1. PULSED FIELD GEL
ELECTROPHORESIS(PFGE)
COURSE TEACHER
PRESENTED BY
DR. N. ERANNA MONOJ
SUTRADHAR
ASST PROF JR. M.SC,PANT
BIOTECH
Dept of Plant Biotechnology University of Agricultural Science(UAS),Bangalore
PALB 3243

2. PULSE FIELD GEL
ELECTROPHORESIS
• Pulsed field gel electrophoresis is a technique
used for the separation of large
deoxyribonucleic acid (DNA) , RNA, or protein
molecules by applying to a gel matrix an electric
field that periodically changes direction.

3. CONTI………..
o Conventional gel electrophoresis of DNA
molecules is carried out by placing DNA in a
solid matrix (i.e. agarose or polyacrylamide) and
inducing the molecules to migrate through the
gel under a static electric field.
o DNA fragments from 100 to 200 bp up to 50
kilobase pairs (kb) are separated by
conventional gel electrophoresis techniques.
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4. LIMITATIONS
• 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 this method.

5. PFGE-INTRODUCTION
 In 1982, Schwartz introduced the concept that DNA
molecules larger than 50 kb can be separated by
using two alternating electric fields.
PFGE separates DNAs from a few kb
to over 10 Mb pairs
 In conventional gels, the current is applied in a
single direction (from top to bottom).
 But in PFGE, the direction of the current is altered
at a regular interval.

7. 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

8. DESIGNS
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)

10. PARTS OF PFGE SYSTEM
1)Gel box
2)High voltage power supply
3)Switch unit
4)Computer system

11. 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.

12. CONTIII
 Temperature: In conventional gel
electrophoresis, DNA molecules were run at
room temperature.(PFGE-4oC-15OC)
 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
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13. APPLICATIONS OF
PFGE
 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.

14.  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

15. THANK YOU ALL
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