- Email: [email protected]
[11] Pulsed-field gel electrophoresis
[ 1 1]
PULSED-FIELDGEI. ELECTROPHORESIS
255
usually kept outside the safety box (not shown in Fig. 6) and is connected to the electrolyzer via a port at the base of the box. An example of the unique purification capability of this instrument is given in Fig. 7: A highly purified preparation of recombinant human superoxide dismutase (SOD) was found to consist of at least three isoforms, with the following isoelectric points: 4.80, 4.92, and 5.07. When monitoring the purification progress, it is seen that a pure pI 5.07 form collects in chamber 5, with the lower pl species focusing in the chambers delimited by the proper set of two isoelectric membranes.
Acknowledgments Supported by grants from Radius in Biotechnology (ESA, Paris) to P.G.R.
[1 1] By J A N E T
Pulsed-Field
Gel Electrophoresis
C. WRESTLER, B A R B A R A
D.
LIPES, B R U C E W . B I R R E N ,
a n d E R I C LAI
Introduction Pulsed-field gel electrophoresis (PFGE) of agarose gels enables the reproducible separation of large DNA fragments. ~ In concept, PFGE is an extension of conventional electrophoresis, in which two alternating (or pulsed) electric fields are used instead of the traditional single slatic field. Separation occurs when these fields are oriented at an obtuse angle to one another. In a pulsed-field gel, the end of each molecule migrates in a new direction with each change of the electric fields. The DNA molecules thus migrate through the agarose matrix in a zigzag motion. The tardiness of the larger molecules in turning corners (e.g., in PFGE) or in running forward and backward [e.g., in field-inversin gel electrophoresis (FIGE), see below] separates them from the smaller size fragments. The effectiveness of PFGE, however, is not limited to the separation of very large DNA molecules. PFGE can improve the resolution of DNA molecules of only a few hundred bases and permits separation up to 12,000 kilobase pairs (kb). 2 Figure 1 shows the effectiveness of PFGE in separating yeast chromosomes from 200 to 2000 kb. J D. C. Schwartz and C. R. Cantor, Cell 37, 67 (1984). = M. Bellis, M. Pages, and G. Roizes, Nuclei~ AcMs Res. 15, 6749 (1987).
MKrlIODS IN ENZYMOLOGY.VOL. 270
Copyrighl ru 1996by Academic P~css.Inc. All rightsol reproduction m any lorm icscrvcd.
256
ELECTROPHORESIS 15 sec
3 0 sec
4 5 sec
[ 111 1 min
1.25 rain
960 914 836
- 273 -214
214
48.5
1.5 rain
1.75 min
2 min
2.5 min
5min
2200 1125 1600
214 48.5
214
FIG. 1. Separation of 50- to l{}00-kb DNAs with different switch intervals, a ladders and chromosomes of the yeast Saccharomyces cerevisiae (strain YNN295) were separated in gels, using identical conditions except for the different switch intervals. All gels were 1% SeaKem LE agarose in 0.5× TBE run at 14° in 6-V/cm fields with a reorientation angle of 120°. With each increase in switch interval the new size limit for resolution (based on the separation of the markers) is indicated.
A n u m b e r o f m o d e l s a n d t h e o r i e s h a v e b e e n p r o p o s e d to e x p l a i n s o m e o f t h e m o r e c o m p l e x b e h a v i o r o f D N A m o l e c u l e s in P F G E . 3 H o w e v e r , b i o l o g i s t s r a r e l y n e e d to c o n s u l t t h e s e p h y s i c a l m o d e l s o r equations for practical PFGE applications. This chapter outlines optimum PFG electrophoretic conditions for the separation of DNA fragments f r o m 1 to 6000 kb.
3 E. Lai and B. W. Birren (eds.), "Current Communications in Cell and Molecular Biology," Vol. 1: Electrophoresis of Large DNA Molecules: Theory and Applications. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1990.
[1 11
PULSED-FIELDGEL ELECTROPHORESIS
257
Unique F e a t u r e s PFGE of high molecular weight DNA involves additional factors that are not encountered in conventional agarose gels. We outline some of these factors and the modifications required for running PFGE.
Sample Preparation, Gel Loading, and DNA Recovery High molecular weight DNA samples for PFGE must be carefully prepared to minimize degradation resulting from mechanical breakage or nuclease activity. This is achieved by using a method developed by Schwartz and Cantor of embedding intact cells in an agarose matrix prior to lysis. ~ This provides support for the DNA while allowing digestion of membranes and subsequent diffusion of non-DNA particles. The DNA can then be separated in gels or subjected to restriction digestion. The DNA plugs can be loaded directly into the wells or placed in front of a comb before pouring the gel. Molecules as large as 12 megabases (Mb) have been successfully prepared by these methods, 2 which have been extensively reviewed? ++ DNA recovery from PFGE can be achieved by using low melting point agarose and treatment with agarase after electrophoresis.
Electrophoretic Parameters" The presence of at least two electric fields introduces many electrophoretic variables that are unique to PFGE. Chief among these is the switch time (or switch interval), which is the duration of each of the alternating electric fields. In general, the larger the D N A molecules, the longer the switch times required for separation. This is because larger molecules take longer to reorient before they begin migrating with each field switch. Thus, large molecules that spend a great portion of each switch interval reorienting will not migrate sufficiently to be resolved. The switch interval is chosen according to the size range of fragments to be resolved. This effect is clearly demonstrated in Fig. 1, in which lengthening the switch time separates larger fragments. One problem associated with PFGE is band inversion. 7 This refers to the migration of certain fragments at a faster rate than some smaller fragments, resulting in an inverted fragment size order in specific regions of the gel. 4 C. L. Smith, P. E. Warburton, A. Gaal, and C. R. Cantor+ 0l "Genetic Engineering" (J. K. Setlow and A. Hollaender, eds.), Vol. 8, p. 45. Plenum, New York, 1986. O. f. Carle and M. V. Olson, Methods Enzymol. 155, 468 (1987). ~+B. Birren and E. Lai, "'Pulsed Field Gel Electrophoresis: A Practical Guide," ist Ed. Academic Press, Orlando. FL, 1993. : G. F. Carle, M. Frank. and M. V. Olson, Science 232, 65 (1986).
258
ELECTROPHORESIS
[ 11]
This is particularly apparent when the reorientation angle between the fields is 180° (i.e., field inversion; see as follows). A technique for minimizing band inversion and improving the linearity of resolution in a gel is switch time ramping. 7 This refers to a progressive increase in the switch interval during the duration of a gel run. DNA fragment migration will reflect the average of the mobilities over all the different switch intervals used. In this way, resolution will be improved by employing favorable separation conditions for each portion of the size range of DNA molecules being separated for at least part of the run. Other variables that must be adjusted in accordance with DNA size are the voltage gradient and the reorientation angle, s The voltage gradient is the electrical potential applied to the gel, measured in volts (V) per centimeter (cm). Gradients from 6 to 10 V/cm are suitable for molecules up to 1 Mb. If large molecules such as Schizosaccharomyces pombe yeast chromosomes (3.5, 4.7, and 5.7 Mb) are to be separated, gradients no higher than 2 V/cm can be used. The reorientation angle, which is the angle between the direction of the pulsed fields, may also be varied to affect resolution and the rate of separation. Angles between 105 and 165 ° give comparable resolution of molecules smaller than 1 Mb. The most common angle is 120 °, which is suitable for all applications. Smaller reorientation angles are desired for separation of very large DNA fragments (>1000 kb) because this increases mobility.
Pulsed-Field Gel Electrophoresis Systems A number of pulsed-field systems have been described, {' which differ mainly in the electrode geometry and method of reorientation of the electric fields. Despite these differences, the theory and the mechanism of PFGE separation are similar among all systems. In this chapter, we discuss only asymmetric voltage field-inversion gel electrophoresis (AVFIGE) ~),~° and contour-clamped homogeneous electric field (CHEF) l~ electrophoresis systems because of their superior resolution and widespread usage. Systems such as the transverse alternating-field electrophoresis (TAFE) 12 gel box S. M. Clark, E. Lai, B. W. Birren, and L. Hood, Science 241, 12(13 (1988). ~ M. Y. Graham, 3". Otani, 1. Boime, M. V. Olson, G. F. Carle, and D. D. Chaplin, Nucleic Acids Res. 15, 4437 (1987). 10 C. Turmel, E. Brassard, R. Forsyth, K. Hood, G. W. Slatcr, and J. Noolandi, in "Current Communications in Cell and Molecular Biology," Vol. 1: Electrophoresis of Large D N A Molecules: Theory and Applications (E. Lai and B. W. Birren, eds.), p. 101. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1990. i1 O. Chu, D. Vollrath, and R. W. Davis, Science 234, 1582 (1986). 12 K. Gardiner, W. Laas, and W. Patterson. Somatic Cell. Mol. Genet. 12, 185 (1986).
