PFGE is an advance version of Electrophoresis Technique. Direction of current is altered at regular interval is its major advantage over Electrophoresis. The main barrier in the electrophoresis Resolution and the Molecular Size can be overcome by this advance version of PFGE.

1. PulseFieldGelElectrophoresis
PFGE
Represented by : Mansi
Patel
Roll no. : 09

2. Content
 Electrophoresis
 Types
 Introduction
 Basic difference between PFGE and GE
 Parts of PFGE
 Working of PFGE
 Various designs of PFGE
 Applications
 Limitations
 References

3. What is electrophoresis

7. Introduction
 In 1982, Schwartz introduced the concept
that DNA molecules >50kb can be separated
by using two alternating fields.
 It is subtype of gel electrophoresis.
 Separation : few kb to over 10Mb
 Direction of the current : altered at a regular
interval.

8. How the word PFGE coined...
• Uniform potential
difference.
Homogenous
Electric field
• Potential
difference varies.
Non-
homogenous
Electric field
Earlier PFGE was known as “Pulse Field Gradient
Electrophoresis”

9. (contd...)
• Non-
homogenous
field
Field strength
• Reorientation
angle
Curved lanes
• Band
sharpening
effect
Complicated
conditions
Later on it was modified to “Pulse Field Gel Electrophoresis”

10. Why to carry out PFGE
instead of GE
 Resolution : increased by two fold then the
conventional GE.
 Separates small, natural linear chromosomal DNAs
ranging in size from 50-kb parasite micro chromosomes
to multimillion-bp yeast chromosomes.
 Intact human chromosomes range 50Mb - 250Mb can
also be studied.

11. How PFGE differs from
conventional GE
PFGE GE
Direction of current is altered at
regular interval.
Current is applied in single direction.
(i.e. From top to bottom)
DNA from few kb to over 10 Mb is
separated.
DNA fragment from 100-200bp up to
50 kb are separated.
Large fragments above 50 kb can run
and highly resolved bands are
obtained.
Above 50 kb, because of the size of the
molecules, the sieving action of the gel
is lost, and fragments run as a broad,
unresolved band.

12. Parts of PAGE system
Gel box
High
voltage
power
supply
Switch
unit
Computer
system

16. Various designs of PFGE
 FIGE
 OFAGE
 TAFE
 RGE
 CHEF
 PHOGE
 PACE

17. Field inversion gel electrophoresis
(FIGE)
 1986, Carle, Frank and Olson
 Reorientation angle : 180°
 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.
 Separation resolution : 800kb

19. Orthogonal field altering gel
electrophoresis (OFAGE)
 Carle and Olson in 1984
 Electric fields : two non-homogeneous
 Arrangement of electrodes was symmetric and
their lengths were unequal:
LARGE electrode : Cathode
SMALL electrode : Anode
 Reorientation angle : >90° - < 180°
 Separation : 1,000kb-2,000kb
 Mammalian genome mapping difficult.
 Used for : lower organisms like yeast

21. Drawbacks

22. Transverse-Alternating Field Gel
Electrophoresis (TAFGE)
 Gardiner et al 1986
 Earlier was known as “TheVertical Pulsed field
system” later known asTAFGE
 TRANSVERSE is referred as the orientation of
the vector of field plane with respect to the gel
plane.
 Gel : vertically in the chamber and alternating
the field of orientation not along but across the
gel.

24. (contd...)
 Array of four electrodes is placed in front and
back of gel (not on plane)
 Sample molecules are forced to move in ZIGZAG
fashion through gel and all lanes face the same
effect , so bands are straight.
 As reorientation angle increased from top to
bottom from 115°-165° (limited field strength at
bottom of gel)
 Separation : Large fragments of DNA 1,600kb

25. Major Advantage of TAFE
 TAFE technology, with regular and sharp
separation of DNA bands, will be of special
advantage in the study of genetics of many
pathogenic protozoan , where such analysis
was impossible before.

