Protocol:Denaturing gradient gel electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis (DGGE)

Principle
Denaturing Gradient Gel Electrophoresis or DGGE, as originally described by Myers et al, 1987 (42), allows the separation, and thus detection, of DNA molecules that differ by as little as a single nucleotide. The electrophoretic separation is based upon the melting properties of the double-stranded DNA molecule. The two complimentary strands of DNA separate (melt-out) under conditions of increased temperature or under the influence of certain chemicals (termed denaturants). An increase in the temperature and/or concentration of chemical denaturant around a DNA molecule will cause it to melt out along its length in discrete segments or regions, called melting domains. Melting domains vary in length between about 25 up to several hundred bases (bp), and each melts cooperatively at a distinct temperature called a Tm. The Tm of a melting domain is highly dependant on its nucleotide sequence and even a single-base substitution is sufficient to alter the Tm of a domain.

DGGE involves the electrophoresis of double stranded DNA molecules (up to 400-500bp for efficient analysis) through a vertical polyacrylamide gel that contains a linear gradient (from top to bottom) of increasing chemical denaturant concentration (as opposed to temperature, which is practically more difficult to control). As a DNA fragment enters the concentration of denaturant where its lowest-temperature melting domain begins to denature, the domain ‘opens’ forming a DNA molecule with a branched structure. This branched DNA has a greatly retarded mobility in the gel matrix. If a DNA sample contains a point mutation in this domain which differs from the normal sequence, the Tm of the domain will be different, the DNA molecule will melt out at a slightly different Tm and thus will have an altered mobility in the gel compared to control DNA. DGGE can be used to detect single base changes in all but the highest-temperature melting domain of a DNA fragment. To facilitate the detection of mutations along the complete length of a DNA fragment under study, a GC-rich segment may be attached to one end of the fragment during the PCR reaction (by the addition of a GC-rich sequence to the 5’end of one of the primer pair). This GC-rich segment, or GC-clamp (usually 30-60bp long) has a very high Tm, and in comparison the remaining DNA fragment will have a single and lower Tm (43), as shown in Figure 5.12.

Prior to the discovery of PCR, DNA fragments for DGGE analysis were generated by cloning. Now with appropriate PCR primers it is possible to generate fragments of any gene of interest. If a DNA sample is heterozygous for a point mutation, the PCR reaction will generate a mixture of double-stranded molecules and up to four bands will be visible after running the DGGE gel. There will be homoduplexes from the wild and the mutant sequences and in addition there will be two types of heteroduplex DNA molecules (which migrate at a slower rate), representing hybridised mismatched wild-type and mutant DNA strands, generated by reassorting of DNA strands during the PCR reaction. If the sample is either homozygous for the normal (wild) sequence or for a mutation, it will only have one homoduplex molecule (figure 5.13).

In order to select the optimal position for the primers used to amplify a region of DNA for DGGE analysis, and to estimate the range of chemical denaturant required, it is useful to have prior knowledge of the melting behaviour of the fragment under study. This can be done using the computer algorithm (Melt ’87) developed by Lerman and Silverstein, 1987 (44). The chemical gradient most appropriate for the fragment is calculated as the Tm of the lowest (preferably main) domain ±10 oC whereby 1 oC is equivalent to 3% denaturant (see calculations).

If this is not possible, the chemical gradient can be analysed empirically by running the fragment on a gel that has a gradient perpendicular to the direction of electrophoresis (Figure 5.14).

Much of the equipment that is required for making and running of DGGE gels is quite specialized. A gradient maker for small volumes is necessary for creating the vertical chemical gradient in the gel (Figure 5.15). Furthermore the gels have to be run at high temperature (usually 60 oC) for >12 hours, which requires equipment that can heat the electrophoresis enivironment (buffer), maintain a steady temperature and recirculate the buffer to replenish that at the cathode during the run. Thus the electrophoresis has to be carried out in a tank large enough to fit the gel and a heater, with an external pump buffer for recirculation (Figure 5.16). There are a few commercially-available DGGE systems, the most comprehensive being the D-Gene system from Biorad.

