In-gel DNA radiolabelling and two-dimensional pulsed field gel electrophoresis procedures suitable for fingerprinting and mapping small eukaryotic genomes.

Abstract

A simple method for complete genome radiolabelling is described, involving long-wave UV exposure of agarose-embedded chromosomal DNA and [alpha-(32)P]dCTP incorporation mediated by the Klenow fragment. Experiments on the budding yeast genome show that the labelling procedure can be coupled with two new two-dimensional pulsed field gel electrophoresis (2D-PFGE) protocols of genome analysis: (i) the KARD (karyotype and restriction display)-PFGE which provides a complete view of the fragments resulting from a single restriction of the whole genome and (ii) the DDIC (double digestion of isolated chromosome)-PFGE which is the eukaryotic counterpart of complete/complete 2D-PFGE in bacterial genomics.