Method: Denaturing Gradient Gel Electrophoresis (DGGE)
update July 1, 1990
C. Helms
Purpose:
Denaturing gradient gels are used to detect non-RFLP polymorphisms.
The small (200-700 bp) genomic restriction fragments are run on a
low to high denaturant gradient acrylamide gel; initially the
fragments move according to molecular weight, but as they progress
into higher denaturing conditions, each (depending on its sequence
composition) reaches a point where the DNA begins to melt. The
partial melting severely retards the progress of the molecule in
the gel, and a mobility shift is observed. It is the mobility shift
which can differ for slightly different sequences (depending on the
sequence, as little as a single bp change can cause a mobility shift).
Alleles are detected by differences in mobility.
Most of the following is taken from a procedure written in June of
1987 by Mark Gray while he was a post-doctoral student in Welcome
Bender's Laboratory (Harvard). There are many variations of the
protocol; some of them are mentioned here. Refer to the literature
citations list for more information.
Principle:
Denaturing gradient gel electrophoresis has been shown to detect
differences in the melting behavior of small DNA fragments (200-700
bp) that differ by as little as a single base substitution. When
a DNA fragment is subjected to an increasingly denaturing physical
environment, it partially melts. As the denaturing conditions
become more extreme, the partially melted fragment completely
dissociates into single strands. Rather than partially melting in
a continuous zipper-like manner, most fragments melt in a step-wise
process. Discrete portions or domains of the fragment suddenly
become single-stranded within a very narrow range of denaturing
conditions. The rate of mobility of DNA fragments in acrylamide gels
changes as a consequence of the physical shape of the fragment.
Partially melted fragments migrate much more slowly during
electrophoresis through the polyacrylamide matrix than completely
double-stranded fragments. When a double-stranded fragment is
electrophoreses into a gradient of increasingly denaturing
conditions, it partially melts and undergoes a sharp reduction in
mobility because it changes shape. In practice, the denaturants
used are heat (a constant temperature of 60 degrees C) and a fixed
ratio of formamide (ranging from 0-40%) and urea (ranging from 0-7
M). The position in the gradient where a domain of a DNA fragment
melts and thus nearly stops migrating is dependent on the nucleotide
sequence in the melted region. Sequence differences in otherwise
identical fragments often cause them to partially melt at different
positions in the gradient and therefore 'stop' at different
positions in the gel. By comparing the melting behavior of the
polymorphic DNA fragments side-by side on denaturing gradient gels,
it is possible to detect fragments that have mutations in the first
melting domain.
Many fragments can be analyzed simultaneously on a single
denaturing gel in which the direction of electrophoresis is
perpendicular to that of the denaturing gradient. When a large
number of different fragments is electrophoresed, the fragments can
be identified by their molecular weight in the low denaturant side
of the gel. By following the S-shaped curves, the characteristic
denaturant concentration at which the first domain melts can be
determined. When two nearly identical sets of fragments are mixed
together and electrophoresed into a 'perpendicular' denaturing
gradient gel, the melted domains that have sequence differences
between each other will melt at slightly different positions and
produce double bands.
Sequence differences are often easily detected in DNA fragments
when nearly identical digests are electrophoresed in the same
direction as that of the denaturing gradient. These 'parallel' gels
permit the simultaneous comparison of as many sets of fragments as
there are lanes on the gel, unlike the perpendicular gels. The
procedures below refer almost entirely to parallel denaturing
gradient gels.
DGGE blotting
Denaturing gradient gel blots of genomic DNA can be used to detect
most single base differences (which may occur as frequently as every
400 bp). Genomic DNA is digested with an enzyme combination that
cuts the DNA into fragments of average size 200-700 bp. Enzymes with
4-base recognition sites, such as AluI, HaeIII, HhaI, MspI, and RsaI
are the most useful. The samples are electrophoresed long enough
for many fragments to reach a position in the gradient at which the
first melting domain denatures. After electrophoresis, the DNA
fragments are transferred to a nylon blot. The blots are hybridized
overnight with a radioactive probe, washed, and exposed to X-ray
film for 1-5 days. Each lane is examined for fragments that have
mobility shifts.
