Gel ElectrophoresisReiner Westermeier, Serva Electrophoresis GmbH, Heidelberg, Germany

Gel electrophoresis is the core separation technique for

genetic analysis and purification of nucleic acid frag-

ments for further studies. In an electric field the nega-

tively charged deoxyribonucleic acid (DNA) and

ribonucleic acid (RNA) fragments migrate through a

porous gel matrix toward the positive electrode, the

anode. Because of the sieving effect of the gel, shorter

fragments move faster than larger ones. In this way the

DNA or RNA samples are separated according to their

molecular sizes into distinct zones, which can be detected

by specific visualisation methods. The most frequently

used technique is the separation of DNA fragments in

agarose gels in simple flatbed boxes combined with the

detection of stained bands under ultraviolet light. Poly-

acrylamidegelsareemployed,whensmall fragmentshave

to be analysed or very high resolution down to one single

base pair is required. In contrast to capillary electro-

phoresis the substrate does not need to be prelabelled.

Principle of Gel Electrophoresis

Electrophoresis is the migration of charged particles ormolecules in an electric eld. This occurs when the sub-stances are in aqueous solution. The speed of migration isdependent on the applied electric eld strength and thecharges of the molecules. Thus, dierently charged mol-ecules will form individual zones while they migrate. Tokeepdiusionof the zones to aminimum, electrophoresis iscarried out in an anticonvective medium such as a viscousuid or a gel matrix. Therefore, the speed of migration isalso dependent on the size of the molecules. In this wayfractionation of a mixture of substances is achieved withhigh resolution.

Electrophoretic mobility

The electrophoretic mobility is dependent on external fac-tors like electric eld strength, viscosity, gel concentration

and temperature and intrinsic properties of themolecule likecharge density, size and hydrophobicity.Although proteins can be separated according to their

net charges or their sizes, nucleic acid molecules are onlydistinguishable on size-based separations in which theproperties of the separation medium have a large inuenceon the distribution of the zones.

Buffers

Electrophoretic separation is performed in buers with aconstant pH value and constant ionic strength. For nucleicacid separation the buer must have a basic pH value toensure that the sample molecules are suciently charged.During electrophoresis, the buer ions are carried throughthe gel just like the sample ions: negatively chargedions toward the anode, positively charged ones towardthe cathode. To guarantee constant pH and buer con-ditions, the supply of electrode buers must be sucient.For nucleic acids the mostly used buer is composed oftris(hydroxymethyl)-aminoethane (Tris), borate andethylenediaminetetraacetic acid (EDTA;TBE). TheseTBEbuers are used in concentrations from 45 to 90mMTrisborate and 1 to 2mM EDTA and have a pH of 8.08.3.

Joule heat

Someof the electrical energy is transformed into Joule heat.Development of Joule heat is increased with high buerconcentrations. To prevent overheating eects, buerstrength and electric eld strength must be limited and mostly for polyacrylamide gels thermostating of the gelsprovides a homogeneous temperature distribution. Whenthe conditions are not chosen correctly, a so-called smilingeect will occur: the electrophoretic mobilities of ions arehigher in the hot centre of the gel plate than at the coolerlateral sides.

Gel medium

The gel medium prevents diusion and thermal convectionof the zones, and serves as a molecular sieve. Two gel typesare employed: agarose and polyacrylamide gels. Agarosegels are used as thick layers in atbed chambers mainly forpreparative purposes, whereas polyacrylamide gels areapplied in thin layers in vertical or cooled atbed systems,mainly for high-resolution techniques like sequencing andgenotyping.

Advanced article

Article Contents

. Principle of Gel Electrophoresis

. Agarose Gel Electrophoresis

. Polyacrylamide Gel Electrophoresis

Online posting date: 15th February 2013

eLS subject area: Molecular Biology

How to cite:Westermeier, Reiner (February 2013) Gel Electrophoresis. In: eLS.John Wiley & Sons, Ltd: Chichester.

