Nucleic acid electrophoresis is an analytical technique used to separate
DNA or
RNA fragments by size and reactivity. Nucleic acid molecules which are to be analyzed are set upon a viscous medium, the
gel
A gel is a semi-solid that can have properties ranging from soft and weak to hard and tough. Gels are defined as a substantially dilute cross-linked system, which exhibits no flow when in the steady-state, although the liquid phase may still dif ...
, where an
electric field induces the nucleic acids (which are negatively charged due to their sugar-
phosphate
In chemistry, a phosphate is an anion, salt, functional group or ester derived from a phosphoric acid. It most commonly means orthophosphate, a derivative of orthophosphoric acid .
The phosphate or orthophosphate ion is derived from phosph ...
backbone) to migrate toward the
anode
An anode is an electrode of a polarized electrical device through which conventional current enters the device. This contrasts with a cathode, an electrode of the device through which conventional current leaves the device. A common mnemonic ...
(which is positively charged because this is an
electrolytic
An electrolyte is a medium containing ions that is electrically conducting through the movement of those ions, but not conducting electrons. This includes most soluble salts, acids, and bases dissolved in a polar solvent, such as water. Upon di ...
rather than
galvanic cell
A galvanic cell or voltaic cell, named after the scientists Luigi Galvani and Alessandro Volta, respectively, is an electrochemical cell in which an electric current is generated from spontaneous Oxidation-Reduction reactions. A common apparatus ...
). The separation of these fragments is accomplished by exploiting the mobilities with which different sized molecules are able to pass through the gel. Longer molecules migrate more slowly because they experience more resistance within the gel. Because the size of the molecule affects its mobility, smaller fragments end up nearer to the anode than longer ones in a given period. After some time, the voltage is removed and the fragmentation gradient is analyzed. For larger separations between similar sized fragments, either the voltage or run time can be increased. Extended runs across a low voltage gel yield the most accurate resolution. Voltage is, however, not the sole factor in determining electrophoresis of nucleic acids.
The nucleic acid to be separated can be prepared in several ways before separation by electrophoresis. In the case of large DNA molecules, the DNA is frequently cut into smaller fragments using a DNA
restriction endonuclease
A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
(or restriction enzyme). In other instances, such as
PCR amplified samples, enzymes present in the sample that might affect the separation of the molecules are removed through various means before analysis. Once the nucleic acid is properly prepared, the samples of the nucleic acid solution are placed in the wells of the gel and a voltage is applied across the gel for a specified amount of time.
The DNA fragments of different lengths are visualized using a
fluorescent dye specific for DNA, such as
ethidium bromide
Ethidium bromide (or homidium bromide, chloride salt homidium chloride) is an intercalating agent commonly used as a fluorescent tag ( nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis. It ...
. The gel shows bands corresponding to different nucleic acid molecules populations with different molecular weight. Fragment size is usually reported in "nucleotides", "base pairs" or "kb" (for thousands of base pairs) depending upon whether single- or double-stranded nucleic acid has been separated. Fragment size determination is typically done by comparison to commercially available
DNA markers containing linear DNA fragments of known length.
The types of gel most commonly used for nucleic acid electrophoresis are
agarose
Agarose is a heteropolysaccharide, generally extracted from certain red seaweed. It is a linear polymer made up of the repeating unit of agarobiose, which is a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose. Agarose is ...
(for relatively long DNA molecules) and
polyacrylamide
Polyacrylamide (abbreviated as PAM) is a polymer with the formula (-CH2CHCONH2-). It has a linear-chain structure. PAM is highly water-absorbent, forming a soft gel when hydrated. In 2008, an estimated 750,000,000 kg were produced, mainly f ...
(for high resolution of short DNA molecules, for example in
DNA sequencing). Gels have conventionally been run in a "slab" format such as that shown in the figure, but
capillary electrophoresis
Capillary electrophoresis (CE) is a family of electrokinetic separation methods performed in submillimeter diameter capillaries and in micro- and nanofluidic channels. Very often, CE refers to capillary zone electrophoresis (CZE), but other elect ...
has become important for applications such as high-throughput DNA sequencing. Electrophoresis techniques used in the assessment of
DNA damage
DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as radiation can cause DNA d ...
include
alkaline gel electrophoresis
In chemistry, an alkali (; from ar, القلوي, al-qaly, lit=ashes of the saltwort) is a basic, ionic salt of an alkali metal or an alkaline earth metal. An alkali can also be defined as a base that dissolves in water. A solution of a so ...
and
pulsed field gel electrophoresis
Pulsed field gel electrophoresis is a technique used for the separation of large DNA molecules by applying to a gel matrix an electric field that periodically changes direction.
