The Pat Brown DNA Microarray Chip

The Pat BrownThe Pat BrownDNA Microarray ChipDNA Microarray Chip

-first developed by Pat Brown and colleagues at Stanford University

-developed for high-throughput measurement of expression patterns of thousands of genes

-simplest realization of this technology is the

What is a cDNA microarray? A set of cloned cDNAs

spotted on a membrane or a glass slide, and hybridized to labeled RNA or DNA

How it worksHow it works1) High speed, high precision robot is used

to spot thousands of DNA samples onto glass slides

2) The slides are then probed with florescent labeled cDNAs

Goal: to measure gene expression by measuring the number of RNA messages

(mRNAs) being produced for each gene.

I. To measure mRNA messages, the spotted cDNA fragments represent the distinct genes to be measured.

II. These will selectively hybridize with their associated mRNA (or cDNA) made from a test sample of cells or tissue, and thus can give a measurement of expression of these target genes in sample.

1A. Prepare your DNA chip using your chosen target DNAs.

2. Hybridize mixture containing fluorescently labeled cDNAs with your DNA chip.

3. Detect bound cDNA using laser technology and store data in a computer.

4. Analyze data using computational methods

1B. Generate a hybridization solution containing a mixture of fluorescently labeled cDNAs.

Overview

I. Printing DNA MicroarraysI. Printing DNA Microarrays The DNA fragments to be arrayed (spotted) The DNA fragments to be arrayed (spotted)

are first amplified by PCR to get sufficient are first amplified by PCR to get sufficient quantity quantity

The reactions should largely yield The reactions should largely yield single single bandsbands in the 400—2000 bp size range in the 400—2000 bp size range

Plain glass microscope slides are either Plain glass microscope slides are either coated with silane-derivative or with poly-L-coated with silane-derivative or with poly-L-Lysine to give the surface DNA binding Lysine to give the surface DNA binding capacity capacity

Preparing Print Tips for PrintingPreparing Print Tips for Printing

Robotic PrintingRobotic PrintingThe “print tip” is a needle-like steel pin The “print tip” is a needle-like steel pin

with a split tip that takes up several with a split tip that takes up several hundred nanoliters of fluid by capillary hundred nanoliters of fluid by capillary actionaction

The tip dips into a well, takes up DNA solution, and The tip dips into a well, takes up DNA solution, and then releases a nanoliter sized drop each time it is then releases a nanoliter sized drop each time it is touched down on a slide. touched down on a slide.

The droplet dries in a few seconds, leaving the The droplet dries in a few seconds, leaving the DNA spot behind as a “stain” DNA spot behind as a “stain”

The spots are laid on the slides in a rectangular The spots are laid on the slides in a rectangular grid pattern.grid pattern.

10 000 gene array could be completed in a single 13 10 000 gene array could be completed in a single 13 hour printing run, yielding on the order of 100 printed hour printing run, yielding on the order of 100 printed arrays. This level of speed typically requires 32 print arrays. This level of speed typically requires 32 print tips.tips.

Post-Processing of SlidesPost-Processing of Slides:: UV crosslink DNA to glass UV crosslink DNA to glass Chemically treated to “finish” Chemically treated to “finish”

the surface i.e. bind the DNA the surface i.e. bind the DNA and make the surface non-and make the surface non-sticky so that it does not non-sticky so that it does not non-specifically bind labeled DNA specifically bind labeled DNA during the later hybridization. during the later hybridization.

Return plates to –20C for Return plates to –20C for long term storage. Plates long term storage. Plates can typically be arrayed from can typically be arrayed from 2-4 times2-4 times

II. Simple Probe Preparation and II. Simple Probe Preparation and HybridizationHybridization

Total RNA and mRNA from tissues or cell lines Total RNA and mRNA from tissues or cell lines is extracted and purified is extracted and purified

