PERSPECTIVE: ANALYTICAL BIOTECHNOLOGY
Pulsed Field Gel Electrophoresis Katheleen Gardiner Eleanor Roosevelt Institute for Cancer Research, 1899 Gaylord Street, Denver, Colorado 80206
The development of pulsed fleld gel electrophoreslr has Increased by 2 orders of magnltude the slze of DNA molecules that can be routinely fractknated and analyzed. Thk limease Is of malor Importance to molecular blology because H slmplHles many prevlously laborious lnvestlgatlons and makes possible many new ones. Its range of appllcatlon spans all organlsms, from bacteria and vlruses to mammals.
INTRODUCTION Much of the rapid progress that is being made in molecular biology today depends upon the ability to separate, size, and visualize DNA molecules. The most common technique for this purpose is that of standard agarose gel electrophoresis. Introduced 20 years ago (11, it employs a constant, uniform electric field and uses the sieving action of the porous agarose matrix to separate double-stranded DNA fragments in a size-dependent manner-the larger the molecule, the more it is retarded in its passage through the pores of the gel. Fragments from 100 to 200 base pairs (bp) up to 50 kilobase pairs (kbp) are routinely separated by this technique (DNA molecules are generally described by their length; 1bp = 660 D, or 1 kbp = 660 kDa). Above 50 kbp, because of the size of the molecules, the sieving action of the gel is lost, and fragments run as a broad, unresolved band with anomalously high mobility. Although larger fragments (up to 750 kbp) have been resolved by this technique (21,the gels used are extremely fragile due to the very low agarose concentrations, and the separation is not adequate for most applications. The effective upper limit then on the size of molecules that can be examined by constant electric fields remains at a low tens of kilobase pairs. The development over the last few years of pulsed field gel electrophoresis has been a major advance for molecular biology: it provides the means for routine separation of fragments exceeding 6000 kbp (3-14). The intent of this perspective is to provide the interested nonmolecular biologist with an overview of the technique of pulsed field electrophoresis. Included are discussions on the basic design of the apparatus, rationalizations of why it works, and some important applications. More detailed reviews and discussions can be found in refs 31 and 67 and in several articles in a special volume of Electrophoresis Journal (Vol. 10, 1989). For those interested in a brief description of standard agarose gel electrophoresis or definitions of some molecular biological terms (such as restriction mapping, nucleic acid hybridization, etc.), these can be found in ref 37. A Focus article on this subject appeared in this journal in 1989 (see ref 68). Need for Large Fragment Resolution. Limiting resolution to fragments of less than 50 kbp did not hamper innumerable kinds and numbers of invaluable experiments (for 0003-2700/91/0363-0658$02.50/0
example, the construction and analysis of genomic and cDNA libraries from all types of organisms), but it did preclude, or at least make extremely laborious, many other investigations. Consider the magnitude of the problem confronting the biologist interested in the genome organization of any of a wide range of organisms. The genome of E. coli (the most widely studied bacterial organism and the workhorse of the recombinant DNA laboratory) is 4600 kbp in size, and that of the medically relevant Herpes virus is about 150 kbp (1416). The yeast S. cerevisiae, responsible for bread and beer, maintains its 17 000-kbp genome in 16 chromosomes that range in size from about 200 to about 2000 kbp (17);the fungus, N . crassa, that molds our bread has seven chromosomes ranging from about 4000 to apparently much greater than 6000 kbp (11). Mammalian parasites such as those causing malaria have a large number of chromosomes, varying in size from strain to strain, all greater than 500 kbp and many greater than 2000 kbp (18). All these are, of course, the simpler organisms. At the higher end in size (and possibly interest) is the mammalian genome, at roughly 3000000 kbp. The human genome consists of 22 pairs of chromosomes plus the X and Y. The smallest of these is chromosome no. 21, containing approximately 50000 kbp of DNA (19). Mammalian genomes have the added distinction of possessing many extremely large genes. Although the dystrophin gene (deletions in which cause Duchenne muscular dystrophy) is so far unique in being greater than 2000 kbp in size (20),many genes are known to exceed 100 kbp, and it is not uncommon for genes to exceed 50 kbp (21, 22).