It must be noted that DGGE is not a method for direct characterization of point mutations. However, it is extremely useful for:


 * 1) screening gene regions (of up to several hundred base pairs) in order to localise a potential mutation(s), simultaneously excluding regions without a mutation,
 * 2) directing the use of direct mutation assays, such as ARMS or RE-PCR, for those mutations known to occur within the targeted gene region, since most nucleotide changes are associated with a distinct DGGE banding pattern.
 * 3) investigating the genotype status in a prenatal diagnosis sample, alongside positive and negative controls for the parental mutations.

Protocols for mutation detection throughout the length of most of the globin genes have been described (45-48). Here we will focus on a protocol appropriate for the b-globin gene.

Steps in DGGE analysis of the b-globin gene
The steps of DGGE analysis include:


 * 1) PCR of the fragment(s) for analysis
 * 2) gel preparation
 * 3) gel running
 * 4) gel staining and viewing.

Exact details of each step will vary slightly according to the equipment available in the lab, but the description here is a solid guideline

PCR amplification of b-globin gene regions
The PCR protocols in this manual are based on those described by Losekoot et al, 1990 (45) and Kleiman et al, 1994 (49), and are suitable for the detection of the majority of common point mutations underlying b-thalassemia throughout the world.

The b-globin gene has been divided into 8 different fragments (named A-H), which span the complete coding sequence and the flanking regions at the 5’ and 3’ end of the gene (Figure 5.17). The optimal position of the primers used to amplify each fragment and the position and length of the GC-clamp was achieved by predicting the fragment melt-map using the computer algorithm (44), examples of which are depicted in Figure 5.12. The sequence of some PCR primer have been modified by using the Amplify version 2.0 computer software (Bill Engels, 1992-1995, Freeware, US), in order to minimize non-specific PCR products (50). The primer sequences for the amplification of fragments A-H are listed in Table 5.15. The fragments should be screened in the order most appropriate for the frequency of mutations in the population under investigation. Fragment E is a region that does not contain many common mutations, but it contains 3 of the 5 polymorphic sites that predict the framework of the b-globin gene (Figure 5.18), which are potentially useful for linkage analysis when appropriate (51).

Reagents for PCR
For PCR of each b-globin gene fragment, using a 50 μl reaction, add the following to an eppendorf tube, on ice:


 * 1) 0.5 μg genomic DNA
 * 2) 15-20 pmol of each primer (forward and reverse for relevant fragment)
 * 3) H2O up to 50 μl final volume
 * 4) 5 μl of 10x reaction buffer (usually provided with the Taq polymerase by the manufacturer, containing 500 mM KCl, 100mM Tris-HCl pH 8.0, 25 mM MgCl2, 2 mg/ml BSA)
 * 5) 2 mM dNTP’s
 * 6) 1 unit of Taq polymerase
 * 7) Overlay with 50 μl paraffin oil.

PCR conditions
The following cycling conditions are appropriate for the majority of PCR machines for all fragments, although some minor adjustments may be required to optimise the PCR reaction:


 * 1) Denaturation at 95 oC for 60 sec
 * 2) Annealing at 58-60 oC for 60 sec (may differ between PCR machines)
 * 3) Extension at 72 oC for 90 sec,

repeated for 35-40 cycles.

Following the PCR reaction, 5 μl may be run on a 1.5% agarose gel to check efficiency and specificity. The expected sizes of fragments A-H are included in Table 5.16. To prepare samples for loading on to the DGGE gel, mix approximately 10 μl of PCR product with 5 μl of loading dye.

