老化原因(英文版)课件

SAGE TECHNOLOGY AND ITS APPLICATIONSPRESENTED BY Dr.R.A.Siddique&Dr.Anand Kumar Animal Biochemistry Division N.D.R.I.,Karnal(Haryana)India,132001 E-mail:WHAT IS SAGE?nSerial analysis of gene expression(SAGE)is a powerful tool that allows digital analysis of overall gene expression patterns.nProduces a snapshot of the mRNA population in the sample of interest.nSAGE provides quantitative and comprehensive expression profiling in a given cell population.SAGE invented at Johns Hopkins University in USA(Oncology Center)by Dr.Victor Velculescu in 1995.An overview of a cells complete gene activity.Addresses specific issues such as determination of normal gene structure and identification of abnormal genome changes.Enables precise annotation of existing genes and discovery of new genes.NEED FOR SAGE.nGene expression refers to the study of how specific genes are transcribed at a given point in time in a given cell.nExamining which transcripts are present in a cell.nSAGE enables large scale studies of DNA expression;these can be used to create expression profiles.nAllows rapid,detailed analysis of thousands of transcripts in a cell.nBy comparing different types of cells,generate profiles that will help to understand healthy cells and what goes wrong during diseases.THREE PRINCIPLES UNDERLIE THE SAGE METHODOLOGY:A short sequence tag(10-14bp)contains sufficient information to uniquely identify a transcript provided that the tag is obtained from a unique position within each transcript Sequence tags can be linked together to from long serial molecules that can be cloned and sequencedQuantitation of the number of times a particular tag is observed provides the expression level of the corresponding transcript.PRE REQUISITES:Extensive sequencing techniques Deep bioinformatic knowledge Powerful computer software(assemble and analyze results from SAGE experiments)Limited use of this sensitive technique in academic research laboratories STEPS IN BRIEF.1.Isolate the mRNA of an input sample(e.g.a tumour).2.Extract a small chunk of sequence from a defined position of each mRNA molecule.3.Link these small pieces of sequence together to form a long chain(or concatamer).4.Clone these chains into a vector which can be taken up by bacteria.5.Sequence these chains using modern high-throughput DNA sequencers.6.Process this data with a computer to count the small sequence tags.SAGE FLOWCHARTSAGE TECHNIQUE(in detail)Trap RNAs with beads Messenger RNAs end with a long string of As(adenine)Adenine forms very strong chemical bonds with another nucleotide,thymine(T)Molecule that consists of 20 or so Ts acts like a chemical bait to capture RNAs Researchers coat microscopic,magnetic beads with chemical baits i.e.TTTTT tails hanging out When the contents of cells are washed past the beads,the RNA molecules will be trapped A magnet is used to withdraw the bead and the RNAs out of the soup cDNA SYNTHESISDouble stranded cDNA is synthesized from the extractedmRNA by means of biotinylated oligo(dT)primer.cDNA synthesized is immobilised to streptavidin beads.ENZYMATIC CLEAVAGE OF cDNA.nThe cDNA molecule is cleaved with a restriction enzyme.nType II restriction enzyme used.nAlso known as Anchoring enzyme.E.g.NlaIII.n Any 4 base recognising enzyme used.nAverage length of cDNA 256bp with sticky ends created.The biotinylated 3 cDNA are affinity purified using strepatavidin coated magnetic beads.LIGATION OF LINKERS TO BOUND cDNAnThese captured cDNAs are divided into two halves,then ligated to linkers A and B,respectively at their ends.nLinkers also known as docking modules.nThey are oligonucleotide duplexes.nLinkers contain:NlaIII 4-nucleotide cohesive overhangType IIS recognition sequence PCR primer sequence(primer A or B).Type IIS restriction enzyme tagging enzyme.Linker/docking module:PRIMER TE AE TAGCLEAVAGE WITH TAGGING ENZYMEnTagging enzyme,usually BmsFI cleave DNA 14-15 nucleotides,releasing the linker adapted SAGE tag from each cDNA.nRepair of ends to make blunt ended tags using DNA polymerase(Klenow)and dNTPs.FORMATION OF DITAGSnWhat is left is a