Molecular Biotechnology

, Volume 41, Issue 3, pp 247–253

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

DOI: 10.1007/s12033-008-9131-y

Cite this article as:
Bogdanova, E.A., Shagina, I.A., Mudrik, E. et al. Mol Biotechnol (2009) 41: 247. doi:10.1007/s12033-008-9131-y

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 depletionDuplex-specific nucleaseKamchatka crabFull-length cDNAExpression cloning

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)

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