Protocol DGGE

Copyright G. Zwart and J. Bok, dept. of Microbial Ecology, Center for Limnology, Netherlands Institute of Ecology (NIOO-KNAW). Updated March 2004

This protocol has been made as part of deliverables D2 and D4 (molecular tools) of the EU project BIOMAN EVK2-1999-00309. We have found that success of DGGE (reproducible gels with straight bands and a high resolution) rests in many small details of the electrophoresis, therefore we have tried to include most of the details of the procedure as we use it. In addition, the quality of the DGGE is determined by the quality of the PCR products. This is not covered here. Double bands most often are a PCR problem, not an electrophoresis problem. Our partner at the Laboratory of Protistology and Aquatic Ecology, University of Gent (Belgium) is using the same method but uses the DCODE system of BIORAD instead of the Protean II system we use.

Fig. 1: Greasing a spacer.

DGGE is an electrophoretic separation method based on differences in melting behaviour of double stranded DNA fragments (Fisher and Lermann, 1979). The electrophoresis takes place in a vertically placed polyacrylamide gel in a gradient of denaturants. It is important that the buffer in the upper buffer compartment is circulated to prevent exhaustion of buffer components. In addition the gel should be kept at constant homogeneous temperature (we use 60°C). To accomplish this we place the gel(s) in an aquarium containing the buffer. For stability the gel (attached to the Protean II gelcore) is placed in the plastic Protean II tank (the tank is not essential). Buffer is circulated by the thermostatpump to the upper buffer compartment and to the tank. Via overflow it is circulated to the aquarium in which the pump is hanging. To facilitate overflow from the upper compartment we have drilled two little holes in the upper part of the core (in the ‘ears’). [In the DCODE system, the mixing of the buffer (and temperature homogeneity) is improved by using a magnetic stirring bean, driven by a stirrer which is placed under the DCODE system.]

Fig.: Fig.:

Fig. 2: Greasing another spacer.

Casting the gel
Gradient choice To set up a new DGGE (new primer set, new sample type/habitat) we usually start with a relatively broad gradient (20-80% denaturant). However, we soon focus to the area of interest which should include the highest and lowest bands in different samples. For the Eukaryotic DGGE described by Van Hannen et al. (1998) as well as for the bacterial DGGE described by Muyzer et al. (1993) we use a denaturant gradient of 30 to 55%. The gel is 1.5 mm thick and contains 8% polyacrylamide.
Fig. 3: Assembly of gelchamber

Preparation • Assemble gelchamber: o Clean glass plates with detergent (e.g. Decon90) and 97% ethanol. Grease from prior runs should be removed completely. o Grease both sides of the spacers with as little as possible silicon grease to cover the full length of the spacer but only a quarter of the spacer width (Fig. 1 and 2). Place the greased side of the spacers at the outer sides of the glass plates. o Place the glass plates and spacers together with the sandwich clamps in the casting stand in which a rubber strip is placed at the bottom to prevent leakage. (Fig. 3 and 4), and check for leakage by filling it with milliQ water. When no leakage occurs, empty the gelchamber and then dry the glass plates with Whatman paper (Fig. 4). • Check the gradient maker and flush with milliQ. Empty pump tubing and attach pipette tip at the outlet tube to the top-middle of the gelchamber with tape (Fig. 5). When no silicon grease is used on the spacers, the DGGE bands will curve down at the sides of the gel (weeping bands instead of smiling researcher) (Brinkhoff and van Hannen, 2001). Using too much grease will deteriorate the quality of the gel. Making acrylamide solutions Chemicals: 0% and 100% acrylamide, APS and TEMED • Create the proper concentrations by mixing 0% and 100% acryl so that the end volumes are 23 ml of acryl with the highest denaturant concentration (e.g. acryl high, e.g. 55%) and 23 ml of acryl with the lowest denaturant concentration (e.g. acryl-low, e.g. 30%). • Add APS (100 µl) and TEMED (8 µl) to the acryl high and low solutions and mix well immediately.

Fig. 4: Drying gelchamber with Whatman paper.

Protocol DGGE


Fig. 5: Gradient maker, pump and casting stand.

