High-Throughput
Screening
Assays on Solid Supports or Chips
-> Array Technology
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Types of Arrays – Applications:

DNA Microarrays:
Expression profiles, disease research (cancer), DNA sequencing,
mutation analysis, gene discovery, diagnosis, drug discovery,…

RNA Microarrays:
RNA-protein interactions, biological function of proteins, drug
discovery,…

Protein Microarray (chips):
Enzyme profiling, Protein-protein Interaction, Protein-ligand
interaction ,...
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What is Microarray ?
Microarrays are fabricated by high-speed robotics, generally on glass but sometimes on nylon
substrates, for which probes with known identity are used to determine complementary
binding, thus allowing massively parallel screening studies.
An experiment with a single DNA chip can provide researchers information on thousands of
genes simultaneously - a dramatic increase in throughput.
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Importance for High Troughput Screening
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Microarray Landscape
Slide
spot
pingroup
subarray
Gene/Protein
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DNA Microarrays – Applications:
• Monitor gene expression
–
–
–
–
Study regulatory networks
Drug discovery - mechanism of action
Diagnostics - tumor diagnosis
etc.
• Genomic DNA hybridizations
–
–
–
–
Explore microbial diversity
Whole genome comparisons - genome evolution
Identify DNA binding sites
Diagnostics - tumor diagnosis
• ?
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DNA Microarray Technologies:
Microarray allows to simultaneously analyze 1000s of sequences
 Developed in early 1990s


Two ways to construct microarrays:
1. DNA chips (developed at Affymetrix, Inc. ): an array of
oligonucleotide (20~80-mer oligos) probes is synthesized either in situ
(on-chip by photolithography) or by conventional synthesis followed by
on-chip immobilization. The array is exposed to labeled sample DNA,
hybridized, and the identity/abundance of complementary sequences
are determined.
2. DNA Microarrays (developed at Stanford University): Obtain
cDNA from cDNA sequencing projects, libraries,… -> ssDNA of cDNA
spotted onto glass slides (around 20,000 spotes/cm) -> not so high
density possible -> hybridization more specific
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Affymetrix GeneChip
Oligonucleotide:
•11-25 mer (short oligo)
•50-70 mer (long oligo)
Steps in the design and implementation of the
microarray experiment
1) Probe
(cDNA/oligo
with known
identity)
Small oligos,
cDNAs,
chromosome,
...
(whole
organism on
a chip?)
2) Chip
fabrication
(Putting
probes on the
chip)
Photolithogr.,
pipette, droptouch,
piezoelectric
(ink-jet),
electric, ...
3) Target
(fluorecently
labeled
sample)
RNA,
(mRNA==>)
cDNA
4) Assay
Hybridization,
long, short,
ligase, base
addition,
electric, MS,
electrophores
is,
fluocytometry
, PCRDIRECT,
TaqMan, ...
5) Readout
6)
Informatics
Fluorescence,
probeless
(conductance,
MS,
electrophores
is), electronic,
...
Robotics
control,
Image
processing,
DBMS,
WWW,
bioinformatic
s, data mining
and
visualization
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DNA microarray experiment:
Drought stress
(Kathiresan, 2005)
Water is a major limiting factor in
agricultural production systems in
several parts of the world. Due to
their sessile nature, plants have had to
develop efficient strategies to cope
with limited water. Tolerant plants
adapt to drought by invoking a battery
of changes in their physiological and
metabolic activities
-> Microarrays used to find metabolic
changes (gene expression changes)
Field experimental procedure
Azucena
Apo
IR64
control
Assess drought-induced expression among
three varieties of rice (IR64, Apo, Azucena)
Stress was applied by withdrawing water from
the plots for 9 days.
Not watered
1 2 3 5 6 7 8 9 12
Fl
(Katherisan, 2005).
18 20 days
Apo, Azucena
IR64
cDNA synthesis and fluorescent dye labeling
1. Total RNA
isolation
2. cDNA Synthesis
3. Dyes are
incorporated
Cy3 (green) -> cyber green
Control/ well-watered
Cy5 (red) -> cyber red
Experimental/ stressed
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Construction of cDNA library
IR64 wellwatered
panicles
IR64 stress
extract
Total RNA
cDNA library
PCR 15 cycles
genes
(Katherisan, 2005).
Spotting of cDNA library on slides
Microtiter plates
PCR products
from >9000 genes
Glass slides
3000 spots per slide
Images by Dr. John Bennett, IRRI
Hybridize process
GeneTAC Hyb station
Image processing by Laser scanning
The microscope slide containing the
microarray is placed inside a
microarray scanner, where the slide
is scanned with two lasers to detect
the bound green and red cDNAs.
Slides Scanning
59 K oligo array from BGI, Beijing
10K rice panicle cDNA library printed at IRRI
22K chips from Agilent
Images by Dr. John Bennett, IRRI
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Data analysis
What accounts for the varying colors?
These actually correspond to the amount of cDNA that binds to the
complementary strands on the spot.
Data analysis
Induced
Expressed
in both
conditions
Repressed
Merged
images
R
G
Expression ratio - Normalization
∆ Gene expression
R
G
600
1200
0.07
Test/ Experimental
G
Reference/ Control
T=
5600 11600 13000 15500 18000
17500 16500 13500
0.03
R
0.4
10900 6500
1.0
2.0
2500
800
6.2
22.5
0/0
Reporting your results
Microarray
Gene
The expression ratios for every gene can be organized into a table where each
column is a microarray and each row is a gene.
This representation however is overwhelming in experiments involving thousands of
genes and data.
DNA Chips (Microarray) made by Photolithography
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DNA Chips (Microarray) made by Photolithograpgy
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DNA Chips (Microarray)
Recent improvments:
-> use ink-jet printer technology (Agilent) to deliver nucleotides for
synthesis
-> ”maskless synthesis” by photolitography (NimbleGen Systems)
-> ultra-high density
-> new technologies: produce longer oligos (50-100 bp)
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DNA Microarray
Direct comparision of
expression patterns
possible
-> expression level of
which genes changes
during wound healing
-> which genes are
involved in which
biochemical and
cellular pathway
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Gene expression and cancer

Hierarchical clustering



Method for analyzing
microarray data
Gene level analysis
Experiment level
analysis
DNA diagnostics

Uses of microarrays in cancer research and
diagnosis.