Many polymorphisms are present in portions of a restriction
fragment not in the first melting domain. Sequence differences are
undetectable unless they are within the first melting domain. To
avoid this problem, it is often possible to 'move' the mutation into
the first melting domain by using different restriction enzyme
combinations. Frequently, at least one of 4-5 different digests
achieves this result. Occasionally, a mobility shift is observed
in more than one restriction digest. By aligning on a restriction
map the partially overlapping fragments that have altered melting
behavior, the region containing the the base difference can be
narrowed down to 100 bp of less.
DGGE- finding polymorphisms with PCR amplified DNA
Although we have yet to try it in our lab, the DGGE should provide
an excellent method of finding polymorphisms when DNA samples
amplified from different individuals are run on parallel denaturing
gradient gels. The mobility shifts of these amplified fragments are
visible with ethidium staining, eliminating the need for
autoradiography. The polymorphisms detected in such a screen could
be pursued with genotypic data collection in the CEPH pedigrees.
Richard Myers advocates the PCR amplification of larger fragments,
e. g. 2- 3 kb in size, followed by restriction digestion of the
amplified products with two different frequent cutting enzymes such
as HaeIII and Sau3AI, then running the samples on two different
denaturing gradient gels (0-50% denaturant and 40-80% denaturant)
with different running times. The gels are then examined under UV
illumination after staining with ethidium bromide. Myers estimates
as many as 75% of the base pair changes in a 2-3 kb fragment could
be detected in this screen.
Special Equipment:
1. DGGE apparatus (Green Mountain Scientific Supply)
2. Electroblotter (Hoefer)
3. BioRad gradient maker
Special solutions:
1. 0% denaturing acrylamide stock
2. 100% denaturing acrylamide stock
3. 50X TAE buffer
4. BstN I digest of pBR322 DNA (gel standards)
Time Required:
After the preparation of restriction digests/ or PCR amplification:
1.
2.
3.
4.
Day 1: 2-4 hours to prepare, load, and start the electrophoresis
Day 2: 1-2 hours to stain and photograph
4 hours for electrotransfer
2 hours for baking the blots
Procedures:
General Notes:
The 25 ml gels are poured with the aid of a gradient maker. The
general strategy for detecting and following polymorphisms is first
to use a DGGE gel which has wide range denaturing conditions,
typically 20% to 80% denaturant. Once a polymorphism is detected,
the distance separating the mobility shifts is amplified by
narrowing the range of the denaturing conditions on the gel to an
appropriate range of denaturing conditions, e. g. 20-50% or 40-80%
denaturant, and the rest of the data collection is done under the
optimized conditions.
The denaturing gradient gels are electrophoresed in an apparatus
purchased from Green Mountain Lab Supply, Inc. (86 Central Street,
Waltham, MA 02154). Parallel denaturing gels can be poured in the
gel frame supplied (perpendicular gradient gels must be prepared
outside the gel frame; this procedure does not cover perpendicular
DGGE). The glass plates (one notched and one unnotched), spacers
and gel combs are supplied with the basic apparatus, and will
produce gels approximately 5 x 5 inches. After the gel is poured
and the acrylamide polymerized, it is immersed in the tank (filled
with 1X TAE buffer equibrated to temperature of 60 degrees C with
the Polyscience constant temperature regulator/circulator). The
buffer is circulated through the tank with a peristatic pump. The
anode is permanently mounted in the gel tank; the cathodes are
graphite rods which are immersed in the upper electrophoresis
chamber while electrophoresis proceeds.
Restriction digestion of genomic DNA samples:
Choose restriction enzymes that will produce an average fragment
size in the 200-700 bp range. The most reliable and inexpensive
4-base recognition site enzymes include AluI, HaeIII, HhaI, MspI,
and RsaI. Cut 7 to 10 ug human genomic DNA for each lane to be run.