collection of short tags taken from each molecule.Two groups of cDNAs are ligated to each other,to create a“ditag”with linkers on either end.nLigation using T4 DNA ligase.PCR AMPLIFICATION OF DITAGSnThe linker-ditag-linker constructs are amplified by PCR using primers specific to the linkers.ISOLATION OF DITAGSThe cDNA is again digested by the AE.Breaking the linker off right where it was added in the beginning.This leaves a“sticky”end with the sequence GTAC(or CATG on the other strand)at each end of the ditag.CONCATAMERIZATION OF DITAGSTags are combined into much longer molecules,called concatemers.Between each ditag is the AE site,allowing the scientist and the computer to recognize where one ends and the next begins.CLONING CONCATAMERS AND SEQUENCINGLots of copies are required-So the concatemers are put into bacteria,which act like living copy machines to create millions of copies from the original These copies are then sequenced,using machines that can read the nucleotides in DNA.The result is a long list of nucleotides that has to be analyzed by computer Analysis will do several things:count the tags,determine which ones come from the same RNA molecule,and figure out which ones come from known,well-studied genes and which ones are newQuantitation of gene expression And data presentationHow does SAGE work?1.Isolate mRNA.2.(b)Synthesize ds cDNA.2.(a)Add biotin-labeled dT primer:4.(a)Divide into two pools and add linker sequences:4.(b)Ligate.3.(c)Discard loose fragments.3.(a)Bind to streptavidin-coated beads.3.(b)Cleave with“anchoring enzyme”.5.Cleave with“tagging enzyme”.6.Combine pools and ligate.7.Amplify ditags,then cleave with anchoring enzyme.8.Ligate ditags.9.Sequence and record the tags and frequencies.Vast amounts of data is produced,which must be sifted and ordered for useful information to become apparent.Sage reference databases:nSAGE mapnSAGE Geniehttp:/www.ncbi.nlm.nih.gov/cgapWhat does the data look like?FROM TAGS TO GENESnCollect sequence records from GenBank nAssign sequence orientation(by finding poly-A tail or poly-A signal or from annotations)nExtract 10-bases-adjacent to 3-most CATGnAssign UniGene identifier to each sequence with a SAGE tagnRecord(for each tag-gene pair)n#sequences with this tagn#sequences in gene cluster with this tagMaps available at http:/www.ncbi.nlm.nih.gov/SAGEDIFFERENTIAL GENE EXPRESSION BY SAGEnIdentification of differentially expressed genes in samples from different physiological or pathological conditions.nApplication of many statistical methodsPoisson approximationBayesian methodChi square test.nSAGE software searches GenBank for matches to each tagnThis allows assignment to 3 categories of tags:nmRNAs derived from known genes nanonymous mRNAs,also known as expressed sequence tags(ESTs)nmRNAs derived from currently unidentified genes SAGE VS MICROARRAYnSAGE An open system which detects both known and unknown transcripts and genes.COMPARISONSAGEnDetects 3 region of transcript.Restriction site is determining factor.nCollects sequence information and copy no.nSequencing error and quantitation bias.MICROARRAYnTargets various regions of the transcript.Base composition for specificity of hybridization.nFluorescent signals and signal intensity.nLabeling bias and noise signals.ContdFeaturesSAGEMicroarrayDetects unknown transcriptsYesNoQuantificationAbsolute measureRelative measureSensitivityHighModerateSpecificityModerateHighReproducibilityGood for higher abundance transcriptsGood for data from intra-platform comparisonDirect cost5-10X higher than arrays.5-10 X lower than SAGERECENT SAGE APPLICATIONSAnalysis of yeast transcriptomeGene Expression Profiles in Normal and Cancer Cell Insights into p53-mediated apoptosis Identification and classification of p53-regulated genes Analysis of human transcriptomes Serial microanalysis of renal transcriptomes Genes Expressed in Human Tumor Endothelium Analysis of colorectal metastases(PRL-3)Characterization of gene expression in colorectal adenomas and cancer Using the transcriptome to analyze the genome(Long SAGE)LIMITATIONS Does not measure