Functional Genomics - Lecture 3 - Sacramento State Genomics - Lecture 3 Slides borrowed from: Debbie...
Transcript of Functional Genomics - Lecture 3 - Sacramento State Genomics - Lecture 3 Slides borrowed from: Debbie...
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Functional Genomics -Lecture 3
Slides borrowed from:
Debbie Laudencia-Chingcuanco, USDA-ARSCheryl Seaton, USDA-ARS
Victoria Carrollo, USDA-ARSZjelka McBride, UC Davis
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Functional Genomics
• “Expression” = Transcription (mRNA)AND/OR Translation (protein)
• “True” Expression results in a functionalgene product
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Functional Genomics• mRNA (transcription) function
– Northern blotting– Complementary DNA (cDNA) libraries
• Expressed sequence tag (EST) libraries• cDNA Microarrays (can be genomic / oligo)
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Functional Genomics• Protein function
• SDS PAGE• Western• Antibody-based arrays• Good ol’ structure/function determination
– X-ray crystallization, NMR, etc.
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GENOME
RNA
PROTEIN
TRANSCRIPTOMEPROTEOMETRANSCRIPTION
TRANSLATION
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What is cDNA ?
Genomic DNA
hnRNA
mRNA
cDNA
Exon 1 Exon 2 Exon 3
splicing
Reverse transcription
transcription
protein
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• Orderly grid or matrixof genes (probes)
• Genes at each spotidentified
• Interrogated withlabeled RNA
• Expression patterns incontrol vs. treated canbe discerned using 2dyes
What is a microarray?
RED - OVEREXPRESSED
GREEN - UNDEREXPRESSED
YELLOW - NO CHANGE
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Gene expression analysis using aDNA microarrays
DNA microarray
TEP 1cDNA
mRNA
P.O.Brown & D.Botstein, Nature Genetics Supplement, Vol 21, Jan 1999, pp 33-37
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Wheat development
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Wheat Grain
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Starch and Sucrose Metabolism
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Microarray schematic
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PRINTING• Omnigrid
microarrayer
• 100 slides
• 74 384 wellplates
• 24 pins
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PRINTING CONSIDERATIONS(METHOD DEVELOPMENT)
• PINS• TYPE OF SLIDE (COATING)• DNA SOLVENT (SSC OR DMSO)• PATTERN DESIRED
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Pins
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Type of Pin
Microspotting pin allowsmultiple stamps(>100stamps/dip)
Dips into solution – pick up0.2-1.0 ul (depends onthe size of the pin)
Delivers – approx. 1nl ofsolution
We will use a DNAconcentration of 0.5ug/ulgiving a spot of 0.5ng.
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1. Non-covalent
2. Covalent
Substrate Coupling Chemistry
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Spot Morphology
Aldehyde, 2XSSC
SLID E CO ATIN G
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Spot Morphology vs. Slide Coating(2X SSC)
polylysine silane
aldehyde
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LIST OF CONTROLSStarch Synthase IIStarch Synthase IIIStarch Branching Enzyme IStarch Branching Enzyme IIADP glucose pyrophosphorylase small subunitADP glucose pyrophosphorylase large subunitUDP glucose pyrophosphorylaseAlpha 1,4-glucan phosphorylaseVacuolar invertaseCell-wall invertaseSucrose synthase ISucrose synthase IISucrose phosphate synthetaseSucrose phophataseSucrose transporterSucrose fructan 6 -fructosyltransferaseSucrose UDP glucosyltransferaseHistone 1Histone 2Histone 3Histone 4
Housekeeping GenesActinAlpha-Tubulin 1Alpha-Tubulin 2Alpha-Tubulin 3Cytochrome P-450 monooxygenaseElongation Factor 1-alphaEF 1-betaEF1-gammaEF 2EnolaseGlutathione S-transferaseHeat Shock Protein (hsp70)Mitochondrial F-1-ATPase subunit 2Ribosomal Protein L3RP L7RP S7RP S4S-adenolsylmethionine decarboxylaseS-adenolsylmethionine synthetaseUbiquitinPolyubiquitinLipid Transfer ProteinStarch SynthaseStarch Synthase I
Rubisco small subunitBeta-amylasePlasma membrane Ca2+-ATPaseH+ transporting ATPaseAldolaseCytoplasmic aldolase Tissue Specific MarkersAlpha/Beta gliadinGamma gliadinOmega gliadinHMW gluteninLMW gluteninFerredoxinPS II proteinPS I proteinOxygen-evolving complexChlorophyll a/b binding complex
