- Email: [email protected]
An asymmetric field inversion gel electrophoresis method for the separation of large DNA molecules
178,172-176
ANALYTICALBIOCHEMISTRY
(1989)
An Asymmetric Field Inversion Gel Electrophoresis Method for the Separation of Large DNA Molecules’ Nicholas
Denko,
Amato
Giaccia,
Bruce Peters,
and Thomas
D. Stamato’
The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pennsylvania 19104
Received
September
6,1966
An electrophoretic method for separating large DNA molecules which uses periodically inverted electric fields of different magnitude in the two directions is described. Net DNA migration is either in the high field direction or in the low field direction, depending on the relative duration of the pulses. With this approach, molecules of up to 1.6 million base pairs can be separated in parallel lanes after a single run under fixed timing conditions. An inexpensive switching unit is the only device needed in addition to the conventional gel box. 0 1989 Academic Press, Inc.
When pulsed field electrophoresis was introduced, it greatly increased the usable range of length-dependent separations of DNA in agarose gels (1). By periodically pulsing electrical fields that are oriented at roughly right angles, separations of over 2000 kilobase pairs (kb)3 are possible (2,3). A major disadvantage of this approach is that the DNA migrates along a distorted course, making size comparisons difficult. Since the original study, however, new gel boxes that have improved the geometry of DNA tracks have been introduced (4,5). Carle et ~2. (6) adapted pulsing of the electrical fields to a one-dimensional scheme in which the polarity of a constant field is periodically reversed, a method termed field inversion gel electrophoresis (FIGE). Net migration is achieved by using a longer pulse in the forward direction. Here the problems of distortion are eliminated because the samples run in straight lanes. However, under a given set of conditions, there is not a unique relationship between migration and size of DNA; i.e., very i This work was supported in part by USPHS Grants ES-02274, ES02470, CA-45277, and CA-10815 from the National Institutes of Environmental Health Sciences and the National Cancer Institute. ’ To whom correspondence should be addressed. 3 Abbreviations used: AFIGE, asymmetric field inversion gel electrophoresis; kb, kilobase pairs; FIGE, field inversion gel electrophoresis; Mb, million base pairs.
large DNAs run at velocities similar to those of much smaller fragments, and intermediate sizes have close to zero mobility. To obtain length-dependent separation over the entire range of sizes, it is necessary to continuously change the pulse times during the run. Recently, LaLande et al. (7) have applied the “biased reptation” model of electrophoresis to FIGE to explain the observed migrations. This theory assumes reptilelike end-to-end migration of DNA through the gel, but is limited to casesof low voltage gradients (7,14) and pulse sequences with necessarily unequal times and fields (7) and predicts well only for relatively small molecules (~500 kb) (7). In a modified FIGE system, he separates yeast chromosomes by using low fields (+2 and - 1 V/ cm) and a constant timing ratio (+2.5 and -1.0) that is controlled by a computer timer and driven with multiple power supplies. With the relatively similar pulse durations and fields, it was necessary to change the running conditions three times during a 72-h run to correctly resolve all the yeast chromosomes. After observing the problems experienced by the FIGE system, we decided to explore different field magnitudes as well as pulse durations to allow for more flexibility in ascertaining the best pulse cycle. In this report, we describe a simple, fixed, one-dimensional pulse sequence that can be adapted to a wide range of size separations. This asymmetric field inversion gel electrophoresis (AFIGE) approach relies on radically different field strengths and pulse times such that a constant set of conditions can be used over the entire course of a run. Using the conditions described in this paper, molecules of up to 1.6 million base pairs (Mb) in size can be separated on a conventional gel box, using a single power supply and a simple switching unit. MATERIALS
AND
METHODS
Apparatus. A diagram of the device is shown in Fig. 1. The apparatus consists of a commercially available horizontal gel electrophoresis system (BRL Model Hl), power supply (40W Heathkit Model SP-2717), and re-
172 All
Copyright 0 1989 rights of reproduction
0003-2697/89 by Academic in any form
$3.00
Press, Inc. reserved.
ELECTROPHORETIC
SEPARATION
Switching timer
FIG. 1. paratus.
