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Restriction Landmark Genome Scanning

Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 200)

Abstract

Restriction landmark genomic scanning (RLGS) is a method that provides both a quantitative genetic and epigenetic (cytosine methylation) assessment of thousands of CpG islands in a single gel without prior knowledge of gene sequence (1). The method is a two-dimensional separation of radiolabeled genomic DNA into nearly 2,000 discrete fragments that have a high probability of containing gene sequences and are ideal in length for cloning and sequence analysis. Genomic DNA is digested with an infrequently cutting restriction enzyme such as NotI, radiolabeled at the cleaved ends, digested with a second restriction enzyme, and then electrophoresed through a narrow, 60 cm-long agarose tube-shaped gel. The DNA in the tube gel is then digested by a third, more frequently cutting restriction enzyme and electrophoresed, in a direction perpendicular to the first separation, through a 5% nondenaturing polyacrylamide gel, and the gel is autoradiographed. Radiolabeled NotI sites are frequently used as “landmarks” because Note can not cleave methylated sites and since an estimated 89% of NotI sites are within CpG islands (2). Using a methylation-sensitive enzyme, the technique has been termed RLGS-M (3). The resulting RLGS profile displays both the copy number and methylation status of the CpG islands. These profiles are highly reproducible and are therefore amenable to inter- and intra-individual DNA sample comparisons.