Clearly, then, the simplest of organisms already presents a genome size that vastly exceeds the limits of standard agarose gel electrophoresis; even the number and size of chromosomes cannot be determined. (The number and approximate sizes of higher eukaryote chromosomes, in particular those of mammals, have been known for quite some time; these chromosomes are large enough to be directly visible in the light microscope.) Furthermore, regardless of the size of an individual gene, investigations into long-range genome organization (e.g., order and physical proximity of genes) generally require resolution of fragments greatly exceeding 50 kbp, if they are to be done in a timely fashion. Many pressing problems of a practical and of a fundamental nature (including the cure and prevention of disease (viral, parasitic, and genetic) and the efficient growth, management, and manipulation of food sources) can benefit from more detailed knowledge of the number, size, and organization of individual genes and the functional significance of their physical proximity. Pulsed field gel electrophoresis offers the increase in maximum size of resolved molecules that is necessary to make such investigations practical. Currently, the largest fragments that can be separated are in excess of 6000 kbp, 2 orders of magnitude larger than that possible by standard electrophoresis. 0 1991 American Chemical Society
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Figure 1. Concept of pulsed field electrophoresis. An agarose gel is represented by the box: the short horizontal lines indicate the wells, where the DNA is loaded. A and B represent two sets of electrodes. When the A electrodes are activated, the DNA is driven downward and to the right, as indicated by the first arrow. When the A electrodes are turned off, the B electrodes are immediately activated. The DNA now moves downward and to the left. The path of the DNA in the center lane with continued alternation of field direction is shown by the
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CONCEPTS OF PULSED FIELD ELECTROPHORESIS Pulsed field gel electrophoresis differs from standard electrophoresis in that it does not use a single constant electric field. Rather, it exposes the DNA to electric fields that regularly change direction throughout the gel run, and it is this alternation in field direction, with or without a change in field strength, that effects the resolution of very large DNA molecules. A schematic diagram of the general concept employed in pulsed field systems is shown in Figure 1. This is based on the original design of the “orthogonal field” device (OFAGE) of Carle and Olson ( 5 ) and has been chosen for illustrative purposes because of its visual and conceptual simplicity. Two electrode arrays, A and B, are shown. When the A electrodes are activated, DNA (being negatively charged because of the backbone of phosphate groups throughout the molecule) migrates downward and to the right; when these electrodes are turned off, the B electrodes are activated, and the DNA now proceeds to migrate downward and to the left. This regular alternation of field direction continues throughout the gel run and causes the DNA to follow a zigzag path, shown by the arrows in the center of the gel, with a net mobility in the downward direction. Actually, pulsed field electrophoresis is rather a misnomer (with its implication of a “dead time” between field applications; this is rarely used) and would more accurately be called alternating field electrophoresis. Mechanism. The development of the theory of alternating field electrophoresis lags behind the practice (for some discussions, see refs 23-25). However, there are both intuitive models and some very insightful experiments that help in understanding the general principles behind the separation. Current theory and experiment for the migration of DNA through an agarose gel suggests that when an electric field is applied, a DNA molecule elongates and stretches out parallel to the field (26,27)1 It can then be moved through successive pores in a snakelike fashion, a process described as reptation. If the field direction changes, however, the molecule must reorient in the new field before it can begin to move through a new pore. If the field direction repeatedly changes, the elongated DNA molecule must repeatedly change directions. Intuitively, smaller molecules are able to reorient, turn comers, and begin moving in a new direction rather quickly. Larger molecules, however, require more time to change directions and therefore have less time to actually migrate, for any given pulse time. Southern et al. (9) presented a variation of this idea, suggesting that when the field direction changes (by an angle >go”), the trailing end of the molecule more rapidly
