DSN-normalization of full-length-enriched cDNA

Whole transcriptome analysis is one of the general requirements to address many basic biological questions. Large-scale EST sequencing is a major approach that provides direct information on the transcriptome and indirect information on the relationship between the genome and different phenotypes. The recent development of novel parallel sequencers (e.g., 454 Life Sciences GS 20) has made it possible to obtain substantial sequence information quickly and at a significantly reduced cost.

However, the major limitation of EST projects is associated with significant fluctuations in the concentrations of different transcripts within cells. Within a population of eukaryotic cellular mRNAs, transcript copy numbers vary by orders (Alberts et al., 1994). Hence, sequencing of the complete eukaryotic transcriptome may require the analysis of about ~10⁸ clones from each cDNA library to identify rare sequences, whereas transcripts of medium and high abundance are sequenced several times.

Methods to decrease the prevalence of highly abundant transcripts and equalize mRNA concentrations in a cDNA library are designated ‘cDNA normalization’. Normalization is utilized to enhance the gene discovery rate of a cDNA library and facilitate the identification and analysis of rare transcripts. This approach is imperative for EST sequencing of the complete transcriptome, and useful in other applications, such as functional screening, construction of specific RNA libraries, and Transcript End Sequence Profiling.

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; 2005). Both total and poly(A)+ RNA can be used as a starting material. 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.

DSN-normalization is performed prior to library cloning, and involves the denaturation of ds cDNA flanked with known adapters, its subsequent renaturation, and enzymatic degradation of the ds DNA fraction (formed by abundant transcripts) by DSN. The equalized ss cDNA fraction remains intact, and is amplified by PCR.

	Schematic outline of DSN-normalization. Black lines represent abundant transcripts, grey line – rare transcripts. Rectangle represents adapter sequence and its complement. Enlarge scheme

DSN isolated from kamchatka crab exhibits a strong preference for ds DNA as a substrate, and is stable under elevated temperatures (Shagin et al., 2002). Maximal DSN activity is observed at 60-65°C, and about 25% activity is retained even after incubation at 70°C for 20 min. These properties allow the effective removal of ds DNA, while ss fraction remains intact.

Owing to DSN thermostability, DSN-based degradation of ds DNA 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.

DSN-normalization includes a step involving amplification of the normalized ss cDNA fraction. To overcome the PCR tendency to amplify shorter DNA fragments more efficiently than longer fragments, regulation of the average length of complex PCR products by partial PCR suppression is used (Shagin et al., 1999). To attain the PCR suppression effect, the cDNA to be amplified should contain inverted terminal repeats.

Adapter sequences can be introduced into cDNA ends by various means, for example, adapter ligation or during cDNA synthesis. Depending on the flanking adapters used in cDNA synthesis, normalized cDNA can be cloned directionally or non-directionally. A size-separation procedure is recommended to remove short cDNA fragments before cloning.

In addition to amplified ds DNA normalization, mostly required when only total RNA is available as a starting material, poly(A+) RNA-first-strand cDNA intermediates generated during first-strand cDNA synthesis are normalized using this method (Zhulidov et al., 2004). In this case, inverted terminal repeats at the ends of first-strand cDNA are required for subsequent PCR amplification.

Detailed protocols of DSN-normalization modifications are available in the book Nucleic Acids Hybridization Modern Applications (Shcheglov et al., 2007).