Pulsed Field Gel Electrophoresis (pfge)
Shomu's Biology
Pulse Field Gel Electrophoresis (PFGE)
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This lecture explains the process of pulse field gel electrophoresis (PFGE), which is used to separate large DNA fragments with better resolution by shifting the electric field at specific time intervals.
Introduction
Pulsed field gel electrophoresis (PFGE) is a procedure used to separate massive DNA molecules by applying an electric field that periodically changes course to a gel matrix. While ordinary gel electrophoresis is effective for separating smaller DNA molecules, it struggles to separate very large DNA molecules.
Development of PFGE
In 1984, David C. Schwartz and Charles Cantor developed a new protocol known as pulsed-field gel electrophoresis (PFGE) at Columbia School. This method introduced an alternating voltage gradient to enhance the resolution of larger DNA molecules. PFGE significantly increased the range of resolution for DNA fragments.
The PFGE Process
The process of PFGE is similar to regular gel electrophoresis, but instead of continuously running the voltage in one direction, the voltage is periodically switched between three directions: one running through the central axis of the gel and two running at an angle of 60 degrees on either side. The pulse times are equal for each direction, resulting in a net forward migration of the DNA.
Switching Interval Ramps
For very large DNA molecules (up to approximately 2 MB), switching interval ramps can be used. These ramps increase the pulse time for each direction over several hours. For example, the pulse time can be increased linearly from 10 seconds at zero hours to 60 seconds at 18 hours. This longer process is necessary due to the size of the fragments being resolved and the non-linear movement of DNA through the gel.
Separation of DNA Fragments
While small fragments can easily navigate through the gel matrix, a threshold size exists (around 30-50 kb) where all larger fragments will run at the same rate and appear as a single diffuse band on the gel. However, with the periodic changing of the electric field direction, different lengths of DNA react to the change at varying rates. Over time, with the continuous change of directions, each band will separate more and more, even at very large lengths. This allows for the separation of very large DNA fragments using PFGE.
