Pulsed Field Electrophoresis for Separation of Large DNA

Barbara Joppa, Research Technician
Samantha Li, Research Technician
Scott Cole, Research Technician
Sean Gallagher, Director
HSI Laboratories, Hoefer Scientific Instruments
San Francisco, CA

Manipulating and analyzing DNA are fundamentals in the field of molecular biology. Indeed, separating complex mixtures of DNA into different sized fragments by electrophoresis was a well established technique by the early 1970's.

Typically, DNA was isolated intact and then treated with restriction enzymes to generate pieces small enough to resolve by electrophoresis in agarose or acrylamide. Although this procedure still forms the core of DNA separation and analysis in today's laboratories, the rules of the separation have changed.

In 1984, Schwartz and Cantor described pulsed field gel electrophoresis (PFGE), introducing a new way to separate DNA. In particular, PFGE resolved extremely large DNA for the first time, raising the upper size limit of DNA separation in agarose from 30-50 kb to well over 10 Mb (10,000 kb).

After this initial report, a succession of papers described new and improved instrumentation and methods. As a result, routine procedures and several commercial pulsed field units are currently available. Now, instead of cloning a large number of small fragments of DNA, PFGE permits cloning and analysis of a smaller number of very large pieces of a genome.

Applications

Applications of PFGE are numerous and diverse (Gemmill, 1991; Birren and Lai, 1990, 1993; and Van Daelen and Zabel, 1991). These include cloning large plant DNA using yeast artificial chromosomes (YAC's) (Ecker, 1990; see also Probe, Vol. 1, No. 1/2; and Butler, et al., 1992) and P1 cloning vectors (see Probe, Vol. 1, No. 3/4); identifying restriction fragment length polymorphisms (RFLP's) and construction of physical maps; detecting in vivo chromosome breakage and degradation (Elia, et al., 1991); and determining the number and size of chromosomes ("electrophoretic karyotype") from yeasts, fungi, and parasites such as Leishmania, Plasmodium, and Trypanosoma.

Theory

Although the theory of pulsed field electrophoresis is a matter of debate, qualitative statements can be made about the movement of DNA in agarose gels during PFGE. During continuous field electrophoresis, DNA above 30-50 kb migrates with the same mobility regardless of size. This is seen in a gel as a single large diffuse band. If, however, the DNA is forced to change direction during electrophoresis, different sized fragments within this diffuse band begin to separate from each other.

With each reorientation of the electric field relative to the gel, smaller sized DNA will begin moving in the new direction more quickly than the larger DNA. Thus, the larger DNA lags behind, providing a separation from the smaller DNA.

Currently, there are three models that attempt to describe the behavior of DNA during PFGE (reviewed by Chu, 1990), the biased reptation model (BRM), the chain model, and, most recently, the bag model (Chu, 1990, 1991).

Instrumentation

Although many types of PFGE instrumentation are available (fig. 1), they generally fall into two categories. The simplest equipment is designed for field inversion gel electrophoresis (FIGE) (Carle, et al., 1986). FIGE works by periodically inverting the polarity of the electrodes during electrophoresis. Because FIGE subjects DNA to a 180ø reorientation, the DNA spends a certain amount of time moving backwards. Only an electrical field switching module is needed; any standard vertical or horizontal gel box that has temperature control can be used to run the gel.

Although more complex in its approach, zero integrated field electrophoresis (ZIFE) (Turmel, et. al, 1990) also falls into this first category. Compared with simple FIGE, ZIFE is very slow. However, ZIFE is capable of resolving larger DNA and giving a larger useful portion of the gel.

The other category contains instruments that reorient the DNA at smaller oblique angle, generally between 96 and 120ø. This causes DNA to always move forward in a zigzag pattern down the gel. For a similar size range under optimal conditions, these separations are faster, resolve a wider size range, and give a larger useful portion of the gel compared to FIGE.

Figure 1:Electrode configuration of commonly
used pulsed field gel electrophpresis units.

Contour-clamped homogeneous electric field (CHEF) (Chu, et al., 1986, 1990); transverse alternating field electrophoresis (TAFE) (Gardiner, et al., 1986) and its relative ST/RIDEtm (Stratagene); and rotating gel electrophoresis (RGE) (Southern, et al., 1987; Anand and Southern, 1990; Gemmill, 1991; and Serwer and Dunn, 1990) are all examples of commonly used transverse angle reorientation techniques for which instrumentation is available. In a further elaboration of the above procedures, Lai and coworkers developed the programmable autonomously controlled electrophoresis (PACE) unit which allows complete control over reorientation angle, voltage, and switch time (Clark, et al., 1988; and Birren, et al., 1989). In contrast with FIGE, these systems require both a special gel box with a specific electrode and gel geometry, and the associated electronic control for switching and programming the electrophoresis run.

Ideally, the DNA should separate in straight lanes to simplify lane-to-lane comparisons. The original pulsed-field systems used inhomogeneous electric fields that did not produce straight lanes, making interpretation of gels difficult (Schwartz and Cantor, 1984). Again, the simplest approach to straight lanes is FIGE, which uses parallel electrodes to assure a homogeneous electric field.

