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Normalization of Full-Length-Enriched cDNA

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Book cover cDNA Libraries

Part of the book series: Methods in Molecular Biology ((MIMB,volume 729))

Abstract

A well-recognized obstacle to efficient high-throughput analysis of cDNA libraries is the differential abundance of various transcripts in any particular cell type. Decreasing the prevalence of clones representing abundant transcripts before sequencing, using cDNA normalization, may significantly increase the efficacy of random sequencing and is essential for rare gene discovery. Duplex-specific nuclease (DSN) normalization allows the generation of normalized full-length-enriched cDNA libraries to permit a high gene discovery rate. The method is based on the unique properties of DSN from the Kamchatka crab and involves denaturation–reassociation of cDNA, degradation of the ds-fraction formed by abundant transcripts by DSN, and PCR amplification of the remaining ss-DNA fraction. The method has been evaluated in various plant and animal models.

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Acknowledgments

This work was supported by Evrogen JSC (Moscow, Russia) and by the program “State Support of the Leading Scientific Schools” (NS-5638.2010.4).

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Authors and Affiliations

  1. Shemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russia

    Ekaterina A. Bogdanova, Ekaterina V. Barsova, Alexander Scheglov, Veronika Anisimova, Laura L. Vagner, Sergey A. Lukyanov & Dmitry A. Shagin

  2. Evrogen JSC, Moscow, Russia

    Irina A. Shagina, Veronika Anisimova & Laura L. Vagner

Authors
  1. Ekaterina A. Bogdanova
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  2. Ekaterina V. Barsova
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  3. Irina A. Shagina
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  4. Alexander Scheglov
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  5. Veronika Anisimova
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  6. Laura L. Vagner
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  7. Sergey A. Lukyanov
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  8. Dmitry A. Shagin
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Corresponding author

Correspondence to Ekaterina A. Bogdanova .

Editor information

Editors and Affiliations

  1. , Dept. Plant Sciences & Plant Pathology, Montana State University, Bozeman, 59717-3150, Montana, USA

    Chaofu Lu

  2. Institute of Biological Chemistry, Washington State University, Pullman, 99164-6340, Washington, USA

    John Browse

  3. Institute of Biological Chemistry, Washington State University, Pullman, 99164-6340, Washington, USA

    James G. Wallis

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Bogdanova, E.A. et al. (2011). Normalization of Full-Length-Enriched cDNA. In: Lu, C., Browse, J., Wallis, J. (eds) cDNA Libraries. Methods in Molecular Biology, vol 729. Humana Press. https://doi.org/10.1007/978-1-61779-065-2_6

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  • DOI: https://doi.org/10.1007/978-1-61779-065-2_6

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-064-5

  • Online ISBN: 978-1-61779-065-2

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