Topic intro slides More complete coverage of components involved in gene expression More information...
-
Upload
arabella-bates -
Category
Documents
-
view
220 -
download
0
Transcript of Topic intro slides More complete coverage of components involved in gene expression More information...
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 ExpressionTechnologyDisplay of Expression Information
Genomic DNA
Promoter Protein coding sequence Terminator
What is a gene?
rRNAtRNAmRNAsnRNAsRNA
Genomic DNA
Types of genes
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
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
Activators
Genome-wide Gene Expression (mRNA) can be Measured with DNA Microarrays
GeneRNAPIITFIIH
Transcription apparatus
mRNA
mRNAlabel
hybridization ATGC
TACG
Some Additional Numbers
Yeast:6200 genes~200 transcriptional regulators
Human:30,000 - 50,000 genes~1700 transcriptional regulators>100 cell types
Genes and Gene ExpressionTechnologyDisplay 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 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
Array Scanning
Laser based - fluorescent emission
Spot Finding
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
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
Rosetta
Ink-jet printingNon-proprietary phosphoamidite oligonucleotide synthesis29,000 oligos/1X3 inches40-60 nucleotides/oligo
More information: Hughes et al., Nature Biotechnology 2001
Genes and Gene ExpressionTechnologyDisplay of Expression Information
alp
ha
0’al
ph
a 7’
alp
ha
14’
alp
ha
21’
alp
ha
28’
alp
ha
35’
alp
ha
42’
alp
ha
49’
alp
ha
56’
alp
ha
63’
alp
ha
70’
alp
ha
77’
alp
ha
84’
alp
ha
91’
alp
ha
98’
alp
ha
105’
alp
ha
112’
alp
ha
119’
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
>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
600 Conditions/Mutations
620
0 G
ene
s
Single-gene MutationsEnvironment
Exercising the Genome
Visualization: Relative vs. absolute expression