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:
Assays on Solid Supports or Chips
-> Array Technology
Expression profiles, disease research (cancer), DNA sequencing, mutation analysis, gene discovery, diagnosis, drug discovery,…
RNA-protein interactions, biological function of proteins, drug discovery,…
Enzyme profiling, Protein-protein Interaction, Protein-ligand interaction ,...
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.
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
(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)
from >9000 genes
3000 spots per slide
Images by Dr. John Bennett, IRRI
GeneTAC Hyb station
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.
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
What accounts for the varying colors?
These actually correspond to the amount of cDNA that binds to the complementary strands on the spot.
Expressed in both conditions
∆ Gene expression
-> use ink-jet printer technology (Agilent) to deliver nucleotides for
-> ”maskless synthesis” by photolitography (NimbleGen Systems)
-> ultra-high density
-> new technologies: produce longer oligos (50-100 bp)
Direct comparision of expression patterns possible
-> expression level of which genes changes
during wound healing
-> which genes are involved in which biochemical and
-> to analyze mechanism of
control of gene expression
-> binding of transcription
factors (DNA binding
ChIP used to analyse a lot of TF at the same time
->In vivo experiment
-> can be used to test complexity of the cell
Iyer et al. 2001 Nature, 409:533-538
Different type arrays
Small molecule array
Templin et al. 2002 Trend in Biotch. Vol 20
Mainly hydrophobic interactions
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
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].
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
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].
Often weaker than covalent methods
Antibodies can be produced against any antigen
Site-specific approaches easier proper orientation
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