Russian Journal of Bioorganic Chemistry

, Volume 31, Issue 2, pp 170–177 | Cite as

A method for the preparation of normalized cDNA libraries enriched with full-length sequences

  • P. A. Zhulidov
  • E. A. Bogdanova
  • A. S. Shcheglov
  • I. A. Shagina
  • L. L. Wagner
  • G. L. Khazpekov
  • V. V. Kozhemyako
  • S. A. LukyanovEmail author
  • D. A. Shagin


We developed a new method for the preparation of normalized cDNA libraries enriched with full-length sequences. It is based on the properties of the recently characterized duplex-specific nuclease from the hepatopancreas of the Kamchatka crab. The duplex-specific nuclease is thermostable, effectively cleaves double-stranded DNA, and is inactive toward single-stranded DNA (Shagin et al., Genome Res., 2002, vol. 12, pp. 1935–1942). Our method enables the normalization of cDNA samples enriched with full-length sequences without use of laborious and ineffective stages of physical separation. The efficiency of the method was demonstrated in model experiments using cDNA samples from several human tissues.

Key words

cDNA normalization duplex-specific nuclease normalized cDNA libraries 


ds cDNA

double-stranded cDNA


duplex-specific nuclease


inverted terminal repeat


polymerase chain reaction


switching mechanism at the 5′-end of the RNA transcript

ss cDNA

single-stranded cDNA


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  1. 1.
    Young, B.D. and Anderson, M., in Nucleic Acids Hybridization: a Practical Approach, Hames, B.D.. and Higgins, S.J., Eds., Oxford-Washington, DC: IRL, 1985, pp. 47–71.Google Scholar
  2. 2.
    Ko, M., Nucleic Acids Res., 1990, vol. 18, pp. 5705–5711.PubMedGoogle Scholar
  3. 3.
    Patanjali, S.R., Parimoo, S., and Weissman, S.M., Proc. Natl. Acad. Sci. U.S.A., 1991, vol. 88, pp. 1943–1947.PubMedGoogle Scholar
  4. 4.
    Swaroop, A., Xu, J., Agarwal, N., and Weissman, S.M., Nucleic Acids Res., 1991, vol. 19, p. 1954.PubMedGoogle Scholar
  5. 5.
    Coche, T. and Dewez, M., Nucleic Acids Res., 1994, vol. 22, pp. 4545–4546.PubMedGoogle Scholar
  6. 6.
    Li, W.-B., Gruber, C.E., Lin, J.-J., Lim, R., D’Alessio, J.M., and Jessee, J.A., BioTechniques, 1994, vol. 16, pp. 722–729.PubMedGoogle Scholar
  7. 7.
    Sasaki, Y., Ayusawa, D., and Oishi, M., Nucleic Acids Res., 1994, vol. 22, pp. 987–992.PubMedGoogle Scholar
  8. 8.
    Soares, M., Bonaldo, M., Jelene, P., Su, L., Lawton, L., and Efstratiadis, A., Proc. Natl. Acad. Sci. U.S.A., 1994, vol. 91, pp. 9228–9232.PubMedGoogle Scholar
  9. 9.
    Bonaldo, M.F., Lennon, G., and Soares, M.B., Genome Res., 1996, vol. 6, pp. 791–806.PubMedGoogle Scholar
  10. 10.
    Luk’yanov, K.A., Gurskaya, N.G., Matts, M.V., Khaspekov, G.L., D’yachenko, L.B., Chenchik, A.A., Il’evich-Stuchkov, S.G., and Luk’yanov, S.A., Bioorg. Khim., 1996, vol. 22, pp. 686–690.PubMedGoogle Scholar
  11. 11.
