Pulsed-field Gel Electrophoresis (PFGE)
What is PFGE?
PFGE is a technique used by scientists to generate a DNA fingerprint for a bacterial isolate.
How does PFGE work?
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. Once the DNA is released then the agarose and DNA suspension, also known as a plug, is treated with restriction enzymes. 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.
Advantages of PFGE
- 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
- 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