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      Simple cDNA normalization using kamchatka crab duplex-specific nuclease.

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          Abstract

          We developed a novel simple cDNA normalization method [termed duplex-specific nuclease (DSN) normalization] that may be effectively used for samples enriched with full-length cDNA sequences. DSN normalization involves the denaturation-reassociation of cDNA, degradation of the double-stranded (ds) fraction formed by abundant transcripts and PCR amplification of the equalized single-stranded (ss) DNA fraction. The key element of this method is the degradation of the ds fraction formed during reassociation of cDNA using the kamchatka crab DSN, as described recently. This thermostable enzyme displays a strong preference for cleaving ds DNA and DNA in DNA-RNA hybrid duplexes compared with ss DNA and RNA, irrespective of sequence length. We developed normalization protocols for both first-strand cDNA [when poly(A)+ RNA is available] and amplified cDNA (when only total RNA can be obtained). Both protocols were evaluated in model experiments using human skeletal muscle cDNA. We also employed DSN normalization to normalize cDNA from nervous tissues of the marine mollusc Aplysia californica (a popular model organism in neuroscience) to illustrate further the efficiency of the normalization technique.

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          Author and article information

          Journal
          Nucleic Acids Res
          Nucleic acids research
          Oxford University Press (OUP)
          1362-4962
          0305-1048
          Feb 18 2004
          : 32
          : 3
          Affiliations
          [1 ] Shemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117871 Moscow, Russia.
          Article
          32/3/e37
          10.1093/nar/gnh031
          373426
          14973331
          51fb3d0d-ca6f-40be-adce-4e98d1f43ee4
          History

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