Historical background
Standard gel electrophoresis techniques for s ...
.
For short DNA segments such as 20 to 60 bp double stranded DNA, running them in polyacrylamide gel (PAGE) will give better resolution (native condition). Similarly, RNA and single-stranded DNA can be run and visualised by PAGE gels containing denaturing agents such as urea.
PAGE gels are widely used in techniques such as DNA foot printing,
EMSA EMSA may stand for:
Acronyms
* Egyptian Medical Students' Association
* Electron Microscopy Society of America former name of the Microscopy Society of America
* Electrophoretic mobility shift assay
* Emergency Medical Services Alliance, in Mario ...
and other DNA-protein interaction techniques.
The measurement and analysis are mostly done with a specialized gel analysis software. Capillary electrophoresis results are typically displayed in a trace view called an
electropherogram
An electropherogram, or electrophoregram, can also be referred to as an EPG or e-gram. It is a record or chart produced when electrophoresis is used in an analytical technique, primarily in the fields of forensic biology, molecular biology and ...
.
Factors affecting migration of nucleic acids
A number of factors can affect the migration of nucleic acids: the dimension of the gel pores, the voltage used, the ionic strength of the buffer, and the concentration
intercalating dye such as ethidium bromide if used during electrophoresis.
Size of DNA
The gel sieves the DNA by the size of the DNA molecule whereby smaller molecules travel faster. Double-stranded DNA moves at a rate that is approximately inversely proportional to the
logarithm
In mathematics, the logarithm is the inverse function to exponentiation. That means the logarithm of a number to the base is the exponent to which must be raised, to produce . For example, since , the ''logarithm base'' 10 of ...
of the number of base pairs. This relationship however breaks down with very large DNA fragments and it is not possible to separate them using standard
agarose gel electrophoresis
Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the ...
. The limit of resolution depends on gel composition and field strength. and the mobility of larger circular DNA may be more strongly affected than linear DNA by the pore size of the gel.
Separation of very large DNA fragments requires
pulse field gel electrophoresis
Pulsed field gel electrophoresis is a technique used for the separation of large DNA molecules by applying to a gel matrix an electric field that periodically changes direction.
Historical background
Standard gel electrophoresis techniques for s ...
(PFGE). In field inversion gel electrophoresis (FIGE, a kind of PFGE), it is possible to have "band inversion" - where large molecules may move faster than small molecules.
Conformation of DNA
The
conformation of the DNA molecule can significantly affect the movement of the DNA, for example,
supercoiled DNA
DNA supercoiling refers to the amount of twist in a particular DNA strand, which determines the amount of strain on it. A given strand may be "positively supercoiled" or "negatively supercoiled" (more or less tightly wound). The amount of a st ...
usually moves faster than relaxed DNA because it is tightly coiled and hence more compact. In a normal plasmid DNA preparation, multiple forms of DNA may be present, and gel from the electrophoresis of the plasmids would normally show a main band which would be the negatively supercoiled form, while other forms of DNA may appear as minor fainter bands. These minor bands may be nicked DNA (open circular form) and the relaxed closed circular form which normally run slower than
supercoiled DNA
DNA supercoiling refers to the amount of twist in a particular DNA strand, which determines the amount of strain on it. A given strand may be "positively supercoiled" or "negatively supercoiled" (more or less tightly wound). The amount of a st ...
, and the single-stranded form (which can sometimes appear depending on the preparation methods) may move ahead of the supercoiled DNA. The rate at which the various forms move however can change using different electrophoresis conditions, for example linear DNA may run faster or slower than supercoiled DNA depending on conditions,
and the mobility of larger circular DNA may be more strongly affected than linear DNA by the pore size of the gel.
Unless supercoiled
DNA markers are used, the size of a circular DNA like plasmid therefore may be more accurately gauged after it has been linearized by
restriction digest
A restriction digest is a procedure used in molecular biology to prepare DNA for analysis or other processing. It is sometimes termed ''DNA fragmentation'' (this term is used for other procedures as well). Hartl and Jones describe it this way:
...
