Molecular Biotechnology

, Volume 41, Issue 3, pp 247–253 | Cite as

DSN Depletion is a Simple Method to Remove Selected Transcripts from cDNA Populations

  • Ekaterina A. Bogdanova
  • Irina A. Shagina
  • Elena Mudrik
  • Igor Ivanov
  • Peter Amon
  • Laura L. Vagner
  • Sergey A. Lukyanov
  • Dmitry A. Shagin
Research

Abstract

A novel DSN-depletion method allows elimination of selected sequences from full-length-enriched cDNA libraries. Depleted cDNA can be applied for subsequent EST sequencing, expression cloning, and functional screening approaches. The method employs specific features of the kamchatka crab duplex-specific nuclease (DSN). This thermostable enzyme is specific for double-stranded (ds) DNA, and is thus used for selective degradation of ds DNA in complex nucleic acids. DSN depletion is performed prior to library cloning, and includes the following steps: target cDNA is mixed with excess driver DNA (representing fragments of the genes to be eliminated), denatured, and allowed to hybridize. During hybridization, driver molecules form hybrids with the target sequences, leading to their removal from the ss DNA fraction. Next, the ds DNA fraction is hydrolyzed by DSN, and the ss fraction is amplified using long-distance PCR. DSN depletion has been tested in model experiments.

Keywords

cDNA depletion Duplex-specific nuclease Kamchatka crab Full-length cDNA Expression cloning 

Notes

Acknowledgment

This work was supported by grant from Rosnauka 02.512.11.2216 and by NS-2395.2008.4.

Supplementary material

12033_2008_9131_MOESM1_ESM.doc (34 kb)
ESM1 (DOC 33 kb)
12033_2008_9131_MOESM2_ESM.doc (370 kb)
ESM2 (DOC 370 kb)