Schematic
diagram
of the asymmetric
field inversion
gel ap-
circulation pump (Gorman Rupp Model 11968). The heart of the switching circuit (Fig. 2) is an Eagle timer (Model DAlOO) which contains a double-pole, doublethrow relay whose position is controlled by two user-adjustable timers. These timers independently set the duration of the forward and reverse pulses by timing the up and down positions, while the relay reverses the field. The magnitude of the applied fields is controlled by the variable resistors Rl and R2 (Ohmite, 50K and 5K ohms) with less than 5% drift over the course of a run. To allow for adequate cooling, 2-mm glass rods are placed beneath the agarose gel with their long axis parallel to the direction of migration and the buffer height adjusted so that 2.5 mm of liquid is above the gel (Fig. 1). The buffer is recirculated through an ice bath at a rate of 120 ml/min with the direction of flow opposite to the direction of DNA migration. This cooling system maintains a gel temperature of 9-14°C. The electrophoresis buffer is 0.5X TBE (45 mM Tris base, 45 mM boric acid, 1.5 mM EDTA, pH 8.2). The gels are l.O-1.5% agarose as indicated in the figure legends and made in 0.5X TBE containing 0.5 fig/ml ethidium bromide and cast in a 22 X 24-cm gel box to a thickness of 0.5 cm. DNA preparation. Saccharomyces cerevisiae strain Kt458, kindly provided by K. Tachell, was grown to stationary phase at 30°C in 25 ml of YPD medium: 1 g yeast extract, 2 g dextrose, and 2 g bactopeptone in 100 ml of distilled water. High-molecular-weight DNA was prepared by the method of Schwartz and Cantor (2). Yeast cells were washed twice at 0°C with 10 ml of 0.05 M EDTA/lO mM Tris, pH 7.8, and resuspended at a density of 4 X 10’ cells/ml in the same buffer at 42°C containing 1% agarose (SeaKern GTG). Seventy milligrams per milliliter lyticase (900 units/ml, Sigma) was added to the cell suspension, mixed, pipetted into 3-mm-diameter tubes, and allowed to cool to room temperature. Inserts
OF
LARGE
DNA
173
MOLECULES
were incubated for 24 h in 50 ml of SB buffer (0.5 M EDTA/O.Ol M Tris, pH 7.5) at 37°C and for 48 h in 50 ml DSP buffer (0.5 M EDTA, 0.01 M Tris, pH 7.5, 1% sarkosyl, 1 mg/ml proteinase K) at 50°C. Inserts were stored in SB buffer and dialyzed against 0.5X TBE buffer before use. Bacteriophage G was grown using standard methods (8). Phage G was generously provided by W. L. Fangman. Intact phage at a DNA concentration of 6 mg/ml in TE buffer (10 mM Tris, pH 7.8,l mM EDTA) at 42°C was mixed with an equal volume of the same buffer containing 2% agarose, and inserts were prepared as described above. The inserts were incubated for 16 h at 50°C in a 10X vol of DSP buffer and dialyzed against a 100X vol of 0.5X TBE buffer for immediate use or against SB buffer for long-term storage. Bacteriophage X DNA and DNA cut with Hind111 restriction enzyme were obtained from International Biotechnologies, Inc. X ladders were prepared by incubating 3 pg of DNA for 16 h at 12°C in a 20-~1 reaction containing 1 unit of T4 DNA ligase (New England Biolabs) 1 mM ATP, 1 mM DTT, 10 mM MgC&, 50 mM Tris, pH 7.5. Southern blots. Standard protocols (8) were used to transfer DNA from gels to nylon membranes except that the DNA was depurinated before the alkali denaturation step by incubating the gels in 0.25 N HCl for 30 min at room temperature. Hybridization probes were prepared by nick-translation of a pBR322 plasmid JC-1203-2 using [32P]dCTP to >lO’ dpm/pg. This probe was kindly provided by J. Cannon and contains sequences from yeast genes adel, trpl, and RASl which are on chromosomes I, IV, and XV, respectively (9). Field inversion gel electrophoresis. FIGE was performed on a Green Mountain Lab Supply Model PPI100 apparatus using a switching-interval ramp in which the forward-migration time varied linearly from 9 s at the start of the run to 60 s at 20 h with a constant 3:l ratio between the forward and the reverse times (6). RESULTS
Net migration of DNA in the asymmetric FIGE system is either in the high field direction or in the low field
FIG.
2.
Schematic
diagram
of the switching
unit.
174
DENKO
ET
AL.
pulsed field technique, the longer pulses resolve larger DNAs, up to 840 kb at 60-s pulses. The most notable difference arises when the pulse duration is above 60 s because there is actually a decrease in the size of the DNA resolved. Figure 4B describes the physical movement, and therefore the “usable” portion of the gel, under the same conditions described above. The value reported is the ratio of the mobilities of X DNA (48.5 kb) and the largest DNA resolved on that run. At 45 s, the mobility of h is five times that of yeast chromosome XI (690 kb), so that after a 20-cm run, these two markers would be separated by 16 cm. Separation of smaller fragments can also be increased by increasing the -2 V/cm pulse. For example, in a pulse sequence of 1.5 s at 10 V/cm and 10 s at -2 V/ cm, the 150-kb fragments will move only a few millimeters, so that the whole gel can be used for separations of 2-150 kb. This excellent physical separation enables delineation of two bands that are almost the same size (e.g., restriction fragments). Migration
FIG. 3. Separation of DNA by migration in the high field direction. DNA (2 pg) Yeast in an agarose insert, 1 pg X ligation ladder, and 1 pg HindIII-cut X were subjected to a pulse cycle of 10 V/cm for 30 s and -2 V/cm for 30 s in a 1.0% gel for a total run of 12 h.
in the Low Field Direction
After reaching the 840-kb peak of resolution using high field migration, we attempted to increase the upper size limit by changing net migration to the low field direction. We reasoned that under a low driving field the
A
Maxlmum
E 1
Migration
0 600 F
’ I
I
2 600 Y F
direction, depending on the relative duration of the pulses. Pulses of approximately equal duration determine net migration in the high field direction, but much longer low field pulses can yield migration in the other direction. We first examined high field migration because of the intuitive ease of achieving net migration.