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Negative controlsSalmon sperm DNAEndochitinaseExochitinaseTrichothecene acetylaseGFP fused to DON mimotope
AFGC Control SetTransgenesBT: B. Thuringiensis cry1 ACBAR: Phosphinothricin acetyltransferaseGFP: Green Fluorescent ProteinGlobinGUS: Beta-GlucuronidaseHPH: Hygromycin B phosphotransferaseLuc: LuciferaseNptII: Kanamycin/Neomycin phosphotransferase Spiking ControlsB-cell receptor proteinMyosin heavy chainMyosin Reg. Light chainInsulin-like growth factorFLJ109 fisHSPC 120Beta2 microglobulinPhosphoglycerate kinaseTyrosine phosphataseG10 homolog
MORE CONTROLS
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NSF Project ITEC GenBank Private Collection
Processed ESTs
Gene Assembly: Phrap
cDNAs
Clusters
Gene Assembly: Phrap
TS TC
Probe Selection
Blast
First EST
Re-array
Blast
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Miniprep DNA
Disrupted whole cells
PCR
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Wheat Endosperm ESTs: Test Array Set
Download Processed ESTs
Gene Assembly:Phrap
Tentative Singlets (TS) Tentative Contigs (TC)
Blast Blast
First EST
Re-array
(Remove vector, ribosomal, ct and mt DNA)
Mini-prep DNA
Verify I.D. (re-sequencing)
PCR amplify inserts
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Array Scanner
ScanArray 5000XL(from Packard BioChip Technologies)
Five lasers (488 nm-633nm excitation range)Eleven emission filters (500 nm -700 nm)
Up to 5 um resolutionSensitivity to < 0.1 molecule fluor/um2
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Cy3/Cy5 Spectra
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GeneChip Expression AnalysisProcess
GeneChip expressionanalysis probe array
Each probe cell containsmillions of copies of a specificoligonucleotide probe
Biotinylated RNAtarget from experi-mental sample
Streptavidin-phycoerythrinconjugate
Image of hybridized probe array
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GeneChip Expression Array Design
Perfect match probe cells
Mismatch probe cells
mRNA
DNA probe pairsReference Sequence
Lipshutz, RJ, Fodor, SPA, Gingeras, TR & DJ Lockhart, Nature Genetics Supplement, Vol 21, Jan 1999, pp 20-24
Fluorescence Intensity Image
Perfect Match OligoMismatch Oligo
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Light directed oligonucleotidesynthesis
Lipshutz, R.J., Fodor, S.P.A., Gingeras, T.R. & D. LockhartNature Genetics Supl., Vol 21, Jan 1999, pp. 20-24
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Applied GenomicsExploiting the human genome
• Molecular diagnostics of cancer• SNPs and Personal pills• Pharmacogenomics and new drugs• Structural genomics and new targets• and many more
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SNP - single nucleotidepolymorphism
• Variations in the gene sequence, resulting in theamino acid change in the protein, which results inthe altered function
• Genotyping of oncogenes - cancer causing or permitting genes
• Identifying and genotyping drug response gene variants
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Genotyping Arrays
120,000 probes for3,000 biallelic loci
Allele AAllele B
MismatchPerfect MatchPerfect MatchMismatch
GenotypeA/A B/B A/B
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Biochip• Microarrays• Microfluidics - “Lab on the Chip”
Saeks, J, GEN, Vol 21, May 15, 2001
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On-Line Resources
Image of hybridized probe array
• Affymetrix: http://www.affymetrix.com
• Stanford: http://genome-www.stanford.edu/
• Silicon Genetics:http:www.signetics.com/GeneSpring/Overview.htm
• Brown Lab, Stanford University:http://cmgm.stanford.edu/pbrown/explore
• MicroArray Project, NIH:http://www.nhgri.nih.gov/DIR/LCG/15K/HTML/dbase.html
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Affymetrix: http://www.affymetrix.comStanford: http://genome-www.stanford.edu/Silicon Genetics:http:www.signetics.com/GeneSpring/Overview.htmBrown Lab, Stanford University:http://cmgm.stanford.edu/pbrown/exploreMicroArray Project, NIH:http://www.nhgri.nih.gov/DIR/LCG/15K/HTML/dbase.html
On-Line Resources
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Course Evaluations (cont’d)
Satisfaction with the Course
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The End