Restriction Landmark Genome Scanning for the Detection of Mutations

summary

There is a continuing need for more efficient methods to examine mutations in humans and other species resulting from exposures to environmental toxins and radiation. Environmental genomic studies, which often utilize nonmodel system species and as a result, there is a particular need for a method that does not rely on the availability of genome sequence information. Restriction landmark genome scanning (RLGS) is a two-dimensional electrophoresis (2-DE) of end-labeled DNA fragments. A vertical giant gel 2-DE system has been developed and applied to the RLGS. On a single RLGS pattern of mouse or human DNA, approx 2000 DNA fragments (spots) varying in size from 1.0 to 5.0 kb in the first dimension and 0.2 to 3.0 kb in the second dimension are visualized. In principle, this system will detect genomic alterations of two types: (1) that due to gain or loss of a cut site for the three restriction fragment enzymes employed in the study and (2) that due to insertion/deletion/ rearrangement (I/D/R) events. After optimization of the sample preparation and electrophoresis conditions, the gel quality reached a level such that the electrophoresis patterns derived from a single DNA sample gave distribution patterns of spots able to be superimposed. This technology can visualize up to 3000 DNA fragments per gel without using any probes, and thus should be highly efficient in monitoring for mutations resulting in I/D/R events in DNA fragments distributed throughout the genome. This method relies on direct labeling of DNA fragments rather than hybridization and therefore precise information on genome sequences is not required. As a result this method is applicable to any species.