Sequence Analysis & Gene Expression GRET workshop, Columbia, MO, June 2005 Bohnert, UIUC) [email protected]m selection: genome size – why – what is the benefit - po ns: apping first, “shotgun sequencing”, BAC alignment/sequencing BAC – bacterial artificial chromosome; also YAC (yeast)] sequence: raw sequence – confirmed sequence ene models – verification ation: is the gene model transcribed? Yes/no/perhaps ubiquitous” gene, family specific, homolog - ortholog - paral ipt profiles: when – how much [abundant] – where ranscript “variants” – inducible by condition X?
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Sequence Analysis & Gene Expression
MUPGRET workshop, Columbia, MO, June 2005 (HJ Bohnert, UIUC)
The Plant Genome ControlsControls for Gene Expression – many Switchboards
• Chromatin condensation state
• Local chromatin environment• Transcription initiation• Transcript elongation• mRNA splicing • mRNA export• mRNA place in the cell• RNA half-life• Killer microRNAs• Ribosome loading• Protein transport/targeting• Protein modifications• Protein turnover
Cells from condition ACells from condition ACells from condition ACells from condition A Cells from condition BCells from condition BCells from condition BCells from condition B
mRNA
Label Dye 2
Ratio of expression of genes from two sources
Label Dye 1
cDNA
equal over under
Mix
ScanArray 3000 Fluorescent Scanner
Overlay Images
Slide 2Cy5 over-expressed
Slide 1Cy3 over-expressed
Reverse Labeling
Universal vs. Universal (control v. control)
Problem area atlow intensity readings
LungvsControl
Cholesterol Biosynthesis
Cell Cycle
Immediate Early Response
Signaling and Angiogenesis
Wound Healing and Tissue Remodeling
Clustered display of data from time course of serum stimulation of primary human fibroblasts.
Eisen et al. Proc. Natl. Acad. Sci. USA 95 (1998) pg 14865
Hierarchical Clustering: 14 Tissues7653 Genes
• One sample, one chipOne sample, one chip• Single Color ScansSingle Color Scans• Labeling by incorporating Biotin into cRNA Labeling by incorporating Biotin into cRNA not not Cy3Cy3 or or Cy5 Cy5 dyesdyes• Oligonucleotides instead of full-length cDNAsOligonucleotides instead of full-length cDNAs• Higher Density ArraysHigher Density Arrays
–Feature sizes down to 18 Feature sizes down to 18 m instead of ~100 m instead of ~100 mm
–Non-contact Creation of ArraysNon-contact Creation of Arrays
Differences in TechnologyDifferences in Technology
Affymetrix
GeneChips
Affy Technology OverviewAffy Technology Overview
• Photolithography and Photolithography and combinatorial combinatorial chemistrychemistry– Technology from Technology from
• Apply required Apply required nucleotide base to nucleotide base to arrayarray
• Apply new mask to de-Apply new mask to de-protect different protect different featuresfeatures
• Stack nucleotides on Stack nucleotides on top of one anothertop of one another
• Repeat with bases and Repeat with bases and masks until 25-mer masks until 25-mer oligonucleotides are oligonucleotides are built directly onto arraybuilt directly onto array
OOOOO
Light(deprotection)
HO HO OOO TTOOO
TTCCO
Light(deprotection)
TTOOO
CATATAGCTGTTCCG
MaskMask
SubstrateSubstrate
MaskMask
SubstrateSubstrate
T T ––
C C ––REPEATREPEAT
OOOOO
Light(deprotection)
OOOOO
Light(deprotection)
HO HO OOOHO HO OOO TTOOOTTOOO
TTCCOTTCCO
Light(deprotection)
TTOOO
Light(deprotection)
TTOOO
CATATAGCTGTTCCG
CATATAGCTGTTCCG
MaskMask
SubstrateSubstrate
MaskMask
SubstrateSubstrate
T T ––
C C ––REPEATREPEAT
Technology Final StepsTechnology Final Steps
• Silicon “wafers” of 90 arrays are cutSilicon “wafers” of 90 arrays are cut
• Glass substrate is then added to plastic Glass substrate is then added to plastic cartridge for:cartridge for:
– Safe handlingSafe handling
– Easy storageEasy storage
– Easy hybridizationEasy hybridization
– Easy scanning Easy scanning
Easy, convenientExpensive (very much so)No confirmation of qualityErroneous data when