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

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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.

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Acknowledgment

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

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Authors and Affiliations

  1. Shemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117871, Moscow, Russia

    Ekaterina A. Bogdanova, Laura L. Vagner, Sergey A. Lukyanov & Dmitry A. Shagin

  2. Evrogen JSC, Miklukho-Maklaya 16/10, 117871, Moscow, Russia

    Ekaterina A. Bogdanova, Irina A. Shagina, Elena Mudrik & Dmitry A. Shagin

  3. GPC Biotech AG, Fraunhoferstr, 20 Martinsried, Munchen, 82152, Germany

    Igor Ivanov & Peter Amon

Authors
  1. Ekaterina A. Bogdanova
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  2. Irina A. Shagina
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  3. Elena Mudrik
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  4. Igor Ivanov
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  5. Peter Amon
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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.

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Bogdanova, E.A., Shagina, I.A., Mudrik, E. et al. DSN Depletion is a Simple Method to Remove Selected Transcripts from cDNA Populations. Mol Biotechnol 41, 247–253 (2009). https://doi.org/10.1007/s12033-008-9131-y

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  • DOI: https://doi.org/10.1007/s12033-008-9131-y

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