[1 1]
PULSED-FIELDGEL ELECTROPHORESIS
259
or the rotating electrophoresis systems L~'Hare too limited in their functions and thus are not recommended. For example, neither system is well suited for separating D N A from l to 100 kb. Asymmetric voltage field-inversion gel electrophoresis involves periodically inverting a uniform electric field (180 ° angle of reorientation) using different voltage gradients in the forward and backward fields. For some switch time and voltage conditions, this is also referred to as zero-integrated field electrophoresis (ZIFE). 1° Schematic diagrams for constructing an A V F I G E apparatus have been published in detaiP and commercial units are available ( C H E F - M A P P E R and Q-Life Autobase; Bio-Rad, Hercules, CA). We do not discuss earlier F I G E systems that use the same forward and reverse voltages (also referred to as standard F I G E ) because of the severe p r o b l e m of band inversion. A V F I G E provides the best possible resolution of fragments in the 1- to 50-kb range ~5 and adequate resolution of molecules in the 50- to 1000-kb range, l° However, it is too slow for separation of fragments larger than 1 Mb. C H E F is the most versatile and widely used P F G E system. This versatility stems from its ability to separate fragments from 1 to 6000 kb without band inversions. Also, C H E F can speed up separation of large fragments by using smaller reorientation angles. ~ A great many electrophoretic data have been generated with the C H E F systems, ~ which guide selection of optimal running conditions. The other reason for the popularity of C H E F apparatuses is that excellent systems are available commercially (Bio-Rad).
Experimental Considerations This chapter is designed to lead someone familiar with conventional electrophoresis through the steps needed to run a PFG. Because various size ranges of D N A require different voltage gradients for optimal separation, the experimental section is divided to cover three size ranges of DNA: l- to 100-kb, 100- to 1000-kb, and 1- to 6-Mb D N A . For each size range, a preferred method and an alternate procedure are provided. Along with the procedure, r e c o m m e n d e d materials and necessary data are given. These protocols provide optimal conditions for running gels in each of the specific size ranges.
~ P. Serwer, Electrophoresis 8, 30l (1987). t4 E. M. Southern, R. Anand, W. R. A. Brown, and D. S. Fletcher, Nucleic Ac,;ds Res. 15, 5925 (1987). ~ B. W. Birren. E. Lai, L. E. Hood, and M. Simon, Anal. Biochem. 177, 282 (1989). i~,B. W. Birren, K. Lai, S. M. Clark, L. Hood, and M. Simon, Nucleic Acids Res. 16, 7563 (1988).
260
ELKCTROPHORESIS
[ 11 ]
Selecting a Size Range One major difference between conventional electrophoresis and PFG electrophoresis is that a D N A size range must be chosen prior to running a PFG. In conventional electrophoresis the same window of resolution (e.g., 0.2 to 20 kb) is nearly always obtained. With PFG electrophoresis there are many parameters that can be altered so as to determine the width of the window of resolution within the continuum of D N A fragment sizes. This window can be fine tuned to obtain high resolution, for example, to separate a 400-kb fragment from a 450-kb fragment. Alternately, the window can be set for lower resolution of a wider size range, as in the separation of a 100-kb fragment from a 700-kb fragment. In general, the narrower the size range of molecules being separated, the higher the resolution obtained. Extremely high resolution requires long runs; usually resolution is compromised to obtain a convenient run time. Every PFG run thus begins with the selection of the desired size range, and this choice determines what fragments will be separated as well as the resolution obtained.
Using Multiple Size Markers The conditions presented in each section are known not to produce band inversion within the range they are designed to separate. However, it is important to use two differently sized markers on every gel to ensure that band inversion has not occurred in a region of interest.
Chemicals To replicate the data in this chapter we recommend using low electroendosmosis ( E E O ) agarose such as SeaKem LE from FMC BioProducts (Rockland, ME). T A E buffer (1 ×) is composed of 40 mM Tris-acetate and 1 mM E D T A . The composition of T B E buffer (0.5×) is 44.5 mM Tris-borate and 12.5 mM E D T A . Resolution of DNA F r a g m e n t s from 1 to 100 kb D N A larger than 20 kb cannot be resolved easily by conventional electrophoresis. Initially, standard F I G E (equal voltage for forward and backward switch times) was the method of choice for separating small D N A because of the simplicity of the equipment. However, severe band inversion problems limit the useflflness of standard FIGE. Asymmetric voltage F I G E ( A V F I G E ) is the preferred method for separating small (1-100 kb) D N A because of superior resolution. Owing to the widespread availability of C H E F boxes in laboratories now, we have also included information on separating small D N A using CHEF.
[ 1 1]
PULSED-FIELDGEl. ELECTROPHORESIS
261
Materials A g a r o s e gel, 1% (w/v) T B E buffer, 0.5× Markers: 1. U n d e r 50 kb: 8.2- to 48.5-kb range (Bio-Rad; B R L , Gaithersburg, M D ) 4.9-49 kb [5 kb ladder; New E n g l a n d Biolabs ( N E B ) , Beverly, M A ] 2. A b o v e 50 kb: 15-291 and 24-291 kb ( M i d - R a n g e I, II; N E B )
Procedures Preferred Method: A VFIGE 1. Set the voltage gradient at 9 V / c m in the forward direction, and at 6 V / c m in the b a c k w a r d direction. 2. Select a size range. 3. C h o o s e switch times from Fig. 2. a. Using the smallest sized D N A f r a g m e n t of interest, choose the initial switch time. (Note: F o r w a r d switch time equals b a c k w a r d switch time). b. Using the largest sized D N A f r a g m e n t of interest, choose., the final switch time for the forward and b a c k w a r d direction. 2.5.
2.0"
/
%, ¢)
.E
1.5
r-
•
1.0.
6O
0.5
0.0 . . . . 0 10
20
30
40
50
60
70
80
90
O0
Size (kb)
FI~. 2. Switch time selection for 1- to 100-kbDNA by AVFIGE. Maximum size of fragments that can be resolved at the different switch times is shown. All data represent migration in 1% agarose gels run at 15° in 0.5x TBE with a 180° reorientation angle, a 9-V/cm forward voltage gradient, and a 6-V/cm reverse voltage gradient.
262
ELECTROPHORESIS
[ 111
TABLE I MOBILITY OF 1- I O 100-kb D N A SEPARA~'ED BY A V F I G E "
Switch time Size (kb)
0.2 sec
0.5 sec
0.8 sec
I sec
1.5 scc
2 sec
1 5 10 15 20 25 30 35 40 45 50 60 75 100
0.58 (/.42 0.24 0.19 0.15
(/.58 (I.40 (I.31 0.28 {).24 0.20 0.18 0.19
0.58 0.45 (I.37 0.34 0.33 0.31 (l.28 0.24 0.20 0.16 0.16
0.58 0.48 0.42 0.40 0.39 0.37 0.34 0.29 0.25 0.20 0.17 0.13
0.58 0.47 0.40 0.38 0.37 0.36 0.35 0.33 0.31 0.28 0.25 0.21 0.15
0.57 0.48 0.41 l).37 0.36 0.36 (/.36 0.36 0.35 0.34 0.32 0.27 0.23 0.2(1
"The mobility (cm/hr) is indicated for D N A of a specific size range separated by a variety of switch times. All data represent migration in 1% agarose gels run at 15° in 0.5× TBE with a 180 ° reorientation angle and a 9-V/cm forward gradient and 6-V/cm reverse gradient.
c. Ramp linearly between the initial and final switch times. 4. Determine run time from Table I. a. Find the mobility of the smallest fragment at the initial switch time. Interpolate if the size or switch time is not explicitly listed in Table I. b. Find the mobility of the smallest fragment at the final switch time. c. Average the mobilities. d. Determine the run time by dividing the desired distance of migration by the average mobility. The desired distance is three-quarters the length of the gel measured from the well.
Example: To Separate DNA Samples in the Range of 10-60 kb 1. Set the voltage gradient at 9 V/cm in the forward direction. In a typical horizontal gel box (30 cm between electrodes) this would be 270 V. Set the voltage gradient at 6 V/cm in the backward direction. In a 30-cm gel box this would be 180 V. 2. Choose switch times from Fig. 2. a. The initial switch time (for forward and backward fields) is 0.2 sec, based on 10 kb being the smallest fragment of interest.
[1 1]
PULSED-FIELD GEL ELECTROPHORESIS
263
b. T h e final switch t i m e (for f o r w a r d a n d b a c k w a r d fields) is 1 sec, b a s e d on 60 k b b e i n g the largest f r a g m e n t of interest. c. Set the r a m p i n g f u n c t i o n of the s w i t c h e r to l i n e a r r a m p . 3, D e t e r m i n e run t i m e f r o m T a b l e I. a. F r o m T a b l e I, a 10-kb f r a g m e n t , u n d e r the influence o f a 0.2-see switch time, has a m o b i l i t y of 0.24 era/hr. b. F r o m T a b l e I, a 10-kb f r a g m e n t , u n d e r the influence of a 1-see switch time, has a m o b i l i t y of 0.42 c m / h r . c. T h e a v e r a g e of t h e s e m o b i l i t i e s is 0.33 c m / h r . d. If the gel is 12.5 cm long, t h e n the 10-kb b a n d s h o u l d m i g r a t e t h r e e - q u a r t e r s the d i s t a n c e f r o m the wells to the e n d of the gel, o r a p p r o x i m a t e l y 9 cm. T o d e t e r m i n e the run time divide 9 cm by 0.33 cm/h. T h e run t i m e s h o u l d be 27.7 hr.
Alternative Method: C H E F 1. Set the v o l t a g e g r a d i e n t at 6 V / c m . 2. Select a size range. 3. C h o o s e switch t i m e s f r o m Fig. 3. a. U s i n g the s m a l l e s t sized D N A f r a g m e n t of interest, c h o o s e the initial switch time. b. U s i n g the largest sized D N A f r a g m e n t of interest, c h o o s e the final switch time. 7.0-
/
6.05.04-.0.