DOI: 10.1002/9780470015902.a0005335.pub2

eLS & 2013, John Wiley & Sons, Ltd. www.els.net 1

Electroendosmosis

The stabilising medium, particularly agarose, can containxed carboxylic and sulfonic groups. In thepresenceofbasicand neutral buers, these groups will become deprotonatedand thus negatively charged. In the electric eld, the xednegative charges are attracted by the anode. They cannotmigrate, because they are apart of thematrix.A counterowof hydrated protons H3O

+ toward the cathode will result incompensation; this eect is termed electroendosmosis. Ingels, electroendosmosis isobservedasaowofwater towardthe cathode,which carries someof the solubilised substancesalong. The electrophoretic and electro-osmotic migrationsare subtractive, which results in blurred zones.Drying of thegel in the area of the anode can also occur.

Agarose Gel Electrophoresis

Properties of agarose gels

Agarose is a polysaccharideobtained fromred seaweed.Thepore size depends on the concentration of agarose (weight ofagarose per volume). Agarose is dissolved in boiling waterand forms a gel during cooling. During this process, doublehelices are built, which are joined laterally to form relativelythick laments. This fact allows the preparation of gels withlarge pore sizes and high mechanical stability. Gels with apore size from 150nm at 1% (w/v) to 500 nm at 0.16% areused.This allows separationof nucleic acid fragment sizes inthe range between 400 and 23000 base pairs (bp).Dierent agarose qualities are available. They are char-

acterised by their gelling temperature (down to 358C),melting point (down to 608C) and the degree of electro-endosmosis. The degree of electroendosmosis is dependenton the number of polar groups remaining fromagaropectin.The 110mm thick gels are cast by pouring the hot

agarose mixed with gel buer onto ultraviolet (UV)-transparent trays. Sample application wells are formed inthe gel surface with inserted plastic combs during gelling(Figure 1a). The gel sizes vary from 5 cm to approximately25 cm separation distances.

Running conditions and properties

Electrophoresis setup

Agarose gels are run in simply designed atbed chambersunder a buer layer to prevent drying due to electro-endosmosis (Figure 1b). The technique is therefore oftencalled submarine electrophoresis. The temperature is onlycontrolled by the applied running conditions. The nucleicacids are separated under native conditions. Quick checksofmultiple samples are performed in 96-well agarose gels inmicrotiter plate format without a buer layer.

Migration of deoxyribonucleic acid fragments

Because of the sieving properties of agarose gels, the rela-tive mobilities of deoxyribonucleic acid (DNA) and

ribonucleic acid (RNA) molecules are dependent on thesizes of the molecules. At a dened pore size of the agarosegel, there is within a certain molecule size range a linearrelationship between the logarithms of the fragmentlengths and the relative migration distances.Under the inuence of the electric eld, nucleic acid

molecules are stretched and migrate through gel pores likea snake with a reptating movement (Noolandie et al.,1989). Above a certain molecule length of approximately20 kbp, the electrophoretic mobilities of DNA moleculesare similar, because these long chains keep to the sameorientation. When the applied eld strength exceeds acertain value, the DNAmolecules are so strongly stretchedthat theybecome rigid rods. This results inpoor separation.

Staining of the bands

The bands are visualised with uorescent dyes that arevisible in UV light ethidium bromide or SYBR Green.SYBR Green is less mutagenic and more sensitive thanethidium bromide. The best results and highest resolutionsare obtained when the gels are stained after the run. Whendyes are added to the gel or the sample during electro-phoresis, the mobilities of the DNA fragments will bemodied and the resolution will suer.Between 100 pg and 1 ng per band are detected. The dyes

intercalate in the helix and stain proportionately tothe length of the molecule. Therefore the sensitivity isdependent on the size of the DNA fragment, and is lowerfor single-stranded DNA and RNA.The new DNA Stain G is a safer alternative to ethidium

bromide and SYBR Green. It is as sensitive as ethidiumbromide and can be used in exactly the sameway in agarose

(b)

(a)

Buffer

+

Figure 1 Schematic drawing of a chamber for agarose gel electrophoresis:

(a) casting tray with comb for forming sample wells and (b) chamber with

gel and buffer. (+) and (2) are the anodal and cathodal platinum electrode

wires. The samples are applied into the sample wells in the gel. The gel is

covered with running buffer.

