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

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Abstract

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.

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Abbreviations

ds cDNA:

double-stranded cDNA

DSN:

duplex-specific nuclease

ITR:

inverted terminal repeat

PCR:

polymerase chain reaction

SMART:

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

ss cDNA:

single-stranded cDNA

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Authors and Affiliations

  1. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997, Russia

    P. A. Zhulidov, E. A. Bogdanova, A. S. Shcheglov, L. L. Wagner, S. A. Lukyanov & D. A. Shagin

  2. ZAO Evrogen, Moscow, Russia

    I. A. Shagina & L. L. Wagner

  3. Cardiological Research Center, Russian Academy of Medical Sciences, Moscow, Russia

    G. L. Khazpekov

  4. Pacific Institute of Bioorganic Chemistry, Far East Division, Russian Academy of Sciences, pr. Stoletiya Vladivostoka 159, Vladivostok, 690022, Russia

    V. V. Kozhemyako

Authors
  1. P. A. Zhulidov
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  2. E. A. Bogdanova
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  3. A. S. Shcheglov
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  4. I. A. Shagina
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  5. L. L. Wagner
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  6. G. L. Khazpekov
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  7. V. V. Kozhemyako
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  8. S. A. Lukyanov
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  9. D. A. Shagin
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Corresponding author

Correspondence to S. A. Lukyanov.

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__________

Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 186–194.

Original Russian Text Copyright © 2005 by Zhulidov, Bogdanova, Shcheglov, Shagina, Wagner, Khazpekov, Kozhemyako, Lukyanov, Shagin.

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Zhulidov, P.A., Bogdanova, E.A., Shcheglov, A.S. et al. A method for the preparation of normalized cDNA libraries enriched with full-length sequences. Russ J Bioorg Chem 31, 170–177 (2005). https://doi.org/10.1007/s11171-005-0023-7

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  • DOI: https://doi.org/10.1007/s11171-005-0023-7

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