~
3.0-
CO
2.0. 1.[?. 0.0
.
0
10
20
30
40
50
60
70
80
90
O0
Size (kb) FI{;. 3. Switch time selection for 1- to 100-kb DNA by CHEF. Maximum size of fragments that can be resolved at the different switch times is shown. All data represent migration in 1% agarose gels run at 15° in 0.5x TBE with a 120° reorientation angle and a 6-V/cm voltage gradient.
264
ELECTROPHORESIS
[ 1 11
TABLE II MomurY OF 1- TO l(/00-kb DNA SEPARATEDBY CHEF" Switch time Size (kb) 1
5 10 20 30 40 50 75 100 150 200 300 400 500 600 700 800 900 1000
0.1-2 see
3 sec
5 sec
1.06
1.06
1.06
0.51 0.38 0.30 0.26 0.23 0.20
0.51 0.38 0.30 0.26 0.23 0.22
0.51 0.38 0.30 0.26 0.23 11.25 0.21
15 sec
30 sec
45 sec
60 sec
75 sec
90 sec
0.32 0.29 0.27 0.22 0.17
0.30 1/.29 0.27 0.25 0.23 0. l 7 0.11
0.30 1/.29 0.28 0.26 0.24 0.21 O.17 0.12 0.07
1/.30 0.29 0.28 0.27 0.26 0.23 0.20 0.17 0.13 0.10
1/.31 0.30 0.31t 0.29 0.28 0.26 0.24 0.21 0.18 0.15 0.12 0.08
0,31 0,30 0,30 0.29 0.28 0.26 0.24 0.22 0.20 0.18 0.15 0.13 O.10
"The mobility (cm/hr) is indicated for DNA of a specific size range subjected to a variety of switch times. All data represent migration in 1% agarose gels run at 15° in 0.5× TBE with a 120° reorientation angle and a 6-V/cm gradient.
.
c. R a m p l i n e a r l y f r o m t h e initial to t h e final s w i t c h t i m e . D e t e r m i n e r u n t i m e f r o m T a b l e II ( s e e p r e v i o u s e x a m p l e ) . a. F i n d t h e m o b i l i t y of t h e s m a l l e s t f r a g m e n t at t h e initial s w i t c h t i m e . I n t e r p o l a t e if t h e size o r s w i t c h t i m e is n o t e x p l i c i t l y listed in T a b l e II. b. F i n d t h e m o b i l i t y o f t h e s m a l l e s t f r a g m e n t at t h e final s w i t c h t i m e . c. A v e r a g e t h e m o b i l i t i e s . d. D e t e r m i n e t h e r u n t i m e by d i v i d i n g t h e d e s i r e d d i s t a n c e o f m i g r a t i o n by t h e a v e r a g e m o b i l i t y . T h e d e s i r e d d i s t a n c e is t h r e e - q u a r t e r s t h e l e n g t h o f t h e gel m e a s u r e d f r o m t h e well.
Resolution of DNA Fragments
from 100 to 1000 kb
S e p a r a t i o n of D N A f r a g m e n t s f r o m 100 to 1000 kb c a n b e s t b e a c h i e v e d u s i n g a c o n t o u r - c l a m p e d h o m o g e n e o u s e l e c t r i c field ( C H E F ) e l e c t r o p h o r e -
[1 1]
PULSED-FIELDGEL ELECTROPHORESIS
265
sis system. Such a system can separate a large n u m b e r of D N A samples in straight lines with shorter run times than other systems. S t a n d a r d field inversion offers g o o d resolution in this range but band inversion can occur with this technique. A s y m m e t r i c voltage F I G E can minimize b a n d inversion, but requires m u c h longer run times than C H E F gels. Protocols for separation of D N A from 100 to 1000 kb by C H E F electrophoresis and by asymmetric voltage F I G E are provided.
Materials A g a r o s e gel, 1% (w/v) T B E buffer, 0.5× Markers: Wild-type A ladders (48.5 to m o r e than 1000 kb; Bio-Rad, B R L , F M C , N E B ) or Saccharomyces cerevisiae c h r o m o s o m e s (2002000 kb; B i o - R a d , B R L , FMC, N E B )
Procedures Preferred Method: CHEF 1. The voltage gradient to be used is 6 V/cm, and the reorientation angle is 120 ° . 2. D e t e r m i n e the size range of D N A fragments to be resolved. 3. D e t e r m i n e switch times, using Fig. 4. 100
/ 80 ¸ %(D
60
E c-
40 O3
20
•
0
;
;
I00
:
l
200
:
',
:
,500 400
500
600
700
800
900 I000
Size (kb) FIG. 4. Switch time selection for 100- to 1000-kb DNA by CHEF. Maximum size of fragments that can be resolved at the different switch times is shown. All data represent migration in 1% agarose gels run at 15° in 0.5x TBE with a 120° reorientation angle and a 6-V/cm voltage gradient.
266
ELECTROPHORESIS
[ 11]
a. D e t e r m i n e the initial switch time from Fig. 4, using the smallest fragment of interest. b. D e t e r m i n e the final switch time f r o m Fig. 4, using the largest fragment of interest. c. R a m p linearly f r o m the initial value to the final value. 4. Calculate the run time using Table I I (see example above). a. D e t e r m i n e the initial velocity of the smallest fragment (i.e., its mobility at the initial switch time) f r o m Table II. Interpolate if the size or switch time is not explicitly listed in Table II. b. D e t e r m i n e the final velocity of the smallest f r a g m e n t (i.e., its mobility at the final switch time) from Table II. c. A v e r a g e these two mobility values. d. Divide the desired migration distance (three-quarters the length of the gel m e a s u r e d f r o m the well) of the smallest f r a g m e n t by the average mobility value to obtain the necessary run time.
Alternative Procedure: A V F I G E 1. Use a forward voltage gradient of 2.8 V / c m and a reverse gradient of 0.8 V/cm. 2. D e t e r m i n e the size range of D N A fragments to be resolved. 3. Select switch times using Fig. 5. a. D e t e r m i n e the initial forward switch time from Fig. 5, using the lOO
"~ 80I ~
so
i_=
~
40
ffl
20
0
100
200
300
400
500
600
700
800
900
1000
Size (kb) FIG. 5. Switch time selection for 100- to 1000-kb DNA by AVFIGE. Maximum size of fragments that can be resolved at the different switch times is shown. All data represent migration in 1% agarose gels run at 15° in 0,5X TBE with a 180° reorientation angle, a 2.8-V/cm forward gradient, and a 0.84-V/cm reverse voltage gradient.
[1 1]
PULSED-FIELD GEL ELECTROPHORESIS
267
TABLE 111 MomLJlY or: 100- TO 1000-kb DNA SEPARAteDBY AVFIGE" Switch time Size (kb) 50 10fi 150 200 30() 400
500 600 700
10 sec
25 sec
4(/sec
50 scc
70 sec
100 sec
0.06 0.04
0.13 0.11 0.09 0.08
0.12 0.11 0.10 0.09
0. l 2 0.i1 0.10 0.09
(1.12 0.11 0.11 0.10
0.12 0.11 0.11 0.10
0.04
0.07 ().05
().fin 0.06
0.08 0.07
O. 10 0.09
0.03
(/.04
0.05
{).08
{}.(}3 0.01
0.04 0,03
0.{}7 {},06
0.{}1
(}.06 (I.05 0.04
800 900 100()
"The mobility (cm/hr) is indicated fl)r DNA of a specific size range subjected Io a variety of switch times. All data represent migration in 1% agarose gels run at 15° in 0.5x TBE with a 180° reorientation angle and a 2.8-V/ cm forward gradient and 0.84-V/cm reversc gradient. smallest f r a g m e n t of this range. T h e initial reverse switch time is 1.4 times the forward switch time. b. D e t e r m i n e the final forward switch time from Fig. 5, using the largest f r a g m e n t of this range. T h e final reverse switch time is 1.4 times the forward switch time. c. R a m p linearly from the initial switch time to the final switch time. . Calculate the run time using T a b l e II1 (see e x a m p l e above). a. D e t e r m i n e the initial velocity of the smallest f r a g m e n t (mobility at the initial forward switch time) from T a b l e III. I n t e r p o l a t e if the size or switch time is not explicitly listed in T a b l e 1II. b, D e t e r m i n e the final velocity of the smallest f r a g m e n t (mobility at the final f o r w a r d switch time) from T a b l e 111. C. A v e r a g e these two m o b i l i t y values. d. D i v i d e the desired m i g r a t i o n distance ( t h r e e - q u a r t e r s the length of the gel m e a s u r e d from well) of the smallest f r a g m e n t by the average m o b i l i t y value to o b t a i n the necessary run time:.
