RLCS, Restriction Landmark cDNA Scanning

  • Takeshi Yaoi
  • Harukazu Suzuki
  • Jun Kawai
  • Sachihiko Watanabe
Part of the Springer Lab Manuals book series (SLM)


It is important to identify and isolate differentially expressed genes whose expression underlies many biological processes such as development, differentiation, and cellular response to various stimuli, and also pathological changes that arise in diseases. Classically, two hybridization methods, differential and subtractive hybridization, have been used to analyze and isolate such genes [1–3]. However, differential hybridization is effective only for mRNAs expressed abundantly in one of the two types of cells. Subtractive hybridization is rather empirical and poor in reproducibility. Furthermore, both methods are time-consuming. Recently, Liang and Pardee have developed a novel method called differential display using an arbitrarily primed reverse transcription-coupled PCR and sequencing gels [4]. This has proved to be a powerful technique for detecting changes in eukaryotic gene expression; it is simple, sensitive, and time-saving. However, PCR-mediated amplification with arbitrary primers requires delicate control, depending on the primers used [5,6], and cDNA probes thus obtained often give false positive signals on Northern blot analysis [7,8]. Furthermore, the differential display seems less sensitive in detecting rare mRNA populations, since it has a strong bias toward higher abundance mRNAs [9].


Differential Display mRNA Species Anchor Primer Restriction Landmark Genomic Scanning Rare mRNA 
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  1. 1.
    Hendrick SM, Cohen DI, Nielsen EA, Davis MM (1984) Isolation of cDNA clones encoding T cell-specific membrane-associated proteins. Nature 308:149–153CrossRefGoogle Scholar
  2. 2.
    Sargent TD, Dawid IB (1983) Differential gene expression in the gastrula of Xenopus laevis. Science 222:135–139PubMedCrossRefGoogle Scholar
  3. 3.
    St John TP, Davis RW (1979) Isolation of galactose-inducible DNA sequences from Saccharomyces cerevisiae by differential plaque filter hybridization. Cell 16:443–452PubMedCrossRefGoogle Scholar
  4. 4.
    Liang P, Pardee AB (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967–971PubMedCrossRefGoogle Scholar
  5. 5.
    Bauer D, Müller H, Reich J, Ahrenkiel V, Warthoe P, Strauss M (1993) Identification of differentially expressed mRNA species by an improved display technique (DDRT-PCR). Nucleic Acids Res 21:4272–4280PubMedCrossRefGoogle Scholar
  6. 6.
    Guimarães MJ, Lee F, Zlotnik A, McClanahan T (1995) Differential display by PCR: novel findings and applications. Nucleic Acids Res 23:1832–1833PubMedCrossRefGoogle Scholar
  7. 7.
    Liang P, Averboukh L, Pardee AB (1993) Distribution and cloning of eukaryotic mRNAs by means of differential display. Nucleic Acids Res 21:3269–3275PubMedCrossRefGoogle Scholar
  8. 8.
    Mou L, Miller H, Li J, Wang E, Chalifour L (1994) Improvements to the differential display method for gene analysis. Biochem Biophys Res Commun 199:564–569PubMedCrossRefGoogle Scholar
  9. 9.
    Bertioli DJ, Schlichter UHA, Adams MJ, Burrows PR, Steinbiβ H-H, Antoniw JF (1995) An analysis of differential display shows a strong bias towards high copy number mRNAs. Nucleic Acids Res 23:4520–4523PubMedCrossRefGoogle Scholar
  10. 10.
    Hatada I, Hayashizaki Y, Hirotsune S, Komatsubara H, Mukai T (1991) A genomic scanning method for higher organisms using restriction sites as landmark. Proc Natl Acad Sci USA 88:9523–9527PubMedCrossRefGoogle Scholar
  11. 11.
    Hayashizaki Y, Hirotsune S, Okazaki Y, Hatada I, Shibata H, Kawai J, Hirose K, Watanabe S, Fushiki S, Wada S, Sugimoto T, Kobayakawa K, Kawara T, Katsuki M, Shibuya T, Mukai T (1993) Restriction landmark genomic scanning method and its various applications. Electrophoresis 14:251–258PubMedCrossRefGoogle Scholar
  12. 12.
    Hayashizaki Y, Hirotsune S, Okazaki Y, Shibata H, Akasako A, Muramatsu M, Kawai J, Hirasawa T, Shiroishi T, Watanabe S, Shiroishi T, Moriwaki K, Taylor BA, Matsuda Y, Elliott RW, Manly KF, Chapman VM (1994) A genetic linkage map of the mouse using restriction landmark genomic scanning (RLGS). Genetics 138:1207–1238PubMedGoogle Scholar
  13. 13.
