Spotted DNA microarrays
Workshop on Gene Expression Analysis June 1, 2004
Yoav AravaDepartment of Biology
Technion
DNA microarrays
Synthesis on slide Spotting pre-made DNA
Photolithography(Affymetrix) PCR ProductsSynthesized
oligonucleotidesInkjet
(Agilent)
•20-25 nts oligonucleotides•High density (>106 oligos)
•60 nts oligonucleotides•Up to 50,000 spots
•70 nts oligonucleotides•Up to 50,000 spots
•100-3,000 bp•Up to 50,000 spots
The processSample preparationMicroarray production
RNA isolationOligonucleotidessynthesis
Genome-wide PCR
PCR purification and preparation
Spotting/Printing
Reverse transcription,Labeling
Hybridization
Scanning
Data analysis
DNA microarray
The processMicroarray production
Oligonucleotidessynthesis 50-70-mer oligonucleotides
5’ linker for binding to the slide
Advantages:• No PCR necessary
Less workNo need for DNA libraries (templates)
• Discrimination between splice variants, mRNAs with high homology etc.
Disadvantages:• Cost• Chances for cross-hybridization
The processMicroarray production
Genome-wide PCROligonucleotidessynthesis
Universal primers•Simple•Cheap
Expression libraries •Only expressed sequences•Redundancy•No full coverage•Expensive
Gene-Specific primers•Expensive•Design
Genomic DNA•Same template for all samples•Simple•Cheap•Sequenced genome•Problem with intervening sequences (e.g. introns)
PrimersTemplate
The processMicroarray production
Genome-wide PCROligonucleotidessynthesis
Set PCR reactions in 96-wells plates (using liquid-handler)
Run PCR reactions (using machine equipped with 4 x 96-wells head).
The processMicroarray production
Oligonucleotidessynthesis
Genome-wide PCR
PCR purification and preparation•Check PCR success by gel electrophorasis,
verify single product of the expected size.
•Ethanol percipitate each sample and transfer to 384-wells printing plates.
Each PCR sample contain enough DNA for thousands of microarrays!
The process
PCR purification and preparation
Spotting/Printing
Microarray production
Oligonucleotidessynthesis
Genome-wide PCR
Non-contact Adjustable spot sizeLow throughputNew technology
Contact High throughputStandard technology
The process
Massive PCR
PCR purification and preparation
Spotting/Printing
Microarray production
Oligonucleotidessynthesis
Section in the print pin
The processSample preparationMicroarray production
RNA isolationOligonucleotidessynthesis
Massive PCR
PCR purification and preparation
Spotting/Printing
Reverse transcription,Labeling
Hybridization
Scanning
Data analysis
DNA microarray
The process
Massive PCR
Microarray productionPolyA (1-2 ug)Total RNA (15-50 ug)PCR amplificationIn vitro transcription
nanograms
Sample preparationRNA isolationOligonucleotides
synthesis
Direct labelingCoupling of fluorescently labeling nucleotides
AminoAllylCoupling of AminoAllyl labeled nucleotide followed by crosslinking of fluorescent dye
Reverse transcription,Labeling
Hybridization
Scanning
Data analysis
DNA microarray
The process
Massive PCR RNA isolation
Microarray production Sample preparation
Oligonucleotidessynthesis
PCR purification and preparation
Spotting/Printing
Hybridization
Scanning
Data analysis
DNA microarray
Reverse transcription,Labeling
The process
Massive PCR
PCR purification and preparation
Spotting/Printing
RNA isolation
Reverse transcription,Labeling
HybridizationDNA microarray
Microarray production Sample preparation
Oligonucleotidessynthesis
Scanning
Data analysis
Massive PCR
PCR purification and preparation
Spotting/Printing
RNA isolation
Reverse transcription,Labeling
HybridizationDNA microarray
Scan of experiment
Scanning
Data analysis
Massive PCR
PCR purification and preparation
Spotting/Printing
RNA isolation
Reverse transcription,Labeling
HybridizationDNA microarray
Microarray production Sample preparation
Oligonucleotidessynthesis
Zoom to one of the blocks
Scanning
Data analysis
Massive PCR
PCR purification and preparation
Spotting/Printing
RNA isolation
Reverse transcription,Labeling
Hybridization
Data analysis
DNA microarray
Microarray production Sample preparation
Oligonucleotidessynthesis
Quantify each spot
Scanning
The processSample preparationMicroarray production
RNA isolationOligonucleotidessynthesis
Genome-wide PCR
PCR purification and preparation
Spotting/Printing
Reverse transcription,Labeling
Hybridization
Scanning
Data analysis
DNA microarray
Top Related