7 Introduction to Pulsed-Field Gel Electrophoresis (PFGE)
99
- No specialgeltank or electrode system is required. A standard electrophoresis system can be easily adapted by adding a switching device
and a buffer circulator.
Disadvantages:
- the upper limit of resolution is -1000 kb;
- DNA molecules of different sizes may exhibit the same mobility at any
given switch time. This occurs when the reverse time is insufficient to
adequately disrupt the conformation oflarger molecules; hence, they
may actually migrate faster than shorter molecules. Therefore, FIGE
works best if the reverse interval is roughly equal to the time it takes
for a molecule to change conformations, and a single reverse interval
separates only a relatively narrow range of sizes. This problern is minimized by progressively changing the switching interval during the
run. The range of sizes resolved by various sets of switching angles
has been empirically determined.
• CHEF (Contour- Clamped Homogeneaus Electric Fields)
Chu et al. (1986) calculated the voltages needed to generate homogeneaus electric fields using multiple electrodes arranged around a closed
contour. This system, like the OFAGE, is capable of resolving very large
molecules (>10Mb); in addition, it also has the advantage ofproducing
straight lanes. This system is currently the most widely used.
• PACE (Programmable Autonomously-Controlled Electrodes)
This is the most recent modification of the pulsed field gel electrophoresis (Clark et al. 1988). In this system, the abilityto alter the reorientation
angle between the fields permits increased speed of separation for large
DNA molecules. DNA fragments as large as 6Mb can be resolved in 24 h.
This is in cantrast to the CHEF where a similar separation would take
around 7 days.
Application of PFGE in gene mapping
In gene mapping, a restriction map expressed in base pairs is required to
guide cloning strategies and precisely define localization of a sequence.
However, standard single-field electrophoresis is only able to resolve fragments up to 40 kb. Genetic markers typically are too far aparttobe linked by
this technique. Likewise, yeast artificial chromosomes (YACs ), bacterial artificial chromosomes (BACs), and P1 clones have inserts that are far larger
than can be analyzed using single-field electrophoresis.
99
- No specialgeltank or electrode system is required. A standard electrophoresis system can be easily adapted by adding a switching device
and a buffer circulator.
Disadvantages:
- the upper limit of resolution is -1000 kb;
- DNA molecules of different sizes may exhibit the same mobility at any
given switch time. This occurs when the reverse time is insufficient to
adequately disrupt the conformation oflarger molecules; hence, they
may actually migrate faster than shorter molecules. Therefore, FIGE
works best if the reverse interval is roughly equal to the time it takes
for a molecule to change conformations, and a single reverse interval
separates only a relatively narrow range of sizes. This problern is minimized by progressively changing the switching interval during the
run. The range of sizes resolved by various sets of switching angles
has been empirically determined.
• CHEF (Contour- Clamped Homogeneaus Electric Fields)
Chu et al. (1986) calculated the voltages needed to generate homogeneaus electric fields using multiple electrodes arranged around a closed
contour. This system, like the OFAGE, is capable of resolving very large
molecules (>10Mb); in addition, it also has the advantage ofproducing
straight lanes. This system is currently the most widely used.
• PACE (Programmable Autonomously-Controlled Electrodes)
This is the most recent modification of the pulsed field gel electrophoresis (Clark et al. 1988). In this system, the abilityto alter the reorientation
angle between the fields permits increased speed of separation for large
DNA molecules. DNA fragments as large as 6Mb can be resolved in 24 h.
This is in cantrast to the CHEF where a similar separation would take
around 7 days.
Application of PFGE in gene mapping
In gene mapping, a restriction map expressed in base pairs is required to
guide cloning strategies and precisely define localization of a sequence.
However, standard single-field electrophoresis is only able to resolve fragments up to 40 kb. Genetic markers typically are too far aparttobe linked by
this technique. Likewise, yeast artificial chromosomes (YACs ), bacterial artificial chromosomes (BACs), and P1 clones have inserts that are far larger
than can be analyzed using single-field electrophoresis.