15). If you want more stability use the casting stand. Fig. • The gelchamber fills slowly (Fig. Renew this buffer after running 4 gels. • Carefully dry the space between the glass plates with Whatman paper. (The comb will be placed later) • Empty the tubing and flush thoroughly with milliQ. This can be a piece of plastic with the same dimensions as the assembled gel. • • Protocol DGGE 2 Fig. Butanol layer • Immediately after pouring.5 h) Prerun • Place the core and tank into the aquarium which is filled with 23 litres 0.5 mm thick plastic plate replaces the gel. tilt the gradient maker slightly to let it out). 9: Placing combs and casting the stacking gel. 15). when running 1 gel. 9). • Stirring bean on (in right leg). 7: Gradient maker. Fig. 16). 10). put a few ml of water-saturated butanol on top to obtain a straight surface. 8: Gelchamber filling slowly. Running the gel (We run at 60V during 15. a buffer dam is placed instead of the second gel. The flow into the upper compartment should be small and gentle. . 6: Casting setup. • Fill the slots with buffer. It is often difficult to see the slots and having extra glass plates or plastic between you and the slots will not help. • Allow the stacking gel to polymerize for at least 15 minutes. Start run • Restart and check the circulation. Fig. Pour the acryl-high in the right leg of the gradient maker (at the pump-side) and the acryl-low in the left leg.5 cm under the comb level. • Let the gel polymerize for at least 60 minutes. Check if buffer overflows from both sides of the tank into the aquarium. • Stop the flow when the acryl is approximately 0. 2 gels can be placed on the core. Start power at 60 V and check the amperage (about 20 mA for one gel). Loading samples • Use gel-saver-tips for loading the samples (mixed with loading buffer) (Fig. 5 µl TEMED and 50 µl APS using a pasteur pipette (Fig. The flow into the tank can be more vigorous. • Place the gelchamber in the cooling core (Fig.Casting the gradient Act quickly! • • Make sure the pump is off and the gradient maker-channel is closed (handle up). • Check buffer level in the aquarium and if needed replenish with demi. Loosen the clamps a quarter counter clockwise to prevent breaking of the sandwich clamps (due to heat-expansion). 10: Flushing slots. Note: if lab temperatures exceed 27°C you may want to bring down the concentration of APS and TEMED. 7). Before Loading the Samples The samples are most easily pipetted into the slots when the gel is standing at the bench. • Remove the comb from the gel and flush the slots with buffer using a syringe (Fig. Make sure the upper compartment fills up and overflows (via the holes). 14) and in the aquarium (Fig. 11. • Load a marker along with the samples. (For determination of band positions and comparability of gels we use 3 marker lanes: 1 in the middle and 2 at each side of the gel). Close the green lid while paying attention to configuration of negative and positive electrode (Fig. • Check immediately if the acryl-low solution mixes with the acryl-high solution (most of the time an air bubble is blocking the channel. Cover the aquarium with plastic food wrap to avoid evaporation (Fig. 12 and 13). Stacking gel and comb • Place the comb and fill up the gelchamber with a mixture of 5 ml acryl 0%. Start circulation (60°) to warm up (takes approx 20 minutes). • Simultaneously: Start the pump (5 ml/min) and move the handle of the gradient maker to horizontal position (channel open) (fig. The gel will stand on the bench by virtue of the gelclamps alone. Elevated temperatures accelerate the polymerisation process Fig. But it can also be a gel chamber (the glassplate sandwich) in which a 1.5x TAE buffer. • After polymerization flush the space between the glass plates by filling it 3 times with milliQ to get rid of the butanol. 6 and 8).

