Pulsed Field Gel Electrophoresis MED 707.pptx

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1. Pulsed Field Gel Electrophoresis By Faisal Naeem and Tayyab ur Rehman

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.

3. Contin ……. 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

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.

6. PFGE(Alternating field electrophoresis)

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 Orthogonal-Field Alternation Gel Electrophoresis (OFAGE) Transverse-Alternating Field Gel Electrophoresis (TAFE) Field inversion gel electrophoresis(FIGE) Rotating Gel Electrophoresis (RGE) Contour-Clamped Homogeneous Electric Fields (CHEF)

9. 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.

10. 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