Although extremely useful for separating relatively small DNA, 4- 1,000 kb (fig. 2), FIGE's reorientation angle of 180ø results in a separation range most useful under 2,000 kb. Furthermore, like other PFGE techniques, FIGE has mobility inversions in which larger DNA can move ahead of smaller DNA during electrophoresis.

Figure 2. Increased separation of the 20-50 kb range with field inversion gel electrophoresis (FIGE). Run conditions: 230 V, 7.9 V/cm, 16 hrs., 50 msec. pulse, forward:reverse pulse ratio = 2.5:1, 1% GTG agarose, 0.5X TBE, 10 C.a) 1 kb ladder, 0.5-12 kb; b) Lambda/Hind III, 0.5-23 kb; and c) High molecular weight markers, 8.3-48.5 kb.

Ramping, where the reorientation pulse length is constantly increased during separation, will minimize inversions. This capability is included in most commercial instrumentation.

Increasing both the separation range and the resolution of large DNA requires smaller reorientation angles, generally 96-140ø, with 120ø most common. Smaller angles (e.g., 100ø) increase the mobility of the DNA generally without seriously affecting resolution. The lower limit is approximately 96ø. Below this, separation is seriously compromised.

TAFE and ST/RIDEtm use a complicated geometry between the electrodes and a vertically placed gel to get straight lanes. CHEF and RGE maintain a homogeneous electric field in combination with a horizontal gel. CHEF changes the direction of the electric field electronically to reorient the DNA by changing the polarity of an electrode array. With RGE the electric field is fixed; to move the DNA in a new direction, the gel simply rotates.

Rotating Gel Electrophoresis

RGE is one of the most recent commercial introductions of pulsed field equipment and combines variable angles with a homogeneous electric field (figs. 3 and 4) (Southern, et al., 1987; Anand and Southern, 1990; Serwer and Dunn, 1990; and Gemmill, 1991). The electrodes are positioned along opposite sides of the buffer chamber with their polarity fixed. Briefly, the gel is cast on a circular running plate and then placed in the buffer chamber. The gel is coupled to a magnetic drive beneath the buffer chamber to eliminate the possibility of leakage that a direct connection might cause.

To force the migrating DNA to a new direction, the magnetic drive simply rotates the gel to the new angle. Because the reorientation angle of the DNA is determined by a straightforward mechanical coupling, RGE offers a lot of flexibility at a reduced cost. Voltage, angle, and pulse times are varied with the program stored into memory of the unit.

Sample Preparation

Along with the ability to separate large DNA came the need for new sample preparation and handling procedures. Large DNA (e.g., yeast chromosomes) is easily sheared and also difficult to pipet due to its high viscosity. The solution to this problem is to first embed the bacteria or yeast in agarose plugs and then treat the plugs with enzymes to digest away the cell wall and proteins, thus leaving the naked DNA undamaged in the agarose. The plugs then are cut to size, treated with restriction enzymes if necessary, loaded in the sample well, and sealed into place with agarose.

Separation Parameters

Several parameters act in concert during PFGE (Southern, et al., 1987; Anand and Southern, 1990; Birren, 1989; and Gemmill, 1991). These will be discussed briefly below as they relate to transverse field instruments such as RGE. The minimum amount of information needed to repeat a separation should include a short description of the pulsed field instrumentation used; applied voltage and field strength (e.g., 180 V at 5.3 V/cm); pulse length (e.g., 87 seconds); reorientation angle (e.g., 120ø); the buffer (0.5X TBE); the agarose type and concentration (SeaKem Gold, 1.1%); the buffer chamber temperature (e.g., 10ø); the type of standards (Clontech S. cerevisiae); and, if possible, the amount of DNA loaded. Although the data listed above is necessary to faithfully reproduce a separation, the information supplied in publications is rarely this complete.

Figure 3. Rotating gel electrophoresis (RGE) separation Saccharomyces cercevisiae chromosomes (245-2190 kb). Run conditions: 180 V, 5.1 V/cm, 34 hrs., 120 angle, 60-120 sec. pulse ramp, 0.5X TBE, 1.2% GTG agarose, 10 C. Two combs were used on the same gel to load 32 samples, a maximum of 72 are possible

Separation Area

Most PFGE systems separate DNA over a relatively small area, limiting the resolution of complex samples. RGE is an exception to this, with a useful separation distance up to 20 cm and a maximum gel size of 18 x 20 cm.

Field Strength

The field strength has a profound effect on pulsed field separations and is a compromise between separation time and resolution of a particular size class. Four to six volts/cm is generally required for resolving DNA up to 2000 kb (e.g., S. cerevisiae chromosomes) in a reasonable period of time (e.g., 1-2 days). However, these field strengths trap and immobilize even bigger DNA in the agarose matrix, and DNA > 3000 kb requires 2 V/cm or less for separation.

Pulse Time

Pulse time primarily changes the size range of separation. Longer pulse times lead to separation of larger DNA. For example, at 5.4 V/cm, the 1.6 Mb and 2.2 Mb chromosomes from S. cerevisiae separate as a single band with 90-second pulse length. Increasing the pulse length to 120 seconds resolves these into two bands (Gemmill, 1991).