Basic equipment for DGGE

 * 1) Two glass plates approximately 18x20 cm, one “eared” and the other plain.
 * 2) Spacers and combs 1mm thick
 * 3) DGGE holder for stabilizing glass plates during gel making
 * 4) Gradient maker, for small volumes (up to approximately 25 ml/chamber), and appropriate tubing
 * 5) Magnetic stirrer and small magnets to fit diameter of gradient maker chambers
 * 6) Tape
 * 7) DGGE holder with electrodes (gel electrophoresis cassette)
 * 8) Transparent aquarium with lid (to avoid excessive evaporation of buffer during electrophoresis).
 * 9) Heating element with thermostat and circulating ability
 * 10) Thermometer to monitor temperature during electrophoresis
 * 11) Power supply
 * 12) Syringe and needles to flush out wells prior to sample loading
 * 13) Fine-ended (duck-billed) tips for sample loading
 * 14) Staining tray
 * 15) UV transilluminator, 256 nm wavelength
 * 16) Camera

acrylamide bis-acrylamide 37.5:1100 g
H2Oup to 250ml

b) 20x TAE, pH 8.0

Tris-HCl800 mM96.912 g

Na2EDTA20 mM 7.445 g

Na Acetate400 mM54.432 g

Adjust pH with acetic acid (about 36 ml)

c) 80% Denaturant/6% acrylamide

75 ml of 40% stock 6% acrylamide

32% formamide

160 ml of 100% stock 5.6 M Urea170 g

1x TAE: 25 ml of 20x TAE stock

H2Oup to 500ml


 * 1) 0% Denaturant/6% acrylamide

75ml of 40% stock 6% acrylamide

1x TAE: 25 ml of 20x TAE stock

H2Oup to 500ml


 * 1) 80% Denaturant/8% acrylamide

100 ml of 40% stock 8% acrylamide

32% formamide 160 ml of 100% stock

5.6 M Urea170 g

1x TAE: 25 ml of 20x TAE stock

H2Oup to 500ml


 * 1) 10% Ammonium peroxydedipersulphate

5 g per 50 ml water

Store in 1ml aliquots at –20 oC. Discard surplus after use.


 * 1) TEMED: N,N,N’,N’-Tetramethylethylenediamine.

Store at 4oC


 * 1) Loading Buffer

0.25% bromophenol blue

0.25% xylene cyanol FF

15% Ficoll-isopaque (6.1%-18.6%)


 * 1) Ethidium bromide10 mg/ml (store in dark bottle)

Preparation of DGGE gel


 * 1) Clean glass plates well, using in succession strong detergent, water and finally acetone or alcohol. Dry well.
 * 2) Place spacers between the plates and tape the sides and bottom, sealing very well to prevent leakage (see Figure 5.15).
 * 3) Place the comb in between the glass plates and put the construction into a “holder” that stabilizes the plates in a vertical position. NOTE: Suitable size of glass plates is approximately 18-20 cm, with spacers and combs 1 mm thick. The volume of gel required is approximately 32 ml.
 * 4) Connect the tubing to a clean and dry gradient maker, closing the communicating channel between the 2 chambers and also the tubing that leads from the gradient maker to the glass plates, with the use of stop-cock or clamps. The tubing that leads from the gradient maker to the glass plates should be about 25-26 cm long.
 * 5) Position the magnetic stirrer about 25 cm above the glass plates and place the gradient maker on top, securing well.
 * 6) In two separate test tubes prepare two denaturing solutions (representing the high and low of the range appropriate for the fragment) of 16 ml* each, and add 10μl TEMED and 130 μl of fresh ammonium peroxydedipersulphate (10% APS). Mix well.


 * For glass plates sized approximately 18 cm x 20 cm with 1mm width spacers; the relative volume of 0% and 80% acrylamides to achieve the upper and lower limits of the range should be calculated as follows:

(% denaturant/80% denaturant) x volume

For example, for a 70% denaturant solution calculate 70/80 x16, ie 14 ml 80% solution + 2 ml 0% solution. For gradient to be as precise as possible the volume of acrylamide should be the volume between plates plus 1-2 cm spare in case of slight leakage compensation.