R e s o l u t i o n of DNA F r a g m e n t s f r o m 1 to 6 Mb S e p a r a t i o n of m e g a b a s e D N A involves a d d i t i o n a l p r o b l e m s c o m p a r e d to the previously described s e p a r a t i o n of smaller D N A . Most notably,
268
ELECTROPHORESIS
111]
the voltage gradient must be decreased and the switch times increased. Consequently, to keep run times within reasonable time frames other parameters must be altered. The data presented in this section were collected from gels made of SeaKem LE. To decrease gel run times lower E E O agarose, such as BioRad C G A or FMC SeaKem G O L D PFG-agarose DNA, can be used. To ensure proper use of such special agaroses check with the manufacturer for decreases in run time. To reduce further the time required to separate megabase-sized DNA, the concentration of agarose can be decreased. For 1- to 3-Mb DNA, 1% gels provide acceptable separation in a reasonable amount of time while retaining the mechanical strength of the gel. However, the ease of handling a 1% gel can be sacrificed in order to separate larger D N A more quickly by using 0.6-0.7% gels. Megabase D N A moves extremely slowly. Decreasing the reorientation angle to 106° lessens the sideways migration of DNA and thus shortens the run times. For gel boxes lacking the ability to change the reorientation angle, 1- to 6-Mb D N A can be separated using a 120° angle. The gel will run approximately 25% longer with this modification. D N A in the megabase size range is best separated using C H E F setups. Owing to the very long run times required, A V F I G E is not recommended for separation of D N A over 1000 kb. We present two different protocols that employ different voltage gradients depending on the maximum size of the D N A to be separated. Materials" Agarose, 1% (w/v) T A E buffer, 1× Markers: S. cerevisiae chromosomes (0.2-1.9 Mb; Bio-Rad, FMC, BRL, NEB) Candida albicans chromosomes (1-3 Mb; Clontech, Palo Alto, CA) Hansenula wingei chromosomes (1-3.3 Mb; Bio-Rad, BRL) Procedure CHEF Separation of 1- to 3-Mb DNA l. Use a reorientation angle of 106 ° and a voltage gradient of 3 V/cm. 2. Select a size range. 3. Select switch times from Fig. 6. a. Choose the initial switch time based on the smallest sized D N A fragment of interest.
[ 111
PULSED-FIELD GEL ELECTROPHORESIS
269
10
I
g. 8 ¸
-¢_
7i 6-
k--
5-
-~
4-
m
5 2
I I000
, 1250
I 1500
, 1750
Size
I 2000
~ 2250
I 2500
, 2750
3000
(kb)
FI(}. 6. Switch time selection for 1- to 3-Mb D N A by C H E F . M a x i m u m size of fragments that can be resolved at the different switch times is shown. All data represent migration in 0.8% agarose gels run at 15 ° in 1× T A E with a 106 ° reorientation angle and a 3-V/cm voltage gradient.
b. C h o o s e the final switch time based on the largest sized D N A fragment of interest. c. Ramp linearly between the initial and the final switch time. . Determine the run time from Table IV (see example above). T A B L E IV MOBII.IIY OF 1- TO 3-Mb D N A SEPARATISI) BY C H E F " Switch times Size (kb)
2.5 min
4 rain
6 min
8 rain
10 rain
1000 1250 1500 1750 2000 225(/ 2500 2750 3000
0.06
().l 1 0.0g (I.04
0.14 /I.12 0.11 0.10
0.14 0.13 0.12 0.11 0.10 0.08
0.15 0.14 (I.13 0.13 0.12 0. I 1 0. I 1 0.09 0.09
" T h e ,nobility ( c m / h r ) is indicated for D N A of a specific size range subjected to a variety of switch times. All data reprcsent migration in 0.8% agarose gels run at 15° in 1 x T A E with a 106 ° reorientation angle and a 3-V/cm gradient.
270
ELECTROPHORESIS
[11]
a. F i n d the m o b i l i t y of the s m a l l e s t f r a g m e n t at the initial switch time. I n t e r p o l a t e if the size or switch t i m e is not explicitly listed in T a b l e IV. b. F i n d the m o b i l i t y of the s m a l l e s t f r a g m e n t at the final switch time. c. A v e r a g e the mobilities. d. D e t e r m i n e the run time by dividing the d e s i r e d d i s t a n c e of m i g r a tion b y the a v e r a g e mobility. T h e d e s i r e d d i s t a n c e is t h r e e - q u a r t e r s the length of the gel m e a s u r e d from the well.
Materials A g a r o s e , 0.8% (w/v) T A E buffer, 1 × M a r k e r s : S. pombe c h r o m o s o m e s (3.5-5.7 Mb; B i o - R a d ) ; Hansenula wingei c h r o m o s o m e s ( 1 - 3 . 3 Mb; B i o - R a d , B R L )
Procedure CHEF Separation of 3- to 6-Mb DNA l. U s e a r e o r i e n t a t i o n angle of 106 ° and a v o l t a g e g r a d i e n t of 2 V / c m . 2. Select a size range. 3. Select switch t i m e s from Fig. 7.
40-
"~
35.
-5
30.
4--'
25"
20
3000
,
I
,
4-000
I
5000
'
6000
Size (kb)
El(;. 7. Switch time selection for 3- to 6-kb DNA by CHEF. Maximum size of fragments that can be resolved at the different switch times is shown. All data represent migration in ().8~ agarose gels run at 15° in 1× TBE with a 1(}6° reorientation angle and a 2-V/cm voltage gradient.
[1 l ]
271
PULSKD-FI ELI) GEL ELECTROPHORKSIS
TABLE V MOBILrFYOF 3- TO 6-Mb DNA SEPARATEDBY CHEF" Switch time Size (kb)
20 min
25 mm
30 min
35 min
2500 3000 3500 4(100 5000 6000
0.06 0.04
0.06 0.05 0.05
0.06 0.06 0.05 0.04 0.03 0.01
0.07 0.06 0.05 0.05 0.03 0.02
"The mobility (cm/hr) is indicated for DNA of a specific size range subjected to a variety of switch times. All data represent migration in 0.8% agarose gels run at 15° in 1× TAE with a 106° reorientation angle and a 2-V/cm gradient.
a. U s i n g the s m a l l e s t sized D N A f r a g m e n t of interest, c h o o s e the initial switch time. b. U s i n g the largest sized D N A f r a g m e n t of interest, c h o o s e the final switch time. c. R a m p l i n e a r l y b e t w e e n the initial a n d final switch times. 4, D e t e r m i n e the r u n t i m e f r o m T a b l e V (see e x a m p l e a b o v e ) . a. F i n d the m o b i l i t y o f the s m a l l e s t f r a g m e n t at the initial switch time. I n t e r p o l a t e if the size o r switch time is n o t explicitly listed in the table. b. F i n d the m o b i l i t y o f the s m a l l e s t f r a g m e n t at the final switch time. c. A v e r a g e the mobilities. d. D e t e r m i n e the run t i m e by dividing the d e s i r e d d i s t a n c e of m i g r a tion b y the a v e r a g e mobility. T h e d e s i r e d d i s t a n c e is t h r e e - q u a r t e r s the l e n g t h of the gel m e a s u r e d f r o m t h e well.
Future Directions In this c h a p t e r we h a v e s u m m a r i z e d the c u r r e n t o p t i m u m e l e c t r o p h o retic c o n d i t i o n s for the s e p a r a t i o n of D N A f r a g m e n t s f r o m 1 to 6000 kb. H o w e v e r , we b e l i e v e that t h e r e a r e m a n y i m p r o v e m e n t s to be m a d e . F o r e x a m p l e , we still d o not u n d e r s t a n d the basis for the a p p a r e n t limit in sizes that can b e effectively s e p a r a t e d . N o r d o we u n d e r s t a n d fully why h i g h e r v o l t a g e s c a n n o t be used to s e p a r a t e large D N A molecules. M o s t p u b l i c a tions have c o n c e n t r a t e d on P F G s e p a r a t i o n s using o n l y two electric fields.
272
ELECTROPHORESlS
[ 121
In theory, PFG separations involving multiple fields should be even more advantageous. The conditions described in this chapter were determined experimentally from large numbers of gel runs. A more logical approach for the future would be the development of a comprehensive model that can correctly simulate all the observed behavior of large DNA molecules under PFGE. A number of computer models consistent with some aspects of PFG separation have been published. However, no current model can explain all the aspects of the behavior of DNA under various PFG conditions. For example, none of the models explain the dependence of separation on the reorientation angle, the voltage gradient limits for the resolution of large DNA, or the effect of using multiple electric fields. Direct microscopic observation of individual DNA molecules in the gel has provided a better picture of how DNA molecules move in electrophoresis. In the future, real-time optimization of DNA electrophoresis might provide major improvements over existing separation procedures.
[ 12]
Migration of DNA through
By GARY W.
SLATER, PASCAl_ MAYER,
Gels
and GuY DROUIN
Introduction Gel electrophoresis has become a major laboratory tool for separating biological macromolecules. For example, large (megabase) double-stranded DNA (dsDNA) molecules can readily be separated on agarose gels using pulsed-field gel electrophoresis (PFGE), while subkilobase single-stranded DNA (ssDNA) molecules can be sequenced on polyacrylamide gels (see [14], [16], and [17] in this volume, and [17] in Vol. 271 of this series1). These two techniques are essential to map and sequence the human genome. Although great technological advances have been made, the process itself, i.e., the electrophoretic migration of large flexible polyelectrolytes in pseuJ N. Matsubara and S. Terabe, Methods Enzymol. 270, Chap. 14, 1996 (this volume); T. Wehr, M. Zhu, and R. Rodriguez, Methods Enzymol. 270, Chap. 16, 1996 (this volume); L. Kfivfinkovfi, P. Gebauer, and P. Bo6ek, Methods, Enzymol. 270, Chap. 17, 1996 (this volume); W. S. Hancock, A. Apfell, J. Chakel, C. Sounders, T. M'Timkulu, E. Pungor, Jr., and A. W. Guzetta, Methods Enzyrnol. 271, 403 (1996).
M E T H O D S IN E N Z Y M O L O G Y , VOL. 270
Copyright k' 1996 by Academic Press, Inc. All rights of reproduction in any form resolved.
