- Instead
of using salts, it may be desirable to use isoelectric precipitation by
lowering the pH. Isoelectric precipitation can be performed when the pH
reaches at isoelectric point (pI) of the target protein. Each protein has
a different pI value, meaning that isoelectric precipitation can be used
as a fractionation method as well. Generally, this method is also very
simple since a mineral acid or trichloroacetic acid (TCA) is titrated
until the target protein is obtained through precipitation. When changing
the pH or salting out is not preferable, polymers such as polyethylene
glycol or organic solvents such as methanol or acetone can be used to
promote precipitation. If required, a cocktail of precipitating reagents
(e.g., a mixture of acetone and TCA) can be developed.
Purification of proteins
- Methods
for purifying target proteins from dirty mixtures vary widely, but
preparation-grade purification is most commonly achieved using
chromatography. Proteins are usually purified by liquid chromatography
(LC), and fast protein LC and high-performance LC can be chosen depending
on whether the goal is preparation or quantitative analysis. For proteins,
it is possible to use the following techniques either in a single step or
sequentially: hydrophobic interaction column chromatography, size
exclusion chromatography, ion exchange column chromatography, and affinity
chromatography.
- If it
is not necessary to prepare a large quantity of the target protein,
electrophoresis is a possibility. Electrophoresis separates proteins
according to their molecular size. If the molecular weight of the target
protein is known, the approximate band position on the gel can be cut and
be used for further analysis like mass spectrometry. In general, sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) provides
1-dimensional (1D) results when analyzing proteins by size. Instead of
directly loading the protein sample when performing SDS-PAGE, it is
possible to perform 2-dimensional (2D) electrophoresis by performing
isoelectric focusing and loading the resultant gel tube with proteins
separated according to their pI values.
- If
the target protein is too small for the band (1D) or spot (2D) to be
easily stained after electrophoresis, a Western blot using antibodies can
be performed. In order to perform Western blotting, it is necessary to
have an antibody specific to the target protein.
- It is
also possible to separate and purify proteins using antibodies.
Immunoprecipitation is a good enrichment method for proteins. Since
antibody binding is specific, most of the final products are obtained only
from the target protein of the antibody. Either protein A-conjugated or
protein G-conjugated agarose, which bind to most antibodies, can be used
in most cases, meaning that virtually any kind of antibody can be applied
to obtain a small amount of target protein through several rounds of
centrifugation.
- The
enzyme-linked immunosorbent assay (ELISA) technique is a sensitive assay
method using antibodies. ELISA is an enzyme-amplified reaction for
antigens present in trace amounts in bodily fluids. Using ELISA, both the
presence of the target protein and quantitative information about it can
be obtained without purification.
References
Boom,
R. et al. (1990) Rapid and simple method for purification of
nucleic acids. J. Clin. Microbiol. 28, 495–503.
Walker,
J.A. et al. (2003) Quantitative intra-short interspersed element
PCR for species-specific DNA identification. Anal. Biochem.316,
259–69.
Birnboim,
H.C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening
recombinant plasmid DNA. Nucl. Acids Res.7, 1513–23.
Wang,
Z. and Rossman, T.G. (1994) Isolation of DNA fragments from agarose gel by
centrifugation. Nucl. Acids Res.22, 2862–3.