Be sure to include an extra ug of genomic DNA for the test digest
(with 1 ug bacteriophage lambda DNA, see procedure "human genomic
DNA digests" for more details on restriction digestion). Use the
manufacturer's recommended restriction buffer and digest the
samples overnight.
Run the test digest and lambda controls on a high percentage agarose
gel (e. g. 1.5%) to determine whether or not the human sample was
completely digested. Once complete, concentrate the DNA (by ethanol
precipitation) to approximately 1 ug DNA/祃 TE, and add the
appropriate amount of 10 X Stop Mix (the lab's standard
glycerol-based loading dye).
For a DGGE polymorphism screen of unknown DNA sequences, prepare
digests of at least 4 unrelated individuals with 3 or more different
restriction enzymes. The DNA 'molecular weight 'standard used to
monitor the progress of fragments in the acrylamide gels is a BstN
I digest of pBR322 DNA [fragment sizes are: 1857, 1060, 929, 383,
121, and 13 bp].
Day 1
Pouring and electrophoresis of parallel denaturing gradient gels:
1. Prepare 150 ml of 1.5% TAE agarose for each gel frame used: Weigh
2.25 g agarose, add 3 ml 50X TAE buffer and 150 ml dH2O. Boil in
the microwave until dissolved, then cool to 50 degrees C in a water
bath.
2. Wash the glass plates with hot water and soap, rinse well with tap
water and deionized water, followed by a 95% ethanol rinse. Lay a
clean and dry notched plate down, then place the lightly greased
(with petroleum jelly) spacers along the length of the two sides.
Carefully lay a clean un-notched plate on top. (The apparatus is
designed to accomodate 2 more gels, prepared with layers of notched
plates and spacers, with the last plate being the un-notched plate.)
3. Pick up the stacked plates and using a pasteur pipet, dribble 50
degrees C 1.5 % agarose (prepared in TAE buffer) down the side to
help seal the spacers.
4. Place the plates in the gel frame so that the notched plate faces
the upper buffer compartment. Center the plates so that the small
gaps on the sides are equal. Insert the plexiglass supports between
the screws in the gel frame and the glass plates. Twist the screws
evenly but firmly enough so that the neoprene gasket between the
first notched plate and the gel frame is compresses enough to form
a waterproof seal.
5. Pour 1.5 % TAE agarose into the trough at the bottom of the gel frame
so that the agarose is drawn up between the plates about 1 cm. Let
the agarose plug harden, then fill with more agarose, and pour
agarose down the side gaps of the gel frame to ensure the seal
between glass plates and spacers is tight. It is important to be
ready to pour the gel soon after the agarose hardens, because as
the agarose dries, it shrinks, and gaps will form causing the
acrylamide to leak before it can polymerize.
The linear gradient is made by placing the high denaturant mix (e.
g. 80%) in the downstream side of the gradient maker, and the low
denaturant mix (e. g. 20%) in the upstream side of the gradient maker.
When the gradient maker is allowed to flow, as long as the downstream
chamber is continuously mixed (using a small magnetic stirrer), the
20% mix flows into the 80% mix forming the linear gradient of
decreasing denaturant concentration.
The gel volume will vary depending on the thickness of the spacers
used. For 0.7 mm spacers and combs, each gel holds approximately
20 ml. Prepare 12.5 ml of the two gel mixes to be used for each gel
using the 0% and 100% denaturing stocks (the total volume is 25 ml,
an excess of 5 ml). The following table gives the proportions of
each denaturant to use in preparing typical gel mixes:
% denaturant
mls 0% denaturant
stock
mls 100% denaturant
stock
volume
20%
10.0
2.5
12.5 ml
40%
7.5
5.0
12.5 ml
60%
5.0
7.5
12.5 ml
80%
2.5
10.0
12.5 ml
The urea in the 100% denaturant stock will drop out of solution at
4 degrees C, (be sure to warm the stock to redissolve the urea before
mixing the desired denaturants).