PULSED-FIELDGEI. ELECTROPHORESIS
255
usually kept outside the safety box (not shown in Fig. 6) and is connected to the electrolyzer via a port at the base of the box. An example of the unique purification capability of this instrument is given in Fig. 7: A highly purified preparation of recombinant human superoxide dismutase (SOD) was found to consist of at least three isoforms, with the following isoelectric points: 4.80, 4.92, and 5.07. When monitoring the purification progress, it is seen that a pure pI 5.07 form collects in chamber 5, with the lower pl species focusing in the chambers delimited by the proper set of two isoelectric membranes.
Acknowledgments Supported by grants from Radius in Biotechnology (ESA, Paris) to P.G.R.
[1 1] By J A N E T
Pulsed-Field
Gel Electrophoresis
C. WRESTLER, B A R B A R A
D.
LIPES, B R U C E W . B I R R E N ,
a n d E R I C LAI
Introduction Pulsed-field gel electrophoresis (PFGE) of agarose gels enables the reproducible separation of large DNA fragments. ~ In concept, PFGE is an extension of conventional electrophoresis, in which two alternating (or pulsed) electric fields are used instead of the traditional single slatic field. Separation occurs when these fields are oriented at an obtuse angle to one another. In a pulsed-field gel, the end of each molecule migrates in a new direction with each change of the electric fields. The DNA molecules thus migrate through the agarose matrix in a zigzag motion. The tardiness of the larger molecules in turning corners (e.g., in PFGE) or in running forward and backward [e.g., in field-inversin gel electrophoresis (FIGE), see below] separates them from the smaller size fragments. The effectiveness of PFGE, however, is not limited to the separation of very large DNA molecules. PFGE can improve the resolution of DNA molecules of only a few hundred bases and permits separation up to 12,000 kilobase pairs (kb). 2 Figure 1 shows the effectiveness of PFGE in separating yeast chromosomes from 200 to 2000 kb. J D. C. Schwartz and C. R. Cantor, Cell 37, 67 (1984). = M. Bellis, M. Pages, and G. Roizes, Nuclei~ AcMs Res. 15, 6749 (1987).
MKrlIODS IN ENZYMOLOGY.VOL. 270
Copyrighl ru 1996by Academic P~css.Inc. All rightsol reproduction m any lorm icscrvcd.
256
ELECTROPHORESIS 15 sec
3 0 sec
4 5 sec
[ 111 1 min
1.25 rain
960 914 836
- 273 -214
214
48.5
1.5 rain
1.75 min
2 min
2.5 min
5min
2200 1125 1600
214 48.5
214
FIG. 1. Separation of 50- to l{}00-kb DNAs with different switch intervals, a ladders and chromosomes of the yeast Saccharomyces cerevisiae (strain YNN295) were separated in gels, using identical conditions except for the different switch intervals. All gels were 1% SeaKem LE agarose in 0.5× TBE run at 14° in 6-V/cm fields with a reorientation angle of 120°. With each increase in switch interval the new size limit for resolution (based on the separation of the markers) is indicated.
A n u m b e r o f m o d e l s a n d t h e o r i e s h a v e b e e n p r o p o s e d to e x p l a i n s o m e o f t h e m o r e c o m p l e x b e h a v i o r o f D N A m o l e c u l e s in P F G E . 3 H o w e v e r , b i o l o g i s t s r a r e l y n e e d to c o n s u l t t h e s e p h y s i c a l m o d e l s o r equations for practical PFGE applications. This chapter outlines optimum PFG electrophoretic conditions for the separation of DNA fragments f r o m 1 to 6000 kb.
3 E. Lai and B. W. Birren (eds.), "Current Communications in Cell and Molecular Biology," Vol. 1: Electrophoresis of Large DNA Molecules: Theory and Applications. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1990.
[1 11
PULSED-FIELDGEL ELECTROPHORESIS
257
Unique F e a t u r e s PFGE of high molecular weight DNA involves additional factors that are not encountered in conventional agarose gels. We outline some of these factors and the modifications required for running PFGE.
Sample Preparation, Gel Loading, and DNA Recovery High molecular weight DNA samples for PFGE must be carefully prepared to minimize degradation resulting from mechanical breakage or nuclease activity. This is achieved by using a method developed by Schwartz and Cantor of embedding intact cells in an agarose matrix prior to lysis. ~ This provides support for the DNA while allowing digestion of membranes and subsequent diffusion of non-DNA particles. The DNA can then be separated in gels or subjected to restriction digestion. The DNA plugs can be loaded directly into the wells or placed in front of a comb before pouring the gel. Molecules as large as 12 megabases (Mb) have been successfully prepared by these methods, 2 which have been extensively reviewed? ++ DNA recovery from PFGE can be achieved by using low melting point agarose and treatment with agarase after electrophoresis.
Electrophoretic Parameters" The presence of at least two electric fields introduces many electrophoretic variables that are unique to PFGE. Chief among these is the switch time (or switch interval), which is the duration of each of the alternating electric fields. In general, the larger the D N A molecules, the longer the switch times required for separation. This is because larger molecules take longer to reorient before they begin migrating with each field switch. Thus, large molecules that spend a great portion of each switch interval reorienting will not migrate sufficiently to be resolved. The switch interval is chosen according to the size range of fragments to be resolved. This effect is clearly demonstrated in Fig. 1, in which lengthening the switch time separates larger fragments. One problem associated with PFGE is band inversion. 7 This refers to the migration of certain fragments at a faster rate than some smaller fragments, resulting in an inverted fragment size order in specific regions of the gel. 4 C. L. Smith, P. E. Warburton, A. Gaal, and C. R. Cantor+ 0l "Genetic Engineering" (J. K. Setlow and A. Hollaender, eds.), Vol. 8, p. 45. Plenum, New York, 1986. O. f. Carle and M. V. Olson, Methods Enzymol. 155, 468 (1987). ~+B. Birren and E. Lai, "'Pulsed Field Gel Electrophoresis: A Practical Guide," ist Ed. Academic Press, Orlando. FL, 1993. : G. F. Carle, M. Frank. and M. V. Olson, Science 232, 65 (1986).
258
ELECTROPHORESIS
[ 11]
This is particularly apparent when the reorientation angle between the fields is 180° (i.e., field inversion; see as follows). A technique for minimizing band inversion and improving the linearity of resolution in a gel is switch time ramping. 7 This refers to a progressive increase in the switch interval during the duration of a gel run. DNA fragment migration will reflect the average of the mobilities over all the different switch intervals used. In this way, resolution will be improved by employing favorable separation conditions for each portion of the size range of DNA molecules being separated for at least part of the run. Other variables that must be adjusted in accordance with DNA size are the voltage gradient and the reorientation angle, s The voltage gradient is the electrical potential applied to the gel, measured in volts (V) per centimeter (cm). Gradients from 6 to 10 V/cm are suitable for molecules up to 1 Mb. If large molecules such as Schizosaccharomyces pombe yeast chromosomes (3.5, 4.7, and 5.7 Mb) are to be separated, gradients no higher than 2 V/cm can be used. The reorientation angle, which is the angle between the direction of the pulsed fields, may also be varied to affect resolution and the rate of separation. Angles between 105 and 165 ° give comparable resolution of molecules smaller than 1 Mb. The most common angle is 120 °, which is suitable for all applications. Smaller reorientation angles are desired for separation of very large DNA fragments (>1000 kb) because this increases mobility.
Pulsed-Field Gel Electrophoresis Systems A number of pulsed-field systems have been described, {' which differ mainly in the electrode geometry and method of reorientation of the electric fields. Despite these differences, the theory and the mechanism of PFGE separation are similar among all systems. In this chapter, we discuss only asymmetric voltage field-inversion gel electrophoresis (AVFIGE) ~),~° and contour-clamped homogeneous electric field (CHEF) l~ electrophoresis systems because of their superior resolution and widespread usage. Systems such as the transverse alternating-field electrophoresis (TAFE) 12 gel box S. M. Clark, E. Lai, B. W. Birren, and L. Hood, Science 241, 12(13 (1988). ~ M. Y. Graham, 3". Otani, 1. Boime, M. V. Olson, G. F. Carle, and D. D. Chaplin, Nucleic Acids Res. 15, 4437 (1987). 10 C. Turmel, E. Brassard, R. Forsyth, K. Hood, G. W. Slatcr, and J. Noolandi, in "Current Communications in Cell and Molecular Biology," Vol. 1: Electrophoresis of Large D N A Molecules: Theory and Applications (E. Lai and B. W. Birren, eds.), p. 101. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1990. i1 O. Chu, D. Vollrath, and R. W. Davis, Science 234, 1582 (1986). 12 K. Gardiner, W. Laas, and W. Patterson. Somatic Cell. Mol. Genet. 12, 185 (1986).
