Gel Electrophoresis
Gel Electrophoresis
Reiner Westermeier, Amersham Biosciences Europe GmbH, Freiburg, Germany
Nucleic acids are separated and displayed using various modifications of gel
electrophoresis and detection methods. Gel electrophoresis is the core technique for
genetic analysis and purification of nucleic acids for further studies. Gel electrophoretic
methods provide the highest resolution of all protein separation techniques.
Principle of Gel Electrophoresis
Electrophoresis is the migration of charged particles or
molecules in an electric field. This occurs when the
substances are in aqueous solution. The speed of
migration is dependent on the applied electric field
strength and the charges of the molecules. Thus,
differently charged molecules will form individual
zones while they migrate. In order to keep diffusion
of the zones to a minimum, electrophoresis is carried
out in an anticonvective medium such as a viscous
fluid or a gel matrix. Therefore, the speed of migration
is also dependent on the size of the molecules. In this
way fractionation of a mixture of substances is
achieved with high resolution.
Electrophoretic mobility
The electrophoretic mobility is dependent on external
factors like electric field strength, viscosity, gel
concentration and temperature, and intrinsic proper-
ties of the molecule like charge density, size and
hydrophobicity.
While proteins can be separated according to their
net charges or their sizes, nucleic acid molecules are
only distinguishable on size-based separations in
which the properties of the separation medium have
a large influence on the distribution of the zones.
Buffers
Electrophoretic separation is performed in buffers
with a constant pH value and constant ionic strength.
During electrophoresis, the buffer ions are carried
through the gel just like the sample ions: negatively
charged ions toward the anode, positively charged
ones toward the cathode. To guarantee constant
pH and buffer conditions, the supply of electrode
buffers must be sufficient. For nucleic acids the
mostly used buffer is composed of tris(hydroxy-
methyl)-aminoethane, borate and ethylenediamine-
tetraacetic acid (EDTA).
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Principle of Gel Electrophoresis

Agarose Gel Electrophoresis

Polyacrylamide Gel Electrophoresis
doi: 10.1038/npg.els.0005335
Joule heat
Some of the electrical energy is transformed into Joule
heat. Development of Joule heat is increased with high
buffer concentrations. In order to prevent overheating
effects, buffer strength and electric field strength must
be limited, and – mostly for polyacrylamide gels –
thermostating of the gels provides a homogeneous
temperature distribution. When the conditions are not
chosen correctly, a so-called ‘smiling effect’ will occur:
the electrophoretic mobilities of ions are higher in
the hot center of the gel plate than at the cooler
lateral sides.
Gel medium
The gel medium prevents diffusion and thermal con-
vection of the zones, and serves as a molecular sieve.
Two gel types are employed: agarose and polyacryla-
mide gels. Agarose gels are used as thick layers in
flatbed chambers mainly for preparative purposes,
whereas polyacrylamide gels are applied in thin layers
in vertical or cooled flatbed systems, mainly for high-
resolution techniques like sequencing and genotyping.
Electroendosmosis
The stabilizing medium, particularly agarose, can
contain fixed carboxylic and sulfonic groups. In the
presence of basic and neutral buffers, these groups
will become deprotonated and thus negatively charged.
In the electric field, the fixed negative charges are
attracted by the anode. They cannot migrate, because
they are a part of the matrix. A counterflow of
hydrated protons H3Oþ toward the cathode will result
in compensation; this effect is termed electroendos-
mosis. In gels, electroendosmosis is observed as a flow
of water toward the cathode, which carries some of
the solubilized substances along. The electrophoretic
and electroosmotic migrations are subtractive, which
results in blurred zones. Drying of the gel in the area
of the anode can also occur.
1
 Gel Electrophoresis

(a)
Agarose Gel Electrophoresis
Properties of agarose gels
Agarose is a polysaccharide obtained from red seaweed.
The pore size depends on the concentration of agarose
(weight of agarose per volume). Agarose is dissolved in
boiling water and forms a gel during cooling. During (b)
this process, double helices are built, which are joined – +
laterally to form relatively thick filaments. This fact
allows the preparation of gels with large pore sizes and
high mechanical stability. Gels with a pore size from
150 nm at 1% (w/v) to 500 nm at 0.16% are used. This
allows separation of nucleic acid fragment sizes in the
range between 400 and 23 000 base pairs (bp).
Different agarose qualities are available. They are
characterized by their gelling temperature (down to Buffer
35 C), melting point (down to 60 C) and the degree of
electroendosmosis. The degree of electroendosmosis is
dependent on the number of polar groups remaining Figure 1 Schematic drawing of a chamber for agarose gel
electrophoresis: (a) casting tray with comb for forming sample
from agaropectin. wells; and (b) chamber with gel and buffer.
The 1–10 mm thick gels are cast by pouring the hot
agarose mixed with gel buffer onto ultraviolet (UV)-
chains keep to the same orientation. When the applied
transparent trays. Sample application wells are formed
field strength exceeds a certain value, the DNA
in the gel surface with inserted plastic combs during
molecules are so strongly stretched that they become
gelling (see Figure 1a). The gel sizes vary from 5 cm to
rigid rods. This results in poor separation.
about 25 cm separation distance.