Gene expression profiling



2733 papers published on microarrays and cancer
1038 papers published on microarrays, gene expression,
cancer diagnosis
Identify genes involved in cancer diagnosis.
Identify gene expression patterns that are associated
with disease outcome.
Gene content analysis

Identify genomic regions that are lost or amplified in
tumors.
Identifying replication origins in yeast

Only 5% of the genome
previously screened for
replication origins.

Used known replication
initiation factors to perform
ChIP/chip analysis

Identified hundreds of
additional replication origins in
a single experiment.
Chromatin immunoprecipitation assay (ChIP)
-> to analyze mechanism of
control of gene expression
-> binding of transcription
factors (DNA binding
proteins)
ChIP used to analyse a lot of
TF at the same time
->In vivo experiment
-> can be used to test
complexity of the cell
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Chromatin immunoprecipitation assay (ChIP)
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DNA – Protein Interaction - Microarray
• Identification of DNA
regions bound by a protein.
• Compare a wild-type strain
to a ∆gene (DNA-binding
protein).
• Do not need any prior
knowledge of the sequence
the protein binds.
Iyer et al. 2001 Nature, 409:533-538
DNA Microarray
Problems:
1. signal based on hybridization -> cross-hybridization (specially with
DNA chips)
2. If expression level high in cell -> maybe not enough targets for
hybridization available -> signal not proportional to mRNA level
-> better method to analyse expression level:
High-Throughput Sequencing of cDNA or small fragments of cDNA
-> Sequences are analyzed -> number of times a sequence is present ->
expression level
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Why study proteins?

They are the machines that make cells function.

RNA levels do not always accurately predict
protein levels.



Often processes are regulated at the transcriptional
level.
Some processes are controlled post-transcriptionally.
Proteins are the targets of drugs.
Different type arrays
Protein-protein array
Small molecule array
Protein-activity array
Protein microarrays

Analysis of thousands of proteins at one time.

Many different types

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Antibody arrayed - detect many proteins
Proteins arrayed - detect interacting proteins
Proteins arrayed - detect interacting small molecules
Proteins arrayed - detect enzymatic activity (Enzyme
profiling)
Peptides arrayed – substrate for enzymes, interaction
Small molecules arrayed – detect enzymatic activity
(enzyme profiling)
Etc.
Requirements
•
•
•
•
•
•
Proper folding and orientation of immobilized proteins or small
molecules
Solvent (presence of ions, hydrophilicity etc.)
Cofactors
pH
Temperature
This makes protein/peptide immobilization more difficult than
DNA immobilization
Comparison DNA-Protein Microarray:
Templin et al. 2002 Trend in Biotch. Vol 20
Quality Images
Actual Spots Boundary, Regional Background Correction
Noncovalent Immobilization
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Physical adsorption
•
•
•
Mainly hydrophobic interactions
Nonspecific
Capturing methods
•
•
Biotin-Avidin  site-specific
Antibody-antigen  site-specificity depends on method
• Antibody developed against enzyme
• Antibody developed against antigen bound to enzyme
• Polyclonal  several epitopes; monoclonal  one epitope
•
•
His-tag binding to nickel-nitrilotriacetic acid
Entrapment in gels
•
•
Immobilized molecules in aqueous environment
Long incubation time required
Biotin-Streptavidin
Binding of biotin to streptavidin.
The ureido group of biotin is
polarized during binding, whereby
the acquired negative charge of
oxygen can be stabilized in the
oxyanion hole formed by Asn23,
Ser27 and Tyr43. Other polar
residues in streptavidin form
hydrogen bonds to biotin to
stabilize the binding further [Weber
et al., 1992].
Covalent Immobilization
•
Chemical immobilization
•
•
•
•
•
EDC/NHS
Thiol–thiol (formation of disulphide) or thiol-gold
Michael reaction between nucleophile and ,β-unsaturated carbonyl
compound
Many others exist, including variations of the techniques mentioned
above
Blocking agents (e.g. BSA) can be used to hinder unspecific binding
EDC/NHS Immobilization
In the first step, EDC reacts with the carboxyl group attached to the carrier surface (left). This creates an
unstable O-acyl isourea intermediate, and to avoid hydrolysis of the intermediate, NHS is added (middle).
Hereby, a stable activated NHS ester is formed and a soluble urea byproduct is released (right) [Vaughan et
al., 1999].
When a biomolecule (Bm) is introduced to the activated NHS ester, NHS is replaced by the biomolecule,
and immobilization is completed [Vaughan et al., 1999].
Noncovalent vs Covalent
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Noncovalent
•
•
•
•
Often weaker than covalent methods
Usually nonspecific
Antibodies can be produced against any antigen
Covalent
•
•
Strong immobilization
Site-specific approaches easier  proper orientation
Covalent Biotinylation
•
Intein-mediated site-specific biotinylation
•
•
Intein at protein C-terminus
When the intein is spliced out a thioester is created at the C-terminus, which is then able to react
with cysteine-biotin
Method A: In vitro in cells (cell lysed before biotinylation)
- Chitin-binding domain on intein functions as affinity tag before biotinylation
Method B: In vivo in cells (biotinylation inside the cells)
Method C: Cell-free system
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End Part 1