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Restriction landmark genomic scanning

Nature Protocols volume 1, pages 2774–2783 (2006)Cite this article

Abstract

Restriction landmark genomic scanning (RLGS) is a method to detect large numbers of restriction landmarks in a single experiment. It is based on the concept that restriction enzyme sites can serve as landmarks throughout a genome. RLGS uses direct end-labeling of the genomic DNA digested with a rare-cutting restriction enzyme and high-resolution two-dimensional electrophoresis. Compared with the conventional gene-detection technologies, such as Southern blot analysis and PCR, RLGS has the following advantages even though it needs specially designed instruments: high-efficiency scanning capacity, scanning extensibility by using alternate restriction enzyme combinations, applicability to any organism, a spot intensity that reflects the copy number of restriction landmarks, and the ability, by using a methylation-sensitive enzyme, to screen the methylated state of genomic DNA. The RLGS protocol can be accomplished in 5 days to 2 weeks.

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Figure 1: Procedure for genomic scanning by two-dimensional gel electrophoresis.
Figure 2: Schematic representation of first-dimension electrophoretic apparatus.
Figure 3: Schematic representation of second-dimension gel casting.
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References

  1. Hatada, I., Hayashizaki, Y., Hirotsune, S., Komatsubara, H. & Mukai, T. A genomic scanning method for higher organisms using restriction sites as landmarks. Proc. Natl. Acad. Sci. USA 88, 9523–9527 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Hayashizaki, Y. et al. Restriction landmark genomic scanning method and its various applications. Electrophoresis 14, 251–258 (1993).

    Article  CAS  PubMed  Google Scholar 

  3. Okazaki, Y. et al. A genetic linkage map of the mouse using an expanded production system of restriction landmark genomic scanning (RLGS Ver.1.8). Biochem. Biophys. Res. Commun. 205, 1922–1929 (1994).

    Article  CAS  PubMed  Google Scholar 

  4. Hayashizaki, Y., Hirotsune, S., Okazaki, Y., Muramatsu, M. & Ashikawa, J. Restriction landmark genomic scanning method. In Molecular Biology and Biotechnology: a comprehensive desk reference. (ed. Meyers, R.) 813–817 (VCH, Weinheim, 1995).

    Google Scholar 

  5. Hayashizaki, Y. & Watanabe, S., eds. Restriction landmark genomic scanning (RLGS). (Springer, Berlin, 1997).

    Book  Google Scholar 

  6. Kawai, J., Hayashizaki, Y., Okazaki, Y., Suzuki, H. & Watanabe, S. Restriction landmark genomic and cDNA scanning. In Encyclopedia of Analytical Chemistry: Instrumentation and Applications. (ed. Meyers, R.) 5196–5222, (John Wiley & Sons Ltd, Chichester, 2000).

    Google Scholar 

  7. Southern, E.M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503–517 (1975).

    Article  CAS  PubMed  Google Scholar 

  8. Saiki, R.K. et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354 (1985).

    Article  CAS  PubMed  Google Scholar 

  9. Shi, H. et al. Oligonucleotide-based microarray for DNA methylation analysis: principles and applications. J. Cell. Biochem. 88, 138–143 (2003).

    Article  CAS  PubMed  Google Scholar 

  10. Asakawa, J. et al. Quantitative and qualitative genetic variation in two-dimensional DNA gels of human lymphocytoid cell lines. Electrophoresis 16, 241–252 (1995).

    Article  CAS  PubMed  Google Scholar 

  11. Kawai, J. et al. Methylation profiles of genomic DNA of mouse developmental brain detected by restriction landmark genomic scanning (RLGS) method. Nucleic Acids Res. 21, 5604–5608 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Dahl, C. & Guldberg, P. DNA methylation analysis techniques. Biogerontology 4, 233–250 (2003).

    Article  CAS  PubMed  Google Scholar 

  13. Akama, T.O. et al. Restriction landmark genomic scanning (RLGS-M)-based genome-wide scanning of mouse liver tumors for alterations in DNA methylation status. Cancer Res. 57, 3294–3299 (1997).

    CAS  PubMed  Google Scholar 

  14. Watanabe, S. et al. Accessibility to tissue-specific genes from methylation profiles of mouse brain genomic DNA. Electrophoresis 16, 218–226 (1995).

    Article  CAS  PubMed  Google Scholar 

  15. Dai, Z. et al. An AscI boundary library for the studies of genetic and epigenetic alterations in CpG islands. Genome Res. 12, 1591–1598 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hayashizaki, Y. et al. A genetic linkage map of the mouse using restriction landmark genomic scanning (RLGS). Genetics 138, 1207–1238 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Hirotsune, S. et al. Spot mapping on the standard profile of restriction landmark genomic scanning (RLGS) of sorted chromosome 20 using methylation-insensitive enzyme. Genomics 24, 593–596 (1994).

    Article  CAS  PubMed  Google Scholar 

  18. Okuizumi, H. et al. Genetic mapping of restriction landmark genomic scanning loci in the mouse. Electrophoresis 16, 233–240 (1995).

    Article  CAS  PubMed  Google Scholar 

  19. Hirotsune, S. et al. Construction of high-resolution physical maps from yeast artificial chromosomes using restriction landmark genomic scanning (RLGS). Genomics 37, 87–95 (1996).

    Article  CAS  PubMed  Google Scholar 

  20. Okazaki, Y. et al. A genetic linkage map of the Syrian hamster and localization of cardiomyopathy locus on chromosome 9qa2.1-b1 using RLGS spot-mapping. Nat. Genet. 13, 87–90 (1996).

    Article  CAS  PubMed  Google Scholar 

  21. Hayashizaki, Y. et al. Identification of an imprinted U2af binding protein related sequence on mouse chromosome 11 using the RLGS method. Nat. Genet. 6, 33–40 (1994).