Running conditions and properties Staining of the bands

Electrophoresis setup
Agarose gels are run in simply designed flatbed
chambers under a buffer layer to prevent drying due
to electroendosmosis (see Figure 1b). The temperature
is only controlled by the applied running conditions.
The nucleic acids are separated under native condi-
tions. Quick checks of multiple samples are performed
in 96-well agarose gels in microtiter plate format
without a buffer layer.
Migration of deoxyribonucleic acid fragments
Because of the sieving properties of agarose gels, the
relative mobilities of deoxyribonucleic acid (DNA)
and ribonucleic acid (RNA) molecules are dependent
on the sizes of the molecules. At a defined pore size of
the agarose gel, there is – within a certain molecule size
range – a linear relationship between the logarithms of
the fragment lengths and the relative migration
distances.
Under the influence of the electric field, nucleic acid
molecules are stretched and migrate through gel pores
like a snake with a reptating movement (Noolandie
et al., 1989). Above a certain molecule length of about
20 kilobase pairs (kbp), the electrophoretic mobilities
of DNA molecules are similar, because these long
The bands are visualized with fluorescent dyes that
are visible in UV light – ethidium bromide or SYBR
Green. SYBR Green is less mutagenic and more
sensitive than ethidium bromide. The best results and
highest resolutions are obtained when the gels are
stained after the run. When dyes are added to the gel or
the sample during electrophoresis, the mobilities of the
DNA fragments will be modified and the resolution
will suffer.
Between 100 pg and 1 ng per band are detected. The
dyes intercalate in the helix and stain proportionately
to the length of the molecule. Therefore the sensitivity
is dependent on the size of the DNA fragment, and
is lower for single-stranded DNA and RNA. For a
permanent record of the separation, instant photos are
taken on a UV table or video documentation systems
are employed.
Agarose electrophoresis is the standard method for
DNA restriction fragment analysis and purification of
DNA and RNA fragments. Figure 2 shows ethidium
bromide stained bands in an agarose gel.
Blotting and hybridization
For restriction fragment length polymorphism
(RFLP) analysis, the separated DNA fragments are

2

23 130 bp
2322 bp
1057 bp
612 bp
335 bp
Figure 2 Separation result of agarose gel electrophoresis.
DNA fragments are detected with ethidium bromide.
transferred onto an immobilizing membrane followed
by hybridization with radiolabeled probes (Southern,
1975). The molecules are transferred onto nitrocellu-
lose or nylon membranes with capillary forces. The
fragments are probed with radioactive DNA or RNA.
The bound complementary nucleic acids are detected
by autoradiography.
Recovery of DNA fragments from gels
Several different procedures are used for the isolation
of nucleic acids from agarose gels: electroelution,
absorption to DEAE paper, absorption to glass
powder or resins, digestion of agarose with enzymes.
For preparative electrophoresis, it is very important
to use highly purified agarose that is free from
polymerase and other enzyme inhibitors. Since the
advent of polymerase chain reaction (PCR) technol-
ogy, tiny amounts of DNA fragments can easily be
amplified for further experiments.
Pulsed field gel electrophoresis
DNA fragments longer than about 20 kb cannot be
resolved in conventional agarose gel electrophoresis
because long DNA molecules align themselves as rods
and migrate with a mobility that is independent of their
length. In pulsed field gel electrophoresis (PFGE), the
molecules are subjected to two alternating electrical
fields that are applied on the gel at an angle between
110 and 180 . The DNA fragments must change their
orientation with changes in the electric field: their helical
structure is first compressed and then stretched. The
‘viscoelastic relaxation time’ is dependent on the size
of the molecule (Schwartz and Cantor, 1984). In addi-
tion, large molecules need more time to change their
direction than small ones. Because of the longer time
Gel Electrophoresis
Contour homogeneous electric field Field inversion gel
electrophoresis electrophoresis
+ + + + +
– + + –
– + + –
+ –
– + + –
– + + –
– + + –
+ +
+ +
+ +
+ + – +
+ +
+ + + + +
Figure 3 Schematic drawing of the principle of pulsed field gel
electrophoresis.
needed for stretching and reorientation, larger mol-
ecules have less time left for migration in the electric field.
In PFGE, the resulting electrophoretic mobilities depend
on the pulse time: DNA molecules with fragment
sizes up to about 10 megabases (Mb) can be resolved.
Pulse times of 1 s to 90 min are applied, depending
on the length of the DNA molecules being analyzed.
Large molecules are better separated with long pulse
times, small molecules need short pulse times. Separa-
tions can take several days.
In order to prevent chromosome-size molecules
breaking by shear forces during pipetting, sample prep-
aration including cell disruption is carried out inside
little agarose blocks. These agarose blocks are inserted
into preformed sample wells of the separation gel.
Pulsed field gel electrophoresis at different angles
The directions of the applied electric fields must differ
at least by an angle of 110 . This is achieved by
different arrangements: inhomogeneous fields created
with point electrodes, hexagonal electrode sets, turning
electrodes or turning gel tables. The resulting migra-
tion direction is diagonal. Figure 3 shows the principle
for two types of PFGE.
Field inversion gel electrophoresis
Field inversion gel electrophoresis (FIGE) is performed
in a standard agarose gel electrophoresis apparatus.
The electric fields are just alternating in the direction
of 180 . The resulting migration in one direction is
achieved by applying a higher field strength or a longer
pulse time in the separation direction. The advant-
age of this method is the simple design. The dis-
advantage is the long separation time, because the
molecules migrate backwards for part of the time. A
wide range of sizes of DNA molecules can be resolved in
such gels.
3
 Gel Electrophoresis

