Download Pulsed-Field Gel Electrophoresis

Pulsed-Field Gel Electrophoresis
UNIT 2.5B
DNA molecules longer than 25 kb are poorly resolved by standard agarose gel electrophoresis (UNIT 2.5A). These longer molecules can be resolved using several techniques that
periodically change the direction of the electric field in the gel. The simplest and most
generally useful of the pulsed-field techniques is field inversion electrophoresis (see Basic
Protocol), which can be tuned to resolve molecules from ∼10 to 2000 kb (or more with
specialized equipment). To resolve molecules beyond the range of field inversion, it is
necessary to use some sort of field-angle alternation electrophoresis such as CHEF
(contour-clamped homogeneous electric field; see Alternate Protocol). High-molecularweight DNA samples and size markers to be resolved by these techniques can be prepared
while embedded in agarose blocks (see Support Protocol).
FIELD-INVERSION ELECTROPHORESIS
Materials
1% agarose gel, standard or pulsed-field grade (e.g., SeaKem FastLane;
FMC Bioproducts)
GTBE buffer (see recipe) or 0.5× TBE buffer (APPENDIX 2)
Samples embedded in agarose (Support Protocol), or liquid samples
BASIC
PROTOCOL
Peristaltic pump (Cole-Parmer Masterflex or equivalent)
Programmable switching device (MJ Research PPI-200 or equivalent)
Additional reagents and equipment for agarose gel electrophoresis (UNIT 2.5A)
NOTE: Some power supplies have pulsed-DC rather than constant-voltage output and are
unacceptable for pulsed-field gels. These can usually be recognized because their output
is fixed, or adjustable in steps, rather than continuously variable.
1. Prepare a 1% agarose gel for a horizontal gel apparatus using GTBE or 0.5× TBE
buffer.
Make the gel only as thick as necessary for the samples so that it will consume little power
and heat up as little as possible.
Ethidium bromide can be incorporated into the gel, but is recommended only for gels
resolving fragments <100 kb (see Critical Parameters).
2. Allow gel to set, then carefully remove comb. Insert into wells any samples that have
been prepared in agarose blocks (see Support Protocol).
If blocks fit tightly into wells, it may be easiest to draw them down into the wells by inserting
a pipettor with a 0.4-mm (or thinner) gel-loading tip into the back of the well to remove
the air from under the blocks. If the blocks do not fit tightly, add melted agarose (55°C in
gel buffer) to the well to hold the block in place.
3. Place gel into gel box, cover with buffer to a depth of 2 to 3 mm, and load any samples
that are in liquid. To avoid shearing DNA >100 kb, cut ∼5 mm off ends of pipet tips
with a razor blade and pipet gently. At least one lane should contain bromphenol blue.
4. Adjust peristaltic pump for an appropriate flow (5 to 10 ml/min for a minigel and 20
to 50 ml/min for a large gel). Connect tubing ends to recirculation ports of gel box
or place directly in buffer tanks.
5. Paying careful attention to polarity, connect programmable switching device to a
constant-voltage DC power supply and connect gel apparatus to switching device.
Contributed by Michael Finney
Current Protocols in Molecular Biology (2000) 2.5B.1-2.5B.9
Copyright © 2000 by John Wiley & Sons, Inc.
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Set switching device for an appropriate switching regime but don’t begin switching
yet. Start gel running.
See Critical Parameters, including Table 2.5B.2, for a guide to time intervals, voltage, and
other parameters. The most commonly used ratio of forward to reverse time is 3:1.
6. Allow bromphenol blue to migrate 1 cm, then start switching device and peristaltic
pump.
7. Complete run and stain gel with ethidium bromide (UNIT 2.5A). Photograph as for a
standard agarose gel (Fig. 2.5B.1).
The gel may be Southern blotted (UNIT 2.9A); note that the acid depurination step is essential
for transfer.
ALTERNATE
PROTOCOL
CHEF ELECTROPHORESIS
It is possible to resolve DNA molecules several million bases in length by periodically
changing the angle of the electric field in the gel. There are a number of variations to the
basic setup but all require specially constructed gel boxes, which can be quite expensive.
