A column gel electrophoresis-coupled genomic DNA subtractive hybridization technique.

We have developed a DNA subtractive hybridization technique especially designed for mammalian genome comparison. The core of this protocol is a newly devised denaturant-containing polyacrylamide gel formed in a glass-column. In this gel system, the following DNA manipulation steps are carried out sequentially: size separation by electrophoresis, heat-denaturation, renaturation, and recovery. In the first step, a mixture of tester and driver DNA fragments are segregated according to their size whilst keeping their double-stranded forms. This reduces the complexity of the original genomic DNA fragments and also segregates DNA fragments having closely related sequences. In the second step, fractionated DNA fragments are quickly denatured and subjected to successive subtractive hybridization in situ by controlling gel temperature in a water bath. In the third step, DNA fragments are recovered by electrophoresis towards the reverse-orientation and are adsorbed onto ion-exchange beads. Two lines of experiments show that our protocol is able to highly enrich or directly extract differences among genomic DNA samples.