Applications of pulsed field gel electrophoresis

The field of application of this technique includes
chromosome mapping, isolation of intact chromoso-
mal and chromosomal-sized DNA, large restriction
fragment mapping and karyotyping. With PFGE,
physical gene maps are created for the identifica-
tion of genes responsible for hereditary diseases.
Another important area of application is bacterial
taxonomy.

Polyacrylamide Gel Electrophoresis

Properties of polyacrylamide gels
Polyacrylamide gels are prepared by chemical copoly-
merization of acrylamide monomers with a cross-
linking reagent, usually N, N0 -methylenebisacrylamide.
A clear transparent gel is obtained, which is chemically
inert, mechanically stable and without electroendos-
mosis. Polymerization of the acrylamide monomers
and the cross-linker molecules occurs in the presence
of free radicals. These are provided by ammonium
persulfate as catalyst; tertiary amino groups, usually
N, N, N0 , N0 -tetramethylethylenediamine (TEMED),
are required as accelerators.
The pore size is exactly controlled with the total
acrylamide concentration (T) and the degree of cross-
linking (C), which is determined by the amount of
cross-linker relative to the total amount of acrylamide.
The pore size decreases with increasing T value. With
increasing cross-linking, the pore size follows a
parabolic function: at high and low cross-linking, the
pores are large and the minimum pore size is obtained
at 4% cross-linking. Sequencing gels contain 5%
cross-linking and gels for single-strand conformation
polymorphism (SSCP) analysis 2% cross-linking.
Acrylamide monomers are toxic and should be
handled with caution. Because oxygen is a scavenger
of free radicals, polymerization is performed in
closed cassettes. Sample application wells for vertical
gels are formed at the upper edge of the gel during
polymerization with the help of an inserted comb (see
Figure 4). Sample wells for flatbed gels are made by
using self-adhesive tape glued onto one of the glass
plates.
Running conditions and properties
For electrophoresis in vertical systems, the complete
gel cassettes are placed into the buffer tanks; the gels
are in direct contact with the electrode buffers. Gels for
flatbed systems are polymerized on a film support and
removed from the cassette before use. Figure 5a shows
Figure 4 Schematic drawing of a cassette with sample well
comb and a caster for polyacrylamide gels.
an example of a flatbed and Figure 5b a vertical
chamber for polyacrylamide gels.
Native conditions
In nondenaturing polyacrylamide gels, the mobility
of DNA fragments is dependent on both size and
sequence. A- and T-rich nucleic acids migrate faster,
because they undergo fewer hydrophobic interactions
with the gel matrix than C- and G-rich fragments.
Therefore, nondenaturing polyacrylamide gels cannot
be used for the determination of fragment length, but
they are very sensitive to conformation differences of
the secondary structure. Very sharp bands are obtained
(see Figure 6). Single-nucleotide polymorphisms and
point mutations are detected with high sensitivity.
Denaturing conditions
In the presence of high molar formamide or urea,
and at elevated temperature above 50  C, the DNA
molecules are completely denatured and exist as single
strands. In this case, the electrophoretic mobilities are
strictly size dependent. When thin gel layers are used,
the resolution reaches single-base difference within
a range of around 1000–1200 bases, which makes
DNA sequencing possible.