In addition, some types of alternating-angle electrophoresis setups dissipate so much
power in the gel that special cooling equipment must be used, adding to the expense.
Running CHEF or other alternating-angle gels, described here, is very similar to running
field-inversion gels, so the factors to be considered in that protocol apply to this one, as
well. See Critical Parameters for a guide to estimating optimal conditions for running
alternating-angle gels.
Additional Materials (also see Basic Protocol)
CHEF electrophoresis voltage-divider circuitry and gel box (see Background
Information).
1. Prepare a 1% agarose gel for a CHEF gel apparatus using GTBE or 0.5× TBE buffer.
2. Allow gel to set, then carefully remove comb. Insert into wells any samples that have
been prepared in agarose blocks (see Support Protocol).
3. Place gel into gel box, cover with buffer to a depth of 2 to 3 mm, adjust recirculation
to ≥100 ml/min, and monitor buffer temperature. Wait 15 min after buffer has reached
kb
1200
Figure 2.5B.1 Chromosomes of
Saccharomyces cerevisiae
separated by field inversion.
200
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desired running temperature to ensure that the gel has equilibrated at the correct
temperature.
4. Load any samples that are in liquid.
5. Paying careful attention to polarity, connect programmable switching device to
constant-voltage DC power supply, voltage divider circuitry, and gel apparatus. Set
the switching device for an appropriate switching regime and start gel.
6. Complete run and stain gel with ethidium bromide (UNIT 2.5A). Photograph as for a
standard agarose gel.
PREPARATION OF HIGH-MOLECULAR-WEIGHT DNA
SAMPLES AND SIZE MARKERS
SUPPORT
PROTOCOL
Very long DNA molecules are extremely fragile and cannot survive the standard manipulations of molecular biology. These molecules can, however, be prepared and manipulated easily while embedded in agarose blocks. This protocol describes the preparation
of very high-molecular-weight DNA.
Materials
1% agarose
Sample to be prepared (e.g., tissue culture cells, nematode worms, nuclei,
yeast, bacteria, or phage; Table 2.5B.1)
Lysis buffer (see recipe)
Storage buffer (see recipe)
400 mM phenylmethylsulfonyl fluoride (PMSF) in ethanol
10 mM Tris⋅Cl, pH 8.0 (APPENDIX 2)
Appropriate restriction enzyme and buffer (UNIT 3.1)
Block molds or petri plates
1. Prepare block molds (see Fig. 2.5B.2) by sealing one end with tape. If block molds
are unavailable, samples can be prepared in agarose poured as a puddle on the bottom
of a petri dish and blocks cut to size using a razor blade.
2. Suspend sample at room temperature in water or an appropriate buffer or medium at
twice the desired final concentration.
wells
same size and shape as comb used to pour gel
clear plastic block
seal bottom surface
with tape
Figure 2.5B.2 Block molds for high-molecular-weight DNA samples. These can be made in the
laboratory, or may be purchased from pulsed-field gel box manufacturers.
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Table 2.5B.1
Preparation of High-Molecular-Weight DNA Samples and Size Markers
Starting material
Preparation
Bacteria and phage
Resuspend bacteria (UNIT 1.2) or phage particles (UNIT
at a concentration calculated to yield the desired
amount of DNA per lane; e.g., 5 × 108 E. coli per ml will
yield ∼100 ng DNA in an average lane.
Start with a concentrated stock of phage λ particles (UNIT
1.13). This procedure does not work well with some lots
of commercial λ DNA, possibly because of damaged
cohesive ends. Try several dilutions of phage stock to see
which works best. The second incubation in lysis buffer
should be done at 25° rather than 37°C; during this
incubation, the cohesive ends of the molecules will
anneal, giving multimers of varying lengths. These
concatemers will stay together during electrophoresis
provided that the gel is run at <25°C.
Anesthetize worms by resuspending in 10 mM NaN3,
then place in agarose.
