microarray principles & applications. overview technology - differences in platforms utility...
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MicroarrayPrinciples & Applications
Overview
Technology - Differences in platforms Utility & Applications - What will a microarray
do for you? The Future of Microarrays – Where are they
heading…
Genotype AnalysisSNP AnalysisMutation Screening
Proteomics
Gene Expression Analysis
Assays Of Biological Variation
The Good Ol’ Days
Sequencing Gels Northerns Westerns
GenotypingPharmacogenetics
Diagnostics
Multiplex-ELISADiagnostics
Tox StudiesExpression db
Microarrays
One Platform = Multiple Applications
Mainly used in gene discovery
Microarray Development
Widely adopted
Relatively young technology
Evolution & Industrialization
1994- First cDNAs are developed at Stanford.
1995- Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray- Schena et. al.
1996- Commercialization of arrays
1996-Accessing Genetic Information with High Density DNA Arrays-Chee et. al.
1997-Genome-wide Expression Monitoring in S. cerevisiae-Wodicka et. al.
Technology
Definition Microarray- A substrate with bound capture probes
Capture probe An oligonucleotide/DNA with gene/polymorphism of
interest
Fabrication Photolithography-Affymetrix Printing-Incyte, Genometrix
Target Generation One color Two color
Analysis “Scanning” of array Amount of hybridized target is assessed.
Background of Microarrays
Basic Types of Fabrication Photolithographic
» Affymetrix» Oligonucleotide capture probe
Mechanical deposition» Incyte, Molecular Dynamics, Genometrix» cDNA or oligonucleotide capture probes» Ink jets, capillaries, tips
Target Preparation RT of RNA to cDNA RNA amplification
Array Advantages
Efficient use of reagents Small volume deposition Minimal wasted materials
High-throughput capability Assess many genes simultaneously Examine many samples quickly Can be automated
Applications
DiscoveryLeads
PreClinicalClinical
Target Discovery
Target Validation
Screening Validation Optimization
Toxicology Optimization
Genotyping ADE Screens
Medium Density
High Density
Applications in Drug Development
Sam
ple
Thr
ough
put
Genes Interrogated
10000
1000
10
10 1000 10000
Leads
Discovery
Pre-Clinical
Clinical
Array Technology
Array Design & Fabrication Determine genes to be analyzed Design DNA reagents to be arrayed Use automated arraying instrument
Affymetrix Fabrication Process
cDNA Microarray Fabrication
Up to 10,000 elements per array Elements 500 to 5000 bases in length Proprietary surface chemistry Reduced background Cleanroom fabrication facility
Scalable operation
Oligonucleotide Microarray
Immobilized gene specific oligo probes
ACUGCUAGGUUAGCUAGUCUGGACAUUAGCCAUGCGGAUGCCAUGCCGCUU
GACCTGTAATCGGTACGCCTA
Genometrix Array Printer
STORAGE
VESSEL
STANDARD 96/384 W ELL
G AL S S
ARRAY
• Proprietary Delivery Mechanism• Fully Automated• Standard Format Compatible
VistaArray Microarrays
Medium density-up to 250 elements
Preselect genes based on high-density arrays
Can be easily customized
Cost effective
High-throughput capability
Hundreds of samples
Automatable
Probe Labeling
• Optimized one-step fluorescent labeling protocol
• No amplification of RNA
• Starting material 200 ng of polyA mRNA
• Built in controls for sensitivity, ratios and RT quality
Probe Labeling
Array Technology
Sample Preparation Isolate cell, tissue, or DNA samples Generate labeled DNA or cDNA materials
Sample Hybridization Hybridize labeled sample to array
Microarray Hybridization
Two probe populations competitively hybridized 1/100,000 sensitivity across most genes in 200 ng
mRNA Routinely detects two-fold changes in expression
Array Technology
Sample Analysis
CCD/ laser imaging Rapid analysis Highly sensitive Fully automated
Image Analysis
Element regionsBackground
Adjusted Elements
Auto-gridding Edge detection Noise filtering
Background subtraction Auto integration into
database
Applications…
Gene Discovery- Assigning function to sequence Discovery of disease genes and drug targets Target validation
Genotyping Patient stratification (pharmacogenomics) Adverse drug effects (ADE)
Microbial ID
The List Continues To Grow….