 * 1) Without delay, put the solution with the lowest denaturant concentration in the chamber of the gradient maker furthest from the glass plates (chamber A in Figure 5.15). Allow a minimal volume to flow through the connecting channel to avoid blockage by air, and then place the solution with the highest denaturant concentration in the chamber nearest the glass plates (chamber B in Figure 5.15).
 * 2) Put the magnet in this chamber, turn stirrer on and open the connecting tube between chambers to begin mixing the two solutions. Immediately open the connection on the tubing leading to the glass plates and the acrylmide will begin to flow steadily under the force of gravity.
 * 3) As soon as the solution has reached the comb (and slightly overflowed), stop the flow and remove the tubing. Wash the tubing and gradient maker well under running water.
 * 4) The gel will polymerise within 30-45 minutes, depending upon ambient temperature (the warmer the temperature, the faster the polymerisation).

Electrophoresis of the DGGE gel

 * 1) Fill the electrophoresis tank with running buffer (1x TAE, about 15-20 litres depending on apparatus). Heat buffer to 60 oC. (Note: The running buffer can be re-used about 5 times).
 * 2) Once the gel has polymerised, carefully remove the comb and clean away excess acrylamide, taking care not to spoil the wells. Remove the tape from bottom of plates.
 * 3) Place the gel in the gel electrophoresis cassette (Figure 5.15B) and submerge in the buffer in the tank (Figure 5.16). Attach the electrophoresis cables (cathode at top) and pre-run gel for about 30 mins at 40 volts (about 40 mA).
 * 4) Flush the wells with a fine syringe and load about 10-15 μl of each sample, containing loading dye, using thin-ended or duck-billed tip.
 * 5) Run gel about 16 hours (overnight) at 40-50 volts. The gel is ready for viewing when the bromophenol blue dye has run completely out of the gel.

DGGE gel staining and viewing

 * 1) After the run, turn off power supply and remove gel from tank and holder.
 * 2) Unseal the sides of the glass plates and gently remove one of the glass plates, leaving the gel to rest on the other.
 * 3) Place the gel (on the glass plate) in a container with approximately 250 ml of 1x TAE (or water) containing 0.5 μg/ml ethidium bromide and stain for about 15 minutes. (Note: The container should be covered to protect the gel from light).
 * 4) Destain the gel for 5 minutes in water.
 * 5) Place the gel onto a UV transilluminator, sliding it off the plate carefully so it does not fold over or split.
 * 6) Examine the gel under UV light (256 nm wavelength) and photograph.

Troubleshooting and precautions
DGGE is a technically demanding method, and the following points must be noted:

All chemicals used especially the components of the gel (acrylamide, formamide and urea) should be of highest quality and as fresh as possible. Acrylamide solution is not very stable and thus acrylamide powder is recommended which should be made into solution not too long before use

Many of the chemicals used in DGGE are highly toxic (eg acrylamide, formamide) and should be handled with extreme care.

The interpretation of DGGE patterns should be done with careful comparison to controls since any nucleotide change, including polymorphisms, may be potentially detected. It must be noted that DGGE is not a method for direct characterization of point mutations, and any mutation detected should be confirmed by a second direct mutation assay.

.

Calculations for conversion of temperature to denaturant concentration and calculation of DGGE gradient range


 * 1) 100% denaturant = 7 M urea plus 40% formamide.

(Stock solutions of 80% denaturant and 0% denaturant are satisfactory for making most gradient ranges, see Table 5.16).


 * 1) The gradient range should be calculated as the Tm of main melting domain ±10 oC.


 * 1) 1oC is equivalent to 3% denaturant.

Thus for gels run at 60 oC calculate denaturant concentration % as 3 x (Tm-60 oC)

Eg for a fragment with main (lowest) melting domain of 72 oC,

the gradient range should be 62oC – 82 oC.

Where 62oC = 3 x (62-60) = 6%

And 82oC = 3 x (82-60) = 66%.

Figure 5.12



Figure 5.13

Increasing

concentration

of chemical

denaturant

Increasing

concentration

of chemical

denaturantHomodimersHeteroduplexesWT Het MutaG-C A BTm G-C >> Tm A >Tm BTm A > Tm BPCR reactionReverse primerForward primer with GC clampb c

Figure 5.14



Figure 5.15



Figure 5.16



Figure 5.17



Figure 5.18



Figure 5.19



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