Topic intro slides More complete coverage of components involved in gene expression More information...
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Transcript of Topic intro slides More complete coverage of components involved in gene expression More information...
![Page 1: Topic intro slides More complete coverage of components involved in gene expression More information on expression technologies -what would the ideal chip.](https://reader035.fdocuments.net/reader035/viewer/2022062321/56649f265503460f94c3ceb7/html5/thumbnails/1.jpg)
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 ExpressionTechnologyDisplay of Expression Information
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Genomic DNA
Promoter Protein coding sequence Terminator
What is a gene?
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rRNAtRNAmRNAsnRNAsRNA
Genomic DNA
Types of genes
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TFIIB
RNAPII
TFIIATFIID
TFIIH TFIIF
Mediator
TFIIE
Activators
How are Genes Regulated? DNA-binding Activators Are Key To Specific Gene Expression
Gene
Chromatin modification complexes
Transcription initiation apparatus
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TFIIB
RNAPII
TFIIATFIID
TFIIH TFIIF
Mediator
TFIIE
Activators
How are Genes Regulated? DNA-binding activators are key, but there are additional factors
Gene
Chromatin modification complexes
Transcription initiation apparatus activators
repressorscoactivatorscorepressorstranscription apparatuschromatin factorsRNA processingRNA transportRNA degradation
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Activators
Genome-wide Gene Expression (mRNA) can be Measured with DNA Microarrays
GeneRNAPIITFIIH
Transcription apparatus
mRNA
mRNAlabel
hybridization ATGC
TACG
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Some Additional Numbers
Yeast:6200 genes~200 transcriptional regulators
Human:30,000 - 50,000 genes~1700 transcriptional regulators>100 cell types
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Genes and Gene ExpressionTechnologyDisplay of Expression Information
![Page 11: Topic intro slides More complete coverage of components involved in gene expression More information on expression technologies -what would the ideal chip.](https://reader035.fdocuments.net/reader035/viewer/2022062321/56649f265503460f94c3ceb7/html5/thumbnails/11.jpg)
Coated glass microarrays
In situ oligonucleotide synthesis arrays
DNA array technology
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Hybridization and Scanning—Glass slide arrays
- Prepare Cy3, Cy5- labeled ss cDNA
- Hybridize 600 ng of labeled ss cDNA to glass slide array
- Scan
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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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Affymetrix high density oligonucleotide arrays: S. cerevisiae• Oligonucleotides synthesized in situ using light- directed, solid phase combinatorial chemistry
• 6181 ORFs + controls for alignment and normalization
• each ORF: 20 perfect match 25-mers (PM)20 single base mismatch partners (MM)
• mRNA signal = average of differences (PM-MM)
• average number of ORF mRNAs detected = 5460
• dynamic range = 0.1 - 100 mRNA molecules/cell
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Hybridization and Scanning—Affymetrix arrays
Biotin-labeled cRNA target #1
- hybridize 10-40 g of cRNA to Affymetrix array
- Wash and scan
Compare data sets #1 & #2
Biotin-labeled cRNA target #2
- Wash and scan
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Rosetta
Ink-jet printingNon-proprietary phosphoamidite oligonucleotide synthesis29,000 oligos/1X3 inches40-60 nucleotides/oligo
More information: Hughes et al., Nature Biotechnology 2001
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Genes and Gene ExpressionTechnologyDisplay of Expression Information
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Mcm1/Fkh2/Ndd1
Ace2, Swi5, Mcm1
1 3.5 >5
1/3 1 3
Yeast Cell Cycle Gene Expression Program
Spellman et al. and Cho et al., 1998
800 Genes
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>9 >6 >3 1:1 >3 >6 >9Fold repression Fold inductionGenomic Reprogramming in Response to Oxidant010204060120minutes One-third of genome expression is transiently reprogrammed6218 genes
ROS response
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600 Conditions/Mutations
620
0 G
ene
s
Single-gene MutationsEnvironment
Exercising the Genome
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Visualization: Relative vs. absolute expression
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