Protocol DGGE

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

Introduction
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.

General
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 60C). 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 l) and TEMED (8 l) 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.

Casting the gradient

Act quickly!

Make sure the pump is off and the gradient maker-channel is closed (handle up). Pour the acryl-high in the right leg of the gradient maker (at the pump-side) and the acryl-low in the left leg. Stirring bean on (in right leg). Simultaneously: Start the pump (5 ml/min) and move the handle of the gradient maker to horizontal position (channel open) (fig. 7). Check immediately if the acryl-low solution mixes with the acryl-high solution (most of the time an air bubble is blocking the channel, tilt the gradient maker slightly to let it out). The gelchamber fills slowly (Fig. 6 and 8). Stop the flow when the acryl is approximately 0.5 cm under the comb level. (The comb will be placed later) Empty the tubing and flush thoroughly with milliQ. Butanol layer Immediately after pouring, put a few ml of water-saturated butanol on top to obtain a straight surface. Let the gel polymerize for at least 60 minutes. After polymerization flush the space between the glass plates by filling it 3 times with milliQ to get rid of the butanol. Carefully dry the space between the glass plates with Whatman paper. Stacking gel and comb Place the comb and fill up the gelchamber with a mixture of 5 ml acryl 0%, 5 l TEMED and 50 l APS using a pasteur pipette (Fig. 9). Allow the stacking gel to polymerize for at least 15 minutes. Note: if lab temperatures exceed 27C you may want to bring down the concentration of APS and TEMED. Elevated temperatures accelerate the polymerisation process

Fig. 6: Casting setup.

Fig. 7: Gradient maker.

Running the gel

(We run at 60V during 15.5 h)

Prerun Place the core and tank into the aquarium which is filled with 23 litres 0.5x TAE buffer. Renew this buffer after running 4 gels. Check buffer level in the aquarium and if needed replenish with demi. Start circulation (60) to warm up (takes approx 20 minutes). Before Loading the Samples The samples are most easily pipetted into the slots when the gel is standing at the bench. It is often difficult to see the slots and having extra glass plates or plastic between you and the slots will not help. The gel will stand on the bench by virtue of the gelclamps alone. If you want more stability use the casting stand. Remove the comb from the gel and flush the slots with buffer using a syringe (Fig. 10). Fill the slots with buffer. Loading samples Use gel-saver-tips for loading the samples (mixed with loading buffer) (Fig. 11, 12 and 13). 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). 2 gels can be placed on the core, when running 1 gel, a buffer dam is placed instead of the second gel. This can be a piece of plastic with the same dimensions as the assembled gel. But it can also be a gel chamber (the glassplate sandwich) in which a 1.5 mm thick plastic plate replaces the gel. Place the gelchamber in the cooling core (Fig. 14) and in the aquarium (Fig. 15). Loosen the clamps a quarter counter clockwise to prevent breaking of the sandwich clamps (due to heat-expansion).

Fig. 8: Gelchamber filling slowly.

Start run
Restart and check the circulation. The flow into the upper compartment should be small and gentle. Make sure the upper compartment fills up and overflows (via the holes). The flow into the tank can be more vigorous. Close the green lid while paying attention to configuration of negative and positive electrode (Fig. 15). Check if buffer overflows from both sides of the tank into the aquarium. Start power at 60 V and check the amperage (about 20 mA for one gel). Cover the aquarium with plastic food wrap to avoid evaporation (Fig. 16).

Fig. 9: Placing combs and casting the stacking gel.

Protocol DGGE

Fig. 10: Flushing slots.

Run for 15.5 hours. Stop pump and power. Remove gel from gelchamber by removing the clamps and one glass plate leaving the gel on the other (Fig. 17, 18 and 19). Incubate gel on glass plate in 1 x TBE/EthidiumBromide; 60 min, shaking slowly. Make sure the gel is not sticking to the glass plate while shaking. Remove the gel from the glass plate by sliding it off carefully onto the UV illuminator. Wetting the UV illuminator first will make sliding easier. Alternatively you can handle the gel by holding it at the upper corners. Wet your gloves first. By gentle handling (needs some practice) even bigger or thinner gels wont break. Fig.11: Loading samples. Make your picture.

End run

Fig.12: Loading samples on the bench.

Fig. 20:

Result of DGGE on eukaryotic DNA obtained from lake samples.

Fig.13: Sample loading.

Preparation of stock-solutions
Make two stock solutions of acrylamide (0 and 100%). By mixing the two solutions in proper proportions all desired concentration of high-acryl and low-acryl can be made. For a 200 ml acrylamide-solution, 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, just leave out the urea and formamide If urea dissolves poorly, the mixture can be heated cautiously. Polymerization accelerates with temperature, so be careful.

Fig.14: Attaching gelchamber to cooling core.

Fig.15: Cooling core in aquarium, lid placed.

