Topic intro slides More complete coverage of components involved in gene expression More information on expression technologies -what would the ideal chip.

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Presentation transcript:

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 Display of Expression Information

Genomic DNA PromoterProtein coding sequenceTerminator What is a gene?

rRNA tRNA mRNA snRNA sRNA Genomic DNA Types of genes

TFIIB RNAPII TFIIA TFIID 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

TFIIB RNAPII TFIIA TFIID 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 repressors coactivators corepressors transcription apparatus chromatin factors RNA processing RNA transport RNA degradation

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

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

Genes and Gene Expression Technology Display of Expression Information

Coated glass microarrays In situ oligonucleotide synthesis arrays DNA array technology

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

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

Cartesian PixSys 5500 with quill 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 µm in diameter Hybridization volumes: ul

Array Scanning Laser based - fluorescent emission

Spot Finding

Hybridization and Scanning—Affymetrix arrays Biotin-labeled cRNA target #1 - hybridize  g of cRNA to Affymetrix array - Wash and scan Compare data sets #1 & #2 Biotin-labeled cRNA target #2 - Wash and scan

Rosetta Ink-jet printing Non-proprietary phosphoamidite oligonucleotide synthesis 29,000 oligos/1X3 inches nucleotides/oligo More information: Hughes et al., Nature Biotechnology 2001

Genes and Gene Expression Technology Display of Expression Information

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’ Mbp1/Swi6 Swi4/Swi6 Mcm1/Fkh2/Ndd1 Ace2, Swi5, Mcm >5 1/3 1 3 Yeast Cell Cycle Gene Expression Program Spellman et al. and Cho et al., Genes

ROS response

600 Conditions/Mutations 6200 Genes Single-gene MutationsEnvironment Exercising the Genome

Visualization: Relative vs. absolute expression