    Carninci, P., Shibata, Y., Hayatsu, N., Sugahara, Y., Shibata, K., Itoh, M., Konno, H., Okazaki, Y., Muramatsu, M., and Hayashizaki, Y., Genome Res., 2000, vol. 10, pp. 1617–1630.CrossRefPubMedGoogle Scholar
  12. 12.
    Shagin, D.A., Rebrikov, D.V., Kozhemyako, V.B., Altshuler, I.M., Shcheglov, A.S., Zhulidov, P.A., Bogdanova, E.A., Staroverov, D.B., Rasskazov, V.A., and Lukyanov, S., Genome Res., 2002, vol. 12, pp. 1935–1942.CrossRefPubMedGoogle Scholar
  13. 13.
    Zhulidov, P.A., Bogdanova, E.A., Shcheglov, A.S., Vagner, L.L., Khaspekov, G.L., Kozhemyako, V.B., Matz, M.V., Meleshkevitch, E., Moroz, L.L., Lukyanov, S.A., and Shagin, D.A., Nucleic Acids Res., 2004, vol. 32, no.3, p. e37.CrossRefPubMedGoogle Scholar
  14. 14.
    Shagin, D.A., Lukyanov, K.A., Vagner, L.L., and Matz, M.V., Nucleic Acids Res., 1999, vol. 27, p. e23.CrossRefPubMedGoogle Scholar
  15. 15.
    Carninci, P., Kvam, C., Kitamura, A., Ohsumi, T., Okazaki, Y., Itoh, M., Kamiya, M., Shibata, K., Sasaki, N., Izawa, M., et al., Genomics, 1996, vol. 37, pp. 327–336.CrossRefPubMedGoogle Scholar
  16. 16.
    Maruyama, K. and Sugano, S., Gene, 1994, vol. 138, pp. 171–174.CrossRefPubMedGoogle Scholar
  17. 17.
    Chenchik, A., Zhu, Y.Y., Diatchenko, L., Li, R., Hill, J., and Siebert, P.D., in BioTechniques Books, Siebert, P. and Larrick, J., Eds., Natick, MA: np, 1998, pp. 305–319.Google Scholar
  18. 18.
    Matz, M., Shagin, D., Bogdanova, E., Britanova, O., Lukyanov, S., Diatchenko, L., and Chenchik, A., Nucleic Acids Res., 1999, vol. 27, pp. 1558–1560.CrossRefPubMedGoogle Scholar
  19. 19.
    Zhu, Y.Y., Machleder, E.M., Chenchik, A., Li, R., and Siebert, P.D., BioTechniques, 2001, vol. 30, pp. 892–897.PubMedGoogle Scholar
  20. 20.
    Franz, O., Bruchhaus, I.I., and Roeder, T., Nucleic Acids Res., 1999, vol. 27, p. e3.CrossRefPubMedGoogle Scholar
  21. 21.
    Moreno-Palanques, R.F and Fuldner, R.A., in Automated DNA Sequencing and Analysis, Adams, M.D., Fields, C., and Venter, J.C., Eds., London: Academic, 1994, pp. 102–109.Google Scholar
  22. 22.
    Engler-Blum, G., Meier, M., Frank, J., and Muller, G., Anal. Biochem., 1993, vol. 44, pp. 235–244.CrossRefGoogle Scholar
  23. 23.
    Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989.Google Scholar

Copyright information

© MAIK “Nauka/Interperiodica” 2005

Authors and Affiliations

  • P. A. Zhulidov
    • 1
  • E. A. Bogdanova
    • 1
  • A. S. Shcheglov
    • 1
  • I. A. Shagina
    • 2
  • L. L. Wagner
    • 1
    • 2
  • G. L. Khazpekov
    • 3
  • V. V. Kozhemyako
    • 4
  • S. A. Lukyanov
    • 1
    Email author
  • D. A. Shagin
    • 1
  1. 1.Shemyakin-Ovchinnikov Institute of Bioorganic ChemistryRussian Academy of SciencesMoscowRussia
  2. 2.ZAO EvrogenMoscowRussia
  3. 3.Cardiological Research CenterRussian Academy of Medical SciencesMoscowRussia
  4. 4.Pacific Institute of Bioorganic Chemistry, Far East DivisionRussian Academy of SciencesVladivostokRussia

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