Publications (14)171.47 Total impact
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Article: Effect of thiazole orange doubly labeled thymidine on DNA duplex formation.
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ABSTRACT: Nucleic acid oligonucleotides are widely used in hybridization experiments for specific detection of complementary nucleic acid sequences. For design and application of oligonucleotides, an understanding of their thermodynamic properties is essential. Recently, exciton-controlled hybridization-sensitive fluorescent oligonucleotides (ECHOs) were developed as uniquely labeled DNA oligomers containing commonly one thymidine having two covalently linked thiazole orange dye moieties. The fluorescent signal of an ECHO is strictly hybridization-controlled, where the dye moieties have to intercalate into double-stranded DNA for signal generation. Here we analyzed the hybridization thermodynamics of ECHO/DNA duplexes, and thermodynamic parameters were obtained from melting curves of 64 ECHO/DNA duplexes measured by ultraviolet absorbance and fluorescence. Both methods demonstrated a substantial increase in duplex stability (ΔΔG°(37) ∼ -2.6 ± 0.7 kcal mol(-1)) compared to that of DNA/DNA duplexes of the same sequence. With the exception of T·G mismatches, this increased stability was mostly unaffected by other mismatches in the position opposite the labeled nucleotide. A nearest neighbor model was constructed for predicting thermodynamic parameters for duplex stability. Evaluation of the nearest neighbor parameters by cross validation tests showed higher predictive reliability for the fluorescence-based than the absorbance-based parameters. Using our experimental data, a tool for predicting the thermodynamics of formation of ECHO/DNA duplexes was developed that is freely available at http://genome.gsc.riken.jp/echo/thermodynamics/ . It provides reliable thermodynamic data for using the unique features of ECHOs in fluorescence-based experiments.Biochemistry 07/2012; 51(31):6056-67. · 3.42 Impact Factor -
Chapter: Tag-based Next Generation Sequencing
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ABSTRACT: Gene expression is tightly controlled by regulatory elements within promoter regions. Genome-wide promoter identification and measurement of promoter activities is of essential importance for understanding gene expression and its regulation in a biological context. Cap analysis gene expression (CAGE) is a method for the isolation of short sequencing tags from the 5' end of mRNA transcripts that are sequenced at high throughput by next-generation sequencing methods. Mapping back the short sequencing tags to a reference genome allows for reliable identification of transcription start sties (TSS) on a genome-wide scale. ...01/2012: pages 23 - 46; , ISBN: 978-527-32819-2 -
Article: DNA amplification techniques in pharmacogenomics.
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ABSTRACT: The variable predisposition of patients, both to disease susceptibility and drug response, is well established. It is largely attributed to genetic, as well as epigenetic variations between individuals, which may be inherited or acquired. The most common variation in the human genome is the SNP, which occurs throughout the genome, both within coding and noncoding regions. Characterization of SNPs in the context of both inherited and acquired conditions, such as cancer, are a main focus of many genotyping procedures. The demand for identifying (diagnosing) targeted SNPs or other variations, as well as the application of genome-wide screens, is continuously directing the development of new technologies. In general, most methods require a DNA amplification step to provide the amounts of DNA needed for the SNP detection step. In addition, DNA amplification is an important step when investigating other types of genomic information, for instance when addressing repeat, deletion, copy number variation or epigenetic regulation by DNA methylation. Besides the widely used PCR technique, there are several alternative approaches for genomic DNA amplification suitable for supporting the detection of genomic variation. In this article, we describe and evaluate a number of techniques, and discuss possible future prospects of DNA amplification in the fields of pharmacogenetics and pharmacogenomics.Pharmacogenomics 06/2011; 12(6):845-60. · 3.97 Impact Factor -
Article: Exciton Primer-mediated SNP detection in SmartAmp2 reactions.