    Article  CAS  PubMed  Google Scholar 

  22. Shibata, H. et al. The use of restriction landmark genomic scanning to scan the mouse genome for endogenous loci with imprinted patterns of methylation. Electrophoresis 16, 210–217 (1995).

    Article  CAS  PubMed  Google Scholar 

  23. Plass, C. et al. Identification of Grf1 on mouse chromosome 9 as an imprinted gene by RLGS-M. Nat. Genet. 14, 106–109 (1996).

    Article  CAS  PubMed  Google Scholar 

  24. Costello, J.F. & Plass, C. Methylation matters. J. Med. Genet. 38, 285–303 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Rush, L.J. & Plass, C. Restriction landmark genomic scanning for DNA methylation in cancer: past, present, and future applications. Anal. Biochem. 307, 191–201 (2002).

    Article  CAS  PubMed  Google Scholar 

  26. Laird, P. W. & Jaenisch, R. DNA methylation and cancer. Hum. Mol. Genet. 3, 1487–1495 (1994).

    Article  CAS  PubMed  Google Scholar 

  27. Counts, J.L. & Goodman, J.I. Alterations in DNA methylation may play a variety of roles in carcinogenesis. Cell 83, 13–15 (1995).

    Article  CAS  PubMed  Google Scholar 

  28. Vachtenheim, J., Horakova, I. & Novotna, H. Hypomethylation of CCGG sites in the 3′ region of H-ras protooncogene is frequent and is associated with H-ras allele loss in non-small cell lung cancer. Cancer Res. 54, 1145–1148 (1994).

    CAS  PubMed  Google Scholar 

  29. Ohtani-Fujita, N. et al. CpG methylation inactivates the promoter activity of the human retinoblastoma tumor-suppressor gene. Oncogene 8, 1063–1067 (1993).

    CAS  PubMed  Google Scholar 

  30. Ottaviano, Y.L. et al. Methylation of the estrogen receptor gene CpG island marks loss of estrogen receptor expression in human breast cancer cells. Cancer Res. 54, 2552–2555 (1994).

    CAS  PubMed  Google Scholar 

  31. Asaga, S. et al. Identification of a new breast cancer-related gene by restriction landmark genomic scanning. Anticancer Res. 26, 35–42 (2006).

    CAS  PubMed  Google Scholar 

  32. Costello, J.F. et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nat. Genet. 24, 132–138 (2000).

    Article  CAS  PubMed  Google Scholar 

  33. Okazaki, Y. et al. Direct detection and isolation of restriction landmark genomic scanning (RLGS) spot DNA markers tightly linked to a specific trait by using the RLGS spot-bombing method. Proc. Natl. Acad. Sci. USA 92, 5610–5614 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Hirotsune, S. et al. Molecular cloning of polymorphic markers on RLGS gel using the spot target cloning method. Biochem. Biophys. Res. Commun. 194, 1406–1412 (1993).

    Article  CAS  PubMed  Google Scholar 

  35. Ohsumi, T. et al. A spot cloning method for restriction landmark genomic scanning. Electrophoresis 16, 203–209 (1995).

    Article  CAS  PubMed  Google Scholar 

  36. Hayashizaki, Y. et al. A new method for constructing NotI linking and boundary libraries using a restriction trapper. Genomics 14, 733–739 (1992).

    Article  CAS  PubMed  Google Scholar 

  37. Suzuki, H., Kawai, J., Taga, C., Ozawa, N. & Watanabe, S. A PCR-mediated method for cloning spot DNA on restriction landmark genomic scanning (RLGS) gel. DNA Res. 1, 245–250 (1994).

    Article  CAS  PubMed  Google Scholar 

  38. Plass, C. et al. An arrayed human Not I-EcoRV boundary library as a tool for RLGS spot analysis. DNA Res. 4, 253–255 (1997).

    Article  CAS  PubMed  Google Scholar 

  39. Smiraglia, D.J. et al. A new tool for the rapid cloning of amplified and hypermethylated human DNA sequences from restriction landmark genome scanning gels. Genomics 58, 254–262 (1999).

    Article  CAS  PubMed  Google Scholar 

  40. Akiyoshi, S. et al. A genetic linkage map of the MSM Japanese wild mouse strain with restriction landmark genomic scanning (RLGS). Mamm. Genome 11, 356–359 (2000).

    Article  CAS  PubMed  Google Scholar 

  41. Rouillard, J.M. et al. Virtual genome scan: a tool for restriction landmark-based scanning of the human genome. Genome Res. 11, 1453–1459 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Zardo, G. et al. Integrated genomic and epigenomic analyses pinpoint biallelic gene inactivation in tumors. Nat. Genet. 32, 453–458 (2002).

    Article  CAS  PubMed  Google Scholar 

  43. Matsuyama, T. et al. Global methylation screening in the Arabidopsis thaliana and Mus musculus genome: applications of virtual image restriction landmark genomic scanning (Vi-RLGS). Nucleic Acids Res. 31, 4490–4496 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Takamiya, T. et al. Restriction landmark genome scanning method using isoschizomers (MspI/HpaII) for DNA methylation analysis. Electrophoresis 27, 2846–2856 (2006).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank J. Kawai for valuable comments. This work was supported by a Research Grant for the RIKEN Genome Exploration Research Project from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government.

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  1. Functional RNA Research Program, Frontier Research System, and Genome Exploration Research Group, Genomic Sciences Center, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan

    Yoshinari Ando & Yoshihide Hayashizaki

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Correspondence to Yoshihide Hayashizaki.

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Ando, Y., Hayashizaki, Y. Restriction landmark genomic scanning. Nat Protoc 1, 2774–2783 (2006). https://doi.org/10.1038/nprot.2006.350

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