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Topic intro slides More complete coverage of components involved in gene expression

Topic intro slides More complete coverage of components involved in gene expression More information on expression technologies -what would the ideal chip do? -limitations to expression technologies Datasets that have been produced thus far. Genes and Gene Expression Technology

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Topic intro slides More complete coverage of components involved in gene expression

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  1. Topic intro slides More complete coverage of components involved in gene expression More information on expression technologies -what would the ideal chip do? -limitations to expression technologies Datasets that have been produced thus far

  2. Genes and Gene Expression Technology Display of Expression Information

  3. What is a gene? Promoter Protein coding sequence Terminator Genomic DNA

  4. Types of genes rRNA tRNA mRNA snRNA sRNA Genomic DNA

  5. How are Genes Regulated? DNA-binding Activators Are Key To Specific Gene Expression Mediator RNAPII TFIIH TFIIF TFIIE TFIIB TFIIA TFIID Chromatin modification complexes Transcription initiation apparatus Activators Gene

  6. How are Genes Regulated? DNA-binding activators are key, but there are additional factors Mediator RNAPII TFIIH TFIIF TFIIE TFIIB TFIIA TFIID Chromatin modification complexes Transcription initiation apparatus activators repressors coactivators corepressors transcription apparatus chromatin factors RNA processing RNA transport RNA degradation Activators Gene

  7. Genome-wide Gene Expression (mRNA) can be Measured with DNA Microarrays Transcription apparatus mRNA Gene RNAPII TFIIH Activators ATGC TACG hybridization label mRNA

  8. Some Additional Numbers Yeast: 6200 genes ~200 transcriptional regulators Human: 30,000 - 50,000 genes ~1700 transcriptional regulators >100 cell types

  9. Genes and Gene Expression Technology Display of Expression Information

  10. DNA array technology Coated glass microarrays In situ oligonucleotide synthesis arrays

  11. Hybridization and Scanning—Glass slide arrays - Prepare Cy3, Cy5- labeled ss cDNA - Scan - Hybridize 600 ng of labeled ss cDNA to glass slide array

  12. Glass Slide DNA Arrays • Large DNA molecules, PCR products, oligonucleotides, • and proteins can be arrayed • Typically non-specific binding to glass or coated surfaces • High quality coated slides commercially available

  13. Cartesian PixSys 5500 withquill printing technology • 3 x 1 inch microscope slides, • specially coated with amino groups • 50 slides/ bed • Up to 32 pins (quills) • 10,000 spots/slide • Spots are 100-200 µm in diameter • Hybridization volumes: 20-100ul

  14. Array Scanning Laser based - fluorescent emission

  15. Spot Finding

  16. - Wash and scan Compare data sets #1 & #2 Biotin-labeled cRNA target #2 Hybridization and Scanning—Affymetrix arrays Biotin-labeled cRNA target #1 - hybridize 10-40 mg of cRNA to Affymetrix array - Wash and scan

  17. Rosetta Ink-jet printing Non-proprietary phosphoamidite oligonucleotide synthesis29,000 oligos/1X3 inches 40-60 nucleotides/oligo More information: Hughes et al., Nature Biotechnology 2001

  18. Genes and Gene Expression Technology Display of Expression Information

  19. alpha 0’ alpha 7’ alpha 14’ alpha 21’ alpha 28’ alpha 35’ alpha 42’ alpha 49’ alpha 56’ alpha 63’ alpha 70’ alpha 77’ alpha 84’ alpha 91’ alpha 98’ alpha 105’ alpha 112’ alpha 119’ Yeast Cell Cycle Gene Expression Program Mbp1/Swi6 Swi4/Swi6 800 Genes Mcm1/Fkh2/Ndd1 Ace2, Swi5, Mcm1 1/3 1 3 Spellman et al. and Cho et al., 1998 1 3.5 >5

  20. ROS response

  21. 600 Conditions/Mutations Exercising the Genome 6200 Genes Environment Single-gene Mutations

  22. Visualization:Relative vs. absolute expression

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