Keywords

Whatman Paper Restriction Landmark Genomic Scanning Buffer Chamber Restriction Trapper Stopcock Valve 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    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.PubMedCrossRefGoogle Scholar
  2. 2.
    Lindsay, S. and Bird, A. P. (1987) Use of restriction enzymes to detect potential gene sequences in mammalian DNA. Nature 327, 336–338.PubMedCrossRefGoogle Scholar
  3. 3.
    Kawai, J., Hirose, K., Fushiki, S., Hirotsune, S., Ozawa, N., Hara, A., et al. (1994) Comparison of DNA methylation patterns among mouse cell lines by restriction landmark genomic scanning. Mol. Cell. Biol. 14, 7421–7427.PubMedGoogle Scholar
  4. 4.
    Hayashizaki, Y., Shibata, H., Hirotsune, S., Sugino, H., Okazaki, Y., Sasaki, N., et al. (1994) Identification of an imprinted U2af binding protein related sequence on mouse chromosome-11 using the RLGS method. Nature Genet. 6, 33–40.PubMedCrossRefGoogle Scholar
  5. 5.
    Plass, C., Shibata, H., Kalcheva, I., Mullins, L., Kotelevtseva, N., Mullins, J., et al. (1996) Identification of grf 1 on mouse chromosome 9 as an imprinted gene by RLGS-M. Nature Genet. 14, 106–109.PubMedCrossRefGoogle Scholar
  6. 6.
    Kuromitsu, J., Yamashita, H., Kataoka, H., Takahara, T., Muramatsu, M. Sekine, T. et al. (1997) A unique downregulation of h2-calponin gene expression in Down’s syndrome: a possible attenuaton mechanism for survival by methylation at the CpG island in the trisomic chromosome 21. Mol. Cell. Biol. 17, 707–712.PubMedGoogle Scholar
  7. 7.
    Okazaki, Y., Okuizumi, H., Ohsumi, T., Nomura, O., Takada, S., Kamiya, M., et al. (1996) A genetic linkage map of the syrian hamster and localization of the cardiomyopathy locus on chromosome 9qa2.1-b1 using RLGS spot-mapping. Nature Genet. 13, 87–90.PubMedCrossRefGoogle Scholar
  8. 8.
    Okazaki, Y., Hirose, K., Hirotsune, S., Okuizumi, H., Sasaki, N., Ohsumi, T., et al. (1995) Direct detection and isolation of restriction landmark genomic scanning (RLGS) spot DNA markers tightly linked to a specific trait by using the spot-bombing method, Proc. Natl. Acad. Sci. USA 92, 5610–5614.PubMedCrossRefGoogle Scholar
  9. 9.
    Kuick, R. M., Asakawa, J., Neel, J. V., Kodaira, M., Satoh, C., Thoraval, D., et al. (1996) Studies of the inheritance of human ribosomal DNA variants detected in two-dimensional separations of genomic restriction fragments. Genetics 144, 307–316.PubMedGoogle Scholar
  10. 10.
    Akama, T. O., Okazaki, Y., Ito, M., Okuizumi, H., Konno, H., Muramatsu, M., et al. (1997) Restriction landmark genomic scanning (RLGS-M)-based genome-wide scanning of mouse liver tumors for alteratons in DNA methylation status. Cancer Res. 57, 3294–3299.PubMedGoogle Scholar
  11. 10a.
    Costello, J. F., Früwald, M. C., Smiraglia, D. J., Rush, L. J., Robertson, G. P., Gao, X., et al. (2000) Aberrant CpG island methylation has non-random and tumour type-specific patterns. Nature Genetics 25,132–138.CrossRefGoogle Scholar
  12. 11.
    Miwa, W., Yashima, K., Sekine, T., and Sekiya, T. (1995) Demethylation of a repetitive DNA sequence in human cancers. Electrophoresis 16, 227–232.PubMedCrossRefGoogle Scholar
  13. 12.
    Costello, J. F., Plass, C., Arap, W., Chapman, V. M., Held, W. A., Berger, M. S., et al. (1997) Cyclin dependent kinase 6 (CDK6) amplification in human gliomas identified using two dimensional separation of genomic DNA. Cancer Res. 57, 1250–1254.PubMedGoogle Scholar
  14. 13.
    Yoshikawa, H., Delamonte, S., Nagai, H., Wands, J. R., Matsubara, K., and Fujiyama, A. (1996) Chromosomal assignment of human genomic Not I restriction fragments in a two-dimensional electrophoresis profile. Genomics 31, 28–35.PubMedCrossRefGoogle Scholar
  15. 14.
    Hayashizaki, Y., Hirotsune, S., Okazaki, Y., Shibata, H., Akasako, A., Muramatsu, M., et al. (1994) A genetic linkage map of the mouse using restriction landmark genomic scanning (RLGS). Genetics 138, 1207–1238.PubMedGoogle Scholar
  16. 15.
    Larsen, F., Gundersen, G., Lopez, R., and Prydz, H. (1992) CpG islands as gene markers in the human genome. Genomics 13, 1095–1107.PubMedCrossRefGoogle Scholar
  17. 16.
    Sambrook, J., Fritsch, E. F., and Maniatis, T., eds. (1990) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  18. 17.
    Kuick, R. D., Skolnick, M. M., Hanash, S., and Neel, J.V. (1991) A two-dimensional electrophoresis-related laboratory information processing system: spot matching. Electrophoresis 12, 736–746.PubMedCrossRefGoogle Scholar
  19. 18.
    Sugahara, Y., Akiyoshi, S., Okazaki, Y., Hayashizaki, Y., and Tanihata, I. (1998) An automatic image analysis system for RLGS films. Mammalian Genome 9, 643–651.PubMedCrossRefGoogle Scholar
  20. 19.
    Hayashizaki, Y., Hirotsune, S., Hatada, I., Tamatsukuri, S., Miyamoto, C., Furuichi, Y., and Mukai, T. (1992) A new method for constructing NotI linking and boundary libraries using a restriction trapper. Genomics 14, 733–739.PubMedCrossRefGoogle Scholar
  21. 20.
    Suzuki, H., Kawai, J., Taga, C., Ozawa, N., and Watanabe, S. (1994) A PCR-mediated method for cloning spot DNA on restriction landmark genomic scanning (RLGS) gel. DNA Res. 1, 245–250.PubMedCrossRefGoogle Scholar
  22. 21.
    Hirotsune, S., Takahara, T., Sasaki, N., Imoto, H., Okazaki, Y., Eki, T., et al. (1996) Construction of high-resolution physical maps from yeast artificial chromosomes using restriction landmark genomic scanning (RLGS). Genomics 37, 87–95.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  1. 1.Department of Neurological SurgeryUCSF Brain Tumor Research CenterSan Francisco
  2. 2.Division of Cancer GeneticsThe Ohio State UniversityColumbus
  3. 3.Ludwig Institute for Cancer ResearchUniversity of California at San DiegoLa Jolla

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