A DIFFERENTIAL CLONING PROCEDURE FOR REARRANGED OR ALTERED GENOMIC DNA-BASED ON IN-GEL COMPETITIVE REASSOCIATION

Relatively large and complex genomes such as those of higher eukaryotes, where conventional genetic analyses are difficult to apply, have been analyzed by a series of new techniques. For example, sequences well over one megabase can be studied by fluorescent in situ hybridization (FISH) which reveals the location and size of specific DNA sequences on chromosomes, and sub-mega to megabase sequences can be analyzed by pulsed-field gel electrophoresis (1, 2). These techniques have been proven to be useful in the detection of alterations in genomic DNA that accompany rather macroscopic changes on the chromosomes. However, if one wants to directly clone genomic DNA at a limited number of anonymous sites which have been altered as a result of DNA rearrangement or mutation, efficient and practical procedures are yet to be completely established. In pursuit of this goal, we have previously reported a novel differential cloning procedure, without using any specific probes, designed for the cloning of restriction DNA fragments whose sizes may differ between DNA preparations (3, 4). The principle underlying this procedure is the dissociation and reassociation of restriction fragments of genomic DNA (referred to as target DNA) in the presence of a large excess of competitor or reference DNA within a gel, following electrophoresis to subtract DNA species which exist both in the target and reference DNA and enrich the unsubtracted target DNA (in-gel competitive reassociation, IGCR). The procedure, however, had several limitations in implementation for general differential cloning of altered DNA fragments in complex genomic DNA such as mammalian DNA. Accordingly, we have overhauled the previous procedure and established a new one, which eliminates the shortcomings of the original, although the basic principle (IGCR) has been maintained for its unique advantages in several critical aspects. Here, we summarize the new procedure designed especially for cloning altered genomic DNA fragments present in low copy numbers in higher organisms. We also present several examples of the practical application of the procedure in analyzing and cloning altered DNA fragments resulting from DNA rearrangement, mutation and polymorphism.