Isolate as in UNIT 4.10. Approximately 1 µg DNA is
contained in 105 mammalian nuclei.
1.13)
Lambda ladders for size markers
Nematodes
Nuclei
Tissue culture cells
Yeast
Cells should be washed several times in a medium
containing no serum, as serum may inhibit proteinase K.
Saccharomyces cerevisiae cells must have their cell walls
removed before being embedded in agarose as described
in UNIT 13.13, Basic Protocol.
The samples may also be suspended in the buffer in which they were originally prepared,
or in a buffer such as TE buffer (APPENDIX 2). See Table 2.5B.1 for guidelines to preparing
specific starting materials.
3. Add equal volume of 1% agarose, melted and cooled to 50°C. Quickly mix and aliquot
solution into block mold. Let agarose solidify on ice.
4. Remove tape from mold and carefully push hardened blocks into a 50-ml conical tube
containing at least 20 vol lysis buffer. Incubate overnight at 50°C, preferably with
gentle agitation.
5. Pour off lysis buffer, add fresh lysis buffer, and incubate overnight at 37°C.
6. Pour off lysis buffer and replace with 20 vol storage buffer. Store at 4°C or proceed
with steps 7 to 9 for sample to be digested with restriction enzymes.
7. Wash sample three times, 1 hr at room temperature each time, with at least 10 vol
storage buffer supplemented with 1 mM PMSF.
CAUTION: PMSF is a powerful covalent inhibitor of proteases. It is both toxic and volatile
and should always be handled in a hood. It is unstable in aqueous solution, so solutions
should always be freshly prepared from a stock of 400 mM in ethanol stored at −20°C
(PMSF will precipitate at −20°C and redissolve when warmed).
8. Wash sample three times, 30 min at room temperature each time, in at least 10 vol of
10 mM Tris⋅Cl, pH 8.0.
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9. Place sample in 1.5-ml microcentrifuge tube and remove excess liquid. Add an
amount of 3× restriction buffer (containing the restriction enzyme) equal to half the
volume of the block. Incubate at appropriate temperature.
For particular batches of samples and lots of enzyme, it may be necessary to titrate the
amount of enzyme and digestion time to minimize DNA degradation.
REAGENTS AND SOLUTIONS
GTBE buffer
50 ml 10× TBE buffer (APPENDIX 2; 0.5× final)
50 ml 2 M glycine (0.1 M final)
900 ml H2O
Store at room temperature
Lysis buffer
100 mM EDTA, pH 8.0 (APPENDIX 2)
10 mM Tris⋅Cl, pH 8.0 (APPENDIX 2)
1% (w/v) N-lauroylsarcosine sodium salt (Sarkosyl)
100 µg/ml proteinase K (add just before use from a 20 mg/ml stock)
Store at room temperature, without proteinase K
Storage buffer
10 mM Tris⋅Cl, pH 8.0 (APPENDIX 2)
10 mM EDTA, pH 8.0 (APPENDIX 2)
Store at room temperature
COMMENTARY
Background Information
In DNA gel electrophoresis, negatively
charged DNA molecules are pulled through a
gel by an electric field. An agarose gel presents
a DNA molecule with a set of pores of varying
sizes. Small molecules pass easily through
most pores and move rapidly through the gel.
Larger molecules have to “squeeze,” or change
conformation, to get through the smaller pores
and are slowed more than smaller molecules.
The mobility of a molecule is related to the
fraction of pores that it can easily move
through, a process known as “sieving.”
DNA molecules larger than a certain size
must squeeze to get through even the largest
pores and cannot be sieved by the gel. Molecules in this size range all migrate at about the
same rate, called “limiting mobility.” For ordinary agarose gel electrophoresis (UNIT 2.5A),
limiting mobility generally occurs between 20
and 40 kb, depending on the exact conditions
of the gel run. Schwartz and Cantor (1984)
showed that it was possible to resolve molecules that would otherwise run at limiting mobility by cyclically varying the orientation of
the electric field in the gel during the run.