.
DNA damage due to increased
cross-link
In chemistry and biology a cross-link is a bond or a short sequence of bonds that links one polymer chain to another. These links may take the form of covalent bonds or ionic bonds and the polymers can be either synthetic polymers or natural ...
ing will also reduce electrophoretic DNA migration in a dose-dependent way.
Concentration of ethidium bromide
Circular DNA are more strongly affected by ethidium bromide concentration than linear DNA if ethidium bromide is present in the gel during electrophoresis. All naturally occurring DNA circles are underwound, but ethidium bromide which intercalates into circular DNA can change the charge, length, as well as the superhelicity of the DNA molecule, therefore its presence during electrophoresis can affect its movement in gel. Increasing ethidium bromide intercalated into the DNA can change it from a negatively supercoiled molecule into a fully relaxed form, then to positively coiled superhelix at maximum intercalation. Agarose gel electrophoresis can be used to resolve circular DNA with different supercoiling topology.
Gel concentration
The concentration of the gel determines the pore size of the gel which affects the migration of DNA. The resolution of the DNA changes with the percentage concentration of the gel. Increasing the agarose concentration of a gel reduces the migration speed and improves separation of smaller DNA molecules, while lowering gel concentration permits large DNA molecules to be separated. For a standard agarose gel electrophoresis, 0.7% gel concentration gives good separation or resolution of large 5–10kb DNA fragments, while 2% gel concentration gives good resolution for small 0.2–1kb fragments. Up to 3% gel concentration can be used for separating very tiny fragments but a vertical
polyacrylamide gel would be more appropriate for resolving small fragments. High concentrations gel, however, requires longer run times (sometimes days) and high percentage gels are often brittle and may not set evenly. High percentage agarose gels should be run with PFGE or FIGE. Low percentage gels (0.1−0.2%) are fragile and may break. 1% gels are common for many applications.
Applied field
At low voltages, the rate of migration of the DNA is proportional to the voltage applied, i.e. the higher the voltage, the faster the DNA moves. However, in increasing electric field strength, the mobility of high-molecular-weight DNA fragments increases differentially, and the effective range of separation decreases and resolution therefore is lower at high voltage. For optimal resolution of DNA greater than 2kb in size in standard gel electrophoresis, 5 to 8 V/cm is recommended.
[ Voltage is also limited by the fact that it heats the gel and may cause the gel to melt if a gel is run at high voltage for a prolonged period, particularly for low-melting point agarose gel.
The mobility of DNA however may change in an unsteady field. In a field that is periodically reversed, the mobility of DNA of a particular size may drop significantly at a particular cycling frequency.] This phenomenon can result in band inversion whereby larger DNA fragments move faster than smaller ones in PFGE.
Mechanism of migration and separation
The negative charge of its phosphate backbone moves the DNA towards the positively charged anode during electrophoresis. However, the migration of DNA molecules in solution, in the absence of a gel matrix, is independent of molecular weight during electrophoresis, i.e. there is no separation by size without a gel matrix. Hydrodynamic interaction between different parts of the DNA are cut off by streaming counterions moving in the opposite direction, so no mechanism exists to generate a dependence of velocity on length on a scale larger than screening length of about 10 nm. This makes it different from other processes such as sedimentation or diffusion where long-ranged hydrodynamic interaction are important.
The gel matrix is therefore responsible for the separation of DNA by size during electrophoresis, however the precise mechanism responsible the separation is not entirely clear. A number of models exists for the mechanism of separation of biomolecules in gel matrix, a widely accepted one is the Ogston model which treats the polymer matrix as a sieve consisting of randomly distributed network of inter-connected pores. A globular protein or a random coil
In polymer chemistry, a random coil is a conformation of polymers where the monomer subunits are oriented randomly while still being bonded to adjacent units. It is not one specific shape, but a statistical distribution of shapes for all the ch ...
DNA moves through the connected pores large enough to accommodate its passage, and the movement of larger molecules is more likely to be impeded and slowed down by collisions with the gel matrix, and the molecules of different sizes can therefore be separated in this process of sieving.
