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Genomics, Bioinformatics & Medicine http://biochem158.stanford.edu/

Genomics, Bioinformatics & Medicine http://biochem158.stanford.edu/. Gene Expression http://biochem158.stanford.edu/Gene%20Expression.html. Doug Brutlag Professor Emeritus of Biochemistry & Medicine Stanford University School of Medicine. Leveraging Genomic Information. Novel Diagnostics

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Genomics, Bioinformatics & Medicine http://biochem158.stanford.edu/

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  1. Genomics, Bioinformatics & Medicinehttp://biochem158.stanford.edu/ Gene Expression http://biochem158.stanford.edu/Gene%20Expression.html Doug Brutlag Professor Emeritus of Biochemistry & Medicine Stanford University School of Medicine

  2. Leveraging Genomic Information • Novel Diagnostics • DNA Microchips & Microarrays • Gene Expression - RNA • Proteomics - Protein • Understanding • Metabolism • Cell Signaling • Differentiation • Disease

  3. Gene Regulatory Mechanisms • Transcriptional Mechanisms • Type of promoters & RNA polymerase • Control of Transcription • Transcription Factors and TFBS • RNA processing • Capping • Splicing and Alternative Splicing • Poly-Adenylation • RNA export to cytoplasm • RNA degradation rates • Micro RNAs (miRNAs) inhibit translation and degrade mRNA • Silencer RNAs (siRNAs or RNAi) degrading mRNA • Epigenetic Mechanisms • DNA methylation • Histone modifications • Acetylation • Methylation • Phosphorylation, etc. • Chromatin remodeling

  4. Eukaryotic Gene Structure miRNA Target

  5. Multiple Enhancer Sequences © Garland Publishing

  6. Enhanceosome: Exploring the Structurehttp://www.rcsb.org/pdb/education_discussion/molecule_of_the_month/download/Enhanceosome.pdf ATF-2/cJun IRF NF Kappa-B

  7. Enhanceosome: Integrating the Signalhttp://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb122_2.html HAT = Histone Acetyl Transferase

  8. Oct3/4-Sox2 bound to Enhancerhttp://www.pdb.org/pdb/explore/explore.do?structureId=1GT0

  9. Repressor Bound to Silencer Sites

  10. Gene Expression Regulatory Network © Current Opinions in Genetics & Development

  11. Hematopoiesis http://www.biocarta.com/pathfiles/h_stemPathway.asp

  12. IL7 Regulatory Pathwayhttp://www.biocarta.com/pathfiles/h_il7Pathway.asp/ © BioCarta

  13. Growth Hormone Receptor Pathwayhttp://www.biocarta.com/pathfiles/h_ghPathway.asp/ © BioCarta

  14. DNA Microarrays & DNA ChipsAccelerate Gene ExpressionAnalysis • Parallel Analyses • Analyze entire genomes instead of single genes • Analyze expression of entire genome • Analyze genetic polymorphisms (SNPs) • Miniaturization • Automation

  15. Diagnosis Using DNA Arrayshttp://www.affymetrix.com/ © Affymetrix Inc.

  16. Affymetrix Arrayshttp://www.affymetrix.com/ Any collection of 25mers (1,200,000) can be synthesized in 100 steps © Affymetrix Inc.

  17. Light Directed Oligonucleotide Synthesishttp://www.youtube.com/watch?v=ui4BOtwJEXs&feature=related Light (deprotection) Mask Glass 25-mer Repeat GeneChip Microarray © Affymetrix Inc.

  18. Automated DNA Chip Synthesishttp://www.affymetrix.com/ © Affymetrix Inc.

  19. Photolithography Maskshttp://www.affymetrix.com/ © Affymetrix Inc.

  20. Hybridization & Detection labeled probe © Affymetrix Inc.

  21. Cystic Fibrosis Arrayhttp://www.affymetrix.com/ © Affymetrix Inc.

  22. Microarrayer in Pat Brown’s Labhttp://cmgm.stanford.edu/pbrown/

  23. High Precision DNA Printing Arrayit Corporation

  24. Mechanical Spotting Microarrayshttp://www.arrayit.com/ Arrayit Corporation

  25. MicroArray analysis of whole genome gene expression Clustering of genes based on their expression pattern Searching for conserved sequence motifs regulating the expression DNA Chips are used toMeasure Gene Expression