Rasolutlonr
II
’
’ I
-
w 1 E 400
I
I II’ -
.-E’ J
-
= *O”
I r’
I
I
in the High Field Direction
Migration in the high field direction was analyzed for electrical fields of +lO and -2 V/cm with equal duration pulses. This protocol is effective and preferred for molecules of up to approximately 850 kb. Because the field driving the DNA is relatively great (10 V/cm), gel runs can be as short as lo-12 h. Figure 3 shows an example of this type of run with 30-s pulse times. Note that there is separation from the 2.0-kb fragment of HindIII-cut X DNA (13 cm) to the fourth yeast band (chromosome IX ca. 450 kb, 5 cm) (9), above which all the yeast bands comigrate. Figure 4A illustrates the relationship between the pulse duration and the maximum size molecule resolved by this high field migration. The error bars at each point on this graph represent the range of sizes from the largest resolved yeast chromosome to the next largest unresolved chromosome. As reported with nearly every
i;: .Z
5.0 -B P .? 5: 4.0-
.$S Eo, 61 0 p ‘2 5 a$ a
Usable
Fraction
of Gel
3,0-
2.0 l.O-
0’1”““““““““““““~” 0 20 40
60 Pulse
duration
60
100
120
140
‘460 P
(seconds)
FIG. 4. (A) Maximum size of DNA resolved by different pulse times during high field migration. Fields were 10 and -2 V/cm with equal forward and reverse pulse times. (B) Ratio of mobility of X DNA to the largest DNA resolved, for the samples described above. This represents the usable portion of the gel. The largest DNA resolved corresponds to the largest yeast chromosome resolved.
ELECTROPHORETIC
yeast 2x
SEPARATION
DNA lx
III
OF
LARGE
DNA
175
MOLECULES
and the FIGE systems are equally useful at separating yeast DNA molecules. However, by using unequal fields, mobility minimum that is seen in FIGE runs under fixed timing conditions is not observed, and there is no need to ramp the times. The yeast chromosomes (Fig. 6, lane 2) were transferred to Zeta Probe (Bio-Rad) and hybridized to probes that are specific for chromosomes I, XV, and IV which are 260,1200, and 1600 kb (9,10), respectively. Phage G has a genome of 670-750 kb (1,3), and this migrates along band 6 (chromosome XI, 690 kb) (9). Figure 7 characterizes the relationship between pulse duration and range of separation for the low field migrations using the aforementioned fields and times. As the times are increased, resolution of larger molecules improves, and that of the smaller molecules decreases. The 2.2-Mb yeast chromosome XII (10) is the largest fragment resolved using these fields. Larger DNA from Schiztosaccharomyces pombea (3-9 Mb) (11) did not migrate into the gel under these conditions, but did so with lower voltages and even longer pulses (data not shown). DISCUSSION
FIG. 6.
Separation of DNA by migration in the low field direction. Yeast DNA in agarose inserts at 1 rg/lane (1X) and 2 pg/lane (2X) was subjected to a pulse cycle of 5 V/cm for 125 s and -10 V/cm for 15 s in a 1.5% gel for a total run of 33 h.
time required to turn around and move in the opposite direction would be more strongly dependent on molecular weight than under a high driving field. Thus, a ratcheting effect is produced in which molecules of different sizes migrate a similar distance in the backward direction (high field strength), but move forward a distance that is more strongly dependent on the molecular weight of the molecule (low field strength). Migration in the low field direction was examined for electrical fields of +5 and -10 V/cm, with the low field pulses being 8.3 times as long as the high field pulses. These conditions usually required longer pulses and are better suited to the resolution of the larger molecules (0.2-2 Mb). Figure 5 shows an example of a gel run 15 s at -10 V/cm and 125 s at +5 V/cm in which all 12 of the strain-characteristic yeast bands are resolved. Note that the lanes are all geometrically equivalent and intragel comparisons are very easy. Figure 6 compares the AFIGE system to the FIGE system of Carle et al. (6) on a two-dimensional gel to check for band inversions. Yeast molecules were first separated in order of size by the FIGE method as described in (6). The lane was then cut out, turned at right angles, and run in an AFIGE gel. Lanes of yeast and phage DNA were also loaded exclusively for the AFIGE run of 12.5 s at -10 V/cm and 125 s at 5 V/cm. After the two runs, the yeast bands align along a diagonal, indicating that the two methods yield approximately equal mobilities for the chromosomes in this size range. Thus, the AFIGE
The AFIGE approach offers a number of advantages over existing pulsed field separations of large DNA mol-
Y %Jz mm EZ I-
FIGE Yeast DNA
7-a654-
FIG. 6. Comparison of the AFIGE and FIGE systems for the separation of yeast chromosomes. Two micrograms of yeast DNA in an agarose insert was separated in the first dimension by FIGE using a switching-interval ramp of 9 to 60 s in the forward direction, and 3 to 20 s in the reverse direction with a constant 3:l ratio (6). This lane was then cut out, rotated 90”, and separated in the second dimension by the AFIGE method (lane 3). Two micrograms of yeast DNA (lane 2) and 0.2 fig of phage G DNA (lane 1) were also run in the AFIGE dimension. Yeast DNA in lane 2 was transferred to Zeta Probe (BioRad) and hybridized to 32P-labeled sequences from chromosomes I, IV, and XV. The blot was then exposed to X-ray film for 24 h (left panel).
