DSN Depletion is a Simple Method to Remove Selected Transcripts from cDNA Populations

Богданова, Е. А.; Shagina, Irina; Mudrik, Elena S.; Ivanov, Igor; Amon, Peter; Vagner, Laura L.; Lukyanov, Sergey; Shagin, Dmitry A.

doi:10.1007/s12033-008-9131-y

Cited by 21 publications

(16 citation statements)

References 21 publications

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“…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%

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

2010

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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.

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Section: Gene Expression Profilingmentioning

confidence: 99%

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

2010

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“…The hybridization conditions used in the normalization protocol were the same as those used for cDNA normalization, and allow effective hybridization of sequences that are substantially identical. It has previously been shown that cDNA sequences with 87% sequence identity do not cross-hybridize under these conditions (23). Thus, the sequences representing divergent repetitive elements behave as unique sequences under the hybridization conditions used.…”

Section: Resultsmentioning

confidence: 99%

“…In the model experiment, we used a commercially available Cot-1 fraction of human DNA; however, the driver can be prepared from the cloned DNA population of undesired sequences, as described in Bogdanova et al (23). …”

Section: Resultsmentioning

confidence: 99%

Normalization of Genomic DNA Using Duplex-Specific Nuclease

Shagina¹,

Богданова

Mamedov

et al. 2010

BioTechniques

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An application of duplex-specific nuclease (DSN) normalization technology to whole-genome shotgun sequencing of genomes with a large proportion of repetitive DNA is described. The method uses a thermostable DSN from the Kamchatka crab that specifically hydrolyzes dsDNA. In model experiments on human genomic DNA, we demonstrated that DSN normalization of double-stranded DNA formed during C0t analysis is effective against abundant repetitive sequences with high sequence identity, while retaining highly divergent repeats and coding regions at base-line levels. Thus, DSN normalization applied to C0t analysis can be used to eliminate evolutionarily young repetitive elements from genomic DNA before sequencing, and should prove invaluable in studies of large eukaryotic genomes, such as those of higher plants.

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“…Email: endeldw @ uc.edu DNA has also been applied for many techniques using short RNA sequences [16,[18][19][20][21][22][23][24][25][26][27][28][29], but applications for long RNA targets have remained elusive.…”

Section: * Corresponding Authormentioning

confidence: 99%

Rational Design of Duplex Specific Nuclease for One-Step Isothermal Viral RNA Detection

2017

J App Biol Biotech

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RNA viruses are a potent human adversary, evidenced by several global pandemics including the Ebolavirus in West Africa, the emerging Zika virus, and outbreaks of new Influenza strains and Norwalk virus in the food supply and cruise ships. Despite the virulence of these pathogens, there remains a significant limitation for detecting these viruses in a fast, accurate and cost effective manner. To meet this need we present a modified form of the duplex specific nuclease enzyme from the Paralithodes camtschaticus crab capable of generating an RNA-based signal amplification in a fraction of the time required for standard RT-qPCR. The applicability of this enzyme is demonstrated in an assay for Norwalk virus detection with a lower limit of ~100 viral copies per liter of environmental water.

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DSN Depletion is a Simple Method to Remove Selected Transcripts from cDNA Populations

Cited by 21 publications

References 21 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

Normalization of Genomic DNA Using Duplex-Specific Nuclease

Rational Design of Duplex Specific Nuclease for One-Step Isothermal Viral RNA Detection

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