  26. Megacluster of Yeast Gene Expression

  27. T Cells Signaling DNA Damage Fibroblast Growth B Cells Signaling CMV Infection Anoxia Polio Infection Cell Signaling IL4 Growth Hormone Human Gene Expression Signatures Genes Biological Conditions

  28. Pho 5 Pho 8 Pho 81 Pho 84 Pho … Transcription Start Finding Transcription Factor Binding Sites Upstream RegionsCo-expressed Genes GATGGCTGCACCACGTGTATGC...ACGATGTCTCGC CACATCGCATCACGTGACCAGT...GACATGGACGGC GCCTCGCACGTGGTGGTACAGT...AACATGACTAAA TCTCGTTAGGACCATCACGTGA...ACAATGAGAGCG CGCTAGCCCACGTGGATCTTGA...AGAATGACTGGC

  29. Finding Transcription Factor Binding Sites Upstream RegionsCo-expressed Genes GATGGCTGCACCACGTGTATGC...ACGATGTCTCGC CACATCGCATCACGTGACCAGT...GACATGGACGGC GCCTCGCACGTGGTGGTACAGT...AACATGACTAAA TCTCGTTAGGACCATCACGTGA...ACAATGAGAGCG CGCTAGCCCACGTGGATCTTGT...AGAATGGCCTAT

  30. Finding Transcription Factor Binding Sites Upstream RegionsCo-expressed Genes ATGGCTGCACCACGTTTATGC...ACGATGTCTCGC CACATCGCATCACGTGACCAGT...GACATGGACGGC GCCTCGCACGTGGTGGTACAGT...AACATGACTAAA TTAGGACCATCACGTGA...ACAATGAGAGCG CGCTAGCCCACGTTGATCTTGT...AGAATGGCCTAT Pho4 binding

  31. Discovering Transcription Factor Binding Sites is Difficult • Binding sites are short (5-15 base pairs) • Not highly conserved (as little as 50%) • Located in long intergenic regions (>10kb) • Not always present (false positive genes)

  32. Three Algorithms • BioProspector • Presented in 2000 • Extends Gibb’s sampling (stochastic method) • For any cluster of sequences • MDscan • Deterministic approach • Enumerative • Very fast • For sequences with some ranking information • MotifCut and MotifScan • Graph-based • Does not use PSSMs • Novel and sensitive

  33. cDNA Diagnostics Using DNA Microarrays

  34. 72 Breast Cancers Classified by451 Gene Expression Assays 72 Patients 451 Genes Sorlie et al., PNAS 98, 10869-10874.

  35. Breast Cancers Classified by451 Gene Expression Assays Sorlie et al., PNAS 98, 10869-10874.

  36. ERB-B2 in Signal Transduction & Oncologyhttp://www.biocarta.com/pathfiles/h_her2Pathway.asp

  37. Herceptin binds to ERB-B2 (HER-2)Blocking Cell Growth Herceptin = anti-HER2 Fab Fragment HER2 (Erb-2) Human Epidermal Growth Factor Receptor

  38. Regulation of transcriptional activity by PMLhttp://www.biocarta.com/pathfiles/h_pmlPathway.asp © BioCarta

  39. Diagnosis &Gene Expression

  40. Predicting Site of Origin forCancers of Unknown Primary PathWork Diagnostics http://www.pathworkdx.com/ © Pathwork Diagnostics Inc.

  41. PathWork Oncology Suite:Site of Origin • 1844 tumors tested one at a time versus all 18 tissues of origin • Retrospective study on well characterized patient samples • Uses PathChip (functionally similar to Affymetrix HU-133A GeneChip) • 604 specimens used for training • 636 specimens used for test • 604 specimens in reserve for final validation • Reproducibility from lab to lab • Performance based on sensitivity (> 70%) & accuracy (> 95%) © Pathwork Diagnostics Inc.

  42. Production Data Flow • Clinical • Laboratory • Receive sample • Process specimen, mRNA extraction and microarray hybridization • Submit data file to PathWork for processing • Deliver report to oncologist Sample • Receive microarray data file • Check file and data quality • Run model against microarray data file • Generate report file • Deliver report file back to clinical lab Microarray data file I NT E R NE T PDF Report

  43. CONFIDENTIAL © Pathwork Diagnostics Inc.

  44. CONFIDENTIAL © Pathwork Diagnostics Inc.

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