By Tom Berkelman, Veronika Kortisova-Descamps, Theresa Redila-Flores Bio-Rad Laboratories
Conventional, homogenous field agarose gel electrophoresis is only capable of separating and resolving DNA fragments less than ~50 kb in size. Larger DNA fragments exhibit the same mobility and cannot be resolved from one another. Pulsed Field Gel Electrophoresis (PFGE), on the other hand, is a powerful technique that extends the size range over which DNA fragments can be separated up to ~10 Mb.
PFGE arose from the observation that DNA molecules elongate upon application of an electric field and relax to an un-elongated state upon removal of the electric field, with the relaxation rate being dependent on the size of the DNA (1). When the orientation of the electric field is changed during electrophoresis, the DNA molecules must relax prior to reorientation, thus affecting the migration rate. Cantor and Schwartz (2) first demonstrated that this effect can be used to greatly extend the size range over which electrophoretic DNA separations are possible.
There are several variants of PFGE, all of which share the essential characteristic that the orientation of the electric field is periodically changed in a manner that results in net migration of DNA. Cantor and Schwartz originally believed that non-homogeneous electric fields were necessary for optimal separation. The system they developed relied on alternation between a homogeneous electric field and a non-homogeneous field. This technique, termed orthogonal field alternating gel electrophoresis (OFAGE) did not result in straight migration lanes and was therefore largely abandoned when it became clear that high resolution separation was possible using homogeneous fields. Transverse alternating field electrophoresis (TAFE) is performed by orienting the electric fields transverse to the gel (3). The electrodes are placed at either side of the gel mounted in a vertical orientation. Rotating gel electrophoresis (RGE) is a technique in which the electric field is reoriented by physically moving the gel with respect to fixed electrodes. This technique has the advantage of electronic simplicity, but has been largely superseded by techniques in which the gel remains stationary and the field is manipulated through multiple independently controlled electrodes.
Field inversion gel electrophoresis (FIGE) is a simple variant of PFGE in which the orientation of the electric field is switched by 180°. The polarity of the field is simply reversed periodically along the axis of migration. The duration of the pulses is unequal so that there is a net migration in a single direction. FIGE only allows separation up to 750 kb, but it has the advantage that it can be performed with conventional agarose gel electrophoresis equipment. The only additional apparatus required is a pulse controller to periodically change the polarity of the field provided by the power supply.
Currently available advanced PFGE instrumentation utilizes multiple electrodes arranged in a hexagonal array, and a technology called PACE (programmed autonomously controlled electrodes, 5). in which each electrode can be controlled independently, allowing the simulation of virtually any pulsed field technique. The leading technique is referred to as CHEF (clamped homogeneous electric fields, 6) in which some of the electrodes are clamped, or held to intermediate potentials, giving homogeneous electric fields necessary for straight, distortion-free lanes. This instrumentation allows the manipulation of pulse time, field strength and pulse angle, all of which influence the migration rate of DNA through an agarose gel and the resolution of the separation.
|Fig 1a. Voltage clamping by the CHEF Mapper system (Bio-Rad). A. Relative electrode potentials when the +60° field angle is activated. B. Relative electrode potentials when the -60° field angle is activated.|
|Fig 1b. Voltage clamping by the CHEF Mapper system (Bio-Rad) in the FIGE mode. A. Relative electrode potentials when the 0° field vector is activated. B. Relative electrode potentials when the 180° field vector is activated.|
Pulse time is the period of the alternating field to which DNA molecules are subjected to in PFGE. Resolution will be will be optimal when the re-orientation time for the DNA molecules is comparable to the pulse time. DNA migration rate increases as the field strength or voltage increases. However, greater migration is accompanied by decreased band sharpness. In selecting the field strength for an experiment, a compromise between run time and resolution has to be made. Field angle affects separation as well, with the mobility of large DNAs (>1 mb) increasing as the field angle is decreased. This is accompanied by decreased resolution of smaller DNAs. Software is available to aid in the optimization of all three of these parameters to suit specific DNA size ranges.
The ability to separate DNA fragments in the size range from 50 kb to 10 Mb has enabled otherwise difficult analyses. Lower eukaryotes such as Saccharomyces have chromosomes in this size range and chromosomal assignment of genes may be determined simply through the combination of PFGE and Southern blotting (7). Long-range genetic mapping is possible through the use of infrequently cleaving restriction endonucleases in conjunction with PFGE. PFGE has also found wide use in bacterial typing, as the fragment patterns produced with infrequently cleaving restriction endonucleases are highly reproducible and distinctive among bacterial strains.
|Fig 2. Yeast chromosomes separated by PFGE|
The large size of DNA molecules to be separated by PFGE imposes certain constraints on sample preparation and handling. High molecular weight DNA is easily cleaved through shearing and imparts very high solution viscosity. For these reasons, DNA samples for PFGE are generally prepared imbedded in gel medium. Cellular source material is suspended in low gelling agarose and the gelled suspension is poured into molds. All subsequent manipulations (e.g. cell lysis, protein removal, restriction digestion) are performed by diffusing reagents into the resultant gel plugs. The processed gel plugs are then carefully loaded into wells of an agarose gel used for PFGE.