The principle involved in alternating-angle
electrophoresis is relatively simple. As a large
molecule squeezes through a pore, it is forced
into an extended conformation. It takes some
time (from milliseconds to minutes, depending
on the molecule’s size) to squeeze through the
first pore but once the extended conformation
is attained, the molecule can continue to
squeeze rapidly through successive pores,
moving at limiting mobility. If a large molecule
moving through a gel is suddenly forced to
change direction by a change in the orientation
of the electric field, it must first change to a new
conformation that will allow it to move in the
new direction. The larger a molecule is, the
longer it will take to change to the new conformation, and this time difference can be used to
separate molecules. For example, although a
100-kb and a 200-kb molecule move through
the gel matrix at the same rate in the steady state
of limiting mobility, every time the direction of
the field changes the 100-kb molecule will get
a “head start” on the 200-kb molecule.
There are a number of implementations of
alternating-angle electrophoresis. Schwartz
and Cantor (1984) originally used an apparatus
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that resulted in nonuniform electric fields.
Nonuniform fields can focus bands, making
them very sharp, but inevitably cause the lanes
to run in various crooked shapes.
More practical systems use uniform electric
fields, which result in gel lanes that run straight.
Several methods of achieving uniform fields
have been invented, including CHEF (contourclamped homogeneous electric fields; Chu et
al., 1986), and simply running the gel in a
rectangular box and periodically rotating it. The
angle between the two fields must be somewhat
greater than 90°, and may be much greater.
In CHEF electrophoresis, the gel is surrounded by a set of electrodes whose voltages
can be fixed in such a way that a uniform
electric field is set up in the gel. Thus CHEF
gels require two pieces of equipment not necessary for field inversion electrophoresis: a
voltage-divider circuit to set the electrode voltages, and a multi-electrode gel box. Both are
available commercially and plans have been
published (Chu, 1989). Figure 2.5B.3 shows a
popular version of CHEF voltage divider circuitry.
Alternating-angle electrophoresis using a
fixed switching time gives optimum resolution
in a single size range, but does separate DNA
outside this size range, albeit with reduced
resolution (see critical parameters). It is there-
fore advantageous to vary the pulse durations
during a run.
Field-inversion electrophoresis employs the
limiting value for the angle between two uniform fields, 180° (Carle et al., 1986). Analysis
of field-inversion electrophoresis is somewhat
more complicated than that of alternating-angle
electrophoresis because different pulse times
are used in the forward and reverse directions.
Like alternating-angle electrophoresis, field inversion slows molecules that would otherwise
run at limiting mobility by taking advantage of
the time required for molecules to change conformation.
One difference between the two techniques
is that in field inversion a single pulse duration
separates only a relatively narrow range of
sizes. To get resolution over a broad size range,
it is necessary to use a range of reverse times;
this is accomplished with a time ramp—i.e.,
progressively increasing the forward and reverse intervals from a lower limit to an upper
limit. It is preferable to cycle repeatedly
through the range of times rather than to use
one long time ramp, as there is some hysteresis
in the behavior of large molecules (the largest
molecules will move more slowly during short
intervals if they immediately follow longer intervals).
Varying the reverse interval also prevents a
possible artifact of field inversion. If the reverse
resistor
electrodes
DNA migration
gel area
input A
input B
diode
Figure 2.5B.3 Circuitry for clamping electrode voltages of a 24-electrode hexagonal CHEF gel
(redrawn from Chu, 1989). Positions of the 24 electrodes are shown schematically, as well as the
direction of the resulting DNA migration. This circuitry can be driven directly by an inverting gel
controller connected to input A and input B. Power dissipation in the resistors limit this circuit to
∼250 V input. All resistors are 470 Ω, 1% tolerance, 3 W and diodes are type 1N4004 (1 A, 400 V).
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time is not long enough, large molecules may
not be sufficiently disrupted by the reversal, and
may actually migrate faster than shorter molecules. This effect can be completely overcome
by using a time ramp whose longest reverse
time is long enough to disrupt all molecules of
interest.