Profiling Gene Expression
LungTumor
LiverTumor
KidneyTumor
Normal vs. Normal
Normal vs. Tumor
Lung Tumor: Up-Regulated
Lung Tumor: Down-Regulated
Lung Tumor: Up-Regulated
Signal transduction Cytoskeleton
Proteases/Inhibitors Kinases
Lung Tumor: Up-Regulated
Signal transduction Cytoskeleton
Proteases/Inhibitors Kinases
Cyclin B1
Cyclin-dependentkinase
Tumor expression-related protein
Lung Tumor: Down-RegulatedSignal transduction Cytoskeleton
Proteases/Inhibitors Kinases
Genes Common to All 3 Tumors
Up-regulated
Down-regulated
Microarrays and Lead Validation and Optimization
May alleviate current bottlenecks High-throughput Biological relevance (e.g. primary cell lines) Validate more than one target per compound Easy and quick assay to develop (no cell engineering)
Generate toxicity data on compound Database correlation to compound structure
Determine mode(s) of compound/target interaction. Broad functionality to a compound (e.g. ion channel
mod, cell cycle regulator, membrane receptor)
Why would you screen more compounds?
Discovery Manufacturability Lower toxicity Better mode of application Improved efficacy
Optimization with Arrays
-10
-5
0
5
10
15
Competition Lead Optimized Toxin Best Drug
Gene Index
Dif
fere
nti
al E
xp
ress
ion
Ex
pre
ss
ion
Pro
file
Target
Optimization with Arrays
-10
-5
0
5
10
15
Competition Lead Optimized Toxin Best Drug
Gene Index
Dif
fere
nti
al E
xp
ress
ion
Ex
pre
ss
ion
Pro
file
Target
Optimization with Arrays
-10
-5
0
5
10
15
Competition Lead Optimized Toxin Best Drug
Gene Index
Dif
fere
nti
al E
xp
ress
ion
Ex
pre
ss
ion
Pro
file
Target
Optimization with Arrays
-10
-5
0
5
10
15
Competition Lead Optimized Toxin Best Drug
Gene Index
Dif
fere
nti
al E
xp
ress
ion
Ex
pre
ss
ion
Pro
file
Target
From Braxton et al., Curr. Op. Biotech. 1998 (9)
Classical Microarray Experiments Normal vs Disease
Example: Analysis of GE patterns in cancer
- DeRisi et. Al (1996)- Pattern of gene expression-networks- Novel gene association/discovery
Molecular Classification Example:Comparison of Breast Tumors
- Perou et. Al (2000)
- Samples classified into subtypes Genome-Wide Analysis
Example: Genome-wide expression in S. cerevisiae
- Wodicka et. Al (1997) Cross-species comparisons
Arrays for SNP and Mutation Analysis
Analyze many samples on hypothesis-driven array configurations to derive genetic information critical to pharmacogenetic evaluation of drug response or disease risk assessment.
Target analytes are derived by multiplex PCR.
All steps from sample preparation to image analysis can be automated.
DNA
Genotyping: SNP Microarray
Immobilized allele specific oligo probes Hybridize with labeled PCR product Assay multiple SNPs on a single array
TTAGCTAGTCTGGACATTAGCCATGCGGAT
GACCTGTAATCG
TTAGCTAGTCTGGACATTAGCCATGCGGAT
GACCTATAATCG
Genotyping Validation Study
NAT2 polymorphisms
N-acetyltransferase enzyme
Phase II metabolic pathway for converting hydrophobic compounds into water-soluble metabolites
NAT2 polymorphisms associated with differences in response to drug therapy
Concordance
~740 colon cancer patient samples
NAT2 genotyping by PCR/RFLP
NAT2 Polymorphisms
341 481 590 803 857
T/C C/T G/A A/G G/A
282
C/T
191
G/A
FDA Arizona Cancer Center Validation Trial
NAT2/COMT 8-plex (genomic)
FDA/AZCC Concordance Study
Gene
# Concordant with RFLP % Concordance
NAT2 481 685/692 99.0%
NAT2 590 676/682 99.1%
NAT2 857 660/660 100%
sCOMT 16/16 100%
Gene
Genometrix Accurate Call Overall % Accuracy
NAT2 481 6/7 99.86%
NAT2 590 5/6 99.85%
Sequencing of discordant samples
Automated Element Scoring
Allele Scoring GUI
Automation of Allele Discrimination
Each point is one sample and represents signal from both alleles for one SNP.
Homozygous Allele B
Homozygous allele A
Heterozygous
0 2000 4000 6000 8000 10000
Allele A
0
4000
8000
12000
Alle
le B
Allele Scoring – Sample Output
A G A G A G A G A G
Utah (father) Male Utah (mother) Female Utah (child) Male Utah (child) Male Utah (child) Male Utah (child) Female Utah (child) Male Utah (child) Male Utah (child) Male Utah (child) Male Utah (pat G) Male Utah (pat G) Female Utah (mat G) Male Utah (mat G) Female Utah (child) Female Caucasian Female Dutch Female German Male German/Danish Female
SNPBNationality Sex
SNP1 SNP9 SNP14 SNP16
Protein Based Microarrays
Platform may support micro-ELISA format or large scale proteomics projects.
