Microarrays10-810 /02-710Computational GenomicsWhy sequence is not enoughIdentifying genes and control regions is not enough to decipher the inner workings of the cell:• We need to determine the function of genes.• We would like to determine which genes are activated in which cells and under which conditions.• We would like to know the relationships between genes (protein-DNA, protein-protein interactions etc.).•We would like to model the various dynamic systems in the cellMicroarrays for molecular biologyDNAmRNAtranscriptiontranslationProteinsTranscription factorsFDA Approves Gene-Based Breast Cancer Test*“ MammaPrint is a DNA microarray-based test that measures the activity of 70 genes... The test measures each of these genes in a sample of a woman's breast-cancer tumor and then uses a specific formula to determine whether the patient is deemed low risk or high risk for the spread of the cancer to another site.”*Washington Post, 2/06/2007What is gene expression?genesExperiments (over time)100 20 70 80gene 1Higherexpression compared to baselineLower expression compared to baselinebaseline expressionSpellman et al Mol. Biol. Cell 1998Expression = level of gene (protein) in this experimentGenes and Gene ExpressionTechnologyDisplay of Expression InformationGenomic DNAPromoter Protein coding sequence TerminatorWhat is a gene?TFIIBRNAPIITFIIATFIIDTFIIHTFIIFMediatorTFIIEActivatorsHow are Genes Regulated?DNA-binding Activators Are Key To Specific Gene ExpressionGeneChromatin modification complexesTranscription initiation apparatusTFIIBRNAPIITFIIATFIIDTFIIHTFIIFMediatorTFIIEActivatorsHow are Genes Regulated?DNA-binding activators are key, but there are additional factorsGeneChromatin modification complexesTranscription initiation apparatusactivatorsrepressorscoactivatorscorepressorstranscription apparatuschromatin factorsRNA processingRNA transportRNA degradationActivatorsGenome-wide Gene Expression (mRNA) can be Measured with DNA MicroarraysGeneRNAPIITFIIHTranscription apparatusmRNAmRNAlabelhybridizationATGCTACGGenes and Gene ExpressionTechnologyDisplay of Expression InformationMicroarray Hybridization• Watson-Crick base pairing of complementary DNA sequences.• Microarrays have thousands of spots, each representing a piece of one gene, immobilized on a glass slide.• The intensity (or intensity ratio) of each spot indicates the amount of labeled cDNA hybridized, thus, representing the starting mRNA transcript abundance.Two major technologies• cDNA arrays- probes are placed on the slides- allows comparison of different cell types• Oligonucleotide arrays- partial sequences are printed on the array- measure values in one tissue typeHybridization and Scanning— cDNA arrays- Prepare Cy3, Cy5-labeled ss cDNA- Hybridize 600 ng of labeled ss cDNA toglass slide array- ScanCartesian PixSys 5500 withquill printing technology• Complete subsequences are printed on the array•10,000 spots/slide• Spots are 100-200 µm in diameter• Hybridization volumes: 20-100ulArray ScanningLaser based - fluorescent emissionHybridization and Scanning—oligo arrayscDNA vs. Oligo: Pros and ConscDNA• Does not require sequence• Cheap• Direct comparisons• Inaccurate• Cannot measure individual samplesOligo• Can be designed to minimize cross hybridization• Allows for internal control• Both lead to better accuracy• expensive• limited to certain speciesErrorsMicroarrays introduce many errors which should be taken into account when working with measured expression values:• Scanning errors• Spotting errors• Cross hybridization• Errors related to day / reading device / experimentalist• Background differences between slidesError typesMicroarrays introduce many types of errors which should be taken into account when working with measured expression values:• Scanning errors additive + multiplicative• Spotting errors multiplicative• Cross hybridization multiplicative• Errors related to day / reading device / experimentalistadditive + multiplicative• Background differences between slides additiveHandling the Different Errors• Scanning errors• Spotting errors• Cross hybridization• Errors related to day / reading device / experimentalist• Background differences between slidesAnalysis of image data (we assume it was performed)Handling the Different Errors• Scanning errors• Spotting errors• Cross hybridization• Errors related to day / reading device / experimentalist• Background differences between slidesUse ratio instead of individual values:Yi= Ri/ GiHandling the Different Errors• Scanning errors• Spotting errors• Cross hybridization• Errors related to day / reading device / experimentalist• Background differences between slidesFor Oligo arrays, use the match / mismatch spotsMatch / Mismatch • Presence and absent calls can be made using the Match / Mismatch information.• However, it has been reported that in some cases the mismatch was higher than the match.Handling the Different Errors• Scanning errors• Spotting errors• Cross hybridization• Errors related to day / reading device / experimentalist• Background differences between slidesNormalization (later)Binding arrays• Instead of printing the genes on the microarray, we can print the intergenic region (an area upstream of the gene).• We tag a protein of interest (a transcription factor) and fuse all proteins to DNA.• Next, we hybridize the extracted portions of DNA onto the array, resulting in areas that are bound by the TF being spotted on the microarray.Genes and Gene ExpressionTechnologyDisplay of Expression InformationYeast cell cycle expression programgenesExperiments (over time)100 20 70 80gene 1Higherexpression compared to baselineLower expression compared to baselinebaseline expressionSpellman et al Mol. Biol. Cell 1998>9 >6 >3 1:1 >3 >6 >9Fold repression Fold inductionGenomic Reprogramming in Response to Oxidant0 10 20 40 60 120minutes One-third of genome expression is transiently reprogrammed6218 genesROS responseVisualization:Relative vs. absolute expression600 Conditions/Mutations6200 GenesSingle-gene MutationsEnvironmentExercising the GenomeUsing annotation databases• Statistical tests to identify the overlap with various functional categoriesGenome wide bindinggenesexperiments (transcription factors)TF1 TF8gene 1Probably bound by this TFno binding by this TFLee et al Science 2002What you should know• The