Schematic outline of DSN-normalization.
Black lines represent abundant transcripts, grey lines – rare transcripts. Rectangle represents adapter sequence and its complement.
cDNA normalization using duplex-specific nuclease (DSN) is a highly efficient approach that can be applied for normalization of full-length-enriched cDNA (Zhulidov et al., 2004; Zhulidov et al., 2005). The method is based on nucleic acid hybridization kinetics (Young and Anderson, 1985) and unique properties of the duplex-specific nuclease (DSN) specific to the double-stranded (ds) DNA (Shagin et al., 2002).
DSN-normalization is performed prior to library cloning. After denaturation of ds cDNA flanked with known adapters, it is subjected to renaturation. During renaturation, abundant transcripts convert to the ds form more effectively than those that are less frequent. Thus, two fractions are formed, specifically, a ds-fraction of abundant cDNA and a normalized single-stranded (ss) cDNA. The ds cDNA fraction is then degraded by DSN.
| Schematic outline of DSN-normalization.Black lines represent abundant transcripts, grey lines – rare transcripts. Rectangle represents adapter sequence and its complement. |
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DSN is an enzyme from Red King (Kamchatka) crab that displays a strong preference for cleaving ds DNA compared to ss-DNA and RNA, irrespective of the sequence length. DSN is stable under elevated temperatures (Shagin et al., 2002). Maximal DSN activity is observed at 60-65°C, and about 25% of the activity is retained after incubation at 70°C for 20 min. Owing to DSN thermostability, ds DNA degradation is performed under conditions of cDNA renaturation that prevent the formation of secondary structures and non-specific hybridization involving adapter sequences within the ss cDNA fraction.
The remaining normalized ss DNA is amplified by PCR. PCR primers and conditions are optimized to minimize the PCR tendency to amplify shorter fragments more efficiently than longer ones. Normalized cDNA can then be used for library cloning or sequencing.
cDNA suitable for normalization can be prepared on the basis of total or poly(A)+ RNA and should contain known adapter sequences at both ends for PCR amplification. The quality of the RNA is crucial, especially when construction of full-length enriched cDNA library is a goal. The flanking sequences can be introduced to the cDNA ends during cDNA synthesis by various means, for example, adapter ligation or during cDNA synthesis using the template-switching approach.
DSN-normalization has been successfully applied to various animal and plant models (see Bogdanova et al., 2008 for review). The flexibility of this normalization procedure allows simple modifications for various purposes. Detailed protocols of DSN-normalization modifications are available in the book Nucleic Acids Hybridization Modern Applications (Shcheglov et al., 2007), and Current Protocols (Bogdanova et al., 2010).