References

  1. 1.
    Aiba, K., Carter, M. G., Matoba, R., & Ko, M. S. (2006). Genomic approaches to early embryogenesis and stem cell biology. Seminars in Reproductive Medicine, 24(5), 330–339. doi: 10.1055/s-2006-952155.CrossRefGoogle Scholar
  2. 2.
    Hart, C. P. (2005). Finding the target after screening the phenotype. Drug Discovery Today, 10(7), 513–519. doi: 10.1016/S1359-6446(05)03415-X.CrossRefGoogle Scholar
  3. 3.
    Kawakami, Y., Fujita, T., Matsuzaki, Y., Sakurai, T., Tsukamoto, M., Toda, M., et al. (2004). Identification of human tumor antigens and its implications for diagnosis and treatment of cancer. Cancer Science, 95(10), 784–791. doi: 10.1111/j.1349-7006.2004.tb02182.x.CrossRefGoogle Scholar
  4. 4.
    Diatchenko, L., Lau, Y.-F. C., Campbell, A. P., Chenchik, A., Mogadam, F., Huang, B., et al. (1996). Suppression Sabtracive Hybridization, A method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proceedings of the National Academy of Sciences of the United States of America, 93, 6025–6030. doi: 10.1073/pnas.93.12.6025.CrossRefGoogle Scholar
  5. 5.
    Bonaldo, M. F., Lennon, G., & Soares, M. B. (1996). Normalization and subtraction: Two approaches to facilitate gene discovery. Genome Research, 6, 791–806. doi: 10.1101/gr.6.9.791.CrossRefGoogle Scholar
  6. 6.
    Scheetz, T. E., Laffin, J. J., Berger, B., Holte, S., Baumes, S. A., Brown, R., 2nd, et al. (2004). High-throughput gene discovery in the rat. Genome Research, 14(4), 733–741. doi: 10.1101/gr.1414204.CrossRefGoogle Scholar
  7. 7.
    Swaroop, A., Xu, J., Agarwal, N., & Weissman, S. M. (1991). A simple and efficient cDNA library subtraction procedure, Isolation of human retina-specific cDNA clones. Nucleic Acids Research, 19, 1954. doi: 10.1093/nar/19.8.1954.CrossRefGoogle Scholar
  8. 8.
    Carninci, P., Shibata, Y., Hayatsu, N., Sugahara, Y., Shibata, K., Itoh, M., et al. (2000). Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes. Genome Research, 10, 1617–1630. doi: 10.1101/gr.145100.CrossRefGoogle Scholar
  9. 9.
    Shagin, D. A., Rebrikov, D. V., Kozhemyako, V. B., Altshuler, I. M., Shcheglov, A. S., Zhulidov, P. A., et al. (2002). A novel method for SNP detection using a new duplex-specific nuclease from crab hepatopancreas. Genome Research, 12, 1935–1942. doi: 10.1101/gr.547002.CrossRefGoogle Scholar
  10. 10.
    Zhulidov, P. A., Bogdanova, E. A., Shcheglov, A. S., Vagner, L. L., Khaspekov, G. L., Kozhemyako, V. B., et al. (2004). Simple cDNA normalization using kamchatka crab duplex-specific nuclease. Nucleic Acids Research, 32, e37. doi: 10.1093/nar/gnh031.CrossRefGoogle Scholar
  11. 11.
    Zhulidov, P. A., Bogdanova, E. A., Shcheglov, A. S., Shagina, I. A., Wagner, L. L., Khaspekov, G. L., et al. (2005). A method for the preparation of normalized cDNA libraries enriched with full-length sequences. Russian Journal of Bioorganic Chemistry, 31, 170–177. doi: 10.1007/s11171-005-0023-7.CrossRefGoogle Scholar
  12. 12.
    Bogdanova, E. A., Shagin, D. A., & Lukyanov, S. A. (2008). Normalization of full-length enriched cDNA. Molecular BioSystems, 4(3), 205–212. doi: 10.1039/b715110c.CrossRefGoogle Scholar
  13. 13.
    Al’tshuler, I. M., Zhulidov, P. A., Bogdanova, E. A., Mudrik, N. N., & Shagin, D. A. (2005). Application of the duplex-specific nuclease preference method to the analysis of point mutations in human genes. Russian Journal of Bioorganic Chemistry, 31(6), 567–575. doi: 10.1007/s11171-005-0078-5.CrossRefGoogle Scholar
  14. 14.
    Zhao, Y., Hoshiyama, H., Shay, J. W., & Wright, W. E. (2008). Quantitative telomeric overhang determination using a double-strand specific nuclease. Nucleic Acids Research, 36(3), e14. doi: 10.1093/nar/gkm1063.CrossRefGoogle Scholar
  15. 15.
    Matz, M. V. (2003). Amplification of representative cDNA pools from microscopic amounts of animal tissue. Methods in Molecular Biology (Clifton N.J.), 221, 103–116.Google Scholar
  16. 16.
    Barnes, W. M. (1994). PCR amplification of up to 35_kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proceedings of the National Academy of Sciences of the United States of America, 91, 2216–2220. doi: 10.1073/pnas.91.6.2216.CrossRefGoogle Scholar
  17. 17.
    Young, B. D., & Anderson, M. (1985). Quantitative analysis of solution hybridisation. In B. D. Hames & S. J. Higgins (Eds.), Nucleic acids hybridisation, a practical approach (pp. 47–71). Oxford-Washington DC: IRL Press.Google Scholar
  18. 18.
    Matz, M., Shagin, D., Bogdanova, E., Britanova, O., Lukyanov, S., Diatchenko, L., et al. (1999). Amplification of cDNA ends based on template-switching effect and step-out PCR. Nucleic Acids Research, 27(6), 1558–1560. doi: 10.1093/nar/27.6.1558.CrossRefGoogle Scholar
  19. 19.
    Zhu, Y. Y., Machleder, E. M., Chenchik, A., Li, R., & Siebert, P. D. (2001). Reverse transcriptase template switching, a SMART approach for full-length cDNA library construction. BioTechniques, 30, 892–897.Google Scholar
  20. 20.
    Gurskaya, N. G., Diatchenko, L., Chenchik, A., Siebert, P. D., Khaspekov, G. L., Lukyanov, K. A., et al. (1996). Equalizing cDNA subtraction based on selective suppression of polymerase chain reaction: cloning of Jurkat cell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate. Analytical Biochemistry, 240(1), 90–97. doi: 10.1006/abio.1996.0334.CrossRefGoogle Scholar
  21. 21.
    Beavo, J. A. (1995). Cyclic nucleotide phosphodiesterases: Functional implications of multiple isoforms. Physiological Reviews, 75, 725–748.Google Scholar
  22. 22.
    Manganiello, V. C., Murata, T., Taira, M., Belfrage, P., & Degerman, E. (1995). Diversity in cyclic nucleotide phosphodiesterase isoenzyme families. Archives of Biochemistry and Biophysics, 322(1), 1–13. doi: 10.1006/abbi.1995.1429.CrossRefGoogle Scholar

Copyright information

© Humana Press 2008

Authors and Affiliations

  • Ekaterina A. Bogdanova
    • 1
    • 2
  • Irina A. Shagina
    • 2
  • Elena Mudrik
    • 2
  • Igor Ivanov
    • 3
  • Peter Amon
    • 3
  • Laura L. Vagner
    • 1
  • Sergey A. Lukyanov
    • 1
  • Dmitry A. Shagin
    • 1
    • 2
  1. 1.Shemiakin and Ovchinnikov Institute of Bioorganic Chemistry RASMoscowRussia
  2. 2.Evrogen JSCMoscowRussia
  3. 3.GPC Biotech AGMunchenGermany

Personalised recommendations