Pulsed field gel      electrophoresis (PFGE)
Upcoming SlideShare
Loading in...5

Like this? Share it with your network

  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Be the first to comment
    Be the first to like this
No Downloads


Total Views
On Slideshare
From Embeds
Number of Embeds



Embeds 0

No embeds

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

    No notes for slide


  • 1. MANJULESH PAI Jitendra Kumar Mohit Kumar Ram College of Fisheres, Mangalore jitenderanduat@gmail.com
  • 2. Gel Electrophoresis  It is a technique used for the separation of DNA, RNA, or protein molecules using an electric field applied to a gel matrix.  The most common technique for this purpose is that of standard agarose gel electrophoresis. jitenderanduat@gmail.com
  • 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. jitenderanduat@gmail.com
  • 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. jitenderanduat@gmail.com
  • 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. jitenderanduat@gmail.com
  • 6. PFGE(Alternating field electrophoresis) jitenderanduat@gmail.com
  • 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 jitenderanduat@gmail.com
  • 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) jitenderanduat@gmail.com
  • 9. 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 jitenderanduat@gmail.com
  • 10. OFAGE jitenderanduat@gmail.com
  • 11. 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. jitenderanduat@gmail.com
  • 12. TAFE jitenderanduat@gmail.com
  • 13. 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).  jitenderanduat@gmail.com
  • 14. jitenderanduat@gmail.com
  • 15. 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. jitenderanduat@gmail.com
  • 16. 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. jitenderanduat@gmail.com
  • 17. CHEF jitenderanduat@gmail.com
  • 18. Cont..  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. jitenderanduat@gmail.com
  • 19. Parts of PFGE system 1)Gel box 2)High voltage power supply 3)Switch unit 4)Computer system jitenderanduat@gmail.com
  • 20. Running Conditions for Pulse Time. PFGE  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. jitenderanduat@gmail.com
  • 21. 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 jitenderanduat@gmail.com
  • 22. jitenderanduat@gmail.com
  • 23. 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. jitenderanduat@gmail.com
  • 24. Cont..  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 jitenderanduat@gmail.com
  • 25. Thank you all jitenderanduat@gmail.com