Pulse Field Gel Electrophoresis (PFGE)

Its give the information's about PFGE and their methods and how the PFGE works and their advantages

1. Pulsed-Field Gel Electrophoresis PRANALI MARBADE MFSC,IFPGS,OMR Chennai Fish Biotechnology

2. Pulsed-Field Gel Electrophoresis 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 single direction (from top to bottom) But in PFGE,the direction of the current is altered at a regular interval.

3. Pulsed field gel electrophories is a technique used for the separation of larger deoxyribonucleic acid (DNA), RNA,or protein molecule by applying to gel matrix an electric field that periodically changes direction. As DNA larger than 15-20 kb migrating through a gel essentially moves together in size independent manner, the standard gel electrophories technique was unable to separate very larger molecule of DNA effectively which led to the practice of pulsed field gel electrophoresis.

4. Principle of pulsed field gel Electrophoresis (PFGE) While in general small fragments can find their way through the gel matrix more easily than larger DNA fragment a threshold length exists above 30-50 kb where all large fragments will run at the same rate, and appear in gel as a single larger diffuse band. The periodic changing of field direction, the various lengths of DNA react to the change at differing rates. That is, larger pieces of DNA will be slower to realign their charge when field direction is changed, while smaller pieces will be quicker. Over the course of time with the consistent changing of directions, each band will begin to separate more and more even at very large lengths. Thus separation of very large DNA pieces using PFGE is made possible.

5. The Method of Pulsed Field Gel Electrophoresis (PFGE)   t akes bacterial cells from an agar plate mixes bacterial cells with melted agarose and pours into a plug mold . The bacterial cells are broken open with biochemicals, or lysed, so that the DNA is free in the agarose plugs loads the DNA gelatin plug into a gel, and places it in an electric ­field that separates DNA fragments according to their size. The gel is stained, so that DNA can be seen under ultraviolet (UV) light

6. The major steps involved in Pulsed-field gel electrophoresis Lysis  First, the bacterial suspension is loaded into an agarose suspension. This is done to protect the chromosomal DNA from mechanical damage by immobilizing it into agarose blocks. Then the bacterial cells are lysed to release the DNA. The agarose-DNA suspension is also known as plug mold. Digestion of DNA:  The bacterial DNA is treated with unusual cutting restriction enzymes so that it yields less number of larger size DNA fragments . Electrophoresis: The larger pieces of DNA are subjected to pulse field gel electrophoresis by applying electric current and altering its direction at regular intervals . Analysis The fragments of different organisms generated by PFGE are compared to standards manually or by computer software like BioNumerics

7. Limitations of Pulsed Field Gel Electrophoresis (PFGE) Time-consuming. Requires trained and skilled technicians. Pattern results vary slightly between technicians. Can’t optimize separation in every part of the gel at the same time. Don’t really know if bands of the same size are the same pieces of DNA. Bands are not independent. Change in one restriction site can mean more than one band change. “Relatedness” should be used as a guide, not true phylogenetic measure. Some strains cannot be typed by PFGE .

8. Applications of Pulsed Field Gel Electrophoresis (PFGE) PFGE may be used for genotyping or genetic fingerprinting. It is commonly considered a gold standard in epidemiological studies of pathogenic organisms. Subtyping has made it easier to discriminate among strains of  Listeria monocytogenes  and thus to link environmental or food isolates with clinical infections. Since, field gel electrophoresis allows the separation of DNA fragments containing up to 100,000 bp (100 kilobase pairs), characterization of such large fragments has allowed construction of a physical map for the chromosomes from several bacterial species.