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ABSTRACT: Most commonly used intercalating fluorescent dyes in DNA detection are lacking any sequence specificity, whereas so-called Exciton Primers can overcome this limitation by functioning as "sequence-specific dyes." After hybridization to complementary sequences, the fluorescence of Exciton Primers provides sequence-specific signals for real-time monitoring of amplification reactions. Applied to the SmartAmp2 mutation detection process, Exciton Primers show high signal strength with low background leading to a superior specificity and sensitivity compared to SYBR Green I. Signal strength can be further enhanced using multiple dyes within one Exciton Primer or use of multiple Exciton Primers in the same amplification reaction. Here we demonstrate the use of Exciton Primers for genotyping a single nucleotide polymorphism (SNP) in the VKORC1 locus (-1639G>A) relevant for Warfarin dosing as an example for Exciton Primers mediated genotyping by SmartAmp2. The genotyping assay can use only one labeled Exciton Primer for endpoint detection, or simultaneously by real-time monitoring detect wild-type and mutant alleles in a one-tube reaction using two Exciton Primers having different dyes. Working directly from blood samples, Exciton Primer mediated genotyping by SmartAmp2 offers superior solutions for rapid point-of-care testing.Human Mutation 02/2010; 31(2):208-17. · 5.69 Impact Factor -
Article: Genome-wide detection and analysis of hippocampus core promoters using DeepCAGE.
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ABSTRACT: Finding and characterizing mRNAs, their transcription start sites (TSS), and their associated promoters is a major focus in post-genome biology. Mammalian cells have at least 5-10 magnitudes more TSS than previously believed, and deeper sequencing is necessary to detect all active promoters in a given tissue. Here, we present a new method for high-throughput sequencing of 5' cDNA tags-DeepCAGE: merging the Cap Analysis of Gene Expression method with ultra-high-throughput sequence technology. We apply DeepCAGE to characterize 1.4 million sequenced TSS from mouse hippocampus and reveal a wealth of novel core promoters that are preferentially used in hippocampus: This is the most comprehensive promoter data set for any tissue to date. Using these data, we present evidence indicating a key role for the Arnt2 transcription factor in hippocampus gene regulation. DeepCAGE can also detect promoters used only in a small subset of cells within the complex tissue.Genome Research 01/2009; 19(2):255-65. · 13.61 Impact Factor -
Article: Corrigendum: Genome-wide analysis of mammalian promoter architecture and evolution
Nature Genetics 08/2007; 39(9):1174-1174. · 35.53 Impact Factor -
Article: Genome-wide analysis of mammalian promoter architecture and evolution.
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ABSTRACT: Mammalian promoters can be separated into two classes, conserved TATA box-enriched promoters, which initiate at a well-defined site, and more plastic, broad and evolvable CpG-rich promoters. We have sequenced tags corresponding to several hundred thousand transcription start sites (TSSs) in the mouse and human genomes, allowing precise analysis of the sequence architecture and evolution of distinct promoter classes. Different tissues and families of genes differentially use distinct types of promoters. Our tagging methods allow quantitative analysis of promoter usage in different tissues and show that differentially regulated alternative TSSs are a common feature in protein-coding genes and commonly generate alternative N termini. Among the TSSs, we identified new start sites associated with the majority of exons and with 3' UTRs. These data permit genome-scale identification of tissue-specific promoters and analysis of the cis-acting elements associated with them.Nature Genetics 07/2006; 38(6):626-35. · 35.53 Impact Factor -
Article: CAGE: cap analysis of gene expression.
Nature Methods 04/2006; 3(3):211-22. · 19.28 Impact Factor -
Article: Tag-based approaches for transcriptome research and genome annotation.
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ABSTRACT: With the increasing number of whole genome sequences available, genomic research has shifted toward the annotation of functional elements and transcribed regions. Thus, the related field of transcriptome research requires accurate methods for the profiling of genes that are not biased by known sequence information, and that also allow for the identification of promoter regions. Starting with serial analysis of gene expression (SAGE), methods making use of short sequencing tags have greatly contributed to transcriptome studies. Here we review recent developments in the use of short sequencing tags in expression profiling, gene discovery and genome annotation. These tags are obtained from the 5' end of mRNAs, both terminal ends of mRNAs, or genomic regions. The 5' end-specific tags, with their ability to identify transcripts along with their transcriptional start sites, will be of particular interest for gene network studies and may become one of the most important approaches in systems biology.Nature Methods 08/2005; 2(7):495-502. · 19.28 Impact Factor -
Article: Libraries enriched for alternatively spliced exons reveal splicing patterns in melanocytes and melanomas.
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ABSTRACT: It is becoming increasingly clear that alternative splicing enables the complex development and homeostasis of higher organisms. To gain a better understanding of how splicing contributes to regulatory pathways, we have developed an alternative splicing library approach for the identification of alternatively spliced exons and their flanking regions by alternative splicing sequence enriched tags sequencing. Here, we have applied our approach to mouse melan-c melanocyte and B16-F10Y melanoma cell lines, in which 5,401 genes were found to be alternatively spliced. These genes include those encoding important regulatory factors such as cyclin D2, Ilk, MAPK12, MAPK14, RAB4, melastatin 1 and previously unidentified splicing events for 436 genes. Real-time PCR further identified cell line-specific exons for Tmc6, Abi1, Sorbs1, Ndel1 and Snx16. Thus, the ASL approach proved effective in identifying splicing events, which suggest that alternative splicing is important in melanoma development.Nature Methods 01/2005; 1(3):233-9. · 19.28 Impact Factor -
Article: Aptamer-dependent full-length cDNA synthesis by overlap extension PCR.
