Pulsed field gel electrophoresis (PFGE)

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Pulsed field gel electrophoresis(PFGE)

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Pulsed field gel electrophoresis (PFGE)

  1. 1. MANJULESH PAI Jitendra Kumar Mohit Kumar Ram College of Fisheres, Mangalore jitenderanduat@gmail.com
  2. 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. 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. 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. 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. 6. PFGE(Alternating field electrophoresis) jitenderanduat@gmail.com
  7. 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. 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. 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. 10. OFAGE jitenderanduat@gmail.com
  11. 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. 12. TAFE jitenderanduat@gmail.com
  13. 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. 14. jitenderanduat@gmail.com
  15. 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. 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. 17. CHEF jitenderanduat@gmail.com
  18. 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. 19. Parts of PFGE system 1)Gel box 2)High voltage power supply 3)Switch unit 4)Computer system jitenderanduat@gmail.com
  20. 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. 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. 22. jitenderanduat@gmail.com
  23. 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. 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. 25. Thank you all jitenderanduat@gmail.com

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