6. Pipet the desired amount of stocks into two separate, labeled
Erlenmeyer flasks. Degas and put the gel mixes on ice (the degassing
can be omitted).
7. Set up the gradient maker with the stirrer in the downstream chamber
and tubing leading from the gradient maker to the top of the gel
plates. Attach a 25 gauge needle to the tubing and wedge the needle
between the glass plates at the top left side. Close the valve
separating the two sides of the gradient maker, and close the tubing
valve.
8. Add 1/200 volume (63 祃) of a 20% ammonium persulfate (in dH20)
solution and 1/2000 volume (6.3 祃) of TEMED (N, N, N',
N'-tetramethyl-ethylenediamine) to each mix.
9. Pipet 12 ml of the high denaturant mix (e. g. 80%) into the
downstream chamber. Open the valve to the upstream chamber so that
a small amount of 80% mix enters the upstream chamber, then close
the valve. With a pasteur pipet, remove the small amount that
entered the upstream chamber and place it back in the downstream
chamber. This eliminates the air pocket in the passageway between
the two chambers. Pipet in 12 ml of the low denaturant mix (e. g.
20%) into the upstream chamber.
10.Open the valve between the two sides of the gradient maker. Be sure
the stirrer is mixing well in the downstream side. Open the valve
in the tubing that leads to the gel plates. The gel mix should begin
to flow down the tubing to the gel. If it does not flow due to a
bubble near the chamber, remove the needle for a short time to start
the flow, (or use an adjustible flow rate pump to move the gel mix).
The entire gel should be poured over a 5 minute period in order to
avoid disruption og the gradient. During the time the gradient is
being poured, check the gel for leaks, and for any disruption in
the pouring.
11. After the gel is poured, insert the comb without trapping small
bubbles under the wells. If additional gels are being poured, refill
the gradient maker as before, and repeat the entire process.
11.Immediately after the gels are poured, rinse out the gradient maker
with distilled water.
12.Check the top corners of the gels for evidence of leaks. If the level
of the mix drops quickly, there is likely to be a leak between the
agarose plug and the plates. If the leak is slow, the gel is
salvageable because polymerization is usually fast (about 10
minutes). After polymerization, there is usually a small amount of
shrinkage at the edges; this is of no concern. Leave the combs in
place until the gel is ready to be loaded. Fill the upper compartment
with TAE buffer. The gel can be loaded one hour after pouring or
several days later, as long as the agarose does not dry out too much.
13.The temperature of the gel buffer in the tank should be brought to
60 degrees C and the pump should be recirculating the buffer before
loading the samples. Fill the large plexiglass tank with enough TAE
buffer to bring the level to within 3/4 of an inch from the top of
the gel frame (an empty tank takes at least 16 liters). Turn on the
heater before putting the gels into the tank. It may take as long
as 45 minutes for the temperature to reach 60 degrees C.
14.After the temperature reaches 60 degrees C, remove the comb and
examine the wells for any defects. Put the entire gel frame into
the tank, attach the tubing for recirculating the buffer to the gel
frame, and start the pump. Attach the leads, immersing the cathode
graphite rods into the upper buffer chamber of each gel frame, then
'piggy back' the cathode leads together at the power supply if more
than one gel frame is in use. Attach the anode lead, and turn on
the current to check the circuit.
15.When everything is ready, load the samples using a pipetman with
flat tips (made for sequencing gels). Change tips with each sample.
Load all the samples before turning on the power. Turn the power
to 65-75 volts and note the time; electrophorese overnight for 16-17
hours. For samples of approximately 400bp, try running the gel for
1200 volt-hours.