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gel electrophoresis.When it gets attached to the phosphategroups of the nucleic acids it emits green light. Its adsorp-tion maximum is at 530 nm, however, it can be detectedwith an UV illuminator.However, it must be noted that open UV tables are

hazardous to health; great caremust be taken to protect theeyes and skin from contact withUV light. For a permanentrecord of the separation, instant photos are taken on theUV table or video documentation systems are employed.

Figure 2 shows ethidium bromide stained bands in anagarose gel.

Blotting and hybridisation

For restriction fragment length polymorphism analysis,the separated DNA fragments are transferred onto animmobilising membrane followed by hybridisation withradiolabelled probes (Southern, 1975). The molecules aretransferred onto nitrocellulose or nylon membranes withcapillary forces. The fragments are probedwith radioactiveDNA or RNA. The bound complementary nucleicacids are detected by autoradiography. See also: NucleicAcids:Hybridisation; SouthernBlotting for theAnalysis ofHuman Disease

Recovery of DNA fragments from gels

Several dierent procedures are used for the isolation ofnucleic acids from agarose gels: electroelution, absorptionto Diethylaminoethyl (ion exchange) paper, absorptionto glass powder or resins and digestion of agarose withenzymes. For preparative electrophoresis, it is veryimportant to use highly puried agarose that is free frompolymerase and other enzyme inhibitors. Since the adventof polymerase chain reaction (PCR) technology, tinyamounts of DNA fragments can easily be amplied forfurther experiments. See also: Genomic DNA: Purication

Pulsed field gel electrophoresis

DNAfragments longer than approximately 20kb cannot beresolved in conventional agarose gel electrophoresis becauselong DNA molecules align themselves as rods and migratewith amobility that is independent of their length. In pulsedeld gel electrophoresis (PFGE), themolecules are subjectedto two alternating electrical elds that are applied on thegel at an angle between 1108 and 1808. The DNA fragmentsmust change their orientation with changes in the electriceld: their helical structure is rst compressed and thenstretched. The viscoelastic relaxation time is dependent onthe size of the molecule (Schwartz and Cantor, 1984). Inaddition, large molecules need more time to change theirdirection than small ones. Because of the longer time neededfor stretching and reorientation, larger molecules have lesstime left for migration in the electric eld. In PFGE, theresulting electrophoretic mobilities depend on the pulsetime: DNA molecules with fragment sizes up to approxi-mately 10Mb can be resolved.Pulse times of 1 s to 90min are applied, depending on the

length of the DNA molecules being analysed. Large mol-ecules are better separated with long pulse times, smallmolecules need short pulse times. Separations can takeseveral days.To prevent chromosome-size molecules breaking by

shear forces during pipetting, sample preparation includ-ing cell disruption is carried out inside little agarose blocks.These agarose blocks are inserted into preformed samplewells of the separation gel.

PFGE at different angles

Thedirections of the applied electric eldsmust dier at leastby an angle of 1108. This is achieved by dierent arrange-ments: inhomogeneous elds created with point electrodes,hexagonal electrode sets, turning electrodes or turning geltables. The resultingmigration direction is diagonal. Figure 3shows the principle for two types of PFGE.

Field inversion gel electrophoresis

Field inversion gel electrophoresis is performed in astandard agarose gel electrophoresis apparatus. The elec-tric elds are just alternating in the direction of 1808. Theresultingmigration in one direction is achieved by applyinga higher eld strength or longer pulse time in the separationdirection. The advantage of this method is the simpledesign. The disadvantage is the long separation time,because the molecules migrate backwards for part of thetime. A wide range of sizes of DNA molecules can beresolved in such gels.

Applications of PFGE

The eld of application of this technique includeschromosome mapping, isolation of intact chromosomaland chromosomal-sized DNA, large restriction fragmentmapping and karyotyping. With PFGE, physical genemaps are created for the identication of genes responsible

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.

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for hereditary diseases. Another important area of appli-cation is bacterial taxonomy.

