Introduction to microarray technologyOutlineUses and types of microarraysNucleic acid hybridization: here DNA-RNAThe rudiments of hybridization kinetics can be helpfulRudiments, completedA knolwedge of the Polymerase Chain Reaction (PCR) can be helpfulPCR, ctdPowerPoint PresentationSlide 10Slide 11Affymetrix GeneChips in summary (details slightly out of date)cDNA microarrays on glass slides A little more detailcDNA arrays: historycDNA arrays: the processSlide 16Slide 17Slide 18Affymetrix GeneChip expression array designSlide 20Slide 21Slide 22Cartoon version: Before labellingBefore HybridizationAfter HybridizationQuantificationAdffymetrix chip image: low res.Acknowledgments1Introduction to microarray technologyLecture 17, Statistics 246March 18, 20042OutlineA little backgroundTypes of microarrayscDNA arraysAffymetrix GeneChips3Uses and types of microarrays Microarrays are currently used to do many different things: to detect and measure gene expression at the mRNA or protein level; to find mutations and to genotype; to (re)sequence DNA; to locate chromosomal changes (CGH = comparative genomic hybridization), and more. There are many different ways to do these things without microarrays, but microarrays promise a high-throughput approach to the tasks. There are many different types of microarrays (called platforms) in use, but all have a high density and number of biomolecules fixed onto a well-defined surface. Low density means 100s (e.g. protein antibodies), medium density would be 1000s to 10s of 1000s (e.g. cDNA arrays), and high-density is 100s to 1000s of 1000s, i.e.millions (e.g. short oligonucleotide arrays). In general there are five basic aspects of microarrays: a) coupling biomolecules to a platform; b) preparing samples for detection; c) hybridization; d) scanning; and e) analyzing the data. Obviously we’re interested in e), but without some knowledge of a) to d), we’d be dangerous.4Nucleic acid hybridization: here DNA-RNA5The rudiments of hybridization kinetics can be helpful6Rudiments, completed7A knolwedge of the Polymerase Chain Reaction (PCR) can be helpful This reaction is used to amplify specific DNA sequences in a complex mixture when the ends of the sequence are known. The source is heat-denatured into single strands. Two synthetic oligonucleotides complementary to the 3’ ends of the segment of interest are added in great excess to the denatured DNA and the temperature is lowered to 50-60˚C or even lower. The genomic DNA remains denatured, because the complementary strands are at too low a concentration to encounter each other during the period of incubation, but the specific oligonucleotides, which are at a very high concentration, hybridize with their complementary sequences in the genomic DNA.8PCR, ctd The hybridized oligos then serve as primers for DNA chain synthesis, which begins upon addition of a supply of dNTPs and a temperature resistant polymerase such as that from Thermus aquilus (a bacterium that lives in hot springs). This enzyme, called Taq polymerase, can extend primers at temperatures up to 72˚C. When synthesis is complete, the whole mixture is heated further (to 95˚C) to melt the newly formed duplexes. When the temperature is lowered again, a new round of synthesis takes place because excess primer is still present. Repeated cycles of synthesis (cooling) and melting (heating) quickly amplify.910The cDNA and short (25 bp) oligo technologies in brief. Long (60-75 bp) oligo arrays are more like the cDNA ones11cDNA clones(probes)PCR product amplificationpurificationprintingmicroarrayHybridise target to microarraymRNA target)excitationlaser 1laser 2emissionscanninganalysis0.1nl/spotoverlay images and normalisecDNA arrays in summary12 Affymetrix GeneChips in summary (details slightly out of date)24µm24µmMillions of copies of a specificMillions of copies of a specificoligonucleotide probe oligonucleotide probe synthesized in situ (“grown”)synthesized in situ (“grown”) Image of Hybridized Probe ArrayImage of Hybridized Probe Array>200,000 different>200,000 differentcomplementary probes complementary probes Single stranded, Single stranded, labeled RNA targetlabeled RNA targetOligonucleotide probeOligonucleotide probe*****1.28cm1.28cmGeneChipGeneChip Probe ArrayProbe ArrayHybridized Probe CellHybridized Probe CellCompliments of D. Gerhold13cDNA microarrays on glass slidesA little more detailAn overview of the Brown-De Risi- Iyer technology, based on —the 2000 CSH Microarray Course notes, Nature Genetics Supp, Jan 1999, —two books edited by M Schena: DNA Microarrays, A Practical Approach, OUP 1999, and Microarray Biochip Technology, Eaton Publishing, 2000, —DNA Arrays or Analysis of Gene Expression by M. Eisen and P. Brown, and —the experiences of my colleagues.14cDNA arrays: history cDNA microarrays have evolved from Southern blots, with clone libraries gridded out on nylon membrane filters being an important and still widely used intermediate. Things took off with the introduction of non-porous solid supports, such as glass - these permitted miniaturization - and fluorescence based detection. Currently, up to about 30,000 cDNAs are spotted onto a microscope slide.15cDNA arrays: the processBuilding the Chip:MASSIVE PCRPCR PURIFICATION and PREPARATIONPREPARING SLIDES PRINTINGPreparing RNA:CELL CULTURE AND HARVESTRNA ISOLATIONcDNA PRODUCTIONHybing the Chip:POST PROCESSINGARRAY HYBRIDIZATIONPROBE LABELINGDATA ANALYSIS16MASSIVE PCRPCR PURIFICATION and PREPARATIONPREPARING SLIDESPRINTINGBuilding the Chip:Full yeast genome = 6,500 reactions IPA precipitation +EtOH washes + 384-well formatThe arrayer: high precision spotting device capable of printing 10,000 products in 14 hrs, with a plate change every 25 minsPolylysine coating for adhering PCR products to glass slidesPOST PROCESSINGChemically converting the positive polylysine surface to prevent non-specific hybridization17Preparing RNA:CELL CULTURE AND HARVESTRNA ISOLATIONcDNA PRODUCTIONDesigning experiments to profile conditions/perturbations/mutations and carefully controlled growth conditionsRNA yield and purity are determined by system. PolyA isolation is preferable but total RNA is useable. Two RNA samples are hybridized/chip.Single strand synthesis or amplification of RNA can be performed. cDNA production includes incorporation of Aminoallyl-dUTP.18Hybing the Chip:ARRAY HYBRIDIZATIONPROBE LABELINGDATA ANALYSISCy3 and Cy5 RNA
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