Download Development of Bioinformatics

Functional Genomics
Rong-I Hong, D.Phil.
Biomedical Engineering Center
Industrial Technology Research
Institute
Rong-I Hong, D.Phil.
[email protected]
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Outline
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Brief Introduction to Functional Genomics
Molecular Interactions on Microarrays
Making and Reading Microarrays
Expression Profiling
Gene Expression Informatics
Application
Assignments
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Brief Introduction
to
Functional Genomics
Ref: Science., Vol. 278, 1997, 601-602
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Operational Definitions




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A Set of Genes
Biomedical Functions
Systematically
Data Management and
Data Mining
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Impact

BEFORE

In past, we have had functions in search of
sequences.
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AFTER

In the future, pathology and physiology will
become “functionators” for the sequence.
Ref: Daniel Tosteson, Dean of Harvard Medical
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School, 1997.
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HOW?
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Molecular Interactions on
Microarrays
Ref: Nature Genetics Supp., Vol. 21, 1999, 5-9
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Double Helix and Complementarity of DNA
H bond
H
N
R
N
O
O
H
N
H
O
H
H
N
N
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R
N
N
N
N
N
Guanine
N
H
O
H
Cytosine
CH3
N
N
N
R
H
N
R
Thymine
Adenine
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Southern Blot
Nitrocellulose paper with
bound DNA
Stack of absorbent paper
Nitrocellulose
Gel
paper
Unlabeled DNA cut with
restriction enzyme
Ladders (known size DNA)
Agarose gel
Sponge
DNA SIZING BY GEL
Alkali solution
ELECTROPHORESIS
HIBRYDIZATION OF LABELED
DNA PROBE TO SEPARATED DNA
Sealed plastic bag
DNA BLOTTED ONTO
NITROCELLULOSE PAPER
VISUALIZATION OF LABELED DNA PROBE
HYBRYDIZED TO COMPLEMENTARY DNA
Labeled DNA
probe in buffer
Labeled marker
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Labeled bands
AUTORADIOGRAPHY
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DNA Hybridization on Solid Support
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Making and Reading
Microarray
Ref: Nature Genetics Supp., Vol. 21, 1999, 15-19
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Southern& Blot
Surface Treatment
Immobilization
Solid surface
Oligonucleotide
Aldehyde-coated glass
Poly-l-lysin coated glass
Streptavidin-coated glass
Isothiocyanate-coated glass
Aldehyde-coated silanized silicon wafer
Epoxide-coated silicon wafer
Iodoacetamide-coated silicon wafer
NH2-oligomer
NH2-oligomer
Biotinylated oligomer
NH2-oligomer
NH2-oligomer
NH2-oligomer
SH-oligomer
Linker - 5' Oligonucleotide
H2N
DNA probe
Et
O
Si
O
O
O
Si (CH2)3N
CH (CH2)3
CH N
Linker - 5' Oligonucleotide
O
Et
DNA immobilization on aldehyde-coated glass
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Southern
Blot
Microarray
Manufacturing
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Southern
Blot
Spot
Preparation
Pin:
•Spot size 150~200 µm
•Volume ~5 nl
Non Contact Ink-jet Technology:
ŽSpot size 100~150 µm
ŽVolume ~5 nl
“Over 10 times speedy than
Ž
Pin”
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Southern
Blot Data
Collection
of Microarray
Incubation of microarrays
with patient DNA
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Microarray image data collected
with scanner hardware
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Collection of Microarray Data
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Reading Microarray Data
Source: Nature Genetics Supp., Vol. 21, 1999, p.18
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QA/QC
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MANUFACTURE
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Incorporated Dyes
Immobilized DNA molecules
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PROCESS
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Hybridization
Signaling
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Data Mining
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Time Course
Differentiation
Stimulation
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Gene Expression Profiling
Ref: Nature Genetics Supp., Vol. 21, 1999, 10-14
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Experiment Process
Source: Nature Genetics Supp., Vol. 21, 1999, p.11
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Signal Comparison
Source: Nature Genetics Supp., Vol. 21, 1999, p.12
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cDNA Microarray Comparison
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Cell, Tissue and Species
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Time Course
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development process
aging
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Diagnosis
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differentiation at different stages
classification
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Gene Expression Informatics
Ref: Nature Genetics Supp., Vol. 21, 1999, 51-55
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Overview of Information Process
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Source: Nature Genetics Supp., Vol. 21, 1999, p.52
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History
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20th Century
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rice on the field
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21st Century
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gene on the chip
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Analysis
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Scatter Plots
Principle Component Analysis
Clustering Analysis
Classification Analysis
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Data Mining
Source: Nature Genetics Supp., Vol. 21, 1999, p.52
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Applications
Ref: Nature Genetics Supp., Vol. 21, 1999,
33-37 (genome),
38-41 (cancer),
45-48 (drug development)
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Biosensing Principle
Analyte
Receptor
Molecular
Recognition
Transducer
Measurement
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DataRecording
and Display
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Biochip Classification Scheme
Antibody
Enzyme
Bioreceptor
DNA
Cellular System
Cell
Biochips
Biomimetic
Transducer
Non-Enzymetic
Protein
Optical
Electrochemical
Mass-Based
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Other
Fresenius J Anal Chem (2000)366:540-551
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Informatics Flow
Array Fabrication
Image Analysis
Image Analysis
Data Management
Chip
Data
Sequence
Data
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Hybridization
Data
Cell
Data
Detection
Data
Expression
Data
Knowledge-based
Database
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Microarray Application
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Source: Science 286, 487 (1999)
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New Drug Development
Genomics
Human
Genome
Project
Screening
Drug
Targets
Clinicals
Drug
Leads
Drug
Genomics will transform drug discovery
1) Reduce time to market with a new drug
2) Prevent competition through blocking patents
3) Lower R&D costs by reducing false positives
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Assignments
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Assignment I

Assuming each spot is deposited as a
hemisphere, please
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propose a way to calculate the total amount of DNA
sample,
propose a strategy to assure the quality of DNA
spots onto the matrix.
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Assignment II
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Discuss the limitations of target labeling and
hybridization, and then propose possible
ways to overcome them.
For Reference: Nature Genetics Supp., Vol. 21, 1999, p.11
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Assignment III
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List the possible sources of variation in
microarray experiments,
and then propose the ideal statistical
models as you suggest to analyze the
microarray data.
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