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Gel Electrophoresis Gel Electrophoresis Advanced article Article contents 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 properties 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(hydroxymethyl)-aminoethane, borate and ethylenediaminetetraacetic acid (EDTA). 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 convection of the zones, and serves as a molecular sieve. Two gel types are employed: agarose and polyacrylamide 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 highresolution 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 electroendosmosis. 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. ENCYCLOPEDIA OF LIFE SCIENCES & 2005, John Wiley & Sons, Ltd. www.els.net 1 Gel Electrophoresis Agarose Gel Electrophoresis (a) 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 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 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 from agaropectin. The 1–10 mm thick gels are cast by pouring the hot agarose mixed with gel buffer onto ultraviolet (UV)transparent trays. Sample application wells are formed in the gel surface with inserted plastic combs during gelling (see Figure 1a). The gel sizes vary from 5 cm to about 25 cm separation distance. Running conditions and properties 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 conditions. 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 2 (b) + – 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. chains keep to the same orientation. When the applied field strength exceeds a certain value, the DNA molecules are so strongly stretched that they become rigid rods. This results in poor separation. Staining of the bands 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 Gel Electrophoresis Contour homogeneous electric field electrophoresis 23 130 bp + – 2322 bp – – – 1057 bp – + + + + + + + – + + – + + + + + + + + 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) technology, 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 addition, large molecules need more time to change their direction than small ones. Because of the longer time – + + + transferred onto an immobilizing membrane followed by hybridization with radiolabeled probes (Southern, 1975). The molecules are transferred onto nitrocellulose or nylon membranes with capillary forces. The fragments are probed with radioactive DNA or RNA. The bound complementary nucleic acids are detected by autoradiography. – + + + Figure 2 Separation result of agarose gel electrophoresis. DNA fragments are detected with ethidium bromide. – – + + 335 bp + – + 612 bp Field inversion gel electrophoresis + + + + Figure 3 Schematic drawing of the principle of pulsed field gel electrophoresis. needed for stretching and reorientation, larger molecules 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. Separations can take several days. In order to prevent chromosome-size molecules breaking by shear forces during pipetting, sample preparation 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 migration 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 advantage of this method is the simple design. The disadvantage 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 chromosomal and chromosomal-sized DNA, large restriction fragment mapping and karyotyping. With PFGE, physical gene maps are created for the identification 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 copolymerization of acrylamide monomers with a crosslinking reagent, usually N, N0 -methylenebisacrylamide. A clear transparent gel is obtained, which is chemically inert, mechanically stable and without electroendosmosis. 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 (TEMED), N, N, N0 , N0 -tetramethylethylenediamine are required as accelerators. The pore size is exactly controlled with the total acrylamide concentration (T) and the degree of crosslinking (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 4 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. 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 independent 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 intercalating 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 migrating 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 radiolabeling, 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. 6 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 interactions. 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 electrophoresis. 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.