176
DENKO
F E 1.5
-
s 'G < i! 1.0 B L E m 0.5 Good 71 1
“pa.dlO”
compmslon b.“dS
0‘ I
0
50
Forward
100 150 pulse time (seconds)
200
250
FIG. 7.
Range of DNA resolved at varying pulse times during low field migration. Fields were 5 and -10 V/cm with a ratio of times 8.3 in the low field direction to 1.0 in the high field direction.
ecules: DNA migrates in straight tracks, facilitating size comparisons; only a simple switching device and lowwattage power supply are needed, the large horizontal gel (22 X 24 cm) allows a large number of samples to be well separated, and under certain protocols, a gel can be run in lo-12 h. Pulsed field systems introduced since that described by Schwartz and Cantor (2) have primarily had orthogonal electrode arrays, with pulses being oriented at obtuse angles (3-5). Most of the recent developments have been in the geometry of the gel chambers and electrodes in order to produce straighter lanes (4,5,9). However, one constraint of these designs is that the pulses must be equal in both directions so that the DNA will migrate evenly along the diagonal (15). Thus, the potential for increased resolution using different field strengths and pulse times in the two directions cannot be utilized by these systems. The FIGE electrophoresis introduced by Carle et al. (6) employed a 180” pulse angle, so it became necessary to significantly bias one set of pulses to achieve net migration through the gel. The original FIGE system used longer pulses (3:l ratio) of the same magnitude (10 V/ cm) to achieve forward migration. Under a fixed set of conditions, however, there was a minimum of migration, and the resulting pattern was not monotonically increasing in size. There appeared to be a size DNA that would resonate at a certain frequency of pulses and not move. By steadily increasing the pulses’ duration, however, it is possible to start all the fragments moving, producing the desired relationship across the gel. Recently, the rate of reorientation of DNA in a gel has been shown to depend on both field strength and molecu-
ET
AL.
lar mass (12,13). Thus, by using fields and times that are extremely different in the two directions, the resonance observed under a constant set of conditions would not be expected. We have used pulses that are radically different in terms of both duration and field strength so that the result is a monotonic increase of size across the gel without inversion of bands. The use of two sets of conditions in the opposite directions suggeststhe potential of AFIGE in resolving a larger range of fragments under a constant set of conditions than the orthagonal systems which have symmetrical cycles. Each direction can be independently tuned to a different range of resolution. ACKNOWLEDGMENTS We thank Drs. John Cannon for furnishing the pBR322 JC 1203-2 probe, Walton L. Fangman for phage G, and K. Tachell for S. cereuisiae strain Kt458. Note added in proof. AFIGE pulse sequences can be produced by any pulsed field electrophoresis switching unit that has independent timers for the two halves of the pulse cycle, and North-South, EastWest outputs, for example IBI Minipulse. The only modification necessary is the addition of an external resistor on the low field side of the circuit. For example, a high field migration on gel oriented as in Fig. 1. North (+) attached to left side of gel box, South (-) attached to right. East (-) with added variable 50K Ohm resistor attached to left, West (+) attached to right. Set times as indicated, and adjust resistor to achieve the desired field strengths.
REFERENCES 1. Fangman,
W. L. (1978)
Nucleic
Acids
Res. 6,653-665.
2. Schwartz, D. C., and Cantor, C. R. (1984) Cell 37,67-75. 3. Carle, G. F., and Olson, M. V. (1985) Nucleic Acids Res. 5664. 4. Gardiner, K., Laas, W., and Patterson, D. (1986) Somatic Gerzet. 12,185-195.
12,5647Cell Mol.
5. Chu, G., Vollrath, D., and Davis, R. W. (1986) Science 234,15821585. 6. Carle, G. F., Frank, M., and Olson, M. V. (1986) Science 232,6568. 7. LaLande, M., Noolandi, J., Turmel, C., Rousseau, J., and Slater, G. (1987) Proc. N&l. Acad. Sci. USA 84,8011-8015. 8. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982). Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 9. Anand, R. (1986) Trends. Genet. 11,278-283. 10. Mortimer, R. K., and Schild, D. (1985) Microbial. Rev. 49, 181212. 11. Vollrath, D., and Davis, R. W. (1987) Nucleic Acids Res. 15,78657876. 12. Holzwarth, G., McKee, C. B., Steiger, S., and Crater, G. (1987) Nucleic Acids Res. 16,10,031-10,043. 13. Hurley, I. (1986) Biopolymers 25,539-554. 14. Slater, G., and Noolandi, J. (1986) Biopolymers 26,431-454. 15. Southern, E. M., Anand, R., Brown, W. R. A., and Fletcher, (1987) Nucleic Acids Res. 15,5925-5943.
