Two-Dimensional DNA Electrophoresis (2D-DE) for Mammalian DNA

Abstract

Pulsed-field gel electrophoresis (PFGE) has been successfully used to generate physical maps of regions of human chromosomes near genes or single copy probes. However, many regions of the human genome contain gene families that often include pseudogenes. Other regions contain stretches of repeats. Conventional PFGE requires a series of single copy probes, which are sometimes difficult if not impossible to find for clusters of homologous genes or repeats. If probes detect several different genes that are dispersed on a chromosome, standard PFGE techniques will not be useful for the generation of large scale restriction maps. To overcome this problem, we have developed a method of two-dimensional DNA electrophoresis (2D-DE). The 2D-DE method consists of two DNA digestion steps and two electrophoretic separation steps (shown in Fig. 1)):