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Pulse Field Gel Electrophoresis

Pulsed-field gel electrophoresis (PFGE) is a technique used to separate large DNA molecules and generate DNA fingerprints for bacterial isolates. It involves using restriction enzymes to cut bacterial DNA into large fragments, which are then separated in an agarose gel using an electric field that periodically changes direction. PFGE allows discrimination of bacterial strains and is commonly used in epidemiological studies to link clinical infections to environmental or food isolates. While time-consuming, it provides stable, reproducible patterns and has been shown to be more discriminating than other subtyping methods for many bacteria.

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December 17 pankajgaonkar12993@gmail.com 1 -Gaonkar Pankaj P.
Historical background of PGFE December 17 pankajgaonkar12993@gmail.com 2 At Columbia University in 1984, David C. Schwartz and Charles Cantor developed a variation on the standard protocol by introducing an alternating voltage gradient to improve the resolution of larger molecules. This technique became known as pulsed-field gel electrophoresis (PFGE). Standard gel electrophoresis techniques was unable to separate very large molecules of DNA effectively. DNA molecules larger than 15-20kb migrating through a gel will essentially move together in a size-independent manner.
WHAT IS “Pulsed Field Gel electrophoresis ”…??? December 17 pankajgaonkar12993@gmail.com 3 Pulsed field gel electrophoresis is a technique used for the separation of large deoxyribonucleic acid (DNA) molecules by applying to a gel matrix an electric field that periodically changes direction. Fig.-A microbiologist runs a pulsed-field gel electrophoresis test used in bacterial typing. PFGE is a technique used by scientists to generate a DNA fingerprint for a bacterial isolate. Pulsed Field - any electrophoresis process that uses more than one electric field alternating.
How does PFGE work? December 17 pankajgaonkar12993@gmail.com 4 •PFGE uses molecular scissors, called restriction enzymes, to cut bacterial DNA at certain locations known as restriction sites. •These molecular scissors are selected to generate a small number of DNA pieces that can be separated based on size. •Usually these DNA pieces, or restriction fragments, are large and need to be specially treated and separated to generate a DNA fingerprint. •First the bacteria are loaded into an agarose suspension, similar to gelatin, then the bacterial cell is opened to release the DNA.
•The treated plugs are then loaded onto an agarose gel and the restriction fragments are separated based on size using an electric field. •What makes PFGE different from how scientists usually separate DNA is because PFGE can separate several large restriction fragments. To do this an electric field that constantly changes direction to the gel is used to generate a DNA fingerprint. Continued… December 17 pankajgaonkar12993@gmail.com 5
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APPLICATION December 17 pankajgaonkar12993@gmail.com 7 •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.
Advantages of PFGE December 17 pankajgaonkar12993@gmail.com 8 PFGE subtyping has been successfully applied to the subtyping of many pathogenic bacteria and has high concordance with epidemiological relatedness. PFGE has been repeatedly shown to be more discriminating than methods such as ribotyping or multi- locus sequence typing for many bacteria. PFGE in the same basic format can be applied as a universal generic method for subtyping of bacteria. (Only the choice of the restriction enzyme and conditions for electrophoresis need to be optimized for each species.) DNA restriction patterns generated by PFGE are stable and reproducible.
Limitations of PFGE December 17 pankajgaonkar12993@gmail.com 9 •Time consuming. •Requires a trained and skilled technician. •Does not discriminate between all unrelated isolates. •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 same size are 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.
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