D. S.
ANALYTICALBIOCHEMISTRY
(1989)
An Asymmetric Field Inversion Gel Electrophoresis Method for the Separation of Large DNA Molecules’ Nicholas
Denko,
Amato
Giaccia,
Bruce Peters,
and Thomas
D. Stamato’
The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pennsylvania 19104
Received
September
6,1966
An electrophoretic method for separating large DNA molecules which uses periodically inverted electric fields of different magnitude in the two directions is described. Net DNA migration is either in the high field direction or in the low field direction, depending on the relative duration of the pulses. With this approach, molecules of up to 1.6 million base pairs can be separated in parallel lanes after a single run under fixed timing conditions. An inexpensive switching unit is the only device needed in addition to the conventional gel box. 0 1989 Academic Press, Inc.
When pulsed field electrophoresis was introduced, it greatly increased the usable range of length-dependent separations of DNA in agarose gels (1). By periodically pulsing electrical fields that are oriented at roughly right angles, separations of over 2000 kilobase pairs (kb)3 are possible (2,3). A major disadvantage of this approach is that the DNA migrates along a distorted course, making size comparisons difficult. Since the original study, however, new gel boxes that have improved the geometry of DNA tracks have been introduced (4,5). Carle et ~2. (6) adapted pulsing of the electrical fields to a one-dimensional scheme in which the polarity of a constant field is periodically reversed, a method termed field inversion gel electrophoresis (FIGE). Net migration is achieved by using a longer pulse in the forward direction. Here the problems of distortion are eliminated because the samples run in straight lanes. However, under a given set of conditions, there is not a unique relationship between migration and size of DNA; i.e., very i This work was supported in part by USPHS Grants ES-02274, ES02470, CA-45277, and CA-10815 from the National Institutes of Environmental Health Sciences and the National Cancer Institute. ’ To whom correspondence should be addressed. 3 Abbreviations used: AFIGE, asymmetric field inversion gel electrophoresis; kb, kilobase pairs; FIGE, field inversion gel electrophoresis; Mb, million base pairs.
large DNAs run at velocities similar to those of much smaller fragments, and intermediate sizes have close to zero mobility. To obtain length-dependent separation over the entire range of sizes, it is necessary to continuously change the pulse times during the run. Recently, LaLande et al. (7) have applied the “biased reptation” model of electrophoresis to FIGE to explain the observed migrations. This theory assumes reptilelike end-to-end migration of DNA through the gel, but is limited to casesof low voltage gradients (7,14) and pulse sequences with necessarily unequal times and fields (7) and predicts well only for relatively small molecules (~500 kb) (7). In a modified FIGE system, he separates yeast chromosomes by using low fields (+2 and - 1 V/ cm) and a constant timing ratio (+2.5 and -1.0) that is controlled by a computer timer and driven with multiple power supplies. With the relatively similar pulse durations and fields, it was necessary to change the running conditions three times during a 72-h run to correctly resolve all the yeast chromosomes. After observing the problems experienced by the FIGE system, we decided to explore different field magnitudes as well as pulse durations to allow for more flexibility in ascertaining the best pulse cycle. In this report, we describe a simple, fixed, one-dimensional pulse sequence that can be adapted to a wide range of size separations. This asymmetric field inversion gel electrophoresis (AFIGE) approach relies on radically different field strengths and pulse times such that a constant set of conditions can be used over the entire course of a run. Using the conditions described in this paper, molecules of up to 1.6 million base pairs (Mb) in size can be separated on a conventional gel box, using a single power supply and a simple switching unit. MATERIALS
AND
METHODS
Apparatus. A diagram of the device is shown in Fig. 1. The apparatus consists of a commercially available horizontal gel electrophoresis system (BRL Model Hl), power supply (40W Heathkit Model SP-2717), and re-
172 All
Copyright 0 1989 rights of reproduction
0003-2697/89 by Academic in any form
$3.00
Press, Inc. reserved.
ELECTROPHORETIC
SEPARATION
Switching timer
FIG. 1. paratus.