the actual expression level of a gene.Average size of a tag produced during SAGE analysis is ten bases and this makes it difficult to assign a tag to a specific transcript with accuracy Two different genes could have the same tag and the same gene that is alternatively spliced could have different tags at the 3 ends Assigning each tag to an mRNA transcript could be made even more difficult and ambiguous if sequencing errors are also introduced in the process Quantitation bias:Contamination of of large quantities of linker-dimer molecules.low efficiency in blunt end ligation.Amplification bias.Depending upon anchoring enzyme and tagging enzyme used,some fraction of mRNA species would be lost.Advances over SAGEGeneration of longer 3 cDNA from SAGE tags for gene identification(GLGI)Long SAGE Cap Analysis of Gene Expression(CAGE)Gene Identification Signature(GIS)SuperSAGE Digital karyotyping Paired-end ditagLong SAGEnIncreased specificity of SAGE tags for transcript identification and SAGE tag mapping.nCollects tags of 21bpnDifferent TypeII restriction enzyme-MmelnAdapts SAGE principle to genomic DNA.nAllows localisation of TIS and PAS.CAGE (Capped Analysis of Gene Expression)nAims to identify TIS and promoters.nCollects 21 bp from 5 ends of cap purified cDNA.nUsed in mouse and human transcriptome studies.n The method essentially uses full-length cDNAs,to the 5 ends of which linkers are attached.nThis is followed by the cleavage of the first 20 base pairs by class II restriction enzymes,PCR,concatamerization,and cloning of the CAGE tagsAAAAAAAAAABiotinBiotin+MmelxBiotin+Xma JIBiotinBiotinMmel-PCRBiotinUni-PCRXmaJI tag1 tag2 XmaJI ConcatenationCloning SequencingPCR amplificationLigation to second linkerMmeI digestion of dsDNAssDNA capture Second strand synthesisFull strand DNA synthesisssDNA releaseReverse transcriptionMicro SAGEnRequires 500-5000 fold less starting input RNA.nSimplifies by the incorporation of a one tube procedure for all steps.nCharacterization of expression profiles in tissue biopsies,tumor metastases or in cases where tissue is scarce.nGeneration of region-specific expression profiles of complex heterogeneous tissues.nLimited number of additional PCR cycles are performed to generate sufficient ditag.nAn expression profile can be obtained from as little as 1-5 ng of mRNA.nComparison between the twoSAGEMicroSAGEAmount of input material2.5-5 ug RNA1-5 ng of mRNACapture of cDNAStreptavidin coated magnetic beadsStreptavidin coated PCR tubeMultiple tube vs.Single tube reactionnSubsequent reactions in multiple tubesnMultiple PCI extraction and ethanol precipitation stepsnSingle tube reactionnEasy change of buffersnNo PCI extraction or ethanol ppt step.nFewer manipulationsPCR25-28 cycles28 cycles followed by re-PCR on excised ditag(8-15)SuperSAGEnIncreases the specificity of SAGE tags and use of tags as microarray probes.nType III RE EcoP15I tag releasingnCollects 26 bp tagsnHas been used in plant SAGE studies.nStudy of gene expression in which sequence information is not available.Flowchart of superSAGEGene Identification Signature(GIS)nIdentifies gene boundaries.nCollects 20bp LongSAGE tags from 3 and 5 end of the transcript.nApplied to human and mouse transcription studies.DIGITAL KARYOTYPINGnAnalyses gene structure.nIdentification amplification and deletion in several cancers.PAIRED END DITAGnIdentifies protein binding sites in genome.nApplied to identify p-53 binding sites in the human genome.1.SAGE:A LOOKING GLASS FOR CANCERnDeciphering pathways involved in tumor genesis and identifying novel diagnostic tools,prognostic markers,and potential therapeutic targets.nSAGE is one of the techniques used in the National Cancer Institutefunded Cancer Genome Anatomy Project(CGAP).nA database with archived SAGE tag counts and on-line query tools was created-the largest source of public SAGE data.nMore than 3 million tags from 88 different libraries have been deposited on the National Center for Biotechnology Education/CGAP SAGEmap web site(http:/www.ncbi.nlm.nih.gov/SAGE/).nSeveral interesting patterns have emerged.ncancerous