A DIFFERENTIAL CLONING PROCEDURE OF COMPLEX GENOMIC DNA FRAGMENTS

We have developed a differential cloning procedure designed for clonin g of anonymous restriction DNA fragments whose molecular sizes differ between two genomic DNA preparations from higher organisms. The proced ure, which was extensively revised from the original one, consists of several steps as summarized below. (i) Digestion of two DNA preparatio ns (target and reference DNA) with the same restriction enzyme (4 base cutter). (ii) Biotinylation of target DNA and conversion of reference DNA to nonamplifiable form by terminal dephosphorylation. (iii) Elect rophoresis of the two DNA preparations through a synthetic gel with a large excess of reference DNA as a competitor. (iv) In-gel alkaline di ssociation of DNA, followed by reassociation (in-gel competitive reass ociation). (v) Elution of DNA from the gel and PCR after adapter ligat ion and adsorption of DNA onto streptavidin-coated matrix. By repeatin g these steps, we attained substantial enrichment (similar to 10,000-f old) of DNA fragments which were originally present at one copy or les s per complex mammalian genome. The details of the procedure and its u nique characteristics in cloning of altered genomic DNA fragments, par ticularly from mammalian genome, are discussed. 1994 Academic Press, I nc.