[1 1]
PULSED-FIELDGEL ELECTROPHORESIS
259
or the rotating electrophoresis systems L~'Hare too limited in their functions and thus are not recommended. For example, neither system is well suited for separating D N A from l to 100 kb. Asymmetric voltage field-inversion gel electrophoresis involves periodically inverting a uniform electric field (180 ° angle of reorientation) using different voltage gradients in the forward and backward fields. For some switch time and voltage conditions, this is also referred to as zero-integrated field electrophoresis (ZIFE). 1° Schematic diagrams for constructing an A V F I G E apparatus have been published in detaiP and commercial units are available ( C H E F - M A P P E R and Q-Life Autobase; Bio-Rad, Hercules, CA). We do not discuss earlier F I G E systems that use the same forward and reverse voltages (also referred to as standard F I G E ) because of the severe p r o b l e m of band inversion. A V F I G E provides the best possible resolution of fragments in the 1- to 50-kb range ~5 and adequate resolution of molecules in the 50- to 1000-kb range, l° However, it is too slow for separation of fragments larger than 1 Mb. C H E F is the most versatile and widely used P F G E system. This versatility stems from its ability to separate fragments from 1 to 6000 kb without band inversions. Also, C H E F can speed up separation of large fragments by using smaller reorientation angles. ~ A great many electrophoretic data have been generated with the C H E F systems, ~ which guide selection of optimal running conditions. The other reason for the popularity of C H E F apparatuses is that excellent systems are available commercially (Bio-Rad).
Experimental Considerations This chapter is designed to lead someone familiar with conventional electrophoresis through the steps needed to run a PFG. Because various size ranges of D N A require different voltage gradients for optimal separation, the experimental section is divided to cover three size ranges of DNA: l- to 100-kb, 100- to 1000-kb, and 1- to 6-Mb D N A . For each size range, a preferred method and an alternate procedure are provided. Along with the procedure, r e c o m m e n d e d materials and necessary data are given. These protocols provide optimal conditions for running gels in each of the specific size ranges.
~ P. Serwer, Electrophoresis 8, 30l (1987). t4 E. M. Southern, R. Anand, W. R. A. Brown, and D. S. Fletcher, Nucleic Ac,;ds Res. 15, 5925 (1987). ~ B. W. Birren. E. Lai, L. E. Hood, and M. Simon, Anal. Biochem. 177, 282 (1989). i~,B. W. Birren, K. Lai, S. M. Clark, L. Hood, and M. Simon, Nucleic Acids Res. 16, 7563 (1988).
260
ELKCTROPHORESIS
[ 11 ]
Selecting a Size Range One major difference between conventional electrophoresis and PFG electrophoresis is that a D N A size range must be chosen prior to running a PFG. In conventional electrophoresis the same window of resolution (e.g., 0.2 to 20 kb) is nearly always obtained. With PFG electrophoresis there are many parameters that can be altered so as to determine the width of the window of resolution within the continuum of D N A fragment sizes. This window can be fine tuned to obtain high resolution, for example, to separate a 400-kb fragment from a 450-kb fragment. Alternately, the window can be set for lower resolution of a wider size range, as in the separation of a 100-kb fragment from a 700-kb fragment. In general, the narrower the size range of molecules being separated, the higher the resolution obtained. Extremely high resolution requires long runs; usually resolution is compromised to obtain a convenient run time. Every PFG run thus begins with the selection of the desired size range, and this choice determines what fragments will be separated as well as the resolution obtained.
Using Multiple Size Markers The conditions presented in each section are known not to produce band inversion within the range they are designed to separate. However, it is important to use two differently sized markers on every gel to ensure that band inversion has not occurred in a region of interest.
Chemicals To replicate the data in this chapter we recommend using low electroendosmosis ( E E O ) agarose such as SeaKem LE from FMC BioProducts (Rockland, ME). T A E buffer (1 ×) is composed of 40 mM Tris-acetate and 1 mM E D T A . The composition of T B E buffer (0.5×) is 44.5 mM Tris-borate and 12.5 mM E D T A . Resolution of DNA F r a g m e n t s from 1 to 100 kb D N A larger than 20 kb cannot be resolved easily by conventional electrophoresis. Initially, standard F I G E (equal voltage for forward and backward switch times) was the method of choice for separating small D N A because of the simplicity of the equipment. However, severe band inversion problems limit the useflflness of standard FIGE. Asymmetric voltage F I G E ( A V F I G E ) is the preferred method for separating small (1-100 kb) D N A because of superior resolution. Owing to the widespread availability of C H E F boxes in laboratories now, we have also included information on separating small D N A using CHEF.
[ 1 1]
PULSED-FIELDGEl. ELECTROPHORESIS
261
Materials A g a r o s e gel, 1% (w/v) T B E buffer, 0.5× Markers: 1. U n d e r 50 kb: 8.2- to 48.5-kb range (Bio-Rad; B R L , Gaithersburg, M D ) 4.9-49 kb [5 kb ladder; New E n g l a n d Biolabs ( N E B ) , Beverly, M A ] 2. A b o v e 50 kb: 15-291 and 24-291 kb ( M i d - R a n g e I, II; N E B )
Procedures Preferred Method: A VFIGE 1. Set the voltage gradient at 9 V / c m in the forward direction, and at 6 V / c m in the b a c k w a r d direction. 2. Select a size range. 3. C h o o s e switch times from Fig. 2. a. Using the smallest sized D N A f r a g m e n t of interest, choose the initial switch time. (Note: F o r w a r d switch time equals b a c k w a r d switch time). b. Using the largest sized D N A f r a g m e n t of interest, choose., the final switch time for the forward and b a c k w a r d direction. 2.5.
2.0"
/
%, ¢)
.E
1.5
r-
•
1.0.
6O
0.5
0.0 . . . . 0 10
20
30
40
50
60
70
80
90
O0
Size (kb)
FI~. 2. Switch time selection for 1- to 100-kbDNA by AVFIGE. Maximum size of fragments that can be resolved at the different switch times is shown. All data represent migration in 1% agarose gels run at 15° in 0.5x TBE with a 180° reorientation angle, a 9-V/cm forward voltage gradient, and a 6-V/cm reverse voltage gradient.
262
ELECTROPHORESIS
[ 111
TABLE I MOBILITY OF 1- I O 100-kb D N A SEPARA~'ED BY A V F I G E "
Switch time Size (kb)
0.2 sec
0.5 sec
0.8 sec
I sec
1.5 scc
2 sec
1 5 10 15 20 25 30 35 40 45 50 60 75 100
0.58 (/.42 0.24 0.19 0.15
(/.58 (I.40 (I.31 0.28 {).24 0.20 0.18 0.19
0.58 0.45 (I.37 0.34 0.33 0.31 (l.28 0.24 0.20 0.16 0.16
0.58 0.48 0.42 0.40 0.39 0.37 0.34 0.29 0.25 0.20 0.17 0.13
0.58 0.47 0.40 0.38 0.37 0.36 0.35 0.33 0.31 0.28 0.25 0.21 0.15
0.57 0.48 0.41 l).37 0.36 0.36 (/.36 0.36 0.35 0.34 0.32 0.27 0.23 0.2(1
"The mobility (cm/hr) is indicated for D N A of a specific size range separated by a variety of switch times. All data represent migration in 1% agarose gels run at 15° in 0.5× TBE with a 180 ° reorientation angle and a 9-V/cm forward gradient and 6-V/cm reverse gradient.
c. Ramp linearly between the initial and final switch times. 4. Determine run time from Table I. a. Find the mobility of the smallest fragment at the initial switch time. Interpolate if the size or switch time is not explicitly listed in Table I. b. Find the mobility of the smallest fragment at the final switch time. c. Average the mobilities. d. Determine the run time by dividing the desired distance of migration by the average mobility. The desired distance is three-quarters the length of the gel measured from the well.
Example: To Separate DNA Samples in the Range of 10-60 kb 1. Set the voltage gradient at 9 V/cm in the forward direction. In a typical horizontal gel box (30 cm between electrodes) this would be 270 V. Set the voltage gradient at 6 V/cm in the backward direction. In a 30-cm gel box this would be 180 V. 2. Choose switch times from Fig. 2. a. The initial switch time (for forward and backward fields) is 0.2 sec, based on 10 kb being the smallest fragment of interest.
[1 1]
PULSED-FIELD GEL ELECTROPHORESIS
263
b. T h e final switch t i m e (for f o r w a r d a n d b a c k w a r d fields) is 1 sec, b a s e d on 60 k b b e i n g the largest f r a g m e n t of interest. c. Set the r a m p i n g f u n c t i o n of the s w i t c h e r to l i n e a r r a m p . 3, D e t e r m i n e run t i m e f r o m T a b l e I. a. F r o m T a b l e I, a 10-kb f r a g m e n t , u n d e r the influence o f a 0.2-see switch time, has a m o b i l i t y of 0.24 era/hr. b. F r o m T a b l e I, a 10-kb f r a g m e n t , u n d e r the influence of a 1-see switch time, has a m o b i l i t y of 0.42 c m / h r . c. T h e a v e r a g e of t h e s e m o b i l i t i e s is 0.33 c m / h r . d. If the gel is 12.5 cm long, t h e n the 10-kb b a n d s h o u l d m i g r a t e t h r e e - q u a r t e r s the d i s t a n c e f r o m the wells to the e n d of the gel, o r a p p r o x i m a t e l y 9 cm. T o d e t e r m i n e the run time divide 9 cm by 0.33 cm/h. T h e run t i m e s h o u l d be 27.7 hr.
Alternative Method: C H E F 1. Set the v o l t a g e g r a d i e n t at 6 V / c m . 2. Select a size range. 3. C h o o s e switch t i m e s f r o m Fig. 3. a. U s i n g the s m a l l e s t sized D N A f r a g m e n t of interest, c h o o s e the initial switch time. b. U s i n g the largest sized D N A f r a g m e n t of interest, c h o o s e the final switch time. 7.0-
/
6.05.04-.0.
~
3.0-
CO
2.0. 1.[?. 0.0
.
