Functional Genomics -Lecture 3
Slides borrowed from:
Debbie Laudencia-Chingcuanco, USDA-ARSCheryl Seaton, USDA-ARS
Victoria Carrollo, USDA-ARSZjelka McBride, UC Davis
Functional Genomics
• “Expression” = Transcription (mRNA)AND/OR Translation (protein)
• “True” Expression results in a functionalgene product
Functional Genomics• mRNA (transcription) function
– Northern blotting– Complementary DNA (cDNA) libraries
• Expressed sequence tag (EST) libraries• cDNA Microarrays (can be genomic / oligo)
Functional Genomics• Protein function
• SDS PAGE• Western• Antibody-based arrays• Good ol’ structure/function determination
– X-ray crystallization, NMR, etc.
GENOME
RNA
PROTEIN
TRANSCRIPTOMEPROTEOMETRANSCRIPTION
TRANSLATION
What is cDNA ?
Genomic DNA
hnRNA
mRNA
cDNA
Exon 1 Exon 2 Exon 3
splicing
Reverse transcription
transcription
protein
• 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
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
Wheat development
Wheat Grain
Starch and Sucrose Metabolism
Microarray schematic
PRINTING• Omnigrid
microarrayer
• 100 slides
• 74 384 wellplates
• 24 pins
PRINTING CONSIDERATIONS(METHOD DEVELOPMENT)
• PINS• TYPE OF SLIDE (COATING)• DNA SOLVENT (SSC OR DMSO)• PATTERN DESIRED
Pins
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.
1. Non-covalent
2. Covalent
Substrate Coupling Chemistry
Spot Morphology
Aldehyde, 2XSSC
SLID E CO ATIN G
Spot Morphology vs. Slide Coating(2X SSC)
polylysine silane
aldehyde
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
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
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
Miniprep DNA
Disrupted whole cells
PCR
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
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
Cy3/Cy5 Spectra
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
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
Light directed oligonucleotidesynthesis
Lipshutz, R.J., Fodor, S.P.A., Gingeras, T.R. & D. LockhartNature Genetics Supl., Vol 21, Jan 1999, pp. 20-24
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
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
Genotyping Arrays
120,000 probes for3,000 biallelic loci
Allele AAllele B
MismatchPerfect MatchPerfect MatchMismatch
GenotypeA/A B/B A/B
Biochip• Microarrays• Microfluidics - “Lab on the Chip”
Saeks, J, GEN, Vol 21, May 15, 2001
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
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
Course Evaluations (cont’d)
Satisfaction with the Course
The End