4
 Gel Electrophoresis

(a)

Electrodes

Electrode
strips

(b)

Electrode
buffer

Figure 5 Schematic drawing of chambers for polyacrylamide

gel electrophoresis: (a) flatbed chamber with cooling plate, the
electrode reservoirs being contained in disposable polyacrylamide
strips; and (b) vertical chamber using liquid buffer.

Detection of bands
Staining
Ethidium bromide and SYBR Green staining are
rarely used for polyacrylamide gels, because the signals
are weaker than in agarose gels.
With silver staining, very high sensitivity indepen-
dent of molecular size is reached, down to 15 pg per
band (Goldman and Merril, 1982). The staining
method requires several steps; staining automates are
available. The chemicals are less toxic than intercalat-
ing dyes, there is no radioactivity, no UV light and no
photography is needed for inspection of the results.
Silver-stained bands can be directly reamplified with
PCR without any intermediate purification step.
Radioactive labeling
Labeling with radioactive phosphorus (32P) during
transcription or replication is employed for various
applications because of its very high sensitivity of
detection. After the run, the gels are dried and exposed
on X-ray film. The major applications are sequencing,
amplified fragment length polymorphism (AFLP),
differential display reverse transcription (DDRT)
and two-dimensional DNA typing.
Figure 6 Separation result of polyacrylamide gel electrophoresis
of DNA fragment with silver staining.
Fluorescence labeling
Labeling of the DNA fragments with Cy5 and other
fluorophors has replaced radiolabeling for many
applications. It allows online detection of the migrat-
ing zones. The dyes are excited with a laser beam, and
the emitted light – with a different wavelength – is
measured with a diode detector.
DNA sequencing gels
For increasing the reading length, long gels in very thin
layers are optimal. In order to achieve a straight front
and straight band distribution over the entire gel
width, the gels are mostly heated with thermoplates.
Fluorescent labeling has generally replaced radiolabel-
ing, which makes the long ultrathin layer gels (Sanger

5
 Gel Electrophoresis
and Coulson, 1978) and wedge gels unnecessary
(Ansorge and Labeit, 1984).
Denaturing gradient gel electrophoresis
Denaturing gradient gel electrophoresis (DGGE)
affords the detection of single-base exchanges in
segments of DNA (Fischer and Lerman, 1979). Gels
are prepared with a gradient from no additive to
7 mol L1 urea and 40% formamide, and run at about
60 C. The differences in melting cause two fragments
of DNA, which slow down at different levels of the gel.
The obtained pattern displays single-base differences.
Temperature gradient gel electrophoresis
Similar effects to DGGE can be achieved with
temperature gradient gel electrophoresis (TGGE)
(Riesner et al., 1989). In this technique, denaturing
gels are run on a differentially thermostated plate with
a cold side (15 C) at the cathode and a hot side (60 C)
at the anode. The technique is mainly used for
screening purposes.
See also
Capillary Electrophoresis
Genomic DNA: Purification
References
Ansorge W and Labeit S (1984) Field gradients improve resolution
on DNA sequencing gels. Journal of Biochemical and Biophysical
Methods 10: 237–243.
Fischer SG and Lerman LS (1979) Two-dimensional electrophoretic
separation of restriction enzyme fragments of DNA. Methods in
Enzymology 68: 183–191.
Goldman D and Merril CR (1982) Silver staining of DNA in
polyacrylamide gels: linearity and effect of fragment size.
Electrophoresis 3: 24–26.
Noolandie J, Slater DW, Lim HA and Viovy JL (1989) Generalized
tube model of biased reptation for gel electrophoresis of DNA.
Science 243: 1456–1458.
Riesner D, Steger G and Wiese U, et al. (1989) Temperature-gradient
electrophoresis of nucleic acids: analysis of conformational
transitions, sequence variations, and protein–nucleic acid inter-
actions. Electrophoresis 10: 377–389.
Sanger F and Coulson AR (1978) The use of thin acrylamide gels for
DNA sequencing. FEBS Letters 87: 107–110.
Schwartz DC and Cantor CR (1984) Separation of yeast
chromosome-sized DNA by pulsed field gradient gel electro-
phoresis. Cell 37: 67–75.
Southern EM (1975) Detection of specific sequences among DNA
fragments separated by gel electrophoresis. Journal of Molecular
Biology 98: 503–517.
Further Reading
Bova R and Micheli MR (eds) (1997) Fingerprinting Methods Based
on PCR. Heidelberg: Springer.
Landegren U (ed.) (1996) Laboratory Protocols for Mutation
Detection. Oxford, UK: Oxford University Press.
Martin R (1996) Gel Electrophoresis: Nucleic Acids. Oxford, UK:
Bios Scientific Publishers.
Rickwood D and Hames BD (eds) (1982) Gel Electrophoresis of
Nucleic Acids. Oxford, UK: IRL Press.
Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A
Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory Press.