0
10
20
30
40
50
60
70
80
90
O0
Size (kb) FI{;. 3. Switch time selection for 1- to 100-kb DNA by CHEF. Maximum size of fragments that can be resolved at the different switch times is shown. All data represent migration in 1% agarose gels run at 15° in 0.5x TBE with a 120° reorientation angle and a 6-V/cm voltage gradient.
264
ELECTROPHORESIS
[ 1 11
TABLE II MomurY OF 1- TO l(/00-kb DNA SEPARATEDBY CHEF" Switch time Size (kb) 1
5 10 20 30 40 50 75 100 150 200 300 400 500 600 700 800 900 1000
0.1-2 see
3 sec
5 sec
1.06
1.06
1.06
0.51 0.38 0.30 0.26 0.23 0.20
0.51 0.38 0.30 0.26 0.23 0.22
0.51 0.38 0.30 0.26 0.23 11.25 0.21
15 sec
30 sec
45 sec
60 sec
75 sec
90 sec
0.32 0.29 0.27 0.22 0.17
0.30 1/.29 0.27 0.25 0.23 0. l 7 0.11
0.30 1/.29 0.28 0.26 0.24 0.21 O.17 0.12 0.07
1/.30 0.29 0.28 0.27 0.26 0.23 0.20 0.17 0.13 0.10
1/.31 0.30 0.31t 0.29 0.28 0.26 0.24 0.21 0.18 0.15 0.12 0.08
0,31 0,30 0,30 0.29 0.28 0.26 0.24 0.22 0.20 0.18 0.15 0.13 O.10
"The mobility (cm/hr) is indicated for DNA of a specific size range subjected to a variety of switch times. All data represent migration in 1% agarose gels run at 15° in 0.5× TBE with a 120° reorientation angle and a 6-V/cm gradient.
.
c. R a m p l i n e a r l y f r o m t h e initial to t h e final s w i t c h t i m e . D e t e r m i n e r u n t i m e f r o m T a b l e II ( s e e p r e v i o u s e x a m p l e ) . a. F i n d t h e m o b i l i t y of t h e s m a l l e s t f r a g m e n t at t h e initial s w i t c h t i m e . I n t e r p o l a t e if t h e size o r s w i t c h t i m e is n o t e x p l i c i t l y listed in T a b l e II. b. F i n d t h e m o b i l i t y o f t h e s m a l l e s t f r a g m e n t at t h e final s w i t c h t i m e . c. A v e r a g e t h e m o b i l i t i e s . d. D e t e r m i n e t h e r u n t i m e by d i v i d i n g t h e d e s i r e d d i s t a n c e o f m i g r a t i o n by t h e a v e r a g e m o b i l i t y . T h e d e s i r e d d i s t a n c e is t h r e e - q u a r t e r s t h e l e n g t h o f t h e gel m e a s u r e d f r o m t h e well.
Resolution of DNA Fragments
from 100 to 1000 kb
S e p a r a t i o n of D N A f r a g m e n t s f r o m 100 to 1000 kb c a n b e s t b e a c h i e v e d u s i n g a c o n t o u r - c l a m p e d h o m o g e n e o u s e l e c t r i c field ( C H E F ) e l e c t r o p h o r e -
[1 1]
PULSED-FIELDGEL ELECTROPHORESIS
265
sis system. Such a system can separate a large n u m b e r of D N A samples in straight lines with shorter run times than other systems. S t a n d a r d field inversion offers g o o d resolution in this range but band inversion can occur with this technique. A s y m m e t r i c voltage F I G E can minimize b a n d inversion, but requires m u c h longer run times than C H E F gels. Protocols for separation of D N A from 100 to 1000 kb by C H E F electrophoresis and by asymmetric voltage F I G E are provided.
Materials A g a r o s e gel, 1% (w/v) T B E buffer, 0.5× Markers: Wild-type A ladders (48.5 to m o r e than 1000 kb; Bio-Rad, B R L , F M C , N E B ) or Saccharomyces cerevisiae c h r o m o s o m e s (2002000 kb; B i o - R a d , B R L , FMC, N E B )
Procedures Preferred Method: CHEF 1. The voltage gradient to be used is 6 V/cm, and the reorientation angle is 120 ° . 2. D e t e r m i n e the size range of D N A fragments to be resolved. 3. D e t e r m i n e switch times, using Fig. 4. 100
/ 80 ¸ %(D
60
E c-
40 O3
20
•
0
;
;
I00
:
l
200
:
',
:
,500 400
500
600
700
800
900 I000
Size (kb) FIG. 4. Switch time selection for 100- to 1000-kb DNA by CHEF. Maximum size of fragments that can be resolved at the different switch times is shown. All data represent migration in 1% agarose gels run at 15° in 0.5x TBE with a 120° reorientation angle and a 6-V/cm voltage gradient.
266
ELECTROPHORESIS
[ 11]
a. D e t e r m i n e the initial switch time from Fig. 4, using the smallest fragment of interest. b. D e t e r m i n e the final switch time f r o m Fig. 4, using the largest fragment of interest. c. R a m p linearly f r o m the initial value to the final value. 4. Calculate the run time using Table I I (see example above). a. D e t e r m i n e the initial velocity of the smallest fragment (i.e., its mobility at the initial switch time) f r o m Table II. Interpolate if the size or switch time is not explicitly listed in Table II. b. D e t e r m i n e the final velocity of the smallest f r a g m e n t (i.e., its mobility at the final switch time) from Table II. c. A v e r a g e these two mobility values. d. Divide the desired migration distance (three-quarters the length of the gel m e a s u r e d f r o m the well) of the smallest f r a g m e n t by the average mobility value to obtain the necessary run time.
Alternative Procedure: A V F I G E 1. Use a forward voltage gradient of 2.8 V / c m and a reverse gradient of 0.8 V/cm. 2. D e t e r m i n e the size range of D N A fragments to be resolved. 3. Select switch times using Fig. 5. a. D e t e r m i n e the initial forward switch time from Fig. 5, using the lOO
"~ 80I ~
so
i_=
~
40
ffl
20
0
100
200
300
400
500
600
700
800
900
1000
Size (kb) FIG. 5. Switch time selection for 100- to 1000-kb DNA by AVFIGE. Maximum size of fragments that can be resolved at the different switch times is shown. All data represent migration in 1% agarose gels run at 15° in 0,5X TBE with a 180° reorientation angle, a 2.8-V/cm forward gradient, and a 0.84-V/cm reverse voltage gradient.
[1 1]
PULSED-FIELD GEL ELECTROPHORESIS
267
TABLE 111 MomLJlY or: 100- TO 1000-kb DNA SEPARAteDBY AVFIGE" Switch time Size (kb) 50 10fi 150 200 30() 400
500 600 700
10 sec
25 sec
4(/sec
50 scc
70 sec
100 sec
0.06 0.04
0.13 0.11 0.09 0.08
0.12 0.11 0.10 0.09
0. l 2 0.i1 0.10 0.09
(1.12 0.11 0.11 0.10
0.12 0.11 0.11 0.10
0.04
0.07 ().05
().fin 0.06
0.08 0.07
O. 10 0.09
0.03
(/.04
0.05
{).08
{}.(}3 0.01
0.04 0,03
0.{}7 {},06
0.{}1
(}.06 (I.05 0.04
800 900 100()
"The mobility (cm/hr) is indicated fl)r DNA of a specific size range subjected Io a variety of switch times. All data represent migration in 1% agarose gels run at 15° in 0.5x TBE with a 180° reorientation angle and a 2.8-V/ cm forward gradient and 0.84-V/cm reversc gradient. smallest f r a g m e n t of this range. T h e initial reverse switch time is 1.4 times the forward switch time. b. D e t e r m i n e the final forward switch time from Fig. 5, using the largest f r a g m e n t of this range. T h e final reverse switch time is 1.4 times the forward switch time. c. R a m p linearly from the initial switch time to the final switch time. . Calculate the run time using T a b l e II1 (see e x a m p l e above). a. D e t e r m i n e the initial velocity of the smallest f r a g m e n t (mobility at the initial forward switch time) from T a b l e III. I n t e r p o l a t e if the size or switch time is not explicitly listed in T a b l e 1II. b, D e t e r m i n e the final velocity of the smallest f r a g m e n t (mobility at the final f o r w a r d switch time) from T a b l e 111. C. A v e r a g e these two m o b i l i t y values. d. D i v i d e the desired m i g r a t i o n distance ( t h r e e - q u a r t e r s the length of the gel m e a s u r e d from well) of the smallest f r a g m e n t by the average m o b i l i t y value to o b t a i n the necessary run time:.