The Ogston model however breaks down for large molecules whereby the pores are significantly smaller than size of the molecule. For DNA molecules of size greater than 1 kb, a reptation
A peculiarity of thermal motion of very long linear macromolecules in ''entangled'' polymer melts or concentrated polymer solutions is reptation. Derived from the word reptile, reptation suggests the movement of entangled polymer chains as bein ...
model (or its variants) is most commonly used. This model assumes that the DNA can crawl in a "snake-like" fashion (hence "reptation") through the pores as an elongated molecule. At higher electric field strength, this turned into a biased reptation model, whereby the leading end of the molecule become strongly biased in the forward direction, and this leading edge pulls the rest of the molecule along. In the fully biased mode, the mobility reached a saturation point and DNA beyond a certain size cannot be separated. Perfect parallel alignment of the chain with the field however is not observed in practice as that would mean the same mobility for long and short molecules. Further refinement of the biased reptation model takes into account of the internal fluctuations of the chain.
The biased reptation model has also been used to explain the mobility of DNA in PFGE. The orientation of the DNA is progressively built up by reptation after the onset of a field, and the time it reached the steady state velocity is dependent on the size of the molecule. When the field is changed, larger molecules take longer to reorientate, it is therefore possible to discriminate between the long chains that cannot reach its steady state velocity from the short ones that travel most of the time in steady velocity. Other models, however, also exist.
Real-time fluorescence microscopy of stained molecules showed more subtle dynamics during electrophoresis, with the DNA showing considerable elasticity as it alternately stretching in the direction of the applied field and then contracting into a ball, or becoming hooked into a U-shape when it gets caught on the polymer fibres. This observation may be termed the "caterpillar" model. Other model proposes that the DNA gets entangled with the polymer matrix, and the larger the molecule, the more likely it is to become entangled and its movement impeded.
Visualization
The most common dye used to make DNA or RNA bands visible for agarose gel electrophoresis is ethidium bromide
Ethidium bromide (or homidium bromide, chloride salt homidium chloride) is an intercalating agent commonly used as a fluorescent tag ( nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis. It ...
, usually abbreviated as EtBr. It fluoresces under UV light when intercalated into the major groove of DNA (or RNA). By running DNA through an EtBr-treated gel and visualizing it with UV light, any band containing more than ~20 ng DNA becomes distinctly visible. EtBr is a known mutagen, and safer alternatives are available, such as GelRed
GelRed is an intercalating nucleic acid stain used in molecular genetics for agarose gel DNA electrophoresis. GelRed structurally consists of two ethidium subunits that are bridged by a linear oxygenated spacer.
GelRed is a fluorophore, and it ...
, produced by Biotium, which binds to the minor groove.
SYBR Green I
SYBR Green I (SG) is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. The SYBR family of dyes is produced by Molecular Probes Inc., now owned by Thermo Fisher Scientific. SYBR Green I binds to DNA. The resulting DN ...
is another dsDNA stain, produced by Invitrogen
Invitrogen is one of several brands under the Thermo Fisher Scientific corporation. The product line includes various subbrands of biotechnology products, such as machines and consumables for polymerase chain reaction, reverse transcription, cl ...
. It is more expensive, but 25 times more sensitive, and possibly safer than EtBr, though there is no data addressing its mutagenicity or toxicity in humans.
SYBR Safe
SYBR Safe is a cyanine dye used as a nucleic acid stain in molecular biology. SYBR Safe is one of a number of SYBR dyes made by the Life Technologies Corporation. SYBR Safe binds to DNA. The resulting DNA-dye-complex absorbs blue light (λmax = ...
is a variant of SYBR Green that has been shown to have low enough levels of mutagenicity and toxicity to be deemed nonhazardous waste under U.S. Federal regulations. It has similar sensitivity levels to EtBr, but, like SYBR Green, is significantly more expensive. In countries where safe disposal of hazardous waste is mandatory, the costs of EtBr disposal can easily outstrip the initial price difference, however.
Since EtBr stained DNA is not visible in natural light, scientists mix DNA with negatively charged loading buffers before adding the mixture to the gel. Loading buffers are useful because they are visible in natural light (as opposed to UV light for EtBr stained DNA), and they co-sediment with DNA (meaning they move at the same speed as DNA of a certain length). Xylene cyanol
Xylene cyanol can be used as an electrophoretic color marker, or tracking dye, to monitor the process of agarose gel electrophoresis and polyacrylamide gel electrophoresis. Bromophenol blue and orange G
Orange G also called C.I. 16230, Acid ...
and Bromophenol blue
Bromophenol blue (3′,3″,5′,5″-tetrabromophenolsulfonphthalein, BPB), albutest is used as a pH indicator, an electrophoretic color marker, and a dye. It can be prepared by slowly adding excess bromine to a hot solution of phenolsulfonph ...
are common dyes found in loading buffers; they run about the same speed as DNA fragments that are 5000 bp and 300 bp in length respectively, but the precise position varies with percentage of the gel. Other less frequently used progress markers are Cresol Red and Orange G
Orange G also called C.I. 16230, Acid Orange 10, or orange gelb is a synthetic azo dye used in histology in many staining formulations. It usually comes as a disodium salt. It has the appearance of orange crystals or powder.
