HistoryandBackgroundofHuman GenomeProject

HaroldRiethman,Ph.D.
riethman@wistar.org

I.Background&OverviewofTheHuman GenomeProject II.ChromosomeStructureReview

 PhysicalBasisFor GeneticInformation Replication Finite

WatsonandCrick,Nature1953

Human male karyotype

Image from NHGRI Web Site

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

Genomics 1982 1988:

Pulsedfield gel electrophoresis Polymerase Chain Reaction Multipoint Linkage Mapping Global Physical Clone Maps Schwartz and Cantor, 1983 Mullis 1983 Lathrop et al., 1984. Olson et al., 1985 Sulston et al., 1985 DeLisi, DOE, mid80s

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

1. PulsedfieldGel Electrophoresis 2. Maplarge chromosomeregions 3. 2.Largeinsert Cloning

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

Cloneends Clonebased PhysicalMap

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

2nd 5yrplan 19931998

FrancisCollinsandDavidGalas OriginallypublishedinScience 262:4346(1993)

Figure 2 Worldwide human genome sequencing progress is shown (measured as base pairs of finished sequence deposited with GenBank).

F S Collins et al. Science 1998;282:682-689

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

AVisionfortheFutureofGenomicsResearch,Nature, April24, 2003.Collins,Green,Guttmacher &Guyer,NHGRI

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

She et al. Nature 2004

Telomeresarerequiredforchromosomereplicationandstability

Telomerase replicates telomeres

http://www.biologyreference.com/

Subtelomeres
Rapidlyevolving Highlyvariable Duplicatedsequences

Pryde etal,1997. Curr Opin GenetDev

Cellcycle,Meiosis,Mitosis

M=mitosis

CellCycle

S=DNAsynthesis

I=interphase

MitosisandMeiosis

MarstonAL,AmonA.2004.NatRevMolCellBiol.