Simple cDNA normalization using kamchatka crab duplex-specific nuclease

Zhulidov, Pavel A.; Богданова, Е. А.; Shcheglov, Alex S.; Vagner, Laura L.; Khaspekov, George L.; Kozhemyako, Valery B.; Matz, Mikhail V.; Meleshkevitch, Ella A.; Moroz, Leonid L.; Lukyanov, Sergey; Shagin, Dmitry A.

doi:10.1093/nar/gnh031

Cited by 367 publications

(275 citation statements)

References 26 publications

Supporting

Mentioning

275

Contrasting

Order By: Relevance

“…A complementary way of increasing the breadth of the transcriptome coverage is normalization of the cDNA library ( Figure 2a) and the depletion of ribosomal RNA ( Figure 2b). The effect of this is to reduce the representation of very common transcripts (Zhulidov et al, 2004). Although gene discovery may still only be marginally more efficient than for non-normalized libraries ), normalization will increase the depth of coverage for most transcripts, which is very valuable for nucleotide variation profiling and singlenucleotide polymorphism (SNP) discovery.…”

Section: Transcriptome Characterizationmentioning

confidence: 99%

“…There is also an increase in the gene discovery rate in a normalized cDNA library, enhancing the identification and analysis of rare transcripts (Cheung et al, 2006). Several normalization methods are reviewed in Bogdanova et al (2009) but duplex-specific nuclease (DSN) normalization (Zhulidov et al, 2004) has been widely used in recent years. (b) Analysis of RNA with an Agilent 2100 Bioanalyzer (Agilent Technologies) using a pooled RNA sample and the same sample after ribosomal RNA (rRNA) depletion with RiboMinus RNA-seq kit (Invitrogen).…”

Section: Gene Expression Profilingmentioning

confidence: 99%

See 1 more Smart Citation

Applications of next generation sequencing in molecular ecology of non-model organisms

2010

View full text Add to dashboard Cite

As most biologists are probably aware, technological advances in molecular biology during the last few years have opened up possibilities to rapidly generate large-scale sequencing data from non-model organisms at a reasonable cost. In an era when virtually any study organism can 'go genomic', it is worthwhile to review how this may impact molecular ecology. The first studies to put the next generation sequencing (NGS) to the test in ecologically well-characterized species without previous genome information were published in 2007 and the beginning of 2008. Since then several studies have followed in their footsteps, and a large number are undoubtedly under way. This review focuses on how NGS has been, and can be, applied to ecological, population genetic and conservation genetic studies of non-model species, in which there is no (or very limited) genomic resources. Our aim is to draw attention to the various possibilities that are opening up using the new technologies, but we also highlight some of the pitfalls and drawbacks with these methods. We will try to provide a snapshot of the current state of the art for this rapidly advancing and expanding field of research and give some likely directions for future developments.

show abstract

Section: Transcriptome Characterizationmentioning

confidence: 99%

Section: Gene Expression Profilingmentioning

confidence: 99%

Applications of next generation sequencing in molecular ecology of non-model organisms

2010

View full text Add to dashboard Cite

show abstract

“…cDNA was normalized to reduce high abundance transcripts using the duplex-specific nuclease-based normalization technology (Evrogen) (Zhulidov et al, 2004).…”

Section: Rna Isolation Cdna Synthesis and Normalizationmentioning

confidence: 99%

A Root-Expressed l-Phenylalanine:4-Hydroxyphenylpyruvate Aminotransferase Is Required for Tropane Alkaloid Biosynthesis in Atropa belladonna

et al. 2014

View full text Add to dashboard Cite

The tropane alkaloids, hyoscyamine and scopolamine, are medicinal compounds that are the active components of several therapeutics. Hyoscyamine and scopolamine are synthesized in the roots of specific genera of the Solanaceae in a multistep pathway that is only partially elucidated. To facilitate greater understanding of tropane alkaloid biosynthesis, a de novo transcriptome assembly was developed for Deadly Nightshade (Atropa belladonna). Littorine is a key intermediate in hyoscyamine and scopolamine biosynthesis that is produced by the condensation of tropine and phenyllactic acid. Phenyllactic acid is derived from phenylalanine via its transamination to phenylpyruvate, and mining of the transcriptome identified a phylogenetically distinct aromatic amino acid aminotransferase (ArAT), designated Ab-ArAT4, that is coexpressed with known tropane alkaloid biosynthesis genes in the roots of A. belladonna. Silencing of Ab-ArAT4 disrupted synthesis of hyoscyamine and scopolamine through reduction of phenyllactic acid levels. Recombinant Ab-ArAT4 preferentially catalyzes the first step in phenyllactic acid synthesis, the transamination of phenylalanine to phenylpyruvate. However, rather than utilizing the typical keto-acid cosubstrates, 2-oxoglutarate, pyruvate, and oxaloacetate, Ab-ArAT4 possesses strong substrate preference and highest activity with the aromatic keto-acid, 4-hydroxyphenylpyruvate. Thus, Ab-ArAT4 operates at the interface between primary and specialized metabolism, contributing to both tropane alkaloid biosynthesis and the direct conversion of phenylalanine to tyrosine.

show abstract

“…Normalization of the PPN library was carried out using the properties of the enzyme DSN (Duplex-specific nuclease) (Evrogen) as described in Zhulidov et al (2004) but with modification (M.C. Marques and M.A.…”

Section: Plant Materials and CI Measurementsmentioning

confidence: 99%

Development of ChillPeach genomic tools and identification of cold-responsive genes in peach fruit

et al. 2008

View full text Add to dashboard Cite

The ChillPeach database was developed to facilitate identification of genes controlling chilling injury (CI), a global-scale post-harvest physiological disorder in peach. It contained 7,862 high-quality ESTs (comprising 4,468 unigenes) obtained from mesocarp tissues of two fullsib progeny contrasting for CI, about 48 and 13% of which are unique to Prunus and Arabidopsis, respectively. All ESTs are in the GatewayÒ vector to facilitate functional assessment of the genes. The data set contained several putative SNPs and 184 unigenes with high quality SSRs, of which 42% were novel to Prunus. Microarray slides containing 4,261 ChillPeach unigenes were printed and used in a pilot experiment to identify differentially expressed genes in cold-treated compared to control mesocarp tissues, and in vegetative compared to mesocarp tissues. Quantitative RT-PCR (qRT-PCR) confirmed microarray results for all 13 genes tested. The microarray and qRT-PCR analyses indicated that ChillPeach is rich in putative fruit-specific and novel cold-induced genes. A website (http://bioinfo.ibmcp. upv.es/genomics/ChillPeachDB) was created holding detailed information on the ChillPeach database.

show abstract

Simple cDNA normalization using kamchatka crab duplex-specific nuclease

Cited by 367 publications

References 26 publications

Applications of next generation sequencing in molecular ecology of non-model organisms

Applications of next generation sequencing in molecular ecology of non-model organisms

A Root-Expressed l-Phenylalanine:4-Hydroxyphenylpyruvate Aminotransferase Is Required for Tropane Alkaloid Biosynthesis in Atropa belladonna

Development of ChillPeach genomic tools and identification of cold-responsive genes in peach fruit

Contact Info

Product

Resources

About