Polyacrylamide Gel Electrophoresis

Properties of polyacrylamide gels

Polyacrylamide gels are prepared by chemical copolymer-isation of acrylamide monomers with a cross-linkingreagent, usually N,N-methylenebisacrylamide. A cleartransparent gel is obtained, which is chemically inert,mechanically stable and without electroendosmosis.Polymerisation of the acrylamide monomers and thecross-linker molecules occurs in the presence of free rad-icals. These are provided by ammonium persulfate ascatalyst; tertiary amino groups, usually N,N,N,N-tetra-methylethylenediamine, are required as accelerators.The pore size is exactly controlled with the total acryla-

mide concentration (T) and the degree of cross-linking (C),which is determined by the amount of cross-linker relativeto the total amount of acrylamide. The pore size decreaseswith increasing T value. With increasing cross-linking, thepore size follows a parabolic function: at high and lowcross-linking, the pores are large and the minimum poresize is obtained at 4% cross-linking. Sequencing gels con-tain 5% cross-linking and gels for single-strand conform-ation polymorphism analysis 2% cross-linking.Acrylamide monomers are toxic and should be handled

with caution.Because oxygen is a scavenger of free radicals,polymerisation is performed in closed cassettes. Sampleapplication wells for vertical gels are formed at the upperedge of the gel during polymerisation with the help of aninserted comb (Figure 4). Sample wells for atbed gels aremadebyusing self-adhesive tape gluedontooneof the glassplates.

Running conditions and properties

For electrophoresis in vertical systems, the complete gelcassettes are placed into the buer tanks; the gels are in

direct contact with the electrode buers. Gels for atbedsystems are polymerised on a lm support and removedfrom the cassette before use. Figure 5a shows an exampleof a atbed and Figure 5b a vertical chamber for poly-acrylamide gels.

Native conditions

In nondenaturing polyacrylamide gels, the mobility ofDNAfragments is dependent onboth size and sequence.A-and T-rich nucleic acids migrate faster, because theyundergo fewer hydrophobic interactions with the gel mat-rix than C- and G-rich fragments. Therefore, non-denaturing polyacrylamide gels cannot be used for thedetermination of fragment length, but they are very sensi-tive to conformation dierences of the secondary structure.Very sharp bands are obtained (Figure 6). Single-nucleotidepolymorphisms andpointmutations are detectedwith highsensitivity.

Denaturing conditions

In the presence of high molar formamide or urea, and atelevated temperature above 508C, the DNA molecules arecompletely denatured and exist as single strands. In thiscase, the electrophoretic mobilities are strictly sizedependent. When thin gel layers are used, the resolution

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Contour homogeneous electric fieldelectrophoresis

Field inversion gelelectrophoresis

Figure 3 Schematic drawing of the principle of pulsed field gel

electrophoresis.

Figure 4 Schematic drawing of a cassette with sample well comb and a

caster for polyacrylamide gels. For electrophoresis the cassette containing

the gel layer is removed from the caster and inserted into the

electrophoresis chamber (Figure 5).

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reaches single-base dierence within a range of approxi-mately 10001200 bases, which makes DNA sequencingpossible.See also:DenaturingGelElectrophoresis ofRNAand DNA Using UreaPolyacrylamide Gels

Detection of bands

Staining

Ethidium bromide and SYBR Green staining are rarelyused for polyacrylamide gels, because the signals areweaker than in agarose gels.With silver staining, very high sensitivity independent of

molecular size is reached, down to 15 pg per band (Gold-man and Merril, 1982). The staining method requires sev-eral steps; staining automates are available. The chemicals

are less toxic than intercalating dyes, there is no radio-activity, no UV light and no photography is needed forinspection of the results. Silver-stained bands can be dir-ectly reamplied with PCR without any intermediatepurication step. See also: Gel Staining Techniques

Radioactive labelling

Labelling with radioactive phosphorus (32P) during tran-scription or replication is employed for various appli-cations because of its very high sensitivity of detection.After the run, the gels are dried and exposed on X-raylm. The major applications are sequencing, ampliedfragment length polymorphism, dierential display reversetranscription and two-dimensional DNA typing. See also:RadiolabellingNucleic Acids: GeneratingDNAProbes byRandom Priming

Fluorescence labelling

Labelling of the DNA fragments with Cy5 and other uor-ophors has replaced radiolabelling for many applications.

Figure 6 Separation result of polyacrylamide gel electrophoresis of DNA

fragments with silver staining. On the lane on the right edge 5 mL of a100bp ladder, diluted to 10ng, has been applied.