A phenomenon that affects all forms of
pulsed-field electrophoresis is “trapping” of the
largest molecules, preventing them from entering the gel. Trapping is dependent on both
voltage and temperature. At high temperatures
and field strengths, molecules as small as 1 Mb
may be trapped; at low temperatures and field
strengths, molecules >5 Mb will enter the gel.
Critical Parameters
All forms of pulsed-field electrophoresis
depend on differences in the time it takes for
molecules of various sizes to change directions
in a gel; these times are affected by a number
of factors. The effects of some of these factors
have been investigated in detail (Birren et al.,
1988) and are discussed below.
Voltage. Voltage is measured in V/cm of gel
length. It is best to measure this directly by
dipping the two probes of a voltmeter into the
buffer at the ends of a running gel, and dividing
the reading by the length of the gel in centimeters. Most horizontal submarine gel boxes apply ∼80% of the power-supply voltage to the
gel (the rest is lost in the buffer tanks), so
voltage can be estimated by multiplying the
power supply readout by 0.8 and dividing the
result by the length of the gel. Sizes resolved
by pulsed-field gels can be changed over a wide
range merely by changing the voltage. Higher
voltage increases the sizes separated, but also
may result in trapping the largest molecules.
Pulsed-field gels are generally run at 5 to 10
V/cm.
Temperature. Pulsed-field gels can be run
over a broad range of temperatures. A given
time interval will separate larger molecules at
30°C than at 10°C. Choose a temperature that
is convenient and adjust times for best resolution. The upper limit of resolution field-inversion gels is affected by trapping, and at 8 V/cm
varies from 2000 kb at 4°C to ∼1000 kb at 30°C.
Although different temperatures may be
used, it is critical that the entire gel be at the
same temperature to prevent the “smile” effect.
This is best accomplished by recirculating the
buffer using a peristaltic pump. In many cases,
pulsed-field gels can be run on the benchtop
with no cooling. Gels that generate a lot of heat
can often be run in a cold room.
Buffer. Because pulsed-field gels are usually
run at higher voltages than standard gels, they
have the potential to generate quite a bit of heat.
For this reason, they are often run in 0.5× TBE
buffer, which carries little current. This works
well, especially for molecules less than a few
hundred kilobases.
DNA has higher mobility in TAE buffer, but
TAE carries a lot of current, causing gels to heat
Table 2.5B.2 Empirical Equations for Fragment Resolution and Velocity Using
Pulsed-Field Gelsa,b
Field-inversion gels
Maximum resolved sizec (kb)
Minimum resolved size (kb)
0.13 × (T + 40) × V1.1 × (3 − A)0.6 × t 0.875
0.75 × maximum size
Velocity of 10-kb fragment (cm/hr)
0.0016 × (T + 25) × V1.6 × (R – 1)
A × (R + 1)
CHEF or other alternating-angle gels
Maximum well-resolved sizec (kb)
Minimum well-resolved size (kb)
Velocity of 10-kb fragment (cm/hr)
0.034 × (T + 40) × V1.1× (3−A)0.6 × t 0.875
0.75 × maximum size
0.0012 × (T + 25) × V1.6 × cos (θ/2)
A
aEquations assume use of 0.5× TBE buffer; for GTBE and TAE buffers, sizes separated will be slightly larger and gels
will run ∼20% and 30% faster, respectively.
bVariables: T, temperature in °C; V, field strength, volts/cm; A, % agarose (multiply by 0.8 for pulsed-field grade agarose);
t, pulse time (reverse time for field-inversion gels) in sec; R, forward-to-reverse time ratio; θ, reorientation angle.
cField inversion does not resolve fragments outside this range; alternating-angle gels will resolve fragments outside this
range, but not as well.
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up. A good compromise is GTBE buffer, which
is 0.5× TBE with 100 mM glycine added.
GTBE increases the mobility of the DNA without significantly increasing the current.
Ethidium bromide. Ethidium slows the reorientation of molecules, possibly by making
DNA stiffer. Addition of ethidium bromide to
the gel can help resolve molecules <100 kb, but
is not recommended for larger molecules.