Staining
Orange G ...
which run at about 125 bp and 50 bp, respectively.
Visualization can also be achieved by transferring DNA after SDS-PAGE
SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a Discontinuous electrophoresis, discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular m ...
to a nitrocellulose membrane followed by exposure to a hybridization probe
In molecular biology, a hybridization probe (HP) is a fragment of DNA or RNA of usually 15–10000 nucleotide long which can be radioactively or fluorescently labeled. HP can be used to detect the presence of nucleotide sequences in analyzed RNA ...
. This process is termed Southern blotting.
For fluorescent dyes, after electrophoresis the gel is illuminated with an ultraviolet
Ultraviolet (UV) is a form of electromagnetic radiation with wavelength from 10 nm (with a corresponding frequency around 30 PHz) to 400 nm (750 THz), shorter than that of visible light, but longer than X-rays. UV radiation ...
lamp (usually by placing it on a light box, while using protective gear to limit exposure to ultraviolet radiation). The illuminator apparatus mostly also contains imaging apparatus that takes an image of the gel, after illumination with UV radiation. The ethidium bromide
Ethidium bromide (or homidium bromide, chloride salt homidium chloride) is an intercalating agent commonly used as a fluorescent tag ( nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis. It ...
fluoresces reddish-orange in the presence of DNA, since it has intercalated with the DNA. The DNA band can also be cut out of the gel, and can then be dissolved to retrieve the purified DNA.
The gel can then be photographed usually with a digital or polaroid camera. Although the stained nucleic acid fluoresces reddish-orange, images are usually shown in black and white (see figures). UV damage to the DNA sample can reduce the efficiency of subsequent manipulation of the sample, such as ligation and cloning. Shorter wavelength UV radiations (302 or 312 nm) cause greater damage, for example exposure for as little as 45 seconds can significantly reduce transformation efficiency. Therefore if the DNA is to be use for downstream procedures, exposure to a shorter wavelength UV radiations should be limited, instead higher-wavelength UV radiation (365 nm) which cause less damage should be used. Higher wavelength radiations however produces weaker fluorescence, therefore if it is necessary to capture the gel image, a shorter wavelength UV light can be used a short time. Addition of Cytidine
Cytidine (symbol C or Cyd) is a nucleoside molecule that is formed when cytosine is attached to a ribose ring (also known as a ribofuranose) via a β-N1- glycosidic bond. Cytidine is a component of RNA. It is a white water-soluble solid. which ...
or guanosine
Guanosine (symbol G or Guo) is a purine nucleoside comprising guanine attached to a ribose ( ribofuranose) ring via a β-N9- glycosidic bond. Guanosine can be phosphorylated to become guanosine monophosphate (GMP), cyclic guanosine monophosphate ...
to the electrophoresis buffer at 1 mM concentration may protect the DNA from damage. Alternatively, a blue light excitation source with a blue-excitable stain such as SYBR Green
SYBR Green I (SG) is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. The SYBR family of dyes is produced by Molecular Probes Inc., now owned by Thermo Fisher Scientific. SYBR Green I binds to DNA. The resulting ...
or GelGreen
GelGreen is an intercalating nucleic acid stain used in molecular genetics for agarose gel DNA electrophoresis. GelGreen consists of two acridine orange subunits that are bridged by a linear oxygenated spacer.
Its fluorophore, and therefore it ...
may be used.
Gel electrophoresis research often takes advantage of software-based image analysis tools, such as ImageJ
ImageJ is a Java-based image processing program developed at the National Institutes of Health and the Laboratory for Optical and Computational Instrumentation (LOCI, University of Wisconsin). Its first version, ImageJ 1.x, is developed in the pub ...
.
References
{{reflist, 2
Molecular biology
Electrophoresis
DNA