Electrodes

Electrode wicks

Electrodebuffer

(a)

(b)

Figure 5 Schematic drawing of chambers for polyacrylamide gel

electrophoresis: (a) Horizontal flatbed chamber with cooling plate: The gel

is used with an open surface, the samples are applied into the sample wells,

instead of electrode reservoirs disposable wicks are soaked in concentrated

buffer, the electrodes are placed onto these wicks and connected to a

power supply. (b) Vertical chamber using liquid buffer: The samples are

applied into the wells located between the two glass plates, which have

been formed by the comb shown in Figure 4, the upper buffer tank is

located in the central block and contains a cathodal platinum electrode

wire, the lower buffer tank at the bottom contains an anodal platinum

electrode wire, the contacts to the power supply are made via the two

plugs located at the top of the central block.

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It allows online detection of the migrating zones. The dyesare excited with a laser beam and the emitted light with adierent wavelength is measured with a diode detector.

DNA sequencing gels

For increasing the reading length, long gels in very thinlayers are optimal. To achieve a straight front and straightband distribution over the entire gel width, the gels aremostly heatedwith thermoplates. Fluorescent labelling hasgenerally replaced radiolabelling, which makes the longultrathin layer gels (Sanger and Coulson, 1978) and wedgegels unnecessary (Ansorge and Labeit, 1984). DNAsequencing in gels has meanwhile more or less been aban-doned: rst it had been replaced by capillary electro-phoresis in multicapillary systems, then by faster andcheaper gel-free next generation sequencing methods.See also: Capillary Electrophoresis; Capillary Electro-phoresis; Sanger, Frederick

Denaturing gradient gel electrophoresis

Denaturing gradient gel electrophoresis (DGGE) aordsthe detection of single-base exchanges in segments ofDNA(Fischer and Lerman, 1979). Gels are prepared with agradient from no additive to 7mol L21 urea and 40%formamide, and run at approximately 608C. The dier-ences in melting cause two fragments of DNA, which slowdown at dierent levels of the gel. The obtained patterndisplays single-base dierences. See also: Nucleic Acids:Thermal Stability and Denaturation

Temperature gradient gel electrophoresis

Similar eects to DGGE can be achieved with temperaturegradient gel electrophoresis (Riesner et al., 1989). In thistechnique, denaturing gels are run on a dierentially ther-mostated plate with a cold side (158C) at the cathode and ahot side (608C) at the anode. The technique is mainly usedfor screening purposes.See also: CapillaryElectrophoresis;Genomic DNA: Purication

References

Ansorge W and Labeit S (1984) Field gradients improve reso-

lution on DNA sequencing gels. Journal of Biochemical and

Biophysical Methods 10: 237243.

Fischer SG and Lerman LS (1979) Two-dimensional electro-

phoretic separation of restriction enzyme fragments of DNA.

Methods in Enzymology 68: 183191.

Goldman D and Merril CR (1982) Silver staining of DNA in

polyacrylamide gels: linearity and eect of fragment size.

Electrophoresis 3: 2426.

Noolandie J, Slater DW, Lim HA and Viovy JL (1989) General-

ized tube model of biased reptation for gel electrophoresis of

DNA. Science 243: 14561458.

Riesner D, Steger G, Zimmat R et al. (1989) Temperature-gra-

dient electrophoresis of nucleic acids: analysis of conforma-

tional transitions, sequence variations, and proteinnucleic

acid interactions. Electrophoresis 10: 377389.

Sanger F and Coulson AR (1978) The use of thin acrylamide gels

for DNA sequencing. FEBS Letters 87: 107110.

Schwartz DC and Cantor CR (1984) Separation of yeast

chromosome-sized DNA by pulsed eld gradient gel electro-

phoresis. Cell 37: 6775.

Southern EM (1975)Detection of specic sequences amongDNA

fragments separated by gel electrophoresis. Journal of

Molecular Biology 98: 503517.

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 Scientic 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 Clon-

ing: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY:

Cold Spring Harbor Laboratory Press.

Westermeier R (2004) Electrophoresis in Practice, 4th edn.

Weinheim: WILEY-VCH.

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