Protein levels may be correlated with mRNA expression profiles.
ELISA reagents already developed and approved in the diagnostic field.
Protein
Proteomics
Microarrays Mendoza et al (1998)
» Sandwich assay for 7 antigens High-density arrays
Holt et al (2000)» Screened 27K human fetal brain proteins on
membrane McBeath and Schreiber (2000)
» Arrayed 0ver 10,000 proteins and screened for small molecule binding
Haab et al (2001) » Competitive hybridization of proteins on
antibody arrays
High- throughput proteomic analysis
High-density Antibody array
Six to twelve replicates of 114 different antibodies spotted Protein mixes at different concentrations labeled and
detected
Haab et al (2001)
Actual vs observed ratios
Cy5/Cy3 fluorescence ratio calculated at each antigen concentration and plotted against actual ratios
Antigen concentration (ng/ml)
Haab et al (2001)
Applications of Protein arrays
Applications
Screening for- Small molecule targets
Post-translational modifications
Protein-protein interactions
Protein-DNA interactions
Enzyme assays
Epitope mapping
marker protein
cytokine
VEGFIL-10IL-6IL-1 MIX
BIOTINYLATED MAB
CAPTURE MAB
ANTIGEN
Detection system
Cytokine Specific Microarray ELISA
Competing Technologies
Bead-based approaches Illumina-fiber optics Luminex-flow cytometry
Mass spectrometry Ciphergen-protein chips Sequenom-SNP detection
Gel-based Sequencing
Conclusion
Technology is evolving rapidly. Blending of biology, automation, and
informatics. New applications are being pursued
Beyond gene discovery into screening, validation, clinical genotyping, etc.
Microarrays are becoming more broadly available and accepted. Protein Arrays Diagnostic Applications…
Analysis Tools
How to analyze thousands of genes? Linear Plots Clustering Principal Components Analysis
Analysis Tools
How to analyze thousands of genes? Linear Plots Clustering Principal Components Analysis
How to handle error bars across array/sample normalization?
How to analyze thousands of genes across a distribution of time?
How to analyze thousands of genes across a distribution of time and a distribution of samples?
How does a user visualize genetic networks?
Microarray Future
Must go beyond describing differentially expressed genes
Potential Visualization Tools for Time Series
•Regular and extended clusters (combining genes interrelated at the same time)
•Causally related genes (combining genes interrelated at different times)
Yuriy FofanovVictor Polinger
U. Of Nottingham
Microarray Future
Must go beyond describing differentially expressed genes
Inexpensive, high-throughput, genome-wide scan is the end game for research applications
Microarray Future
Must go beyond describing differentially expressed genes
Inexpensive, high-throughput, genome-wide scan is the end game for research applications
Protein microarrays beginning to be used Fundamentally change experimental design Will enhance protein dB construction
Microarray Future
Must go beyond describing differentially expressed genes
Inexpensive, high-throughput, genome-wide scan is the end game for research applications
Protein microarrays being used Publications are now being focused on
biology rather than technology
Microarray Future
Must go beyond describing differentially expressed genes
Inexpensive, high-throughput, genome-wide scan is the end game for research applications
Protein microarrays will be deployed within the next year
Publications are now being focused on biology rather than technology
SNP analysis Faster, cheaper, as accurate as sequencing Disease association studies Population surveys
Microarray Future
Must go beyond describing differentially expressed genes
Inexpensive, high-throughput, genome-wide scan is the end game for research applications
Protein microarrays will be deployed within the next year
Publications are now being focused on biology rather than technology
SNP analysis-population surveys, SNP map Chemicogenomics
Dissection of pathways by compound application Fundamental change to lead validation
Microarray Future
Must go beyond describing differentially expressed genes
Inexpensive, high-throughput, genome-wide scan is the end game for research applications
Protein microarrays will be deployed within the next year
Publications are now being focused on biology rather than technology
SNP analysis-population surveys, SNP map Chemicogenomics Diagnostics
Tumor classification Patient stratification Intervention therapeutics
Microarray Future
Must go beyond describing differentially expressed genes
Inexpensive, high-throughput, genome-wide scan is the end game for research applications
Protein microarrays will be deployed within the next year
Publications are now being focused on biology rather than technology
SNP analysis-population surveys, SNP map Chemicogenomics Diagnostics
Industrialized Biology
Rapid replacement of single-gene experiments
Human genome project ushered in production line sequencing
Biologists in industry-what background is appropriate?