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High-Throughput Screening. Assays on Solid Supports or Chips -> Array Technology. Types of Arrays – Applications:. DNA Microarrays: Expression profiles, disease research (cancer), DNA sequencing, mutation analysis, gene discovery, diagnosis, drug discovery,… RNA Microarrays:

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high throughput screening

High-Throughput Screening

Assays on Solid Supports or Chips

-> Array Technology

types of arrays applications
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 ,...

what is microarray
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.

microarray landscape
Microarray Landscape

Slide

spot

pingroup

subarray

Gene/Protein

dna microarrays applications
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
  • ?
dna microarray technologies
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

affymetrix genechip
AffymetrixGeneChip
  • Oligonucleotide:
  • 11-25 mer (short oligo)
  • 50-70 mer (long oligo)
dna microarray experiment

Drought stress

(Kathiresan, 2005)

DNA microarray experiment:

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

Not watered

1 2 3 5 6 7 8 9 12 18 20 days

Fl

Field experimental procedure

control

Azucena

Apo

IR64

Assess drought-induced expression among three varieties of rice (IR64, Apo, Azucena)

Stress was applied by withdrawing water from the plots for 9 days.

Apo, IR64

Azucena

(Katherisan, 2005).

cdna synthesis and fluorescent dye labeling

Cy3 (green) -> cyber green

Control/ well-watered

Cy5 (red) -> cyber red

Experimental/ stressed

cDNA synthesis and fluorescent dye labeling

1. Total RNA isolation

2. cDNA Synthesis

3. Dyes are incorporated

construction of cdna library

IR64 well-watered panicles

IR64 stress

extract

Total RNA

cDNA library

PCR 15 cycles

genes

Construction of cDNA library

(Katherisan, 2005).

slide22

Spotting of cDNA library on slides

Microtiter plates

Glass slides

PCR products

from >9000 genes

3000 spots per slide

Images by Dr. John Bennett, IRRI

hybridize process
Hybridize process

GeneTAC Hyb station

image processing by laser scanning
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.

slide25

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

data analysis
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 analysis1
Data analysis

Expressed in both conditions

Repressed

Induced

Merged images

R

G

expression ratio normalization
Expression ratio - Normalization

R

Test/ Experimental

T=

∆ Gene expression

G

Reference/ Control

600

1200

5600

11600

13000

15500

18000

R

G

17500

16500

13500

10900

6500

2500

800

0/0

0.07

0.4

1.0

6.2

22.5

0.03

2.0

reporting your results

Microarray

Gene

Reporting your results

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.

slide34

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)

slide35

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

gene expression and cancer
Gene expression and cancer
  • Hierarchical clustering
    • Method for analyzing microarray data
    • Gene level analysis
    • Experiment level analysis
dna diagnostics
DNA diagnostics
  • Uses of microarrays in cancer research and diagnosis.
    • 2733 papers published on microarrays and cancer
    • 1038 papers published on microarrays, gene expression, cancer diagnosis
  • Gene expression profiling
    • 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
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.
slide39

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

slide41

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

slide42

DNA Microarray

  • Problems:
  • signal based on hybridization -> cross-hybridization (specially with DNA chips)
  • 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
why study proteins
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.
protein protein array
Protein-protein array

Different type arrays

Protein-activity array

Small molecule array

protein microarrays
Protein microarrays
  • Analysis of thousands of proteins at one time.
  • Many different types
    • 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
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
slide48

Comparison DNA-Protein Microarray:

Templin et al. 2002 Trend in Biotch. Vol 20

noncovalent immobilization
Noncovalent Immobilization

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
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
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
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
Noncovalent vs Covalent

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
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