A genomic scanning method for higher organisms using restriction sites as landmarks.

Abstract

We have developed a powerful genomic scanning method, termed "restriction landmark genomic scanning," that is useful for analysis of the genomic DNA of higher organisms using restriction sites as landmarks. Genomic DNA is radioactively labeled at cleavage sites specific for a rare cleaving restriction enzyme and then size-fractionated in one dimension. The fractionated DNA is further digested with another more frequently occurring enzyme and separated in the second dimension. This procedure gives a two-dimensional pattern with thousands of scattered spots corresponding to sites for the rust enzyme, indicating that the genome of mammals can be scanned at =1-megabase intervals. The position and intensity of a spot reflect its locus and the copy number of the corresponding restriction site, respectively, based on the nature of the end-labeling system. Therefore, this method is widely applicable to genome mapping or detection of alterations in a genome.Genomic DNA analysis is essential for clarifying the characteristics of mammalian DNA. However, the genomes of these organisms are very large. For example, the mammalian genome is about 3 x 109 base pairs (bp), which is 1000 times that of Escherichia coli. The first step in analyzing large genomes requires the scanning of many landmarks. Southern blotting has been used for this purpose (1); however, usually only one locus on the genome can be detected with a single probe. Therefore, when this method is applied to scanning a genome, it must be repeated many times with many probes. Several Southern blot repetitions might be theoretically sufficient for some purposes, if a repetitive sequence is used as a probe (2, 3). However, bands (spots) corresponding to loci cannot be separated in these systems.In this paper, we introduce a concept termed "restriction landmark," in which each restriction enzyme recognition site can be used as a landmark. Based on this concept, we developed a restriction landmark genomic scanning (RLGS) method, which employs (i) direct end labeling of the genomic DNA digested with a restriction enzyme and (ii) highresolution, two-dimensional electrophoresis. Using this method, we simultaneously separated and detected thousands of signals (spots) derived from restriction sites. Thus, we could locate landmarks on mammalian genomes at intervals averaging -1 megabase pair (Mbp). MATERIALS AND METHODSDNA Preparation. Genomic DNA was extracted from each sample as described (4). 1 Ci = 37 GBq) for 30 min at 370C in 100 Al of 50 mM Tris HCI (pH 7.4), 100 mM NaCI, 10 mM dithiothreitol, 0.16 ,uM dXTP[aS], and 33 tM ddYTP [aS]. To inactivate the enzyme, this reaction mixture was incubated at 650C for 1 hr. When the average size of the DNA fragments was more than several hundred kilobase pairs, an additional digestion was performed using restriction enzyme B. (iv) One microgram ofthe DNA from step iii was fractionated on a 50 x 20 x 0.1 cm agarose gel (0.8-1% Seakem GTG agarose; FMC) and then electrophoresed in 1x TAM buffer (50 mM Tris-acetate, pH 7....

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