Pulsed-field gel electrophoresis (PFGE) is a technique used to separate large DNA molecules by applying an electric field that periodically changes direction. It was developed in 1984 by Schwartz and Cantor to improve resolution of DNA fragments larger than could be separated by conventional gel electrophoresis. PFGE uses switching angles and times to separate DNA fragments from a few kb to over 10 Mb based on their size. It has various applications including genome mapping, fingerprinting of bacteria, and studying DNA damage and repair.

2. WHY PFGE?
 Standard gel electrophoresis techniques (AGE) 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.
 The gels used are extremely fragile due to the very low
agarose concentrations, and the separation is not adequate
for most applications
 DNA(>50kb) cant be separated by agarose gel
electrophoresis method.

3. HISTORICAL BACKGROUND OF PGFE
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).
PFGE separates DNAs from a few kb to over 10 Mb
pairs

4. 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.
Pulsed Field - any electrophoresis
process that uses more than one electric
field alternatingly.

5. HOW DOES PFGE WORK?
 PFGE resolves DNA by alternating the electrical field between spatially distinct
pairs of electrodes.
 This technique results in the separation of DNA fragments of up to ~10 Mb by their
reorientation and movement at different speeds through the pores of an agarose
gel.
 PFGE stemmed from the observation that DNA molecules elongate upon
application of an electric field and return to an unelongated state upon removal of
the electric field; this relaxation rate is dependent on the size of the DNA.
 When the orientation of the electric field is changed during electrophoresis, the
DNA molecules must return to their elongated form prior to reorientation, thus
affecting the migration rate.

6. 4
 This effect can be used to greatly extend the size range over which electrophoretic
DNA separations are possible.
 When the electrical field is applied to the gel, the DNA molecules elongate in the
direction of the electrical field.
 The first electrical field is then switched to the second field according to the run
specifications.
 The DNA must change conformation and reorient before it can migrate in the
direction of this field.
 As long as the alternating fields are equal with respect to the voltage and pulse
duration, the DNA will migrate in a straight path down the gel.

9. RELATED TERMS
Pulsed Field - any electrophoresis process that uses more than one electric field
alternating
Switch Interval - amount of time by which each of the alternating fields is active
Reorientation Angle - acute angle between the two alternating electric fields
Field Inversion - PFGE system in shich the two alternating fields are oriented
opposite each other
Voltage Gradient - electrical potential applied to the gel
Homogeneous Field - electric field that has uniform potential differences
across the whole field

10. DIFFERENT TYPES OF PULSED FIELD GEL
ELECTROPHORESIS
1) Orthogonal-Field Alternation Gel Electrophoresis
(OFAGE)
2) Transverse-Alternating Field Gel Electrophoresis
(TAFE)
3) Field inversion gel electrophoresis(FIGE)
4) Rotating Gel Electrophoresis (RGE)
5) Contour-Clamped Homogeneous Electric Fields (CHEF)

12. 1) ORTHOGONAL-FIELD ALTERNATION
GEL ELECTROPHORESIS (OFAGE):
▪ A similar apparatus that used two nonhomogeneous electric fields
was reported by Carle and Olson in 1984.
▪ The major drawbacks -not uniform.
▪ The angle between the electric field varied across the gel.
▪ DNA molecules migrated at different rates depending on their
location in the gel.
▪ The angle between the electric fields varies from less than 180° and
the more than 90°.
▪ DNA molecules from 1,000 to 2,000 kb can be separated in OFAGE

14. 2)TRANSVERSE-ALTERNATING FIELD
GEL ELECTROPHORESIS (TAFE):
▪ Earlier called The vertical pulsed field system
▪ This form of PFGE allows separation of large DNA fragments.
▪ In TAFE, simple four-electrode array is placed in front and at the back of it.
▪ The angle between the electric fields varies from the top of the gel
(115°) to the bottom (approximately 165°).
▪ TAFE has been used for the separation of fragments up to 1,600kb
fragments.

16. 3)FIELD INVERSION GEL ELECTROPHORESIS
(FIGE)
▪
 In 1986, Carle, Frank and Olson developed a simpler system, FIGE, in
which the two fields were 180° apart. (single pair electrode used)
 Net forward migration is achieved by increasing the ratio of
forward to reverse pulse times to 3:1.
 FIGE is very popular for smaller fragment separations.
 FIGE provides acceptable resolution up to 800 Kb (600-750
kb).
▪

18. 4) ROTATING GEL ELECTROPHORESIS (RGE):
 In England in 1987, Southern described a novel PFGE system where the gel is
mounted on a rotating platform.
 Alternates between 2 orientation(120) apart.
 In RGE, the electric field is uniform and bands are straight because only one set of
electrodes is used.
 Switch times are too long in RGE.
 The DNA molecules migrate in straight lanes, due to the homogeneous fields.
 DNA molecules from 50 kb to 6,000 kb can be separated.

20. 5) CONTOUR-CLAMPED HOMOGENEOUS ELECTRIC
FIELDS (CHEF):
▪ CHEF has twenty-four point electrodes equally spaced around the hexagonal
contour.
▪ In the CHEF system, there are no passive electrodes.
▪ All the electrodes are connected to the power supply via an external loop of
resistors, all of which have the same resistance.
▪ This apparatus produces electric fields that are sufficiently uniform so that all lanes
of a gel run straight.
▪ CHEF uses an angle of reorientation of 120O
▪ Molecules up to 7,000 kb can be separated by CHEF.

22. PARTS OF PFGE SYSTEM

24. RUNNING CONDITIONS FOR PFGE
Pulse Time-
▪ In PFGE, DNA is subjected alternately to two electrical fields at
different angles for a time called the pulse time
▪Different DNA molecules have different pulse time
▪ Electrical Field Strength-
▪ Electrophoretic mobility is defined as the velocity per unit field.
▪ In most ordinary electrophoresis, the mobility is
independent of field strength.

25. ▪Temperature: In conventional gel electrophoresis, DNA
molecules were run at room temperature. (PFGE-4oC-
15O
C)
▪ Switch interval: The highest resolution for molecules of a
given size is obtained by using the shortest switch intervals
▪Agarose Concentration: Faster DNA migration occurs in
gels of lower agarose concentration

26. PFGE has proved to be an efficient method for genome size
estimation
In PFGE DNA fragments obtained by using endonucleases
produce a discrete pattern of bands useful for the fingerprinting
and physical mapping of the chromosome.
The PFGE technique is useful to establish the degree of
relatedness among different strains of the same species.
APPLICATIONS OF PFGE

27. PFGE has proven extremely powerful in the analysis of large DNA molecules
from a variety of sources including intact chromosomal DNAs from fungi (16),
parasitic protozoa.
Yeast Artificial Chromosome (YAC) libraries have been constructed by PFGE.
PFGE has also shown itself useful in the study of radiation-induced DNA
damage and repair, size organization
APPLICATIONS OF PFGE

28. PFGE subtyping has been successfully applied 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 multi- locus sequence typing for many
bacteria.
PFGE in the same basic format can be applied as a universal generic
method for subtyping of bacteria.
(Only the choice of the restriction enzyme and conditions for
electrophoresis need to be optimized for each species.)
DNA restriction patterns generated by PFGE are stable and reproducible.
APPLICATIONS OF PFGE

30.  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.
LIMITATIONS OF PFGE

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