Labeling is carried out by performing reverse Labeling is carried out by performing reverse transcription of the RNA in the presence of dye-transcription of the RNA in the presence of dye-labeled nucleotides labeled nucleotides

resulting cDNA copy has incorporated resulting cDNA copy has incorporated fluorescently labeled bases fluorescently labeled bases

the red (Cy5) and green (Cy3) labeled probes the red (Cy5) and green (Cy3) labeled probes must be produced in separate labeling reactions must be produced in separate labeling reactions so that each incorporates one color of dye so that each incorporates one color of dye

Hybridization: Hybridization: Red and green labeled probe reactions are Red and green labeled probe reactions are

combinedcombined ““Blocking DNA” added and then applied to the Blocking DNA” added and then applied to the

surface of the DNA slides (repetitive and non-surface of the DNA slides (repetitive and non-specific DNA fragments, to block such specific DNA fragments, to block such hybridization sites on the array before they can hybridization sites on the array before they can attach to labeled probe fragments, Poly-A attach to labeled probe fragments, Poly-A fragments)fragments)

microscope cover slip is applied to spread the microscope cover slip is applied to spread the solution uniformly over the array and prevent solution uniformly over the array and prevent rapid evaporation rapid evaporation

Hybridization: about 8—16 hours suffices Hybridization: about 8—16 hours suffices to produce a reasonable amount of to produce a reasonable amount of appropriate hybridization appropriate hybridization

The slide is washed to remove buffer and The slide is washed to remove buffer and unhybridized probe DNAunhybridized probe DNA

Data Processing Data Processing The finished slide is scanned by a The finished slide is scanned by a

fluorescent scanner.fluorescent scanner.

ScannersScannersConfocal Microscopy ScannersConfocal Microscopy Scanners:: The The

most common approach is to scan across most common approach is to scan across the slide with a laser that is tuned/filtered the slide with a laser that is tuned/filtered to predominantly excite a single dye to predominantly excite a single dye

CCD Camera “Scanners”CCD Camera “Scanners”:: An An alternative approach is to simultaneously alternative approach is to simultaneously excite both dyes over the entire slide excite both dyes over the entire slide surface with bright white tungsten filament surface with bright white tungsten filament light (no lasers) light (no lasers)

Images must be quantifiedImages must be quantified numerical measurements of the brightness of the spotsnumerical measurements of the brightness of the spots

NormalizationNormalization --subtract backgroundsubtract background -adjust for variable DNA-adjust for variable DNA -adjust for variable amounts of green and red -adjust for variable amounts of green and red dyesdyesRatioRatio red:green intensityred:green intensity

Identify critical genes to target in viruses and Identify critical genes to target in viruses and bacteriabacteria

Functional relations among genes (assign Functional relations among genes (assign function to genes)function to genes)

Clue to specific physiological processes in an Clue to specific physiological processes in an environment (how are cells surviving)environment (how are cells surviving)

Where a gene product is produced (in which cell)Where a gene product is produced (in which cell) Molecular picture of physiological processesMolecular picture of physiological processes

Analysis of virulence potential of E. coli strains isolated from clinical samples

(A) genomic DNA from avian E. coli isolate

(B) hybridization pattern obtained with genomic DNA from bovine strain

(C) hybridization obtained with genomic DNA from human E. coli isolate

Boxed spots indicate pathotype-specific genes

Application:-Protein needed for cell function-Location for protein function-Cells to target for drug therapy-Genes to target for drug therapy

    Green: Downregulation    Yellow: No Change

Gene expression probing using cDNA microarray revealed genes downregulated transcriptionally with metabolic shift in mammalian cells

reduction in ratio of lactate production rate to glucose consumption rate

Web ReferencesWeb References•http://www.escience.ws/b572/L24/L24.htm•http://cmgm.stanford.edu/pbrown/pdf/Brown_PO_Nat_Genet_1999.pdf •http://genome-www.stanford.edu/nci60/figures.shtml •http://jcm.asm.org/cgi/content/full/41/5/2113 •http://www.genetics.ucla.edu/microarray/ArrayManual 09-21-2001.doc•http://hugroup.cems.umn.edu/Research/Genomics/regulation.htm•http://las.perkinelmer.com/catalog/Product.aspx?ProductID=CBC0000