Reorientation Angle

Any angle between 96 and 165ø produces roughly equivalent separation (Birren, et al., 1988; and Gemmill, 1991). The smaller the angle, however, the faster the DNA mobility. And for separating extremely large DNA, 96 to 105ø is almost a requirement to get a good separation in the shortest possible time.

Buffers

Two buffers are commonly employed for PFGE--TAE and TBE (1x TAE is 40 mM Tris acetate, 1 mM EDTA, pH 8.0; 1x TBE is 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0). Both are used at a relatively low ionic strength to prevent heating and carry the designations of either 0.25 and 0.5x to indicate the dilution relative to the standard concentration. An added benefit to low ionic strength buffers is an increase in DNA mobility. For example, while using RGE to compare various buffers and agaroses, White (1992) found that lowering both TAE and TBE to 0.25 x gave the maximum mobility (40-50% faster than 1x). Below 0.25x, the DNA mobility dropped off.

Agarose

The type of agarose also affects DNA separation, with the fastest mobilities and best resolution achieved in gels made of low electroendosmosis (EEO) agarose (Birren, et al., 1989; and White, 1992). Although most standard electrophoresis grades of agarose are suitable for PFGE (e.g., SeaKem GTG), agarose with minimal EEO will provide a faster separation. Several low EEO "pulsed field grades" are available, including FastLane and Gold (FMC BioProducts), and Megarose (Clontech).

Figure 4. Rotating gel electrophoresis (RGE) separation of 3000 to 6000 kb DNA Schizosaccharomyces pombe chromosomes. Run conditions: 50 V, 1.4 V/cm, 100 hrs, 100 angle, concatamated multiple runs: 2500 sec./50hrs, 3000 sec./50hrs, 0.5X TBE, 0.8% megarose (Clontech), 10 C.

The concentration of agarose affects both the resolution and mobility of the DNA (Birren, et al., 1989; and White, 1992). Higher concentrations of agarose yield sharper, but slower moving bands. And the typical concentrations used (0.8-1.2%) represent a tradeoff between speed and resolution. High percentages of low EEO agarose may improve resolution without sacrificing the speed of separation (White, 1992).

Temperature

Because DNA mobility also depends on the separation temperature, the temperature must be constant both during and between runs. Although higher temperatures increase DNA mobility, it does so at the expense of resolution (Birren, et al., 1989; and Gemmill, 1991).

Conclusion

Since its introduction over 8 years ago, PFGE has evolved into a routine, pragmatic technique for molecular biologists. This is reflected in the present availability of methods chapters and manuals (e.g., Birren and Lai, 1990, 1993; Anand and Southern, 1990; Van Daelen and Zabel, 1991).

What does the future hold? Possibilities include using a new or improved separation material, and going beyond the current size limit of @ 10 Mb. Anecdotal reports suggest separations in the range of 20 Mb or larger are possible, which would further simplify the complex task of genome mapping.

For more information on Hoefer's HulaGeltm rotating gel electrophoresis unit, contact Technical Services at Hoefer Scientific Instruments at (415) 282-2307 or 800-227-4750.

References

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Birren, B., and Lai, E. (1993). Pulsed field electrophoresis: A practical guide. Academic Press, San Diego.

Birren, B., and Lai, E., eds. (1990). "Methods: A Companion to Methods of Enzymology." Pulsed-Field Electrophoresis. Vol. 1, Number 2. Academic Press, San Diego.

Birren, B., Hood, L., and Lai, E. (1989). "Pulsed field gel electrophoresis: Studies of DNA migration made with the programmable, autonomously-controlled electrode electrophoresis system." Electrophoresis 10, pp. 302-309.

Birren, B.W., Lai, E., Clark, S.M., Hood, L., and Simon, M.I. (1988). "Optimized conditions for pulsed field gel electrophoretic separations of DNA." Nucleic Acids Research 16, pp. 7563-7582.

Butler, R., Ogilvie, D.J., Elvin, P., Riley, J.H., Finniear, R.S., Slynn, G., Morten, J.E.N., Markham, A.F., and Anand, R. (1992). Walking, cloning, and mapping with yeast artificial chromosomes: a contig encompassing D21S13 and D21S16.

Carle, G.F., Frank, M., and Olson, M.V. (1986). "Electrophoretic separation of large DNA molecules by periodic inversion of the electric field." Science 232, pp. 65-68.

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Chu, G. (1990). Pulsed-field electrophoresis: theory and practice. In Methods: A Companion to Methods of Enzymology. Pulsed-Field Electrophoresis (B. Birren and E. Lai, eds.), Vol. 1, No. 2, pp. 129-142. Academic Press, San Diego.

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Ecker, J. (1990). PFGE and YAC analysis of the Arabidopsis genome. In Methods: A Companion to Methods of Enzymology. PulsedField Electrophoresis (B. Birren and E. Lai, eds.), Vol. 1, No. 2, pp. 186-194. Academic Press, San Diego.

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