Analysis of Gene ExpressionB o b G r o s s , B i o 3 9 / 1 3 9Gene ExpressionThe current state of the cell can be characterized by its pattern of gene expressionExpression patterns for characterized genes may help to understand uncharacterized genes with a similar pattern of expressionMultiple genes in the same expression pattern family might be part of the same metabolic pathwaysNew metabolic or regulatory pathways can be discovered by looking for similarities in gene expression patterns Two principle methods used today–SAGE analysis–DNA Microarray analysisB o b G r o s s , B i o 3 9 / 1 3 9Potential ImpactsPreventive medicineDesign drugs that treat disease causes rather than symptomsMore targeted drug treatmentsIdentification of complex genetic diseasesPathogen analysisBetter understanding of development by analysis of gene expression over time and in different tissuesGenetic testing and privacyB o b G r o s s , B i o 3 9 / 1 3 9SAGE AnalysisSAGE = Serial Analysis of Gene ExpressionDesigned to take advantage of high-throughput sequencing technology to obtain a quantifiable profile of gene expressionSAGE measures the level of a tag - a short sequence that represents the mRNA levelA tag is a nucleotide sequence of a defined length, directly downstream of the 3'-most restriction site for a particular restriction enzyme. Based on two principles–a short sequence (9-10) bases contains enough information to uniquely identify a transcript, provided it is isolated from a specific position within the transcript–concatenation of short sequence tags allows efficient analysis of transcripts in a serial manner by sequencing multiple tags within a single cloneVelculescu VE, et al, Science. 1995 Oct 20;270(5235):484-7 Zhang L, et al, Science. 1997 May 23;276(5316):1268-72B o b G r o s s , B i o 3 9 / 1 3 9Making Serial TagsFrom Science 270:484 (1995)biotinylated oligo(dT) primerFokI cuts at GGATG..9..|tags arranged serially can be amplified by PCRNlaIII cuts at CATG|after TE cleavage make blunt ended with Klenow DNA polymeraseB o b G r o s s , B i o 3 9 / 1 3 9How SAGE WorksB o b G r o s s , B i o 3 9 / 1 3 9Some Possible Problems•two or more genes share the same tag•one gene has more than one tag•alternative termination of transcription•polymorphism in a population•sequencing errors can have major effect when dealing with such short sequencesB o b G r o s s , B i o 3 9 / 1 3 9SAGE Tags in Human PancreasFrom Science 270:484 (1995)B o b G r o s s , B i o 3 9 / 1 3 9SAGE Gene to TagB o b G r o s s , B i o 3 9 / 1 3 9SAGE Information EntryB o b G r o s s , B i o 3 9 / 1 3 9ListingSAGE Libraries Containing a TagB o b G r o s s , B i o 3 9 / 1 3 9Selecting Libraries for ComparisonB o b G r o s s , B i o 3 9 / 1 3 9SAGE Cancer:NormalB o b G r o s s , B i o 3 9 / 1 3 9SAGE Library DetailsB o b G r o s s , B i o 3 9 / 1 3 9SAGE Library DetailsB o b G r o s s , B i o 3 9 / 1 3 9SAGE Profile NeighborsB o b G r o s s , B i o 3 9 / 1 3 9DNA Microarray TechnologySimultaneously measure the levels of expression for tens of thousands of genesMutation and polymorphism detectionDiscover new gene relationshipsMassively parallel approach allows for new kinds of questions to be askedB o b G r o s s , B i o 3 9 / 1 3 9How DNA Microarrays WorkcDNAs or artificially synthesized “probes” are spotted in a gridded pattern on a filter or on a glass slideThe slide is hybridized with samples made from an mRNA population - usually by RT-PCRThe sample can be radioactive or fluorescently labeledLevels of hybridization are determined for each spot on the slideCan use different populations of probes mixed together with different colored fluorescent tags - this enables relative levels of expression to be determinedB o b G r o s s , B i o 3 9 / 1 3 9Affymetrix Chips 1slideB o b G r o s s , B i o 3 9 / 1 3 9Affymetrix Chips 2slidecoat slide with photosensitive materialcoatingB o b G r o s s , B i o 3 9 / 1 3 9Affymetrix Chips 3slideExpose to light in pattern for ‘A’ nucleotidesto etch away the coating at those spotscoatingB o b G r o s s , B i o 3 9 / 1 3 9Affymetrix Chips 3slideExpose to light in pattern for ‘A’ nucleotidesto etch away the coating at those spotscoatingB o b G r o s s , B i o 3 9 / 1 3 9Affymetrix Chips 4slideAttach an ‘A’ to each exposed spotcoatingB o b G r o s s , B i o 3 9 / 1 3 9Affymetrix Chips 4slideAttach an ‘A’ to each exposed spotcoatingAA A A AB o b G r o s s , B i o 3 9 / 1 3 9Affymetrix Chips 5slideDissolve away the protective coatingAA A A AB o b G r o s s , B i o 3 9 / 1 3 9coatingAffymetrix Chips 6slideApply another protective photosensitive coatingAA A A AB o b G r o s s , B i o 3 9 / 1 3 9Affymetrix Chips 7slideExpose to light in pattern for ‘C’ nucleotidesto etch away the coating at those spotsAA A A AcoatingB o b G r o s s , B i o 3 9 / 1 3 9Affymetrix Chips 7slideExpose to light in pattern for ‘C’ nucleotidesto etch away the coating at those spotsAA A A AcoatingB o b G r o s s , B i o 3 9 / 1 3 9Affymetrix Chips 8slideAdd ‘C’ to all exposed spotsAA A A AcoatingB o b G r o s s , B i o 3 9 / 1 3 9Affymetrix Chips 8slideAdd ‘C’ to all exposed spotsAA A A AcoatingC C C C C C CCB o b G r o s s , B i o 3 9 / 1 3 9Affymetrix Chips 9slideDissolve protective coatingcoatingAA A AC C C C C C CCAB o b G r o s s , B i o 3 9 / 1 3 9Affymetrix Chips 10slideDissolve protective coatingAA A AC C C C C C CCAB o b G r o s s , B i o 3 9 / 1 3 9Affymetrix Chips 11slideApply new protective coatingAA A AC C C C C C CCAB o b G r o s s , B i o 3 9 / 1 3 9coatingAffymetrix Chips 12slideApply new protective coatingAA A AC C C C C C CCAB o b G r o s s , B i o 3 9 / 1 3 9coatingAffymetrix Chips 13slideExpose to light in pattern for ‘G’ nucleotidesto …