Agarose. Low agarose concentration shifts
the sizes of molecules resolved toward the
larger range, and also speeds the migration of
all molecules. Special pulsed-field grade
agarose is available from several companies.
These agaroses make gels with large pore sizes,
so that larger molecules can be resolved, and
with high gel strength, so that low-percentage
gels can be used. A gel poured with 1% pulsedfield grade agarose will give similar results to
a gel poured with 0.8% regular agarose, but will
be much stronger.
Pulse times. For alternating-angle electrophoresis, best resolution is obtained by using
pulse times just long enough to resolve the
largest molecules of interest (or better yet, using
a time ramp with a maximum that is just long
enough).
Pulse times are much more critical for fieldinversion electrophoresis, because a given reverse pulse time resolves a narrower range of
sizes. Use a range of reverse times broad
enough to separate all molecules of interest.
The ratio between forward and reverse times is
generally in the range of 2.5:1 to 3.5:1. Lower
ratios give better separation but make gels run
more slowly. The most commonly used ratio is
3:1.
Table 2.5B.2 lists equations predicting the
sizes resolved and the speed of gel runs for
field-inversion and alternating-angle gels. For
example, for a field inversion gel run at 12° at
8 V/cm in 0.8% agarose with reverse pulses of
10 sec and forward pulses of 30 sec:
Size = 0.13 × (12 + 40) × 81.1 ×
(3 − 0.8)0.6 × 100.875
=0.13 × 52 × 9.85 × 1.6 × 7.5 = 800 kb
Minimum size = 0.75 × 800 = 600 kb
Velocity =
=
0.0016 × (12 + 25) × 81.6 × (3 − 1)
0.8 × (3 + 1)
0.0016 × 37 × 27.9 × 2
= 1.0 cm ⁄ hr
0.8 × 4
Thus, the gel will resolve fragments from
600 to 800 kb, and a 10-kb fragment will move
at 1 cm/hr.
Troubleshooting
Size range resolved too high or too low.
Refer to Table 2.5B.2 and adjust whatever parameter is convenient (typically voltage or
pulse times) to change the sizes resolved.
Bands broadening or disappearing at top of
gel. Decrease trapping by decreasing temperature or voltage.
Bands smeared. Run samples into gel for
longer time, at lower voltage, or both.
Excessive smile. More heat is being produced than can be dissipated effectively. Either
reduce the heating by decreasing voltage, gel
thickness, or buffer depth, or increase buffer
recirculation.
Anticipated Results
Molecules of interest should be well resolved in straight lanes (see Fig. 2.5B.2).
Time Considerations
The time required for running a gel can be
estimated from Table 2.5B.2. The support protocol on sample preparation requires two overnight incubations, and restriction enzyme digestion (if desired) takes most of another day.
Literature Cited
Birren, B.W., Lai, E., Clark, S.M., Hood, L. and
Simon, M.I. 1988. Optimized conditions for
pulsed field electrophoretic separations of DNA.
Nucl. Acids Res. 16:7563-7581.
Carle, G.F., Frank, M., and Olson, M.V. 1986. Electrophoretic separations of large DNA molecules
by periodic inversion of the electric field. Science
232:65-68.
Chu, G. 1989. Pulsed field electrophoresis in contour-clamped homogeneous electric fields for
the resolution of DNA by size or topology. Electrophoresis 10:290-295.
Chu, G., Vollrath, D., and Davis, R.W. 1986. Separation of large DNA molecules by contour
clamped homogeneous electric fields. Science
234:1582-1585.
Schwartz, D.C. and Cantor, C.R. 1984. Separation
of yeast chromosome-sized DNAs by pulsedfield gradient electrophoresis. Cell 37:67-75.
Key Reference
Schwartz and Cantor, 1984. See above.
Describes the original use of pulsed fields to separate large molecules.
Pulsed-Field Gel
Electrophoresis
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Contributed by Michael Finney
MJ Research
Watertown, Massachusetts
Preparation and
Analysis of DNA
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