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Published online 18 February 2004

Nucleic Acids Research, 2004, Vol. 32, No. 3 e37
© 2004 Oxford University Press

Simple cDNA normalization using kamchatka crab duplex-specific nuclease

Pavel A. Zhulidov1, Ekaterina A. Bogdanova1,2, Alex S. Shcheglov1, Laura L. Vagner1,2, George L. Khaspekov3, Valery B. Kozhemyako4, Mikhail V. Matz5, Ella Meleshkevitch5, Leonid L. Moroz5, Sergey A. Lukyanov*,1 and Dmitry A. Shagin1,2

1 Shemiakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, 117871 Moscow, Russia, 2 Evrogen JSC, Miklukho-Maklaya 16/10, 117871 Moscow, Russia, 3 Cardiology Research Centre, Russian Academy of Medical Science, Moscow 121552, Russia, 4 Pacific Institute of Bioorganic Chemistry, RAS Far East Division, Pr. Stoletiya Vladivostoka 159, 690022 Vladivostok, Russia and 5 Whitney Laboratory and Department of Neuroscience, University of Florida, 9505 Ocean Shore Boulevard, St Augustine, FL 32080, USA

*To whom correspondence should be addressed. Tel/Fax: +7 095 330 7056; Email: luk{at}ibch.ru
+BF707524–BF708380, BF713631, BF713632, BI273615–BI273627 and CK327631–CK328797

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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