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ABSTRACT: Sequencing of the human genome in combination with computational annotation has provided tremendous data on predicted genes. However, for most of them, no corresponding cDNAs are available yet. Furthermore, even where cDNA clones were obtained, gene transcripts often have many different splice variants that are not covered by current gene collections. For direct synthesis of cDNA clones corresponding to predicted genes, new splice variants, or any other gene of interest, we established optimal PCR conditions for the direct amplification of exons from genomic DNA, which require a specific Taq aptamer. PCR products comprising differently tagged exons were concatenated by overlap extension into full-length cDNAs. To prove the effectiveness of the approach, the 1900-bp full-length open reading frame of the human mitochondrial aldehyde dehydrogenase (ALDH2) gene was synthesized in a two-step reaction comprising all 13 exons. Thus, our conditions are of general value for in vitro synthesis of cDNAs and alternative splice variants from genomic DNA.BioTechniques 08/2004; 37(1):124, 126, 128-9. · 2.67 Impact Factor -
Article: Absolute expression values for mouse transcripts: re-annotation of the READ expression database by the use of CAGE and EST sequence tags.
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ABSTRACT: The RIKEN expression array database (READ) provides comprehensive gene expression data for the mouse, which were obtained as relative values from microarray double-staining experiments with E17.5 mRNA as common reference. To assign absolute expression values for mouse transcripts within READ, we applied the E17.5 reference sample to CAGE (cap analysis of gene expression) and expressed sequence tag (EST) high-throughput tag sequencing. Newly assigned values within the READ database were validated by comparison to expression data from serial analysis of gene expression, CAGE and EST experiments. These experiments confirmed the great significance of the absolute expression values within the improved READ database. The new Absolute READ database on absolute expression data is available under.FEBS Letters 03/2004; 559(1-3):22-6. · 3.54 Impact Factor -
Article: Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage.
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ABSTRACT: We introduce cap analysis gene expression (CAGE), which is based on preparation and sequencing of concatamers of DNA tags deriving from the initial 20 nucleotides from 5' end mRNAs. CAGE allows high-throughout gene expression analysis and the profiling of transcriptional start points (TSP), including promoter usage analysis. By analyzing four libraries (brain, cortex, hippocampus, and cerebellum), we redefined more accurately the TSPs of 11-27% of the analyzed transcriptional units that were hit. The frequency of CAGE tags correlates well with results from other analyses, such as serial analysis of gene expression, and furthermore maps the TSPs more accurately, including in tissue-specific cases. The high-throughput nature of this technology paves the way for understanding gene networks via correlation of promoter usage and gene transcriptional factor expression.Proceedings of the National Academy of Sciences 01/2004; 100(26):15776-81. · 9.68 Impact Factor -
Article: Genome-wide detection and analysis of hippocampus core promoters using DeepCAGE
[show abstract] [hide abstract]
ABSTRACT: Finding and characterizing mRNAs, their transcription start sites (TSS) and their associated promoters is a major focus in post-genome biology. Mammalian cells have at least 5-10 magnitudes more TSS than previously believed, and deeper sequencing is necessary to detect all active promoters in a given tissue. Here, we present a new method for high throughput sequencing of 5' cDNA tags, DeepCAGE: merging the Cap Analysis of Gene Expression method with ultra-high throughput sequence technology. We apply DeepCAGE to characterize 1.4 million sequenced TSS from mouse hippocampus and reveal a wealth of novel core promoters which are preferentially used in hippocampus: this is the most comprehensive promoter dataset for any tissue to date. Using this data, we present evidence indicating a key role for the Arnt2 transcription factor in hippocampus gene regulation. DeepCAGE can also detect promoters used only in a small subset of cells within the complex tissue. Yes Yes
Top co-authors
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Institutions
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2009
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University of Copenhagen
- Bioinformatics Centre
Copenhagen, Capital Region, Denmark
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2004–2005
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RIKEN
Wako, Saitama-ken, Japan
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