Gel Electrophoresis of DNA
W
ith the advent of gene-sequencing techniques, there has been an increasing demand for analysis and separation of large DNA fragments. One potential technique for this purpose is gel electrophoresis. Unfortunately, for large molecules, the method has not had the resolving power to be of use. However, the introduction of pulsedfield gel electrophoresis (PFGE) has dramatically extended the range of sizes of DNA molecules that can be separated. Here we compare the use of constant and pulsed fields in electrophoretic separations and summarize the recent experiments by Steven Smith and co-workers that describe the visualization of individual DNA molecules during these separations. Conventional agarose gel electrophoresis of DNA is a sieving technique. Separation of molecules smaller than the maximum pore size of the matrix occurs because the smaller the molecule, the larger the fraction of pores it can enter. The size limit for sieving of DNA is a few hundred base pairs (bp) as DNA lengths approach the size of the pores. Once DNA molecules become larger than the pores of the agarose, they have to distort to enter the gel and the sieving power of the gel becomes unimportant. Instead, these larger molecules move through the agarose matrix with a mobility that is dependent on both the electric field strength and the molecular chain length. Resolution is predicted to decrease with the square of the molecular size. Furthermore, at high constant field strengths, electrical orientation effects further reduce the size dependence of mobility and DNA molecules with sizes above approximately 20,000 base pairs (20 kilobase pairs, 20 kb) do not resolve well in conventional agarose gel electrophoresis. In 1983 Schwartz et al. (1) described a technique in which the DNA is subjected alternately to two electrical fields at different angles for a time called the pulse time. The molecules
presumably change direction prior to net translational motion each time the field is switched. Larger molecules take longer to change direction and have less time to move during each pulse, so they migrate more slowly than the smaller molecules. Use of pulsed fields has led to the separation of DNA ranging from 10 to 10,000 kb, a remarkable increase of resolving power over conventional gel electrophoresis. The electrophoretic mobility of DNA is expressed as the distance moved per unit field in unit time. In pulsed-field electrophoresis, only a nominal mobility can be estimated because DNA follows a zigzag path. Therefore the measured distance is less than that actually migrated. In PFGE, the relative or normalized mobility is the relative distance traveled per unit time in the average field direction when
FOCUS compared with a standard size DNA marker. With short pulse times, the molecules probably migrate directly along the diagonal in response to an average field effect, whereas at intermediate pulse times, no net translational motion may occur for a significant proportion of the pulse, and so the elapsed time is an overestimate of the migration time. The size of the largest resolved DNA species increases with increasing pulse time. At a given pulse time, an increase in the electric field should decrease the reorientation time and lead to the resolution of larger fragments. This has indeed been found to be true. The resolution of pulsed-field gel electrophoresis is dramatically affected by the number and configuration of the electrodes used, because they alter the shape of the applied electrical fields. The initial description of the technique, referred to as pulsed-field gel electrophoresis, involved any separation in which the electric fields were
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FOCUS alternated. Subsequently, numerous names have been given to pulsed-field techniques (2,3). Because each of these is simply a variation of the original electrode geometry, it would, perhaps, be more useful to describe the reorien tation angle that was used and whether the fields were homogeneous or inhomogeneous. These names include OFAGE (orthogonal field alternating gel electrophoresis), FIGE (field inver sion gel electrophoresis), TAFE (trans verse alternating field electrophoresis), CHEF (contour-clamped homoge neous electric field), PHOGE (pulsed homogeneous orthogonal field gel elec trophoresis), RFGE (rotating field gel electrophoresis), and PACE (program mable, autonomously controlled elec trodes). Theoretical models
Although use of pulsed fields has led to substantive improvements in the sepa ration of much larger DNA molecules, the underlying motion of the molecules in both constant and pulsed fields has remained somewhat of a mystery. Two theoretical models have been proposed to describe the motion in constant elec tric fields: a "biased reptation" model (4-6) and a direct simulation incorpo rating many degrees of freedom of the DNA (7). Biased reptation makes use of the concept of a segmented tube through which a chain passes. A very long DNA strand snakes its way through gel pores with one end leading and the rest of the molecule following the same path. The path chosen by the head is assumed to be a semirandom walk, biased by the electric field force. The idealized mo tion, referred to as reptation, has been biased by the electric field. The repta tion model saw early success when ap plied to diffusion-controlled processes in the absence of any electric field. Sev eral groups (4, 8) extended this theory to the case of a constant external field. Although several assumptions were made and the extension was not straightforward, experimental data for the constant field case appeared to support the biased reptation-tube the ory. The direct simulation of Deutsch (7) represents the DNA as a set of charged beads connected by freely hinged links and the gel as a two-dimensional lattice of obstacles. There is a short-range re pulsive force between each obstacle and all the beads on the chain that completely prevents the chain from crossing an obstacle. Deutsch's results are shown for DNA in a fairly low con stant field (Figure 1). A chain of DNA moves downward in the direction of the electric field. Panel A shows the chain
Figure 1. The evolution of a DNA molecule in a gel. The parameters in this run were qEllkBT = 0.604, where q is the charge on the bead, Ε is the electric field, / is the persistence length, kB is the Boltzmann's constant, and Γ is the absolute temperature. The relative times for each frame shown are A, 260; B, 270; C, 280; D, 290; E, 330; F, 340; G, 350; and H, 360. The electric field is vertically downward. (Adapted with permission from Reference 7; copyright 1988, AAAS.)