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extends in the new field and becomes the new leading end. This would explain why early efforts to use orthogonal fields failed (the fields in Figure 1 are only nominally at 90’). Because the trailing end of a long molecule will be farther behind the trailing end of a short molecule, the distance migrated will clearly be size dependent. It is also easy to visualize how, at too short pulse times, large molecules will be unable to advance. Such rationalizations, in particular Southern’s, have bearing on direct observations. Smith et al. (28) and Schwartz and Koval (29) have monitored the movement of large DNA molecules (A-DNA, S. cereuisiae chromosomes, and bacteriophage G DNA (750 kbp)) during pulsed field electrophoresis by staining the DNA with fluorescent dyes and using a microscope-mounted electrophoresis device. Observations support the general intuitive idea outlined above but add interesting detail. Molecules do indeed move through the gel lengthwise, generally in an extended configuration, but go through cycles of elongation and condensation. When the field direction changes, apparently both ends of the molecules may start off in the new direction (28). As a result, they sometimes become hooked around obstacles in the gel, appearing in U or J configurations, until the previously trailing end “wins” and the molecule slides off and continues to advance. DNA molecules exhibit great elasticity, extending to essentially the contour length when hung up, and contracting again when liberated and the trailing end catches up with the leading end. The leading end often appears more fluorescent, suggesting that the DNA bunches up as it is blocked by the gel matrix and looks for new pores, or it appears to fork, as different regions near the leading end move through different pores. These pictures provide a rationale for the effects that have been reported of high field strengths on very large molecules (30-32). Because the DNA is uniformly charged, the electric force is not confined to the ends of the molecule. Strong electric fields could conceivably force multiple segments of a large extended molecule simultaneously through different pores, preventing any net migration and causing considerable entanglement with each successive field switch. This “trapping” can account for the effects of high field strengths on the largest chromosomes of S. cereuisiae and can also explain why the large chromosomes of S. pombe do not enter the gel unless very low field strengths are used (see below). Pulsed Field Apparatus. Schwartz et al. introduced the concept of field alternation in 1983 by demonstrating its effectiveness in separating the chromosomes of the yeast S. cereuisiae (3). Within a year, two designs were proposed, using either two sets of diode arrays or two sets of linear electrodes (similar to that shown in Figure 1)to provide the alternating fields ( 4 , 5 ) . Both separated molecules as large as 1500 kbp. The major, and important, drawback to these designs, however, was that the electrode geometries produced nonhomogeneous electric fields (as can be inferred by inspection of Figure l),causing lane-to-lane variations in the speed and trajectory of DNA fragments. For many applications, these distortions in the path severely hinder interpretation of band patterns. As a result, many different pulsed field designs have been developed in attempts to generate uniform fields. (A note on nomenclature: acronyms abound. They are used to refer to some specific aspect of the particular pulsed field apparatus. Commercial development has added additional catchy names, e.g., TAFE, Transverse Alternating Field Electrophoresis). These variations on a theme are illustrated in Figure 2 and discussed here, in chronological order of development. (a) Vertical Pulsed Field System (Now Called TAFE) (6, 7,33). This apparatus is essentially a three-dimensional model of the original Carle and Olson OFAGE (orthogonal
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Flgure 2. Pulsed field configurations producing electric fields that are uniform in all lanes of the gel. (a) The vertical pulsed field apparatus or transverse alternating flelds (TAFE). The gel stands vertically in the center of a large fish tank like box, supported largely by the buffer and held in place by two thin Plexiglas strips at the sides. Electrode pairs, A and B, alternate in activation, as in Figure 1. The resultant path of the DNA is down the center of the gel, as indicated by the arrows. (b) Field inversion. A single pair of electrodes is activated alternately in the A orientation (DNA moves downward) and the B orientation (DNA moves upward). (c) Rotating apparatus. A single pair of electrodes produces a constant electric fdd. The gel is rotated between positions A and B. (d) Contour clamp homogeneous electric field apparatus (CHEF). A hexagonal array of point electrodes in a resistor circuit provides control of the electric field at all points around the gel. Arrow indicates net migration.