By mixing the two solutions in proper proportions all desired concentration of high-acryl and low-acryl can be made.5 hours. Polymerization accelerates with temperature. Fig.11: Loading samples. Fig. Incubate gel on glass plate in 1 x TBE/EthidiumBromide. Remove gel from gelchamber by removing the clamps and one glass plate leaving the gel on the other (Fig. 60 min. Wetting the UV illuminator first will make sliding easier. 18 and 19). Protocol DGGE 3 Fig. Preparation of stock-solutions Make two stock solutions of acrylamide (0 and 100%). Make sure the gel is not sticking to the glass plate while shaking. Wet your gloves first. lid placed. Stop pump and power. shaking slowly. By gentle handling (needs some practice) even bigger or thinner gels wont break. For a 200 ml acrylamide-solution.• • • • • • • Run for 15. the mixture can be heated cautiously. 20: Result of DGGE on eukaryotic DNA obtained from lake samples. Fig.16: Aquarium covered with saran wrap .12: Loading samples on the bench. Fig.15: Cooling core in aquarium. Make your picture. End run Fig. Fig. Alternatively you can handle the gel by holding it at the upper corners.14: Attaching gelchamber to cooling core. 100% denaturant: • 40 ml 40% acrylamide (= 8%) • 84 g ureum • 80 ml 100% formamide • 2 ml 50x TAE • add MilliQ to 200 ml For the 0% denaturant stock solution.13: Sample loading. just leave out the urea and formamide If urea dissolves poorly. Remove the gel from the glass plate by sliding it off carefully onto the UV illuminator. 17. so be careful.

99% Sigma catalog number T-7024 Store at 4°C 98+% Sigma catalog number U5378 • • • Formamide Silicon grease 50X TAE stock (Tris-acetate buffer) Fig. UK Saran. Combs Protocol DGGE 4 . Germany Home made of 1 cm thick glass Whatman.0) 50 µl Ethidiumbromide (10 mg/ml) per liter TBE approx. Central cooling core 3. USA Bio-Rad. Kent. Width: 30cm. Tank and lid 2.5:1) Ammoniumpersulfate Prepare a 10% solution (w/v) in milliQ and store in aliquots at -20° C (e. scientific. 4.0 DL30 Length: 44cm. Middleton. 3030917 Omnilabo art 1090681 No.5 M EDTA (pH 8. No. 19: The glass plate will come off without breaking.1 ml glacial acetic acid o 100 ml 0. First move spacers slightly outward at the top. Hessisch Oldendorf. 5X concentrated stock solution (per liter) o 54 g Tris base o 27.5 g boric acid o 20 ml 0. 171931 Fig.5xTAE buffer Tris-borate (TBE) • • • TBE/EthidiumBromide Temed Urea Fig.Chemicals • • Acryl APS Promega Acrylamide-Bis ready-to-use solution 40% (37.07746. keep the upper plate slightly tilted and let the gel come down slowly on the bottom plate. Hercules. If the gel sticks to the upper plate. USA Bio-Rad. Sandwich clamps 5. Bio-Rad Vertical Electrophoresis System 1.B. DOW BIOzym.g. • • 0. Height: 29 cm Cat.5 M EDTA (pH 8.0) Add 230 ml 50*TAE to 23 L demi. Large plate at top. index fingers behind the larger plate and exert gentle pressure. 400 µl for one gel) 100% Sigma catalog number F7508 Merck catalog number 1. Karlsruhe. Hercules.0100 Per liter o 242 g Tris base o 57.S. 17: Removing glass plate and spacers. with gelchamber assembled. Equipment and materials Tool Gradient Maker Tubing Pump Vertical Electrophoresis System Power Supply Immersion Circulator (thermostat pump) Aquarium Chromotography paper food wrap Gelsaver tips 1-200 µl Company C. USA Haake. USA Gilson. Germany Model GM-100 Minipuls 2 PROTEAN II xi system 220/2. Casting stand. Del Mar. 18: Then place thumb nails on spacer. Alignment card 6.