Protocol DGGE

Fig.16: Aquarium covered with saran wrap

Chemicals
Acryl APS Promega Acrylamide-Bis ready-to-use solution 40% (37.5:1) Ammoniumpersulfate Prepare a 10% solution (w/v) in milliQ and store in aliquots at -20 C (e.g. 400 l for one gel) 100% Sigma catalog number F7508 Merck catalog number 1.07746.0100 Per liter o 242 g Tris base o 57.1 ml glacial acetic acid o 100 ml 0.5 M EDTA (pH 8.0) Add 230 ml 50*TAE to 23 L demi. 5X concentrated stock solution (per liter) o 54 g Tris base o 27.5 g boric acid o 20 ml 0.5 M EDTA (pH 8.0) 50 l Ethidiumbromide (10 mg/ml) per liter TBE approx. 99% Sigma catalog number T-7024 Store at 4C 98+% Sigma catalog number U5378

Formamide Silicon grease 50X TAE stock (Tris-acetate buffer)

Fig. 17: Removing glass plate and spacers. Large plate at top. First move spacers slightly outward at the top.

0.5xTAE buffer Tris-borate (TBE)

TBE/EthidiumBromide Temed

Urea

Fig. 18: Then place thumb nails on spacer, index fingers behind the larger plate and exert gentle pressure.

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 l Company C.B.S. scientific, Del Mar, USA Gilson, Middleton, USA Bio-Rad, Hercules, USA Bio-Rad, Hercules, USA Haake, Karlsruhe, Germany Home made of 1 cm thick glass Whatman, Kent, UK Saran, DOW BIOzym, Hessisch Oldendorf, Germany Model GM-100 Minipuls 2 PROTEAN II xi system 220/2.0 DL30 Length: 44cm, Width: 30cm, Height: 29 cm Cat. No. 3030917 Omnilabo art 1090681 No. 171931
Fig. 19: The glass plate will come off without breaking. If the gel sticks to the upper plate, keep the upper plate slightly tilted and let the gel come down slowly on the bottom plate.

Bio-Rad Vertical Electrophoresis System 1. Tank and lid 2. Central cooling core 3. Casting stand, with gelchamber assembled. 4. Sandwich clamps 5. Alignment card 6. Combs

Protocol DGGE

References: Theory, development and improvement papers: Fisher, S. G., and L. S. Lerman. 1979. Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis. Cell 16:191-200 Myers, R. M., S. G. Fisher, L. S. Lerman, and T. Maniatis. 1985. Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Res. 13:3131-3145. Myers, R. M., T. Maniatis, and L. S. Lerman. 1987. Detection and Localization of Single Base Changes by Denaturing Gradient Gel-Electrophoresis. Method Enzymol. 155:501-527. Sheffield, V. C., D. R. Cox, L. S. Lerman, and R. M. Myers. 1989. 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. Proc. Natl. Acad. Sci. U. S. A. 86:232-236. Abrams, E. S., S. E. Murdaugh, and L. S. Lerman. 1995. Intramolecular DNA Melting between Stable Helical Segments - Melting Theory and Metastable States. Nucleic Acids Res. 23:2775-2783. Brinkhoff, T., and E. J. van Hannen. 2001. Use of silicone grease to avoid "smiling effect" in denaturing gradient gel electrophoresis. J. Rapid Methods Autom. Microbiol. 9:259-261. Cariello, N. F., and T. R. Skopek. 1993. Mutational Analysis Using Denaturing Gradient GelElectrophoresis and Pcr. Mutation Research 288:103-112. Cariello, N. F., J. A. Swenberg, A. Debellis, and T. R. Skopek. 1991. Analysis of Mutations Using Pcr and Denaturing Gradient Gel- Electrophoresis. Environ. Mol. Mutagen. 18:249-254. Collins, M., and R. M. Myers. 1987. Alterations in DNA Helix Stability Due to Base Modifications Can Be Evaluated Using Denaturing Gradient Gel-Electrophoresis. Journal of Molecular Biology 198:737-744. Costes, B., E. Girodon, N. Ghanem, M. Chassignol, N. T. Thuong, D. Dupret, and M. Goossens. 1993. Psoralen-Modified Oligonucleotide Primers Improve Detection of Mutations by Denaturing Gradient GelElectrophoresis and Provide an Alternative to Gc-Clamping. Hum. Mol. Genet. 2:393-397. Cotton, R. G. H. 1989. Detection of Single Base Changes in Nucleic-Acids. Biochem. J. 263:1-10. Wu Y, VM Hayes, J Osinga, IM Mulder, MW Looman, CH Buys, and RM Hofstra. 1998 Improvement of fragment and primer selection for mutation detection by denaturing gradient gel electrophoresis. Nucl. Acids. Res. 26: 5432-5440. VM Hayes, Y Wu, J Osinga, IM Mulder, P van der Vlies, P Elfferich, CH Buys and RM Hofstra 1999. Improvements in gel composition and electrophoretic conditions for broad-range mutation analysis by denaturing gradient gel electrophoresis. Nucl. Acids. Res. 27: e29 Janse, I., J. Bok and G. Zwart. A simple remedy against artifactual double bands in denaturing gradient gel electrophoresis. J. Microbiological Methods 57 (2004) 279-281.

