DNA Microarray Technology May 1st, 2002

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DNA Microarray Technology May 1st, 2002

• Victor Chan, D. Phil.
• Assistant Technical Director
• ManTech Environmental Corp.

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Overview
Microarrays and DNA Hybridization Oligonucleotide
arrays and cDNA arrays Gene Expression
Profiling Research Projects at AFRL/HEST
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DNA Microarrays
An array is an orderly arrangement of
samples. DNA microarrays contain sample spots
less than 0.04 mm2. DNA microarrays are used to
analyze gene expression or gene sequences based
on a 2-phase hybridization reaction between
labeled targets derived from biological specimens
of interest and a large number of ordered sets of
single-stranded DNA probes with known sequences
immobilized on a solid surface.
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DNA Hybridization
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Microarray Hybridization
DNA Microarray
Hybridized Array
Labeled Target
Detection Reagents
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cDNA and Oligonucleotide Microarrays
Type of Microarrays 1. cDNA arrays
(Microspotting) 2. Oligonucleotide arrays
(Photolithographic synthesis, Ink- jet
technology, etc.) Applications Gene
Expression SNPs Mutations Deletions and
insertions Genotyping
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Microarray Fabrication Technologies
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Photolithographic Synthesis of Affymetrix
GeneChips
Lamp
Mask
Chip
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On chip photolithographic synthesis of
oligonucleotides
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Probe Design of Affymetrix Expression GeneChip
Array
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5
mRNA sequence
Perfect Match
Mismatch
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Affymetrix GeneChip
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Affymetrix GeneChip Technology
Specimens
Bioinformatics
Labeled Targets
0
0
0
0
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Target Preparation
cDNA
Specimens
mRNA
Labeled Targets (cDNA or cRNA)
Labeled Fragments
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Rat Genome Chip
8799 Probe Sets
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Rat-Tox Chip
1031 Probe Sets
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Probe Sets
PM
MM
Probe Cell - 24mm x 24mm - 3mm/Pixel - 64
Pixels
Probe Pair
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cDNA Microarrays
cDNA Probes GenBank UniGene dbEST
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cDNA Microarrays
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2-Dye Technology of cDNA Microarrays
Cy5
Test
RT Labeled with Fluor Dyes
Control
Cy3
scanning
cDNA Microarrays
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cDNA Array
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Oligonucleotide Arrays vs. cDNA Arrays
Oligonucleotide Arrays cDNA Arrays High
density Lower density High specificity Cross
-hybridization cannot be
determined Requires small amount of
samples Larger amount of samples is
required Gene expression and Mostly for Gene
Expression Genetic alteration/polymorphism Hi
gh reagent costs Lower reagent
costs Hybridization of target from a single
source Simultaneous hybridization of targets
from two sources Clones for signal
validation have to be Clones available for
signal validation obtained from other
sources Difficult to manufacture arrays in-house
Array manufacture is labor intensive
and initial set-up is expensive High cost per
array Low cost per array Lower
flexibility High flexibility
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Applications of DNA Microarrays
Gene expression profiling Mutation and
polymorphism analysis Genotyping
Traditional A few genes
DNA Microarray Genome-wide Analysis
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Central Dogma of Molecular Biology
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Analysis of Microarray Data
Information
Data
Knowledge
Data Mining
Data Processing
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Data Processing
Intensity Algorithm
Background Subtraction
Normalization
Expression Algorithm
Data Processing
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Processing of Expression Analysis Data
Intensity Algorithm Transforming pixel image
to raw hybridization signals Background
Subtraction Determining specific
hybridization signals (subtract signals of cross
hybridization and nonspecific staining) Expressio
n Algorithm Transforming specific
hybridization signals to gene expression
levels. Normalization Normalizing the
signals to a reference so that Technically
related variations between probe arrays from the
same or different experiments can be minimized.
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Data Mining
Experiments
Genes
Novel genes Novel functions of known genes
Pathway elucidations Biological Processes
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Data Mining
Principal Component Analysis (PCA) Hierarchical
Cluster Analysis (HCA)Multidimensional Scaling
(MDS) k-Means Classification and Regression
Tree (CART)Self-Organizing Maps
(SOM)Correlation Coefficient ClusteringSupport
Vector Machine (SVM)k-Nearest Neighbors
(KNN)Partial Least-Squares Regression
(PLS)Back-Propagation Artificial Neural Network
(BP-ANN)Genetic Algorithm (GA)
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Gene Expression Profiles of human Cancers
Leukemia melanoma breast cancer colon cancer

• cDNA microarray
• 9703 cDNA probes

Scherf et al. Nature Genetics 24 236-244 (2000)
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Applications of Gene Expression Profiling
Disease diagnosis and prognosis Pharmacogenomics
and toxicogenomics Classification of chemical
toxicants Elucidation of cellular pathways,
biological processes and regulatory networks
Identification of biomarkers
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Research Projects at AFRL/HEST
Toxicogenomic Fingerprinting Computational
Biology (Systems Biology)
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Cellular Responses to Toxic Insults
Damages in Cellular Targets
Toxicants
Repair Processes
Cell Cycle Arrest
Beyond Repair
Damages Repaired
Death
Recovery
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Toxicogenomic Fingerprinting
Toxicants
Gene Expression Profiles
Damages in Cellular Targets
Repair Processes
Cell Cycle Arrest
Beyond Repair
Damages Repaired
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Toxicogenomic Fingerprinting

• Toxicants Specific cellular targets and extent of
  damages
• Cellular responses to specific damages
• Up-regulation/Down-regulation of specific genes
• Unique gene expression profiles (fingerprints)
• Early indicators for toxicity
• Database of specific changes in gene
  expression associated with chemical toxicity
• Identification of toxicants
• Classification of chemicals based on the
  pattern of altered gene expression
• Structure-toxicity Relationship
• Predict cellular responses to new chemicals

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Computational Biology
Gene Expression Profiles
Damages in Cellular Targets
Repair Processes
Cell Cycle Arrest
Beyond Repair
Damages Repaired
?
Death
Recovery
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Computational Biology
Gene Expression Profiles
Damages in Cellular Targets
Repair Processes
Cell Cycle Arrest
Beyond Repair
Damages Repaired
?
Death
Recovery
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Multi-level Regulations
Functional Components
Information
Cellular Pathways
Cellular Processes
Cellular Phenotype
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Cellular Pathway
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Multi-pathway network
DNA Repair
Apoptosis
Cell Cycle Arrest
Ubiquitin Proteasome Pathway
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Automated Data Analysis
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Automated Data Analysis
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Automated Data Analysis
Database Query (e.g. blast search)
Sequence Analysis (e.g. Functional Domains)
Biochemical Activity
Biological Activity
Literature Search
Expression Levels of Related Genes on the
Array (Structure, Function, Pathway, etc.)
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Automated Data Analysis
Database Query (e.g. blast search)
Sequence Analysis (e.g. Functional Domains)
Biochemical Activity
Biological Activity
Literature Search
Expression Levels of Related Genes on the
Array (Structure, Function, Pathway, etc.)
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Integrated Data Analysis
Genomics
Proteomics
Metabonomics
Automated Data Analysis
Activities of Cellular Processes
Multi-platform Data Integration and Analysis
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The End
Questions?