Day 2
1. Turn off the power in the morning and note the time again. Turn off
the heater and pump. Disconnect the electrical leads and pump tubing,
and remove the gel frames from the tank. Loosen the screws, remove
the plexiglass supports, and pull the plates out of the frame. Lay
the plates down, keeping track of the order and orientation of the
gels. Separate the glass plates by wedging a spatula between the
plates at a lower corner, and lift off the top plate.
2. Mark the gel for orientation purposes by slicing the upper left hand
corner with a razor blade. Stain the gel on the plate (which should
be placed in a tray) by pouring enough ethidium bromide stain
solution over the gel to cover the surface. Stain for 5-10 minutes,
then carefully pour off or aspirate the staining solution and
destain with distilled water for several minutes. Examine the gel
by UV illumination (first transfer the gel to a platform such as
UV translucent plexiglass or Saran wrap). Genomic DNA digests
should be visible as evenly loaded straight smears; PCR-amplified
fragments should be visible as bands or tight smears. Any unusual
features in some lanes should be noted before photographing the gel.
Electrophoretic transfer of the DNA to nylon blots:
3. After photography, transfer the gel back to a staining tray and
aspirate any excess liquid. Add enough 0.5 N NaOH to cover the gel,
and mix once or twice over the next 5 minutes, then aspirate the
NaOH. This denatures the DNA fragments.
4. Neutralize the gel by pouring in 0.5M Tris-HCl, pH8.0, mixing once
or twice over the next 5 minutes, then remove the Tris solution.
5. Fill the tray with transfer buffer (20 mM Tris-HCl, pH8.0 + 1 mM
EDTA) and let it equilibrate for 10 minutes.
6. Measure the size of the gel after this equilibration, because it
will bave increased in size. Cut a sheet of nylon membrane large
enough to cover the gel, label, and wet it in a tray of transfer
buffer for at least 5 minutes.
7. Fill the electrotransfer chamber with enough transfer buffer to
reach close to the top.
8. Assemble the sandwich in a tray filled with transfer
buffer--bubbles can easily be displaced by buffer when assembling
the immersed sandwich. In the tray, lay down the rigid plastic
support screen, then one of the 'Scotchbrite' pads (supplied with
the electrotransfer apparatus), and then one layer of Whatman 3MM
paper. In order to lay down the fragile gel without tearing or
stretching, attach it to a 3MM paper as follows: Using a piece of
X-ray film, scoop the gel up out of the tray. Gently spread the gel
out flat using a glass pipet; as long as the gel and the film are
thoroughly wet, the gel will not tear easily. After the gel is flat
and the orientation noted, remove all excess buffer by soaking it
up on the edges with paper towels. Roll the glass pipet across the
gel several times to drive the buffer to the edges. Lay down a piece
of dry 3MM paper that is slightly larger than the gel, but small
enough to fit into the electrotransfer apparatus. Lay the paper down
in the middle of the gel first, then spread it toward the edges so
that no air bubbles are trapped. Pick up the gel by the 3MM paper
(the gel should be completely bound to the dry paper). Lay the gel
down on the sandwich with the paper side down. Next lay down the
labeled nylon membrane, watching for the appearance of bubbles. Put
down 2 layers of wet Whatman 3MM paper, then the other thoroughly
wet Scotchbrite pad. Place the second rigid support screen on top
of the sandwich, and quickly insert the sandwich into the guides
of the electrotransfer box. Try to dislodge any newly formed bubbles
by tilting the box from side to side. Note carefully the polarity
of the sandwich; the blot side should be facing the anode (+) side
of the electrotransfer box.
9. Connect the electrodes and turn the current up to 600 mA (about 30
volts). Keep the current at 600 mA for two hours. Usually it is
necessary to adjust the voltage slightly downwards during the two
hours to maintain 600 mA. Do not allow the buffer to heat up too
much; if it does, some plastic parts of the transfer appaatus may
soften and warp. (Do the trasfer in the cold room if heat is too
much of a problem.) If a power supply that can produce 0.6 amps is
unavailable, electrophoresis at 200mA for 4 hours is sufficient.