R e s o l u t i o n of DNA F r a g m e n t s f r o m 1 to 6 Mb S e p a r a t i o n of m e g a b a s e D N A involves a d d i t i o n a l p r o b l e m s c o m p a r e d to the previously described s e p a r a t i o n of smaller D N A . Most notably,
268
ELECTROPHORESIS
111]
the voltage gradient must be decreased and the switch times increased. Consequently, to keep run times within reasonable time frames other parameters must be altered. The data presented in this section were collected from gels made of SeaKem LE. To decrease gel run times lower E E O agarose, such as BioRad C G A or FMC SeaKem G O L D PFG-agarose DNA, can be used. To ensure proper use of such special agaroses check with the manufacturer for decreases in run time. To reduce further the time required to separate megabase-sized DNA, the concentration of agarose can be decreased. For 1- to 3-Mb DNA, 1% gels provide acceptable separation in a reasonable amount of time while retaining the mechanical strength of the gel. However, the ease of handling a 1% gel can be sacrificed in order to separate larger D N A more quickly by using 0.6-0.7% gels. Megabase D N A moves extremely slowly. Decreasing the reorientation angle to 106° lessens the sideways migration of DNA and thus shortens the run times. For gel boxes lacking the ability to change the reorientation angle, 1- to 6-Mb D N A can be separated using a 120° angle. The gel will run approximately 25% longer with this modification. D N A in the megabase size range is best separated using C H E F setups. Owing to the very long run times required, A V F I G E is not recommended for separation of D N A over 1000 kb. We present two different protocols that employ different voltage gradients depending on the maximum size of the D N A to be separated. Materials" Agarose, 1% (w/v) T A E buffer, 1× Markers: S. cerevisiae chromosomes (0.2-1.9 Mb; Bio-Rad, FMC, BRL, NEB) Candida albicans chromosomes (1-3 Mb; Clontech, Palo Alto, CA) Hansenula wingei chromosomes (1-3.3 Mb; Bio-Rad, BRL) Procedure CHEF Separation of 1- to 3-Mb DNA l. Use a reorientation angle of 106 ° and a voltage gradient of 3 V/cm. 2. Select a size range. 3. Select switch times from Fig. 6. a. Choose the initial switch time based on the smallest sized D N A fragment of interest.
[ 111
PULSED-FIELD GEL ELECTROPHORESIS
269
10
I
g. 8 ¸
-¢_
7i 6-
k--
5-
-~
4-
m
5 2
I I000
, 1250
I 1500
, 1750
Size
I 2000
~ 2250
I 2500
, 2750
3000
(kb)
FI(}. 6. Switch time selection for 1- to 3-Mb D N A by C H E F . M a x i m u m size of fragments that can be resolved at the different switch times is shown. All data represent migration in 0.8% agarose gels run at 15 ° in 1× T A E with a 106 ° reorientation angle and a 3-V/cm voltage gradient.
b. C h o o s e the final switch time based on the largest sized D N A fragment of interest. c. Ramp linearly between the initial and the final switch time. . Determine the run time from Table IV (see example above). T A B L E IV MOBII.IIY OF 1- TO 3-Mb D N A SEPARATISI) BY C H E F " Switch times Size (kb)
2.5 min
4 rain
6 min
8 rain
10 rain
1000 1250 1500 1750 2000 225(/ 2500 2750 3000
0.06
().l 1 0.0g (I.04
0.14 /I.12 0.11 0.10
0.14 0.13 0.12 0.11 0.10 0.08
0.15 0.14 (I.13 0.13 0.12 0. I 1 0. I 1 0.09 0.09
" T h e ,nobility ( c m / h r ) is indicated for D N A of a specific size range subjected to a variety of switch times. All data reprcsent migration in 0.8% agarose gels run at 15° in 1 x T A E with a 106 ° reorientation angle and a 3-V/cm gradient.
270
ELECTROPHORESIS
[11]
a. F i n d the m o b i l i t y of the s m a l l e s t f r a g m e n t at the initial switch time. I n t e r p o l a t e if the size or switch t i m e is not explicitly listed in T a b l e IV. b. F i n d the m o b i l i t y of the s m a l l e s t f r a g m e n t at the final switch time. c. A v e r a g e the mobilities. d. D e t e r m i n e the run time by dividing the d e s i r e d d i s t a n c e of m i g r a tion b y the a v e r a g e mobility. T h e d e s i r e d d i s t a n c e is t h r e e - q u a r t e r s the length of the gel m e a s u r e d from the well.
Materials A g a r o s e , 0.8% (w/v) T A E buffer, 1 × M a r k e r s : S. pombe c h r o m o s o m e s (3.5-5.7 Mb; B i o - R a d ) ; Hansenula wingei c h r o m o s o m e s ( 1 - 3 . 3 Mb; B i o - R a d , B R L )
Procedure CHEF Separation of 3- to 6-Mb DNA l. U s e a r e o r i e n t a t i o n angle of 106 ° and a v o l t a g e g r a d i e n t of 2 V / c m . 2. Select a size range. 3. Select switch t i m e s from Fig. 7.
40-
"~
35.
-5
30.
4--'
25"
20
3000
,
I
,
4-000
I
5000
'
6000
Size (kb)
El(;. 7. Switch time selection for 3- to 6-kb DNA by CHEF. Maximum size of fragments that can be resolved at the different switch times is shown. All data represent migration in ().8~ agarose gels run at 15° in 1× TBE with a 1(}6° reorientation angle and a 2-V/cm voltage gradient.
[1 l ]
271
PULSKD-FI ELI) GEL ELECTROPHORKSIS
TABLE V MOBILrFYOF 3- TO 6-Mb DNA SEPARATEDBY CHEF" Switch time Size (kb)
20 min
25 mm
30 min
35 min
2500 3000 3500 4(100 5000 6000
0.06 0.04
0.06 0.05 0.05
0.06 0.06 0.05 0.04 0.03 0.01
0.07 0.06 0.05 0.05 0.03 0.02
"The mobility (cm/hr) is indicated for DNA of a specific size range subjected to a variety of switch times. All data represent migration in 0.8% agarose gels run at 15° in 1× TAE with a 106° reorientation angle and a 2-V/cm gradient.
a. U s i n g the s m a l l e s t sized D N A f r a g m e n t of interest, c h o o s e the initial switch time. b. U s i n g the largest sized D N A f r a g m e n t of interest, c h o o s e the final switch time. c. R a m p l i n e a r l y b e t w e e n the initial a n d final switch times. 4, D e t e r m i n e the r u n t i m e f r o m T a b l e V (see e x a m p l e a b o v e ) . a. F i n d the m o b i l i t y o f the s m a l l e s t f r a g m e n t at the initial switch time. I n t e r p o l a t e if the size o r switch time is n o t explicitly listed in the table. b. F i n d the m o b i l i t y o f the s m a l l e s t f r a g m e n t at the final switch time. c. A v e r a g e the mobilities. d. D e t e r m i n e the run t i m e by dividing the d e s i r e d d i s t a n c e of m i g r a tion b y the a v e r a g e mobility. T h e d e s i r e d d i s t a n c e is t h r e e - q u a r t e r s the l e n g t h of the gel m e a s u r e d f r o m t h e well.
Future Directions In this c h a p t e r we h a v e s u m m a r i z e d the c u r r e n t o p t i m u m e l e c t r o p h o retic c o n d i t i o n s for the s e p a r a t i o n of D N A f r a g m e n t s f r o m 1 to 6000 kb. H o w e v e r , we b e l i e v e that t h e r e a r e m a n y i m p r o v e m e n t s to be m a d e . F o r e x a m p l e , we still d o not u n d e r s t a n d the basis for the a p p a r e n t limit in sizes that can b e effectively s e p a r a t e d . N o r d o we u n d e r s t a n d fully why h i g h e r v o l t a g e s c a n n o t be used to s e p a r a t e large D N A molecules. M o s t p u b l i c a tions have c o n c e n t r a t e d on P F G s e p a r a t i o n s using o n l y two electric fields.
272
ELECTROPHORESlS
[ 121
In theory, PFG separations involving multiple fields should be even more advantageous. The conditions described in this chapter were determined experimentally from large numbers of gel runs. A more logical approach for the future would be the development of a comprehensive model that can correctly simulate all the observed behavior of large DNA molecules under PFGE. A number of computer models consistent with some aspects of PFG separation have been published. However, no current model can explain all the aspects of the behavior of DNA under various PFG conditions. For example, none of the models explain the dependence of separation on the reorientation angle, the voltage gradient limits for the resolution of large DNA, or the effect of using multiple electric fields. Direct microscopic observation of individual DNA molecules in the gel has provided a better picture of how DNA molecules move in electrophoresis. In the future, real-time optimization of DNA electrophoresis might provide major improvements over existing separation procedures.
[ 12]
Migration of DNA through
By GARY W.
SLATER, PASCAl_ MAYER,
Gels
and GuY DROUIN
Introduction Gel electrophoresis has become a major laboratory tool for separating biological macromolecules. For example, large (megabase) double-stranded DNA (dsDNA) molecules can readily be separated on agarose gels using pulsed-field gel electrophoresis (PFGE), while subkilobase single-stranded DNA (ssDNA) molecules can be sequenced on polyacrylamide gels (see [14], [16], and [17] in this volume, and [17] in Vol. 271 of this series1). These two techniques are essential to map and sequence the human genome. Although great technological advances have been made, the process itself, i.e., the electrophoretic migration of large flexible polyelectrolytes in pseuJ N. Matsubara and S. Terabe, Methods Enzymol. 270, Chap. 14, 1996 (this volume); T. Wehr, M. Zhu, and R. Rodriguez, Methods Enzymol. 270, Chap. 16, 1996 (this volume); L. Kfivfinkovfi, P. Gebauer, and P. Bo6ek, Methods, Enzymol. 270, Chap. 17, 1996 (this volume); W. S. Hancock, A. Apfell, J. Chakel, C. Sounders, T. M'Timkulu, E. Pungor, Jr., and A. W. Guzetta, Methods Enzyrnol. 271, 403 (1996).
M E T H O D S IN E N Z Y M O L O G Y , VOL. 270
Copyright k' 1996 by Academic Press, Inc. All rights of reproduction in any form resolved.