has "bunched u p " at the leading edge. As time progresses, a hairpin forms (panel B) that grows in size. The loca tion of the top of the hairpin remains fixed in space, but the entire chain has slid down, so that the location of the hairpin relative to the rest of the chain has moved up. As time progresses, the hairpin envelops the trailing edge of the chain (panel C). The effect of this is to drastically alter the path the upper half of the chain takes. It now slides downward along the path of the hair pin, causing the chain to enter an in verted " U " shape (panel D). Eventual ly one end exhibits a greater mobility (panels Ε & F), and the chain unwinds around the obstacles. It ends up almost fully extended (panel G). In panel H, the chain ends up looking as it did in panel A but is further downstream. The cycle is ready to be repeated over and over again. The lower the electric field, the looser the coiling and the more open the reorientation. This kind of motion differs from the biased reptation model in that the DNA does not appear confined to a tube and forms hairpins. The differ ence between these two models is larg est for pulsed field electrophoresis, where hairpin formation is crucial in predicting the chain motion (9). Deutch's direct simulation is able to reproduce several features of pulse
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field experiments not explainable by the biased reptation model (7). The first direct experimental evi dence concerning molecular orienta tion during both constant and pulsedfield gel electrophoresis was described earlier this year by S. B. Smith, P. K. Aldridge, and J. B. Callis at the Univer sity of Washington, Seattle (10). Their approach was based on the work of M. Yanagida and co-workers (11), who were the first to observe individual DNA molecules that had been stained with a dye under a fluorescent micro scope. Smith and co-workers used ethidium bromide to stain DNA. The stained λ-phage DNA (48.5 kb) was premixed into molten agarose, and then thin layers (10-20 μπι) were cast under a cover slip onto a microscope slide. The sample was excited in green light and viewed in red. An image in tensifier and vidicon camera recorded the image with infrared light from the excitation source blocked. The record ed video images were processed and photographed from the monitor screen. Fine platinum wires placed adjacent to the cover slip formed the electrophore sis electrodes. In the absence of an elec tric field, the DNA molecules embed ded in the agarose were globular in shape and appeared to be quite mo tionless. However, when an electric field was applied (6 V/cm), the series of
Figure 3. Yeast DNA changing direction during OFAGE.
Figure 2. Time sequence of a molecule of λ-phage DNA traveling toward the left in 1.5% agarose with a field of 8 V / c m . The frames are taken 0.4 s apart. The contour length of unstained λ-phage DNA is 15 μιτι, but the inter calating dye lengthens it. Scale bar is 10 μην (Adapted with permission from Reference 10; copyright 1989, AAAS.)
images showed the λ-phage DNA in the agarose migrating in a slow constrained motion, winding through the invisible pores in the agarose, stretching in the direction of the electric field, and con tracting as the tails caught up with the heads (see Figure 2). The molecules of ten encountered obstacles such that both ends moved down field simulta neously, forming a " U " shape. Increas ing the field strength caused the DNA to become more extended and better aligned. "How remarkably similar these observations were to the simula tion of Deutsch," comments Smith. According to Smith, experiments with very long DNA chains such as yeast chromosomal DNA (Saccharomyces cerevisiae, 200-1500 kb) indi cate that the longer chain DNA aligned more exactly in the electric field than did the λ-phage DNA. To avoid any shearing of these DNA molecules, solid agarose beads containing yeast cells treated to remove the cell walls and protein were mixed with the molten agarose containing ethidium bromide and cast into a thin gel. When the elec tric field was applied, one could visual ize the intact chromosomal DNA mole cules streaming into the bulk of the gel from the beads. When the electric field was removed, these molecules con tracted and filled the pores in the aga rose, making the gel voids and larger pores visible for the first time. During pulsed-field gel electropho
resis, size separation appears to be de pendent on the way that molecules re align when the direction of the electric field is changed. Smith and co-workers studied the electrophoretic separation of stained yeast DNA in an electric field that was suddenly rotated 120°. (A CHEF system that produced a ho mogeneous OFAGE field was used.) The molecules reversed their direc tions, according to Smith, and the tails became the heads. These observations (Figure 3) correspond quite well with the switchback model of Southern (12), who predicted this sort of motion and showed how the continual tacking of the field would lead to the resolution of molecules based on chain length. At the same time, microscopy showed the ad ditional affect of alternating chain elongation and contraction was also important in the separation process. Several computer models have re cently been constructed using the rep tation tube theory or a variation of it (10, 13, 14). Tube-type models lack some of the generality of Deutsch's model. The motion is more con strained, and it is not possible for a loop to expand out of the center of the mole cule in a tube model. However, tube models run faster on a computer, re quiring fewer calculations. This is im portant for megabase-sized molecules. Comparison of computer model graph ics with microscopic motion pictures of actual DNA molecules will give a quick
A contour-clamped homogeneous electric field system was constructed on a microscope slide. A computer with a digital-to-analog converter and multiplexer controlled the potential on 16 elec trodes adjacent to the edges of the square cover slip. The direction and magnitude of the electric field were controlled with a joystick or cycled automatically. The electric field of 4 V/cm had changed direction by 120° 5 s before this picture was recorded. Scale bar is 10 μ