field gel electrophoresis; ref 5, and Figure 1)and resulted from the observation that only DNA in the center of the OFAGE gel migrated straight down the gel. In the vertical apparatus, the gel stands upright between two linear electrode pairs and is supported by two thin plastic strips and the buoyancy of the buffer. DNA runs in a straight path in all lanes because the field is uniform across the gel; quite sharp bands are obtained, relating to the field gradient down the gel. The angle between the alternate fields is l l O o a t the wells but is much greater further down the gel. (b) F i e l d I n v e r s i o n ( F I G E ) ( 8 , 3 4 ) . This is the simplest of the pulsed field designs, employing a single pair of electrodes and a standard submarine agarose gel electrophoresis box. As the name implies, during the gel run, the field regularly inverts, first driving the DNA out of the wells and then back toward the wells. Thus, the angle between the fields is 180O. Clearly, either the forward pulse time or the forward field must be greater than the reverse for net forward migration of the DNA. This system works admirably for fragments < 600 kbp but sometimes suffers from band inversion, where larger fragments run ahead of intermediately sized fragments. To avoid these anomalies, "ramping" is frequently used the forward and reverse pulse times (and/or fields) are increased, either gradually or in a step fashion, during the run. (c) Rotating Gels ( 9 , 3 5 ) . Instead of alternating the field direction, the gel is mounted on a rotating platform that then alternates between two orientations (120O apart) within a constant electric field. From the frame of reference of the DNA, this is the same as changing the field direction. ( d ) C o n t o u r C l a m p e d Homogeneous E l e c t r i c F i e l d
(CHEF) D e v i c e ( 1 0 , 11 ). This widely used device uses a hexagonal array of point electrodes in a voltage divider circuit to produce homogeneous fields (oriented a t 120') approxi-
mating that of pairs of infinitely long parallel electrodes. This useful application of electrostatics results in fields that are uniform across all lanes of the gel. Clark et al. modified the CHEF system to use computer-controlled digital/analog converters at each electrode (12, 31). This PACE system retains the hexagonal array and can generate an essentially unlimited variety of field strengths and angles. The apparatus is expensive, and so far, the versatility it provides has not been shown to bring any real practical advantage to pulsed field analysis. All these designs, although differing in kind, number, and arrangement of electrodes, have eliminated major distortions and are capable of resolving fragments exceeding 6000 kpb in size. Resolution in all systems also depends upon the same set of electrophoretic parameters. These include pulse time, field strength, temperature, buffer composition, type and concentration of agarose, and an obtuse angle between the fields. In practice, all of these variables except the pulse time and field strength are generally kept constant, and the size range to be resolved is manipulated by increasing the pulse time to separate larger molecules and decreasing it for smaller molecules. The effects of the two important variables, pulse time and field strength, are discussed in some detail here. E f f e c t o f P u l s e T i m e . The efficacy of field alternation is illustrated in Figure 3, with six different DNA samples. Fragments in the A-Hind111 digest range in size from 2 to 23 kbp; in the X ladder, the bands are concatamers of 48 kbp; S. cereuisiae chromosomes range from 200 to 1600 kbp; S. pombe chromosomes are 3500, 4700, and 5600 kbp; human DNA digested with EcoRI gives fragments averaging about 3 kbp, and with BssHII, about 500 kbp. In panel a, these DNAs are electrophoresed under standard constant field conditions. Only the bands in the X digest and most EcoRI fragments are resolved. The other DNA samples migrate in broad diffuse bands (called compression zones, in particular with reference to pulsed field analysis) with anomalously high mobilities. The effect of field alternation, even at a short (4-9) pulse is dramatic, as shown in panel b. Fragments as large as 150 kbp are clearly well resolved, as shown in the X ladder and S. cereuisiae lanes, and some resolution of BssHII fragments has appeared. Increasing the pulse time to 15 and 60 s (panels c and d) resolves progressively larger fragments to a maximum of approximately 1600 kbp, as measured in the S. cereuisiae DNA. The 60-s resolution has essentially eliminated the compression zone and also gives a better indication of the average size of BssHII fragments. A uniform smear is seen throughout the lane; much material extends above the largest yeast band and also remains in the wells. That this average size is vastly greater than that of the EcoRI fragments has made BssHII (and other enzymes of its class) very important in the mapping of mammalian genomes (see below). Figure 3b-d illustrates a feature common to all pulsed field designs-for given voltage, temperature, and buffer conditions, the longer the pulse time, the larger the fragments that can be resolved. Thus, by judicious choice of pulse time, optimum resolution of different size classes can be controlled. Figure 4 shows the relationship between molecular size and mobility for the gel conditions of Figures 3b-d and 6b (see below). In each case, a t the shorter pulse times, the relationship is linear through a range where excellent calculation of unknown fragment sizes would be possible. This contrasts with standard electrophoresis where mobility of DNA molecules is inversely proportional to the log molecular weight. At the 60-s pulse time, the relationship here is linear above and below approximately 600 kbp. There is a slight inflection at 600 kbp, corresponding to a slight band compression that can be seen in the gel. This effect is more striking in other systems (311.