10.After the electrotransfer, turn off the power, remove and
disassemble the sandwiches, and put the nylon blots in a tray of
2 X SSC for 5 minutes. Let the blots air dry one pieces of Whatman
3MM paper before baking in a vacuum oven at 80 degrees C for 1-2
hours.
After the blots are baked, they can be treated as any other Southern
blot.
Interpretation of results:
If the electrophoresis time was long enough, most of the fragments
should be visible as sharp, focussed bands that do not trail up at
the sides. Some bands will appear as almost invisible long smears
because of the simultaneous melting of more than one domain in the
fragment. Some particularly short fragments (less than 300 bp) and
GC rich fragments will not form sharp focussed bands, usually in
the most denaturing part of the gel. The most common difference
caused by single base changes is a shift in the position of the
fragment, either up or down with respect to a standard. Other types
of changes include: a sharp focussed band will become very diffuse
or almost a diffuse smear, a diffuse unfocused band becomes sharp
and focused, the position of a smear is shifted up or down.
Often it is not clear whether or not a fragment is different from
its counterpart in another lane. In these cases, it is easiest to
reexamine them by repeating the electrophoresis in a denaturing
gradient gel with a much narrower range of denaturant concentration.
For example, if the ambiguous fragment is present at a position 60%
of the distance from the top of a 20-80% gel, then its critical
denaturant concentration is 56% (0.6 x 60 per cent units + 20%).
Repeat the electrophoresis of this sample on a denaturing gradient
gel with a range of 41-715 (15% on either side of that desired).
Any real differences will often be greatly exaggerated in the less
steep gradient. If more than one fragment is different, it is
frequently an artifact. There are many ways to create artifactual
results on denaturing gradient gel blots. Real differences are
reproducible on different gels/blots.
Solutions:

40% Acrylamide stock
(37.5: 1 Acrylamide: bisacrylamide)
Caution: acrylamide is a neurotoxin, especially dangerous in
powder form. Wear a mask when weighing out acrylamide.
Dissolve 194.8 g electrophoresis grade acrylamide and 5.2 g
bisacrylamide in enough deionized water to make a total
volume of 500 ml. Store in the dark at 4 degrees C.

0% denaturant
(6.5 % acrylamide in 1X TAE buffer)
81 ml 40% acrylamide stock
10 ml 50 X TAE buffer
409 ml ddH2O
Total volume = 500 ml; store in the dark at 4 degrees C.

100% denaturant
(6.5 % acrylamide, 40% formamide, 7 M urea, in 1X TAE buffer)
81 ml 40% acrylamide stock
10 ml 50 X TAE buffer
210 g urea
200 ml formamide
adjust final volume to 500 ml
store in the dark at 4 degrees C.

50X TAE Buffer
per liter:
242 g Trizma base
2M Tris
136.1 g sodium acetate 1M sodium acetate
18.6 g Na2EDTA
50 mM EDTA
Add ddH2O to bring volume close to 900 ml
pH to 7.4 with concentrated HCl, then adjust volume to 1 liter

Transfer Buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA)
per liter:
20 ml 1 M Tris-HCl pH8.0
2 ml 0.5 M EDTA
978 ml ddH2O

1.5 % TAE agarose
per 200 ml:
3 g agarose
4 ml 50 X TAE buffer
196 ml ddH2O

20% ammonium persulfate (20 % APS)
2 g ammonium persulfate
+ ddH2O to 10 ml
store at 4 degrees C.
Many scientists feel that making fresh 20 % APS every 1-2
weeks produces better results.
References:
Myers, R. M., Maniatis, T., and L. S. Lerman. (1987). "Detection and
localization of single base pair changes by denaturing gradient gel
electrophoresis." in Meth. Enzymol. 155: 501.
Gray, M., pers. comm. (1987). after his procedure notes "Detection and
localization of single base pair changes by denaturing gradient gel
electrophoresis".
Myers, R. M., pers. comm.