Schematic
diagram
of the asymmetric
field inversion
gel ap-
circulation pump (Gorman Rupp Model 11968). The heart of the switching circuit (Fig. 2) is an Eagle timer (Model DAlOO) which contains a double-pole, doublethrow relay whose position is controlled by two user-adjustable timers. These timers independently set the duration of the forward and reverse pulses by timing the up and down positions, while the relay reverses the field. The magnitude of the applied fields is controlled by the variable resistors Rl and R2 (Ohmite, 50K and 5K ohms) with less than 5% drift over the course of a run. To allow for adequate cooling, 2-mm glass rods are placed beneath the agarose gel with their long axis parallel to the direction of migration and the buffer height adjusted so that 2.5 mm of liquid is above the gel (Fig. 1). The buffer is recirculated through an ice bath at a rate of 120 ml/min with the direction of flow opposite to the direction of DNA migration. This cooling system maintains a gel temperature of 9-14°C. The electrophoresis buffer is 0.5X TBE (45 mM Tris base, 45 mM boric acid, 1.5 mM EDTA, pH 8.2). The gels are l.O-1.5% agarose as indicated in the figure legends and made in 0.5X TBE containing 0.5 fig/ml ethidium bromide and cast in a 22 X 24-cm gel box to a thickness of 0.5 cm. DNA preparation. Saccharomyces cerevisiae strain Kt458, kindly provided by K. Tachell, was grown to stationary phase at 30°C in 25 ml of YPD medium: 1 g yeast extract, 2 g dextrose, and 2 g bactopeptone in 100 ml of distilled water. High-molecular-weight DNA was prepared by the method of Schwartz and Cantor (2). Yeast cells were washed twice at 0°C with 10 ml of 0.05 M EDTA/lO mM Tris, pH 7.8, and resuspended at a density of 4 X 10’ cells/ml in the same buffer at 42°C containing 1% agarose (SeaKern GTG). Seventy milligrams per milliliter lyticase (900 units/ml, Sigma) was added to the cell suspension, mixed, pipetted into 3-mm-diameter tubes, and allowed to cool to room temperature. Inserts
OF
LARGE
DNA
173
MOLECULES
were incubated for 24 h in 50 ml of SB buffer (0.5 M EDTA/O.Ol M Tris, pH 7.5) at 37°C and for 48 h in 50 ml DSP buffer (0.5 M EDTA, 0.01 M Tris, pH 7.5, 1% sarkosyl, 1 mg/ml proteinase K) at 50°C. Inserts were stored in SB buffer and dialyzed against 0.5X TBE buffer before use. Bacteriophage G was grown using standard methods (8). Phage G was generously provided by W. L. Fangman. Intact phage at a DNA concentration of 6 mg/ml in TE buffer (10 mM Tris, pH 7.8,l mM EDTA) at 42°C was mixed with an equal volume of the same buffer containing 2% agarose, and inserts were prepared as described above. The inserts were incubated for 16 h at 50°C in a 10X vol of DSP buffer and dialyzed against a 100X vol of 0.5X TBE buffer for immediate use or against SB buffer for long-term storage. Bacteriophage X DNA and DNA cut with Hind111 restriction enzyme were obtained from International Biotechnologies, Inc. X ladders were prepared by incubating 3 pg of DNA for 16 h at 12°C in a 20-~1 reaction containing 1 unit of T4 DNA ligase (New England Biolabs) 1 mM ATP, 1 mM DTT, 10 mM MgC&, 50 mM Tris, pH 7.5. Southern blots. Standard protocols (8) were used to transfer DNA from gels to nylon membranes except that the DNA was depurinated before the alkali denaturation step by incubating the gels in 0.25 N HCl for 30 min at room temperature. Hybridization probes were prepared by nick-translation of a pBR322 plasmid JC-1203-2 using [32P]dCTP to >lO’ dpm/pg. This probe was kindly provided by J. Cannon and contains sequences from yeast genes adel, trpl, and RASl which are on chromosomes I, IV, and XV, respectively (9). Field inversion gel electrophoresis. FIGE was performed on a Green Mountain Lab Supply Model PPI100 apparatus using a switching-interval ramp in which the forward-migration time varied linearly from 9 s at the start of the run to 60 s at 20 h with a constant 3:l ratio between the forward and the reverse times (6). RESULTS
Net migration of DNA in the asymmetric FIGE system is either in the high field direction or in the low field
FIG.
2.
Schematic
diagram
of the switching
unit.
174
DENKO
ET
AL.
pulsed field technique, the longer pulses resolve larger DNAs, up to 840 kb at 60-s pulses. The most notable difference arises when the pulse duration is above 60 s because there is actually a decrease in the size of the DNA resolved. Figure 4B describes the physical movement, and therefore the “usable” portion of the gel, under the same conditions described above. The value reported is the ratio of the mobilities of X DNA (48.5 kb) and the largest DNA resolved on that run. At 45 s, the mobility of h is five times that of yeast chromosome XI (690 kb), so that after a 20-cm run, these two markers would be separated by 16 cm. Separation of smaller fragments can also be increased by increasing the -2 V/cm pulse. For example, in a pulse sequence of 1.5 s at 10 V/cm and 10 s at -2 V/ cm, the 150-kb fragments will move only a few millimeters, so that the whole gel can be used for separations of 2-150 kb. This excellent physical separation enables delineation of two bands that are almost the same size (e.g., restriction fragments). Migration
FIG. 3. Separation of DNA by migration in the high field direction. DNA (2 pg) Yeast in an agarose insert, 1 pg X ligation ladder, and 1 pg HindIII-cut X were subjected to a pulse cycle of 10 V/cm for 30 s and -2 V/cm for 30 s in a 1.0% gel for a total run of 12 h.
in the Low Field Direction
After reaching the 840-kb peak of resolution using high field migration, we attempted to increase the upper size limit by changing net migration to the low field direction. We reasoned that under a low driving field the
A
Maxlmum
E 1
Migration
0 600 F
’ I
I
2 600 Y F
direction, depending on the relative duration of the pulses. Pulses of approximately equal duration determine net migration in the high field direction, but much longer low field pulses can yield migration in the other direction. We first examined high field migration because of the intuitive ease of achieving net migration.
