Pulsed-field gel electrophoresis (PFGE) is a powerful molecular typing technique by which genomic DNA is isolated from the organism of interest, followed by restriction enzyme analysis. The digestion products are then analyzed on an agarose gel by applying an electric field that periodically changes direction allowing for separation of the larger DNA fragments (entire genomic DNA) and approximate measurement of fragment length.
PFGE can separate large DNA molecules (up to 10 Mb), whereas standard DNA gel electrophoresis commonly resolves fragments up to ∼50 kb. PFGE takes 2–3 days, excluding sample preparation.
PFGE was first developed by Schwartz et al. in yeast. The largest molecule so far resolved by Pulse-Field Gel Electrophoresis is estimated at 14 Mb (Barry and Pollard, 1993)
PFGE is a variation of agarose gel electrophoresis. Some of the major differences between conventional gel electrophoresis and PFGE is tabulated here:
|Property||Conventional gel electrophoresis||Pulsed-field gel electrophoresis|
|Resolving power (Separating power)||Separates DNA fragments up to ∼50 kb in size.||can separate large DNA molecules(up to 10 Mb)|
|Direction of current||Current only runs in a single direction||The direction of the current switches from the primary to the secondary electrodes located at a 60° angle from the centre of the agarose gel.|
Table of Contents
Steps of pulsed-field gel electrophoresis
Preparation of Bacterial cells
Streak bacterial isolates (e.g. Staphylococcus aureus) onto TSA plates and incubate at 37 °C for 18–24 h.
Mix bacterial cells with melted agarose and pour into a plug mold
Bacterial cells are embedded into an agarose gel to prevent shearing of chromosomal DNA, and DNA digestion is performed in situ.
Lysis of Bacterial cells
The bacterial cells are broken open with biochemicals, or lysed, so that the DNA is free in the agarose plugs. The bacterial chromosome is digested using a rare cutting enzyme that recognizes specific DNA sequences ranging from 6 to 8 nucleotides, resulting in a limited number of DNA fragments of varying lengths. After DNA restriction, slices of the agarose blocks are inserted into wells of the agarose gel matrix.
Digested DNA samples are loaded into the DNA gelatin plug into a gel, and are subjected to separation by alternating the electric field between spatially distinct pairs of electrodes
Application of an electric field that is periodically reorienting causes long DNA molecules to reorient during electrophoresis. The time required for reorientation is also inversely proportional to the size of the DNA fragment. As smaller molecules reorient more quickly than larger molecules, separation of various DNA fragment sizes ranging from kilobases to megabases ensues if the gel is run for a sufficient time. This process generates a fingerprint profile that can be digitally imagined.
This system consists of 24 electrodes in an octagon arrangement producing a constant electrophoresis gradient that switches from the primary to the secondary electrodes located at a 60° angle from the centre of the agarose gel. Consequently, this configuration causes the DNA molecules to re-orient in the agarose gel matrix over a 120° angle.
Staining the gel
The gel is stained so that DNA can be seen under ultraviolet (UV) light.
Taking photograph and processing the Image
A digital camera takes a photograph of the gel and stores the picture in the computer. The obtained gel images are normalized and patterns of the DNA fragments are analyzed using various software such as BioNumerics (Applied Maths, Austin, TX, USA), Fingerprinting II (Bio-Rad, Hercules, CA, USA), GelQuest (SequentiX, Klein Raden, Germany), and PyElph (Pavel & Vasile, 2012). These software and similar other softwares help for storing, sharing and analyzing PFGE data.
References and further readings
- Pulsed-field Gel Electrophoresis. Center for Disease Control (CDC)
- Pulse Field Gel Electrophoresis by Batu K. Sharma-Kuinkel, Thomas H. Rude, and Vance G. Fowler, Jr
- Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Schwartz DC, Cantor CR.