Pulsed-Field Gel Electrophoresis

TLDR

Pulsed‑field gel electrophoresis uses alternating electric fields to separate DNA fragments up to megabase size, surpassing conventional agarose gels that resolve only up to ~20–50 kb. PFGE is performed by preparing unsheared DNA, digesting it with a rare‑cutting restriction enzyme, then running it in an alternating electric field where fragment migration time is proportional to size, followed by visualization and interpretation of band patterns. Figure 1 illustrates the PFGE workflow.

Abstract

Pulsed- field gel electrophoresis (PFGE) was first described by Schwartz and Cantor (1) It is now an umbrella term for the alternating of an electric field in more than one direction through a solid matrix to achieve the separation of DNA fragments. The method requires the preparation of unsheared DNA, digestion of the DNA using a rare-cutting restriction endonuclease, separation of fragments by PFGE, and the visualization and interpretation of banding patterns Fig. 1.Conventional agarose gel electrophoresis employs a static field and can resolve DNA fragments up to 50 kilobases (kb), although in practice, fragments larger than 20 kb co-migrate under the conditions usually applied. By introducing a pulse or change in the direction of the electric field, fragments as large as 10 megabases (Mb) can be separated. The time required by DNA fragments of different sizes to reorientate to the new electric field is proportional to their molecular weight and it is this factor that allows the separation and focusing of DNA fragments. Fig 1. Practical steps involved in PFGE.