26. Rotating Gel Electrophoresis
(RGE)
 In England in 1987, Southern described a
novel PFGE system that rotates the gel between
rotating platform.
 Switch times are too long in RGE.
 Bands are straight : One set of electrodes is
used
Homogenous field
 Separation : DNA molecules 50kb -6,000kb

29. Counter-clamped Homogenous electric
field (CHEF)
 CHEF is the most widely used apparatus.
 Electrodes : 24 electrodes but are placed
around equally hexagonal counter.
 No passive electrodes.
 Angle of reorientation : 120°
 Electric field : uniform (run straight)
 Separation : 7,000kb

30. (contd...)
• Electrodes
Power supply
• External
loop of
resistors
Same
resistance
• Migration
of sample
Straight lane

34. Pulsed-Homogeneous Orthogonal Field
Gel Electrophoresis (PHOGE)
 Electric field : Homogeneous
 Reorientation angle : 90°
 Major difference here is reorientation occurs
four times per cycle.
 DNA lanes in PHOGE do not run straight.
 Separation : DNA fragments up to 1Mb

35. Programmable Autonomously-
Controlled Electrodes (PACE)
 A computer-driven system known as PACE,
designed by Lai et al may be the ultimate
PFGE device.
 The system offers precise control over all
electric field parameters by the independent
regulation of the voltages on 24 electrodes
arranged in a closed contour.
 Separation : 100kb to over 6Mb

36. Tools to study :

37. (contd...)
 Pulse time : 10v/s ,0.1 s ,5Mb
03v/s ,1000s ,5-7Mb
 Temperature : 14°C - 22°C
Increase temp. , decrease resolution
 Switch interval : shortest switch interval
highest resolution.
 Agarose concentration : Higher agarose-velocity
decrease- molecule spends more time in gel

38. (contd...)
 Voltage : 6-10v/cm, 1Mb
decrease voltage if >1Mb
(`coz larger mol. ,pulse time increase so mol.
Reorient in gel for longer period)
 Reorientation angle : widening reorientation
angle yields sharper resolution.
 Best resolution b/w 120° - 150 °

39. (contd...)
 The PACE system can perform all previous
pulsed field switching regimens (i.e. FIGE,
OFAGE, PHOGE)

40. Applications
 The ability to separate, isolate and analyze
Mb size fragments of DNA is already
providing insights into the genome
organization of organisms as diverse as
bacteria and humans.

41. (contd...)
 Molecular epidemiology :
PFGE is known as standard “GOLD TECHNQUE”
I. Strain typing in public health and food
safety.
II. Food Quality Control : PFGE is used in
fermentation process in industries to
monitor genetic stability of organisms.

42. (contd...)
 Cancer research :
I. DNA damage and repair studies
II. Apoptosis assay

43. Genomic Application :
Why to study genomic DNA
To study similarity in genetic makeup
(Phylogenetic information)
Check production of protein, hormones
enzymes
 Useful in the study of radiation-induced DNA
damage and repair, size organization and
variation in mammalian centromers.

44. (contd...)
I. Generation of artificial chromosome
libraries
II. Genome mapping

45. (contd...)
 Future application includes protein
separation and sequencing and studies of
DNA topology.

46. Limitations
 Expensive instrumentation, time consuming
(~24 hours), requires high level skill.
 Some strains are untypeable; complex
patterns are difficult to interpret.
 Bands of same size don't guarantee same
DNA; relatedness is only relative and not
absolute.

47. References
 http://link.springer.com/article/10.1134%2FS1
990519X08060
 http://www.researchgate.net/publication/225
4444142_Pulsed_field_gel_electrophoresis_T
heory_instruments_and_application
 http://www.ncbi.nlm.nih.gov/pubmed/20692
376
 http://www.ncbi.nlm.nih.gov/pmc/articles/P
MC312377/

48. (contd...)
 http://sciencelearn.org.nz/Contexts/Enviro-
imprints/Sci-Media/Pulsed-field-gel-
electrophoresis
 http://www.bio.davidson.edu/genomics/meth
od/pulse_field.html
 http://www.bio-rad.com/en-in/life-science-
research/applications-technologies
 http://www.bio-rad.com/en-in/applications-
technologies/pulsed-field-gel-electrophoresis