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Figure 3. Effect of pulse time. Gels are 0.8% agarose and have been stained with ethidium bromide. Gels in b-d were run in the TAFE system (Beckman Instruments); V/cm indicates the downward component of the total field. I n all cases: lanes 1 and 7, phage A-DNA digested with Hind111 to produce fragments of 1.9, 2.05, 4.6, 6.4, 9.6, and 23 kbp; lanes 2 and 3, human genomic DNA digested with EcoRI and BssHII, respectively; lane 4, A-DNA ligated to form concatamers of the basic 48-kbp unit (therefore, the sizes of the bands are: 48, 96, 144, etc., kbp); lane 5, chromosomes from S. cerevisiae strain yPH149 (courtesy of P. Heiter), sizes are determined by comparison with the positions of the bands of the A concatamers; lane 6, chromosomes from S. pombe. (a) Standard constant field electrophoresis: 16 h, 2.25 V/cm. (b) 14 h, 4-s pulse time, 6 V/cm. (c) 14 h, 15-s pulse time, 6 V/cm. (d) 18 h, 60-s pulse time, 6 V/cm.
In no case in Figure 3 were the chromosomes of S. pombe resolved. Conditions to resolve fragments > 1600 kbp are not merely modest extensions of those to resolve < 1600 kbp. First, as expected from the results above, a longer pulse time must be used. However, because the DNA is so very large, the voltage must also be lowered to prevent trapping. This in turn requires a further increase in the pulse time. Thus, to achieve resolution in the megabase range, long slow gel runs are required as shown in Figure 5-several days, a t roughly 1-2 V/cm, with a 30-min pulse time. These conditions are effective, but the long run times are a problem when much data are required (see below). A second feature of Figure 5 is the lack of resolution of fragments of less than 1100 kbp.
Fragments of 2-1100 kbp all migrate in a broad diffuse band, reminiscent of standard constant field electrophoresis. Indeed, long pulse times mimic constant electric fields from the viewpoint of smaller DNA fragments. There are conditions that separate the full range from 50 to 6000 kbp on a single gel (9,12,32),but these generally sacrifice optimum resolution of all size classes. Effect of Field Strength. Increasing the field strength has mixed results. Compare Figure 6a with Figure 3c. Both gels were run with a 15-s pulse time, but the former, run a t 8 V/cm rather than 6, resolves larger fragments, as monitored by the number of bands visible in the A and yeast lanes. In contrast, resolution with a 60-s pulse time at 8 V/cm results
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Figure 5. Resolution of fragments > 1600 kbp. The gel is 0.55% agarose; buffer concentration 2 times that used in Figure 3. Electrophoresis conditions: 12 h, 30-min pulse time, 1.25 V/cm; followed by 60 h, 20-min pulse time, 1.2 V/cm. C: chromosomes from S. cerevisiae. P: chromosomes from S. pombe. 1-4: human DNA digested with NotI, BssHII, MIuI, and NNI restriction enzymes. Note that here the molecular weights are given in millions of base pairs, mbp.
in the nonappearance of some of the largest yeast bands (data not shown). This effect is postulated to be due to the trapping of long DNA molecules in multiple pores of the agarose (see above) and implies there are upper limits on the speed with which pulsed field separations can be carried out. This effect is also likely to be responsible for the conditions required for the separation of the chromosomes of S. pombe. Field strengths suitable for fragments less than 1600 kbp are too high for the separation of the fragments in the megabase ranges. Turmel et al. (36),however, may have ameliorated this problem. They have presented evidence that insertion of spikes of reverse polarity (
