Académique Documents
Professionnel Documents
Culture Documents
HaroldRiethman,Ph.D.
riethman@wistar.org
WatsonandCrick,Nature1953
GenomicsPre1982:
MendelsLawsofInheritance GeneticLinkageMaps PhysicalMaps(Cytogenetic) StructureofDNA MolecularCloning DNASequencing Mendel,1866 Sturtevant,1913 Painter,1933 Watson&Crick,1953 CohenandBoyer,1972 Sanger,Maxam,and Gilbert,Midseventies Botsteinetal.,1980
HumanRFLPMapping
First proposals to sequence human genome Yeast Artificial Chromosome (YAC) Automated DNA Sequencers Mapping and Sequencing the Human Genome
Burke et al., 1987 Hood, midlate 80s National Research Council, 1988
FromRiethmanetal.,GenomeAnalysisVol 1,1997
PolymeraseChainReaction
InvitrosynthesisofDNA Markerstorageanddistribution bycomputer Sensitivescreeningand detection
FromFanningandGibbs,GenomeAnalysisVol 1,1997
MappingandSequencingtheHumanGenome NationalResearchCouncil,1988
Aspecialeffortshouldbeorganizedandfundedtomap,sequence,andincreaseunderstandingofthe HumanGenome. Technologydevelopmentessentialforeveryphaseoftheproject. Earlygoalsshouldbeahighresolutiongeneticmap,acollectionoforderedclones,andaseriesof complementaryphysicalmapsofincreasingresolution.Thenucleotidesequence,theultimategoal,will requiremajoradvancesinDNAhandlingandsequencingtechnologies. Acomparativegeneticapproachisessentialforinterpretingtheinformationinthehumangenome. Competing,peerreviewedprogramsemphasizingtechnologydevelopment. Componentsubprojectsshouldhavethepotentialtoimproveby5to10foldincrementsthescaleor efficiencyofmapping,sequencing,analyzing,orinterpretingtheinformationinthehumangenome. Establishcentralizedfacilitiesforstoringanddistributingclones,andadatacenterforthecomputer basedcollectionanddistributionoflargeamountsofDNAsequenceinformation.
Maps
Landmarks DNA probes STS Fragment Ends Restriction Enzyme Meiosis Radiation Clone ends
UniversalLandmark
SequenceTaggedSite(STS)1989
ReplacesclonedDNAprobemappinglandmarkswithPCRassays. EachSTSisuniquelydescribedbyapairofoligonucleotides,aproductsize,and PCRreactionconditions.Canbestoredanddistributedelectronically. Enablesmergingofmappingdataobtainedfrommanylabsusingmanydifferentmethods intoasingleconsensusmapoflandmarksalongachromosome. Eliminatestheneedforhugecollectionsofclonedprobesegmentsuponwhich priormapsdepended.
MeioticBreaks GeneticLinkageMaps
RadiationinducedBreaks RadiationHybrid(RH)Maps
Thefirst5yearplan(19911995)
GeneticMap: fullyconnected,2to5centimorgan resolution,eachmarkeridentifiedbyanSTS PhysicalMap: 100kbresolutionSTSmap 2Mbcontigs formostofgenome Sequencing: Improve/developtechnologytoreducecostto0.50/base Generatetotalof10Mbofhumansequenceinlargestretchesduringtechnologydevelopment ModelOrganisms: Geneticmapofmouse Generatetotalof20Mbofsequencefrommodelorganisms,focusoncontiguousstretchesof1Mb,during technologydevelopment Informatics: Developsoftwareanddatabasedesignstosupportlargescalemapping&sequencing Createdatabasetoolsthatprovideeasyaccessandpermitcomparisonsofuptodatedatasets Developalgorithmsandanalyticaltoolstointerpretinformation ELSI Ethical,Legal,andSocialImplications Training TechnologyDevelopment Supportinnovative&highriskprojects TechnologyTransfer toindustryandthemedicalcommunity
HGP
Figure 2 Worldwide human genome sequencing progress is shown (measured as base pairs of finished sequence deposited with GenBank).
Published by AAAS
HGP3rd 5yrplan19982003
CapillarySequencers,Automation,19971998
Green,1997
WeberandMeyers,1997
InternationalHumanGenomeSequencingConsortium DraftHumanGenomeSequence:NatureFeb.15,2001
CeleraGenomicsDraftHumanGenomeSequence ScienceFeb.16,2001
InternationalHumanGenomeSequencingConsortium FinishedHumanGenomeSequence:Oct.21,2004
GenomicstoBiology
Comprehensivelyidentifythestructuralandfunctionalcomponents encodedinthehumangenome.Encode. Elucidatetheorganizationofgeneticnetworksandproteinpathwaysand establishhowtheycontributetocellularandorganismalphenotypes. Developadetailedunderstandingoftheheritablevariationinthehuman genome.HapMap Understandevolutionaryvariationacrossspeciesandthemechanisms underlyingit. Developpolicyoptionsthatfacilitatethewidespreaduseofgenome informationinbothresearchandclinicalsettings
Technologydevelopment
DNAsequencing Geneticvariation Thegenome'partslist Proteomics Pathwaysandnetworks Geneticcontributionstohealth,diseaseanddrugresponse Molecularprobesforexploringbasicbiologyanddisease Computationalresourcesforstorage,analysis,andintegrationof hugedatasets
QuantumLeaps
Theabilitytodetermineagenotypeatverylowcost,allowinganassociation studyinwhich2,000individualscouldbescreenedwithabout400,000genetic markersfor$10,000orless. TheabilitytosequenceDNAatcoststhatarelowerbyfourtofiveordersof magnitudethanthecurrentcost,allowingahumangenometobesequenced for$1,000orless. TheabilitytosynthesizelongDNAmoleculesathighaccuracyfor$0.01per base,allowingthesynthesisofgenesizedpiecesofDNAofanysequencefor between$10and$10,000. TheabilitytodeterminethemethylationstatusofalltheDNAinasinglecell. Theabilitytomonitorthestateofallproteinsinasinglecellinasingle experiment.
ChromosomeStructure
Humanmalekaryotype
ImagefromNHGRIWebSite
Gene
GeneticConcept Functionalpieceofachromosome PhysicalConcept Stringofnucleotidesthatencodea discretefunction Proteinencodinggene NoncodingRNAs
Heterochromatin
Highly condensed chromosome regions Large heterochromatic tracts are usually repetitive sequence Can be very difficult to clone and assemble sequences Often gene-poor, low transcription levels Enriched near centromeres and telomeres
Heterochromatin
Heterochromatic Centromere Satellites
Euchromatin
Less condensed chromosome regions Very wide range of DNA sequence types and organization Contains most genes, but very unevenly distributed Mostly accessible to cloning and assembly
Euchromatic Region
FromUCSCWebBrowser
SegmentalDuplicatons
LargesegmentsofrecentlyduplicatedDNA >1kb,>90% Highlyenrichedinpericentromeresand subtelomeres Associatedwithlargepolymorphismsand diseaseassociateddeletions
Hillieretal. Nature2003
ChromosomeFunction
Replication
DNAReplication
CentromeresandTelomeres
Centromeresensurepropersegregationofchromosomes
SequenceOrganizationofHumanCentromereandPericentromere
Telomeresarerequiredforchromosomereplicationandstability
http://www.biologyreference.com/
Subtelomeres
Rapidlyevolving Highlyvariable Duplicatedsequences
Cellcycle,Meiosis,Mitosis
M=mitosis
CellCycle
S=DNAsynthesis
I=interphase
MitosisandMeiosis
MarstonAL,AmonA.2004.NatRevMolCellBiol.