    Okazaki Y, Hirose K, Hirotsune S, Okuizumi H, Sasaki N, Ohsumi T, Yoshiki A, Kusakabe M, Muramatsu M, Kawai J, Watanabe S, Plass C, Chapman VM, Nakao K, Katsuki M, Hayashizaki Y (1995) Direct detection and isolation of RLGS spot DNA marker tightly linked to a specific trait using RLGS spot-bombing method. Proc Natl Acad Sci USA 92:5610–5614PubMedCrossRefGoogle Scholar
  14. 14.
    Kawai J, Hirose K, Fushiki S, Hirotsune S, Ozawa N, Hara A, Hayashizaki Y, Watanabe S (1994) Comparison of DNA methylation patterns among mouse cell lines by restriction landmark genomic scanning (RLGS). Mol Cell Biol 14:7421–7427PubMedGoogle Scholar
  15. 15.
    Kawai J, Suzuki H, Taga C, Hara A, Watanabe S (1995) Correspondence of RLGS-M spot behavior with tissue expression on mouse homologue of DP1/ TB2 gene. Biochem Biophys Res Commun 213:967–974PubMedCrossRefGoogle Scholar
  16. 16.
    Hayashizaki Y, Shibata H, Hirotsune S, Sugino H, Okazaki Y, Sasaki N, Hirose K, Imoto H, Okuizumi H, Muramatsu M, Komatsubara H, Shiroishi T, Moriwaki K, Katsuki M, Hatano N, Sasaki H, Ueda T, Mise N, Takagi N, Plass C, Chapman VM (1994) Identification of an imprinted U2af binding protein related sequence on mouse choromosome 11 using the RLGS method. Nat Genet 6:33–40PubMedCrossRefGoogle Scholar
  17. 17.
    Shibata H, Yoshino K, Muramatsu M, Plass C, Chapman VM, Hayashizaki Y (1995) The use of restriction landmark genomic scanning to scan the mouse genome for endogenous loci with imprinted patterns of methylation. Electrophoresis 16:210–217PubMedCrossRefGoogle Scholar
  18. 18.
    Watanabe S, Kawai J, Hirotsune S, Suzuki H, Hirose K, Taga C, Ozawa N, Fushiki S, Hayashizaki Y (1995) Accessibility to tissue-specific genes from methylation profiles of mouse brain genomic DNA. Electrophoresis 16:218–226PubMedCrossRefGoogle Scholar
  19. 19.
    Kawai J, Hirotsune S, Hirose K, Fushiki S, Watanabe S, Hayashizaki Y (1993) Methylation profiles of genomic DNA of mouse developmental brain detected by restriction landmark genomic scanning (RLGS) method. Nucleic Acids Res 21:5604–5608PubMedCrossRefGoogle Scholar
  20. 20.
    Hirotsune S, Hatada I, Komatsubara H, Nagai H, Kuma K, Kobayakawa K, Kawara T, Nakagawara A, Fujii K, Mukai T, Hayashizaki Y (1992) New spproach for detection of amplification in cancer DNA using restriction landmark genomic scanning. Cancer Res 52:3642–3647PubMedGoogle Scholar
  21. 21.
    Nagai H, Ponglikitmongkol M, Mita E, Ohmachi Y, Yoshikawa H, Saeki R, Yumoto Y, Nakanishi T, Matsubara K (1994) Aberation of genomic DNA in association with human hepatocellular carcinomas detected by 2-dimensional gel analysis. Cancer Res 54: 1545–1550PubMedGoogle Scholar
  22. 22.
    Miwa W, Yashima K, Sekine T, Sekiya T (1995) Demethylation of a repetitive DNA sequence in human cancers. Electrophoresis 16:227–232PubMedCrossRefGoogle Scholar
  23. 23.
    Hirotsune S, Shibata H, Okazaki Y, Sugino H, Imoto H, Sasaki N, Hirose K, Okuizumi H, Muramatsu M, Plass C, Chapman VM, Tamatsukuri S, Miyamoto C, Fumichi Y, Hayashizaki Y (1993) Molecular cloning of polymorphic markers on RLGS gel using the spot target cloning method. Biochem Biophys Res Commun 194:1406–1412PubMedCrossRefGoogle Scholar
  24. 24.
    Suzuki H, Kawai J, Taga C, Ozawa N, Watanabe S (1994) A PCR-mediated method for cloning spot DNA on restriction landmark genomic scanning (RLGS) gel. DNA Res 1:175–180CrossRefGoogle Scholar
  25. 25.
    Suzuki H, Yaoi T, Kawai J, Hara A, Kuwajima G, Watanabe S (1996) Restriction landmark cDNA scanning (RLCS): a novel cDNA display system using two-dimensional gel electrophoresis. Nucleic Acids Res 24:289–294PubMedCrossRefGoogle Scholar
  26. 26.
    Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159PubMedCrossRefGoogle Scholar
  27. 27.
    Kato K (1995) Description of the entire mRNA population by a 3′ end cDNA fragment generated by class IIS restriction enzymes. Nucleic Acids Res 23:3685–3690PubMedCrossRefGoogle Scholar
  28. 28.
    Schaefer BC (1995) Revolutions in rapid amplification of cDNA ends: new strategies for polymerase chain reaction cloning of full-length cDNA ends. Anal Biochem 227:255–273PubMedCrossRefGoogle Scholar

Copyright information

© Springer Japan 1997

Authors and Affiliations

  • Takeshi Yaoi
  • Harukazu Suzuki
  • Jun Kawai
  • Sachihiko Watanabe

There are no affiliations available

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