Pulsed-Field Gel Electrophoresis

(PFGE)
 Historical background of PFGE
• Standard gel electrophoresis techniques was
unable to separate very large molecules of DNA
effectively. DNA molecules larger than 15-20kb
migrating through a gel will essentially move
together in a size-independent manner.
• At Columbia University in 1984, David C.
Schwartz and Charles Cantor developed a
variation on the standard protocol by introducing
an alternating voltage gradient to improve the
resolution of larger molecules.
• This technique became known as pulsed-field
gel electrophoresis (PFGE).
 What is Pulsed Field Gel
Electrophoresis??
 Pulsed field gel electrophoresis is a technique
used for the separation of large deoxyribonucleic
acid(DNA) molecules by applying to a gel matrix
an electric field that periodically changes
direction.
 PFGE is a technique used by scientists to
generate a DNA fingerprint for a bacterial
isolate.
 Pulsed field– Any electrophoresis process that
uses more than one electric field alternating.
 How does PFGE work?
• PFGE uses molecular scissors, called restriction
enzymes, to cut bacterial DNA at certain locations
known as restriction sites.
• These molecular scissors are selected to generate a
small number of DNA pieces that can be separated
based on size.
• Usually these DNA pieces, or restriction fragments,
are large and need to be specially treated and
separated to generate a DNA finger print.
• First the bacteria are loaded into an agarose
suspension, similar to gelatin, then the bacterial cell
is opened to release the DNA.
• Once the DNA is released then the agarose and
DNA are mixed and poured into the plug mold.
• The treated plugs are then loaded onto an
agarose gel and the restriction fragments are
separated based on size using an electric field.
• What makes PFGE different from how scientists
usually separate DNA is because PFGE can
separate several large restriction fragments. To do
this an electric field that constantly changes
direction to the gel is used to generate a DNA
fingerprint.
 APPLICATION
• PFGE may be used for genotyping or genetic
fingertyping.
• It is commonly considered a gold standard in
epidemiological studies of pathogenic
organisms.
• Subtyping has made it easier to discriminate
among strains of Listeria monocytogenes and
thus to link environmental or food isolates with
clinical infections.
 Advantages of PFGE

• PFGE subtyping has been succesfully spplied to

the subtyping of many pathogenic bacteria and
has high concordance with epidemiological
relatedness.
• PFGE has been repeatedly shown to be more
discriminating than methods such as ribotyping
or multilocus sequence typing for many bacteria.
• PFGE in the same basic format can be applied
as a universal generic method for subtyping of
bacteria.
• DNA restriction patterns generated by PFGE are
stable and reproducible.
 Limitations of PFGE
• Time consuming.
• Requires a trained and skilled technician.
• Does not discriminate between all unrelated isolates.
• Pattern results vary slightly between technicians.
• Can’t optimize separation in every part of the gel at the
same time.
• Don’t really know if bands of same size are same pieces
of DNA.
• Bands are not independent.
• Change in one restriction site can mean more than one
band change.
• “Relatedness” should be used as a guide, not true
phylogenetic measure.
• Some strains cannot be typed by PFGE.
THANK YOU