Melting curves: Poland D. 1974 Biopolymers 13:1859-1871. Fixman, M., Freire, J. J. 1977. Theory of DNA melting curves. Biopolymers 16:26932704 Lerman, L. S., and K. Silverstein. 1987. Computational Simulation of DNA Melting and Its Application to Denaturing Gradient Gel-Electrophoresis. Method Enzymol. 155:482-501. Mutation detection (tests of DGGE resolution) Abrams, E. S., S. E. Murdaugh, and L. S. Lerman. 1990. Comprehensive Detection of Single Base Changes in Human Genomic DNA Using Denaturing Gradient Gel-Electrophoresis and a Gc Clamp. Genomics 7:463-475. Mikheev, A., R. S. Cha, and H. Zarbl. 1995. Detection of Point Mutations in Ras in Tumor-Cell Lines by Denaturant Gradient Gel-Electrophoresis, p. 442-451, Small Gtpases and Their Regulators, Pt A, vol. 255. Fodde, R., and M. Losekoot. 1994. Mutation Detection by Denaturing Gradient Electrophoresis (Dgge). Human Mutation 3:83-94. Gejman, P. V., and L. S. Weinstein. 1994. Detection of Mutations and Polymorphisms of G(S)Alpha, Subunit Gene by Denaturing Gradient Gel-Electrophoresis, p. 308-320, Heterotrimeric G Proteins, vol. 237. Nollau, P., and C. Wagener. 1997. Methods for detection of point mutations: Performance and quality assessment. Clin. Chem. 43:1114-1128. Applications in environmental microbiology Muyzer, G., E. C. de Waal, and A. G. Uitterlinden. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes coding for 16S ribosomal RNA. Appl. Environ. Microbiol. 59:695-700.

Protocol DGGE

Ferris, M. J., G. Muyzer, and D. M. Ward. 1996. Denaturing gradient gel electrophoresis profiles of 16S rRNA- defined populations inhabiting a hot spring microbial mat community. Appl. Environ. Microbiol. 62:340-346. Van der Gucht, K., K. Sabbe, L. De Meester, N. Vloemans, G. Zwart, M. Gillis, and W. Vyverman. 2001. Contrasting bacterioplankton community composition and seasonal dynamics in two neighbouring hypertrophic freshwater lakes. Environ. Microbiol. 3:680-690. van Hannen, E. J., M. P. van Agterveld, H. J. Gons, and H. J. Laanbroek. 1998. Revealing genetic diversity of eukaryotic microorganisms in aquatic environments by denaturing gradient gel electrophoresis. Journal of Phycology 34:206-213. Zwart, G., R. Huismans, M. P. van Agterveld, Y. Van de Peer, P. De Rijk, H. Eenhoorn, G. Muyzer, E. J. van Hannen, H. J. Gons, and H. J. Laanbroek. 1998. Divergent members of the bacterial division Verrucomicrobiales in a temperate freshwater lake. FEMS Microbiol. Ecol. 25:159-169.

Other techniques Barros, F., I. Munozbarus, M. V. Lareu, M. S. Rodriguezcalvo, and A. Carracedo. 1994. Double-Strand and Single-Strand Conformation Polymorphism Analysis of Point Mutations and Short Tandem Repeats. Electrophoresis 15:566-571. Ahmed, F. E. 1998. Molecular approaches for detection of mutations. Environ. Carcinog. Ecotoxical. Rev.-Pt. C J. Env. Sci. Health 16:47-80. Wartell, R. M., S. Hosseini, S. Powell, and J. Zhu. 1998. Detecting single base substitutions, mismatches and bulges in DNA by temperature gradient gel electrophoresis and related methods. J. Chromatogr. A 806:169-185. Bjorheim, J., G. Gaudernack, and P. O. Ekstrom. 2002. Melting gel techniques in single nucleotide polymorphism and mutation detection: From theory to automation. J. Sep. Sci. 25:637-647. Another protocol: Muyzer G, Hottentrager S, Teske A and Wawer C. Denaturing gradient gel electrophoresis of PCRamplified rDNA. A new molecular approach to analyse the genetic diversity of mixed microbial communities. Molecular Microbial Ecology Manual 3.4.4: 1-23; Eds: Akkermans ADL, van Elsas JD, de Bruijn FJ, Kluwer, Dordrecht, The Netherlands 1996. SOFTWARE for melting calculations and pcr primer design: A dos programme for melting curves can be found at http://web.mit.edu/osp/www/melt.html (melt94) A programme for primer design can be downloaded at biotechniques: http://www.biotechniques.com/softlib/tggestar.html

Protocol DGGE