A differential cloning procedure for rearranged or altered genomic DNA based on in-gel competitive reassociation

Abstract

We have developed a substantially improved differential cloning procedure designed for cloning anonymous altered restriction DNA fragments from higher organisms. The improvements include (i) in-gel dissociation and reassociation of biotinylated restriction digests of target DNA fragments, (ii) replacement of agarose gel by a synthetic gel material for electrophoresis, (iii) use of a reassociation enhancing reagent (CTAB) for in-gel reassociation, and (iv) introduction of PCR. After several cycles of IGCR, we attained considerable enrichment of altered or rearranged DNA fragments which were originally present at one copy or less per complex eukaryotic genome. Examples of enrichment include those of an exogenously added DNA fragment, a chromosomal DNA sequence that has undergone a deletion, and DNA fragments containing a recombination junction.