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