26: 5432-5440.. 263:1-10. and T.. Lerman. E. Cell 16:191-200 • Myers. Res. Applications in environmental microbiology • Muyzer. R. J.. and R. 1985. B. E. M. and L. IM Mulder. G. Freire. • Fodde. 2001. Mutation Research 288:103-112.Electrophoresis. 1997. p. Acids. • Cariello.. S. 255. and L. Girodon. Microbiological Methods 57 (2004) 279-281. E. 1995. M. MW Looman. vol. Uitterlinden. 13:3131-3145. 1993. 59:695-700. S. Detection and Localization of Single Base Changes by Denaturing Gradient Gel-Electrophoresis. Environ. • Abrams.. P Elfferich. • Cotton. Bok and G. Protocol DGGE 5 . Subunit Gene by Denaturing Gradient Gel-Electrophoresis. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes coding for 16S ribosomal RNA. and T.Melting Theory and Metastable States. S. S. 237. G. Cox. Method Enzymol. L... N. Thuong. 2:393-397. Biochem. and C. 1995. N. E. development and improvement papers: • Fisher. p.. Lerman. 27: e29 • Janse. J Osinga. and L. and M. S. Lerman. Detection of Mutations and Polymorphisms of G(S)Alpha. E. T. • Myers. • Costes. and L. 308-320. S. R. C. Biopolymers 16:26932704 • Lerman. G. Detection of Point Mutations in Ras in Tumor-Cell Lines by Denaturant Gradient Gel-Electrophoresis. U. Theory of DNA melting curves. P. • Nollau. R. Detection of Single Base Changes in Nucleic-Acids. Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis. • Brinkhoff. G. Mol. Genomics 7:463-475. J. Wu Y. J. S. VM Hayes. 1994. Intramolecular DNA Melting between Stable Helical Segments . • Gejman. and E. Nucl.. V. CH Buys. Maniatis. Appl. T. Improvements in gel composition and electrophoretic conditions for broad-range mutation analysis by denaturing gradient gel electrophoresis. Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis. Environ. Losekoot. Chem. 1989. • Mikheev. 23:2775-2783. 1987. J. Ghanem. R... • Sheffield. A. Debellis. Goossens. A. and A. • Fixman. M. IM Mulder. Maniatis. R. J. 155:501-527.. M. Pt A. Wagener. P van der Vlies. Microbiol. Fisher. Mutation Detection by Denaturing Gradient Electrophoresis (Dgge). H. Heterotrimeric G Proteins. A. Psoralen-Modified Oligonucleotide Primers Improve Detection of Mutations by Denaturing Gradient GelElectrophoresis and Provide an Alternative to Gc-Clamping. Mol. S. M. Myers. Mutational Analysis Using Denaturing Gradient GelElectrophoresis and Pcr. Dupret. and L. Acids. Proc. N. T. Microbiol. Res. F. A. P. D. VM Hayes. and H. Sci. Acad. 1974 Biopolymers 13:1859-1871. F. CH Buys and RM Hofstra 1999. Silverstein. 1977.. Cha. I. Mutagen. J. Journal of Molecular Biology 198:737-744. M. de Waal. Zwart. Myers. Nucleic Acids Res. S. Attachment of a 40-Base-Pair GC rich sequence (GC-Clamp) to genomic DNA fragments by the polymerase chain-reaction results in improved detection of single-base changes. Natl. Murdaugh. J. 1979. Zarbl. Analysis of Mutations Using Pcr and Denaturing Gradient Gel.. M.. and M. R. and K. 1998 Improvement of • fragment and primer selection for mutation detection by denaturing gradient gel electrophoresis. Lerman. 155:482-501. Alterations in DNA Helix Stability Due to Base Modifications Can Be Evaluated Using Denaturing Gradient Gel-Electrophoresis. N. Computational Simulation of DNA Melting and Its Application to Denaturing Gradient Gel-Electrophoresis. 1993. R. E. S. • Collins.. Genet. S. J Osinga. and R. and RM Hofstra.. and T. 1991. 1993. van Hannen. S. A simple remedy against artifactual double bands in denaturing gradient gel electrophoresis.. S. R. J. 18:249-254. 43:1114-1128. Clin. • Cariello. 9:259-261. C. Weinstein. Methods for detection of point mutations: Performance and quality assessment. Comprehensive Detection of Single Base Changes in Human Genomic DNA Using Denaturing Gradient Gel-Electrophoresis and a Gc Clamp. Small Gtpases and Their Regulators. Lerman. L. Skopek. Use of silicone grease to avoid "smiling effect" in denaturing gradient gel electrophoresis. 1989. 86:232-236. 1987. 1987. Chassignol. Mutation detection (tests of DGGE resolution) • Abrams. S. Murdaugh. S. Nucl.. Melting curves: • Poland D. 442-451. Method Enzymol. 1994. vol. Swenberg. V. Y Wu. L. 1990. S. Nucleic Acids Res. Skopek. D. G.References: Theory. Human Mutation 3:83-94. Hum. Rapid Methods Autom. Lerman.

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