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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Hatada, I., Hayashizaki, Y., Hirotsune, S., Komatsubara, H., and Mukai, T. (1991) A genomic scanning method for higher organisms using restriction sites as landmarks. Proc. Natl. Acad. Sci. USA 88, 9523–9527.
Asakawa, J., Kuick, R., Neel, J. V., Kodaira, M., Satoh, C., and Hanash, S. M. (1994) Genetic variation detected by quantitative analysis of end-labeled genomic DNA fragments. Proc. Natl. Acad. Sci. USA 91, 9052–9056.
Asakawa, J., Kuick, R., Neel, J. V., Kodaira, M., Satoh, C., and Hanash, S. M. (1995) Quantitative and qualitative genetic variation in two-dimensional DNA gels of human lymphocytoid cell lines. Electrophoresis 16, 241–252.
Kuick, R., Asakawa, J., Neel, J. V., Kodaira, M., Satoh, C., and Hanash, S. M. (1995), High yield of restriction fragment length polymorphisms in two-dimensional separations of human genomic DNA. Genomics 25, 345–353.
Thoraval, D., Asakawa, J., Kodaira, M., Chang, C., Radany, E., Kuick, R., Lamb, B., Richardson, B., Neel, J. V., Glover, T., and Hanash, S. (1996) A methylated human 9-kb repetitive sequence on acrocentric chromosomes is homologous to a subtelomeric repeat in chimpanzees. Proc. Natl. Acad. Sci. USA 93, 4442–4447.
Hayashizaki, Y., Hirotsune, S., Okazaki, Y., Muramatsu, M., and Asakawa, J. (1997) Restriction Landmark Genomic Scanning Method. In Encyclopedia of Molecular Biology and Molecular Medicine, Vol. 5. Editor-in-chief: Meyers, R. A., VCH publisher, Weinheim, New York, Basel, Cambridge, pp. 304–319.
Asakawa, J., Kuick, R., Kodaira, M., Nakamura, N., Katayama, H., Pierce, D., Funamoto, S., Preston, D., Satoh, C., Neel, J. V., and Hanash, S. M. (2004) A genome scanning approach to assess the effects of radiation in mice and humans. Radiat. Res.; 161, 380–390.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Asakawa, Ji. (2008). Restriction Landmark Genome Scanning for the Detection of Mutations. In: Martin, C.C., Martin, C.C. (eds) Environmental Genomics. Methods in Molecular Biology, vol 410. Humana Press. https://doi.org/10.1007/978-1-59745-548-0_10
Download citation
DOI: https://doi.org/10.1007/978-1-59745-548-0_10
Publisher Name: Humana Press
Print ISBN: 978-1-58829-777-8
Online ISBN: 978-1-59745-548-0
eBook Packages: Springer Protocols