2 Copyright noticeMany of the images in this power point presentation of other people. The Copyright belong to the original authors. Thanks!
3 Evolution & Industrialization 1989: First Affymetrix Genechip Prototype1994: First Commercial Affymetrix Genechip1994- First cDNAs arrays were developed at Stanford University.1994: First Commercial Scanner-Affymetrix1996- Commercialization of arrays1997-Genome-wide Expression Monitoring in S. cerevisiae
4 Why use DNA Microarrays for Expression Analysis? Conventional expression analysis only allows the study of the expression of a single gene in a single experimentThe highly parallel nature of microarrays allows the simultaneous study of the expression of thousands or even tens of thousands of different genes in a single experimentMicroarrays allow researchers to undertake global expression analysis that is not feasible with conventional techniques
5 What Microarrays detect? What genes are Present/Absent in a cell?What genes are Present/Absent in the experiment vs. control?Which genes have increased/decreased expression in experiment vs. control?Which genes have biological significance?
6 Why analyze so many genes? Just because we sequenced a genome doesn’t mean we know anything about the genes. Thousands of genes remain without an assigned function.Patterns/clusters of expression are more predictive than looking at one or two prognostic markers – can figure out new pathways
8 The 6 steps of a DNA microarray experiment Manufacturing of the microarray2. Experimental design and choice of reference: what to compare to what?3. Target preparation (labeling) and hybridization
9 The 6 steps of a microarray experiment (cont.) 4. Image acquisition (scanning) and quantification (signal intensity to numbers)5. Database building, filtering and normalization6. Statistical analysis and data mining
10 Different Types of DNA Microarrays How DNA sequences are laid downSpottingPhotolithography (Affymetrix)type of DNA sequencescDNA (Complete sequences)Oligonucleotides
11 Affymetrix Microarrays Involves Fluorescently tagged cRNAOne chip per sampleOne for controlOne for each experimentSpotted MicroarraysInvolves two dyes/one chipRed dyeGreen dyeControl and experiment on same chip
12 Production of Affymetrix Arrays Choice of probes25mer oligonucleotides11-16 per gene3’ biasedDispersedChosen to minimize cross-hybridizationComputer algorithms are used to design photolithographic masks for use in manufacturing
13 Each gene is represented on the probe array by multiple probe pairs Each probe pair consists of a perfect match and a mismatch oligonucleotide
14 Match and mismatch ATGCTGTACAATCGCTTGATACTGG ATGCTGTACAATAGCTTGATACTGG The exact match is a section of the mRNA sequence you wish to probe forThe mismatch is identical except for one base difference from it’s exact match counterpart, and is used to calculate a background.There are typically 11 “probe pairs” scattered around the chip- called a probe set.By combining the expression values for a probe set, a value for the expression of mRNA can be found.ATGCTGTACAATCGCTTGATACTGGATGCTGTACAATAGCTTGATACTGGMismatch probe:Target sequence:Perfect match probe:
15 Why do we have mismatch probes? Mismatch probes (MM) are trying to detect background.The mismatch probes are supposed to detect things that are close but not an exact match.It is assumed that these things also bind to the perfect match (PM), erroneously.
16 Production of Affymetrix Arrays: Photolithographic Synthesis
17 Creating Targets mRNA cDNA cRNA Target Reverse Transcriptase in vitro transcriptioncRNA
18 RNA-DNA Hybridization TargetsRNAprobe setsDNA(25 base oligonucleotides of known sequence)
19 Non-Hybridized Targets are Washed Away (fluorescently tagged)“probe sets” (oligo’s)Non-bound ones are washed away
20 Custom GeneChipsAffymetrix offers over 120 prokaryotic arrays that are manufactured by Nimblegen Inc.Custom GeneChips are also available for both Eukaryotic and Prokaryotic systems.
21 Spotted Arrays Robotically spotted cDNAs or Oligonucleotides Printed on Nylon, Plastic or Glass surfaceDNA probes prepared in 384-well platesGlass slides bought commercially or prepared in houseArrays are spotted with a commercial arrayerReplicatesControls
22 Microarray of thousands of genes on a glass slide
24 bacterial glycerol stocks) Building the chipPCR amplificationDirectly from colonies withSP6-T7 primers in 96-wellplatesConsolidate into384-well platesArrayed Library(96 or 384-well plates ofbacterial glycerol stocks)Slide 4Ideally, you will have a slides consist of all genes in the genomeThe design of the array / template is an imporatnt bioinformatics question. As you are only able to detect expression of genes on this template.Spot as microarrayon glass slides
25 Expression profiling with DNA microarrays cDNA “B”Cy3 labeledcDNA “A”Cy5 labeledLaser Laser 2HybridizationScanningSlide 5You will have another sample of interests consists of a portion of the genes and the questions is to find out what genes are being expressed in this sample. The simplified version of the experiments is to label the sample with a color dye hybridize the sample on the slides and scan the slides under some laser to see which spots that light up. This provides a list of genes and it’s corresponding expression levels in our samples.Our interest is on comparing the expression levels between the two samples.+AnalysisImage Capture
26 Spotted ArraysAffymetrix ArraysHomemadeTailoredCheaper?????Maximum 24,000 features per arrayProne to variabilityCommercially available“Off the rack”More expensive?????Maximum 500,000 features per arrayLess variability