Rasolutlonr
II
’
’ I
-
w 1 E 400
I
I II’ -
.-E’ J
-
= *O”
I r’
I
I
in the High Field Direction
Migration in the high field direction was analyzed for electrical fields of +lO and -2 V/cm with equal duration pulses. This protocol is effective and preferred for molecules of up to approximately 850 kb. Because the field driving the DNA is relatively great (10 V/cm), gel runs can be as short as lo-12 h. Figure 3 shows an example of this type of run with 30-s pulse times. Note that there is separation from the 2.0-kb fragment of HindIII-cut X DNA (13 cm) to the fourth yeast band (chromosome IX ca. 450 kb, 5 cm) (9), above which all the yeast bands comigrate. Figure 4A illustrates the relationship between the pulse duration and the maximum size molecule resolved by this high field migration. The error bars at each point on this graph represent the range of sizes from the largest resolved yeast chromosome to the next largest unresolved chromosome. As reported with nearly every
i;: .Z
5.0 -B P .? 5: 4.0-
.$S Eo, 61 0 p ‘2 5 a$ a
Usable
Fraction
of Gel
3,0-
2.0 l.O-
0’1”““““““““““““~” 0 20 40
60 Pulse
duration
60
100
120
140
‘460 P
(seconds)
FIG. 4. (A) Maximum size of DNA resolved by different pulse times during high field migration. Fields were 10 and -2 V/cm with equal forward and reverse pulse times. (B) Ratio of mobility of X DNA to the largest DNA resolved, for the samples described above. This represents the usable portion of the gel. The largest DNA resolved corresponds to the largest yeast chromosome resolved.
ELECTROPHORETIC
yeast 2x
SEPARATION
DNA lx
III
OF
LARGE
DNA
175
MOLECULES
and the FIGE systems are equally useful at separating yeast DNA molecules. However, by using unequal fields, mobility minimum that is seen in FIGE runs under fixed timing conditions is not observed, and there is no need to ramp the times. The yeast chromosomes (Fig. 6, lane 2) were transferred to Zeta Probe (Bio-Rad) and hybridized to probes that are specific for chromosomes I, XV, and IV which are 260,1200, and 1600 kb (9,10), respectively. Phage G has a genome of 670-750 kb (1,3), and this migrates along band 6 (chromosome XI, 690 kb) (9). Figure 7 characterizes the relationship between pulse duration and range of separation for the low field migrations using the aforementioned fields and times. As the times are increased, resolution of larger molecules improves, and that of the smaller molecules decreases. The 2.2-Mb yeast chromosome XII (10) is the largest fragment resolved using these fields. Larger DNA from Schiztosaccharomyces pombea (3-9 Mb) (11) did not migrate into the gel under these conditions, but did so with lower voltages and even longer pulses (data not shown). DISCUSSION
FIG. 6.
Separation of DNA by migration in the low field direction. Yeast DNA in agarose inserts at 1 rg/lane (1X) and 2 pg/lane (2X) was subjected to a pulse cycle of 5 V/cm for 125 s and -10 V/cm for 15 s in a 1.5% gel for a total run of 33 h.
time required to turn around and move in the opposite direction would be more strongly dependent on molecular weight than under a high driving field. Thus, a ratcheting effect is produced in which molecules of different sizes migrate a similar distance in the backward direction (high field strength), but move forward a distance that is more strongly dependent on the molecular weight of the molecule (low field strength). Migration in the low field direction was examined for electrical fields of +5 and -10 V/cm, with the low field pulses being 8.3 times as long as the high field pulses. These conditions usually required longer pulses and are better suited to the resolution of the larger molecules (0.2-2 Mb). Figure 5 shows an example of a gel run 15 s at -10 V/cm and 125 s at +5 V/cm in which all 12 of the strain-characteristic yeast bands are resolved. Note that the lanes are all geometrically equivalent and intragel comparisons are very easy. Figure 6 compares the AFIGE system to the FIGE system of Carle et al. (6) on a two-dimensional gel to check for band inversions. Yeast molecules were first separated in order of size by the FIGE method as described in (6). The lane was then cut out, turned at right angles, and run in an AFIGE gel. Lanes of yeast and phage DNA were also loaded exclusively for the AFIGE run of 12.5 s at -10 V/cm and 125 s at 5 V/cm. After the two runs, the yeast bands align along a diagonal, indicating that the two methods yield approximately equal mobilities for the chromosomes in this size range. Thus, the AFIGE
The AFIGE approach offers a number of advantages over existing pulsed field separations of large DNA mol-
Y %Jz mm EZ I-
FIGE Yeast DNA
7-a654-
FIG. 6. Comparison of the AFIGE and FIGE systems for the separation of yeast chromosomes. Two micrograms of yeast DNA in an agarose insert was separated in the first dimension by FIGE using a switching-interval ramp of 9 to 60 s in the forward direction, and 3 to 20 s in the reverse direction with a constant 3:l ratio (6). This lane was then cut out, rotated 90”, and separated in the second dimension by the AFIGE method (lane 3). Two micrograms of yeast DNA (lane 2) and 0.2 fig of phage G DNA (lane 1) were also run in the AFIGE dimension. Yeast DNA in lane 2 was transferred to Zeta Probe (BioRad) and hybridized to 32P-labeled sequences from chromosomes I, IV, and XV. The blot was then exposed to X-ray film for 24 h (left panel).
