Topic intro slides

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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
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Genes and Gene Expression Technology Display of
Expression Information
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What is a gene?
Promoter
Protein coding sequence
Terminator
Genomic DNA
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Types of genes
rRNA tRNA mRNA snRNA sRNA
Genomic DNA
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How are Genes Regulated? DNA-binding Activators
Are Key To Specific Gene Expression
Chromatin modification complexes
Transcription initiation apparatus
Activators
Gene
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How are Genes Regulated? DNA-binding activators
are key, but there are additional factors
Chromatin modification complexes
Transcription initiation apparatus
activators repressors coactivators corepressors tr
anscription apparatus chromatin factors RNA
processing RNA transport RNA degradation
Activators
Gene
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Genome-wide Gene Expression (mRNA) can be
Measured with DNA Microarrays
Transcription apparatus
mRNA
Gene
RNAPII
TFIIH
Activators
ATGC TACG
hybridization
label
mRNA
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Some Additional Numbers
Yeast 6200 genes 200 transcriptional
regulators Human 30,000 - 50,000 genes 1700
transcriptional regulators gt100 cell types
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Genes and Gene Expression Technology Display of
Expression Information
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DNA array technology
Coated glass microarrays In situ
oligonucleotide synthesis arrays
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Hybridization and ScanningGlass slide arrays
- Prepare Cy3, Cy5- labeled ss cDNA
- Scan
- Hybridize 600 ng of labeled ss cDNA to
glass slide array
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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

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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

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Array Scanning
Laser based - fluorescent emission
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Spot Finding
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Hybridization and ScanningAffymetrix arrays
Biotin-labeled cRNA target 1
- hybridize 10-40 mg of cRNA to Affymetrix array
- Wash and scan
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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
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Genes and Gene Expression Technology Display of
Expression Information
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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 gt5
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ROS response
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600 Conditions/Mutations
Exercising the Genome
6200 Genes
Single-gene Mutations
Environment
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Visualization Relative vs. absolute expression
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