Applications of Toxicogenomic Technologies to Predictive Toxicology and Risk Assessment (2007) / Chapter Skim
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2 Toxicogenomic Technologies
2 Toxicogenomic Technologies
Pages 22-44
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From page 22... ...
Gene sequencing technologies have also enabled rapid analysis of sequence variation in individual genes, which underlies the diversity in responses to chemicals and other environmental factors. Perhaps the most emblematic technology of the post-genomic era is the microarray, which makes it possible to simultaneously analyze many elements of a complex system.
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From page 23... ...
4. New bioinformatic tools, database resources, and statistical methods will integrate data across technology platforms and link phenotypes and toxicogenomic data.
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Despite the impressive advances in technology, the cost of genome sequencing remains a barrier to the application of whole-genome sequencing to study individual variation in toxic responses and susceptibility. Current costs for wholegenome sequencing in a modern genome sequencing center using Sanger sequencing technology were estimated at approximately $0.004 per bp, which translates to approximately $12M for an entire human genome (Chan 2005)
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SNPs occur on approximately every 2 kilobases in the human genome and have been commonly detected by automated Sanger sequencing as discussed above. The Human DNA Polymorphism Discovery Program of the National Institute of Environmental Health Sciences Environmental Genome Project (EGP)
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Of all the different types of epigenetic modifications, DNA methylation is the most easily measured and amenable to the efficient analysis characteristic of toxicogenomic technologies. DNA methylation refers to addition of a methyl group to the 5-carbon of cytosine in an area of DNA where there are many cytosines and guanines (a CpG island)
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From page 27... ...
Measurements of gene expression have evolved from the single measures of steady-state mRNA using Northern blot analysis to the more global analysis of thousands of genes using DNA microarrays and serial analysis of gene expression (SAGE) , the two dominant technologies (Figure 2-2)
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DNA microarray technology can be used to generate large amounts of data at moderate cost but is limited to surveys of genes that are included in the microarray. In this technology (see Box 2-1 and Figure 2-3)
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From page 29... ...
The actual value reported depends on the microarray technology platform used and the experimental design. For Affymetrix GeneChips, in which each sample is hy bridized to an individual array, expression for each gene is measured as an "aver age difference" that represents an estimated expression level, less nonspecific background.
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(2005) analyzed gene expression in a mouse model of hypertension and compared results obtained using spotted cDNA arrays and Affymetrix GeneChips.
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DNA Microarray Technology Microarray technology (Figure 2-3) fundamentally advanced biology by enabling the simultaneous analysis of all transcripts in a system.
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Relative gene expression levels in the two samples are estimated by measuring the fluorescence intensities for each arrayed probe; a sample expression vector summarizing the expression level of each gene in the patient sample (relative to the control) is reported.
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From page 33... ...
(C) The data from each gene in each sample are collected and these "sample expression vectors" are assembled into a single "expression matrix." Each column in the expression matrix represents an individual sample and its measured expression levels for each gene (the sample expression vector)
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From page 34... ...
Because proteins carry out most functions encoded by genes, analysis of the protein complement of the genome provides insights into biology that cannot be drawn from studies of genes and genomes. MS, gene and protein sequence databases, protein and peptide separation techniques, and novel bioinformatics tools are integrated to provide the technology platform for proteomics (Yates 2000; Smith 2002; Aebersold and Mann 2003)
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. Moreover, modified protein forms are often resolved from unmodified forms, which enable separate characterization and quantitative analysis of each.
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Nevertheless, a continuing challenge of shotgun proteome analyses is the identification of less abundant proteins and modified protein forms. Quantitative Proteomics The application of quantitative analyses has become a critical element of proteome analyses.
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. Use of stable-isotope-labeled standard peptides that uniquely correspond to proteins or protein forms of interest are spiked into proteolytic digests from complex mixtures, and the levels of the target protein are measured relative to the labeled standard.
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. A second tier of bioinformatic tools evaluates outputs from database search algorithms and estimates probabilities of correct protein identifications (Keller et al.
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. Another useful development is the emerging collection of databases of matched peptide and protein sequences and corresponding spectral data that define them (Craig et al.
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Microarray technology approaches include antibody microarrays, in which immobilized antibodies recognize proteins in complex mixtures (de Wildt et al. 2000; Miller et al.
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. This approach is directed to functional analysis of known proteins as opposed to identification and analysis of the components of complex mixtures.
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. 2 Unsupervised analysis methods look for patterns in the data without using previous knowledge about the data; information about treatment or classification supervised methods use this knowledge.
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TECHNOLOGY ADVANCEMENT AND ECONOMY OF SCALE A major determinant of success in genome sequencing projects was achieving economy of scale for genome sequencing technologies (see above)
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From page 44... ...
44 Applications of Toxicogenomic Technologies metabolomic analyses in academic and industry laboratories are done on a small scale to address specific research questions. The evolution of transcriptome profiling and proteomic and metabolomic technology platforms to increase standardization and reduce costs will be essential to maximize their impact.
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