7 Introduction to Pulsed-Field Gel Electrophoresis {PFGE)
97
Parameters affecting PFGE resolution
The PFGE resolution ofDNA is dependent upon several critical parameters,
which are discussed below.
Resolving fragments greater than -1Mb in size requires reducing the voltage from the customary 6- 10 V I cm. Why large molecules fail to be resolved
with higher voltages is not known.
Temperature has a dramatic effect on DNA mobility in PFGE (unlike in
single-field electrophoresis). Although mobility is considerably increased
with increased temperature, gels run at higher temperatures have diminished resolution. Most investigators maintain their bufferat 4- 14 oc during
electrophoresis.
As standard electrophoresis, the concentration of agarose will affect the Separation of molecules. Although bands are sharper with higher concentrations of matrix, the rate of migration is considerably reduced.
In PFGE, for any fixed voltage, the migration rate of DNA molecules varies
depending on the angle between the electric fields (the reorientation angle);
reducing the reorientation angle increases fragment mobility. This effect is
particularly pronounced for very large molecules ( > 1 Mb ). In most systems,
the angle is fiXed. An advantage of the Programmahle Autonomously-Controlled Electrodes (PACE) systemisthat this parameter can also be altered,
allowing rapid resolution of fragments of any desired size (see "PACE").
This is the most important determinant of relative mobilities for DNA molecules. The best resolution is obtained by using the shortest switch interval
that permits separation of the complete size range of the fragments to be
examined. The duration of the pulse time loosely correlates with the size of
the molecules it resolves best. This relationship is not linear, however.
Furthermore, there is a relatively narrow range of sizes for which each interval is best, and PFGE Separations carried out using a single switch interval
have regions of compressed and expanded resolution. Molecules Ionger or
shorter than the ideal range will have reduced resolution. This problern is
circumvented by programming the pulse time to linearly change during the
course of an electrophoretic run, a technique called ramping. The duration
of the run directly correlates with the pulse time. An electrophoretic run
designed to separate yeast chromosomes greater than 3 Mb may take up
to a week to complete.
Voltage
Temperature
Agarose
Reorientation
angle
Switch interval
(pulse times)
97
Parameters affecting PFGE resolution
The PFGE resolution ofDNA is dependent upon several critical parameters,
which are discussed below.
Resolving fragments greater than -1Mb in size requires reducing the voltage from the customary 6- 10 V I cm. Why large molecules fail to be resolved
with higher voltages is not known.
Temperature has a dramatic effect on DNA mobility in PFGE (unlike in
single-field electrophoresis). Although mobility is considerably increased
with increased temperature, gels run at higher temperatures have diminished resolution. Most investigators maintain their bufferat 4- 14 oc during
electrophoresis.
As standard electrophoresis, the concentration of agarose will affect the Separation of molecules. Although bands are sharper with higher concentrations of matrix, the rate of migration is considerably reduced.
In PFGE, for any fixed voltage, the migration rate of DNA molecules varies
depending on the angle between the electric fields (the reorientation angle);
reducing the reorientation angle increases fragment mobility. This effect is
particularly pronounced for very large molecules ( > 1 Mb ). In most systems,
the angle is fiXed. An advantage of the Programmahle Autonomously-Controlled Electrodes (PACE) systemisthat this parameter can also be altered,
allowing rapid resolution of fragments of any desired size (see "PACE").
This is the most important determinant of relative mobilities for DNA molecules. The best resolution is obtained by using the shortest switch interval
that permits separation of the complete size range of the fragments to be
examined. The duration of the pulse time loosely correlates with the size of
the molecules it resolves best. This relationship is not linear, however.
Furthermore, there is a relatively narrow range of sizes for which each interval is best, and PFGE Separations carried out using a single switch interval
have regions of compressed and expanded resolution. Molecules Ionger or
shorter than the ideal range will have reduced resolution. This problern is
circumvented by programming the pulse time to linearly change during the
course of an electrophoretic run, a technique called ramping. The duration
of the run directly correlates with the pulse time. An electrophoretic run
designed to separate yeast chromosomes greater than 3 Mb may take up
to a week to complete.
Voltage
Temperature
Agarose
Reorientation
angle
Switch interval
(pulse times)