and normal cells derived from the same tissue type are very similar.ntumors of the same tissue of origin but of different histological type or grade have distinct gene expression patterns ncancer cells usually increase the expression of genes associated with proliferation and survival and decrease the expression of genes involved in differentiation.nSAGE studies have been performed in patients with colon,pancreatic,lung,bladder,ovarian,and breast cancers.n SAGE experiments validated in multiple tumor and normal tissue pairs using a variety of approaches,including Northern blot analysis,real-time PCR,mRNA in situ hybridization,and immunohistochemistry.nIdentification of an ideal tumor marker.E.g.Matrix metalloprotease1 in ovarian cancer is overexpressed.p53-TUMOR SUPRESSOR GENEnp53 is thought to play a role in the regulation of cell cycle checkpoints,apoptosis,genomic stability,and angiogenesis.nSequence-specific transactivation is essential for p53-mediated tumor suppression.nThe analysis of transcriptomes after p53 expression has determined that p53 exerts its diverse cellular functions by influencing the expression of a large group of genes.nIdentification of Previously Unidentified p53-Regulated Genes by SAGE analysis.nVariability exists with regard to the extent,timing,and p53 dependence of the expression of these genes.2.IMMUNOLOGICAL STUDIESnOnly a few SAGE analysis has been applied for the study of immunological phenomena.nSAGE analyses were conducted for human monocytes and their differentiated descendants,macrophages and dendritic cells.nDC cDNA library represented more than 17,000 different genes.Genes differentially expressed were those encoding proteins related to cell motility and structure.nSAGE has been applied to B cell lymphomas to analyze genes involved in BCR mediated apoptosis.-polyamine regulation is involved in apoptosis during B cell clonal deletion.ContdnLongSAGE has been used to identify genes of T cells with SLE that determine commitment to the disease.nFindings indicate that the immatureCD4+T lymphocytes may be responsible for the pathogenesis of SLE.nSAGE has been used to analyze the expression profiles of Th-1 and Th-2 cells,and newly identified numerous genes for which expression is selective in either population.nContributes to understanding of the molecular basis of Th1/Th2 dominated diseases and diagnosis of these diseases.3.YEAST TRANSCRIPTOMEnYeast is widely used to clarify the biochemical physiologic parameters underlying eukaryotic cellular functions.nYeast chosen as a model organism to evaluate the power of SAGE technology.nMost extensive SAGE profile was made for yeast.nAnalysis of yeast transcriptome affords a unique view of the RNA components defining cellular life.4.ANALYSIS OF TISSUE TRANSCRIPTOMESnUsed to analyze the transcriptomes of renal,cervical tissues etc.nEstablishing a baseline of gene expression in normal tissue is key for identifying changes in cancer.nSpecific gene expression profiles were obtained,and known markers(e.g.,uromodulinin the thick ascending limb of Henles loop and aquaporin-2 inthe collecting duct)were found.REFERENCESnMaillard,Jean-Charles,et al.,Efficiency and limits of the Serial Analysis of Gene Expression.,Veterinary Immunol.and Immunopathol.2005.,108:59-69.nMan,M.Z.et al.,POWER-SAGE:comparing statistical tests for SAGE experiments.,Bioinformatics 2000.,16:953-959.nPolyak,K.and Riggins,G.J.,Gene discovery using the serial analysis of gene expression technique:Implications for cancer research.,J.of Clin.Oncol.2001.,19(11):2948-2958.nTuteja and Tuteja.,Serial Analysis of Gene Expression:Applications in Human Studies.,J.of Biomed.And Biotechnol.2004.,2:113-120.nTuteja and Tuteja.,Serial analysis of gene expression:application in cancer research.,Med.Sci.Monit.2004.,10(6):132-140.nVelculescu,V.E.et al.Serial analysis of gene expression.,Science 1995.,270:484-487.nWing,San Ming.,Understanding SAGE data.,Trends in Genetics 2006.,23:1-12.nYamamoto,M.,et al.,Use of serial analysis of gene expression(SAGE)technology.,J.of Immunol.meth.2001.,250:45-66.