176
DENKO
F E 1.5
-
s 'G < i! 1.0 B L E m 0.5 Good 71 1
“pa.dlO”
compmslon b.“dS
0‘ I
0
50
Forward
100 150 pulse time (seconds)
200
250
FIG. 7.
Range of DNA resolved at varying pulse times during low field migration. Fields were 5 and -10 V/cm with a ratio of times 8.3 in the low field direction to 1.0 in the high field direction.
ecules: DNA migrates in straight tracks, facilitating size comparisons; only a simple switching device and lowwattage power supply are needed, the large horizontal gel (22 X 24 cm) allows a large number of samples to be well separated, and under certain protocols, a gel can be run in lo-12 h. Pulsed field systems introduced since that described by Schwartz and Cantor (2) have primarily had orthogonal electrode arrays, with pulses being oriented at obtuse angles (3-5). Most of the recent developments have been in the geometry of the gel chambers and electrodes in order to produce straighter lanes (4,5,9). However, one constraint of these designs is that the pulses must be equal in both directions so that the DNA will migrate evenly along the diagonal (15). Thus, the potential for increased resolution using different field strengths and pulse times in the two directions cannot be utilized by these systems. The FIGE electrophoresis introduced by Carle et al. (6) employed a 180” pulse angle, so it became necessary to significantly bias one set of pulses to achieve net migration through the gel. The original FIGE system used longer pulses (3:l ratio) of the same magnitude (10 V/ cm) to achieve forward migration. Under a fixed set of conditions, however, there was a minimum of migration, and the resulting pattern was not monotonically increasing in size. There appeared to be a size DNA that would resonate at a certain frequency of pulses and not move. By steadily increasing the pulses’ duration, however, it is possible to start all the fragments moving, producing the desired relationship across the gel. Recently, the rate of reorientation of DNA in a gel has been shown to depend on both field strength and molecu-
ET
AL.
lar mass (12,13). Thus, by using fields and times that are extremely different in the two directions, the resonance observed under a constant set of conditions would not be expected. We have used pulses that are radically different in terms of both duration and field strength so that the result is a monotonic increase of size across the gel without inversion of bands. The use of two sets of conditions in the opposite directions suggeststhe potential of AFIGE in resolving a larger range of fragments under a constant set of conditions than the orthagonal systems which have symmetrical cycles. Each direction can be independently tuned to a different range of resolution. ACKNOWLEDGMENTS We thank Drs. John Cannon for furnishing the pBR322 JC 1203-2 probe, Walton L. Fangman for phage G, and K. Tachell for S. cereuisiae strain Kt458. Note added in proof. AFIGE pulse sequences can be produced by any pulsed field electrophoresis switching unit that has independent timers for the two halves of the pulse cycle, and North-South, EastWest outputs, for example IBI Minipulse. The only modification necessary is the addition of an external resistor on the low field side of the circuit. For example, a high field migration on gel oriented as in Fig. 1. North (+) attached to left side of gel box, South (-) attached to right. East (-) with added variable 50K Ohm resistor attached to left, West (+) attached to right. Set times as indicated, and adjust resistor to achieve the desired field strengths.
REFERENCES 1. Fangman,
W. L. (1978)
Nucleic
Acids
Res. 6,653-665.
2. Schwartz, D. C., and Cantor, C. R. (1984) Cell 37,67-75. 3. Carle, G. F., and Olson, M. V. (1985) Nucleic Acids Res. 5664. 4. Gardiner, K., Laas, W., and Patterson, D. (1986) Somatic Gerzet. 12,185-195.
12,5647Cell Mol.
5. Chu, G., Vollrath, D., and Davis, R. W. (1986) Science 234,15821585. 6. Carle, G. F., Frank, M., and Olson, M. V. (1986) Science 232,6568. 7. LaLande, M., Noolandi, J., Turmel, C., Rousseau, J., and Slater, G. (1987) Proc. N&l. Acad. Sci. USA 84,8011-8015. 8. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982). Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 9. Anand, R. (1986) Trends. Genet. 11,278-283. 10. Mortimer, R. K., and Schild, D. (1985) Microbial. Rev. 49, 181212. 11. Vollrath, D., and Davis, R. W. (1987) Nucleic Acids Res. 15,78657876. 12. Holzwarth, G., McKee, C. B., Steiger, S., and Crater, G. (1987) Nucleic Acids Res. 16,10,031-10,043. 13. Hurley, I. (1986) Biopolymers 25,539-554. 14. Slater, G., and Noolandi, J. (1986) Biopolymers 26,431-454. 15. Southern, E. M., Anand, R., Brown, W. R. A., and Fletcher, (1987) Nucleic Acids Res. 15,5925-5943.
D. S.