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