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TheDNAsequencesmakingupanyorganismcomprise
thebasicblueprintforthatorganism
TheHumanGenomeProject(andothers)
Potentialbenefits
Molecularmedicine
Improveddiagnosisofdisease
Diseasegeneidentificationwillleadtomoreaccuratediagnosis
Earlierdetectionofgeneticpredispositionstodisease
Willbeabletoassessriskforcertaindiseases,e.g.cancer,Type
IIdiabetes,heartdisease
Rationaldrugdesign
Drugsdesignedtotargetspecificgeneproductsthatcause
disease
Genetherapyandcontrolsystemsfordrugs
Replacementofdefectivegenesforcertaindiseases
Pharmacogenomics"customdrugs
Drugtherapybasedononesgenotype
TheHumanGenomeProject(andothers)
Potentialbenefits
Bioarchaeology,anthropology,evolution,and
humanmigration
Studyevolutionthroughgermlinemutationsin
lineages.
Studymigrationofdifferentpopulationgroups
basedonfemalegeneticinheritance.
StudymutationsontheYchromosometotrace
lineageandmigrationofmales.
Comparebreakpointsintheevolutionofmutations
withagesofpopulationsandhistoricalevents.
TheHumanGenomeProject(andothers)
Potentialbenefits
DNAforensics(identification)
IdentifypotentialsuspectswhoseDNAmaymatchevidence
leftatcrimescenes.
Exoneratepersonswronglyaccusedofcrimes.
Identifycrimeandcatastrophevictims.
Establishpaternityandotherfamilyrelationships.
Identifyendangeredandprotectedspeciesasanaidtowildlife
officials(couldbeusedforprosecutingpoachers).
Detectbacteriaandotherorganismsthatmaypolluteair,
water,soil,andfood.
Determinepedigreeforseedorlivestockbreeds.
TheHumanGenomeProject(andothers)
Potentialbenefits
Agriculture,livestockbreeding,and
bioprocessing
Disease,insect,anddroughtresistantcrops.
Healthier,moreproductive,diseaseresistantfarmanimals.
Morenutritiousproduce.
Biopesticides.
Ediblevaccinesincorporatedintofoodproducts
Newenvironmentalcleanupusesforplantsliketobacco.
DNAsequencingmethodologies:
ca.1977
MaxamGilbert
basemodificationby
generalandspecific
chemicals.
depurinationor
depyrimidination.
singlestrandexcision.
notamenableto
automation
Sanger
DNAreplication.
substitutionof
substratewithchain
terminatorchemical.
moreefficient
automation??
MaxamGilbertchemicalmethod
versussynthesisbasedmethods
FredSanger:NobelPrize1980
Insteadoftakingacompletesequenceand
breakingitdown,buildDNAsequencesupand
analyzestepsalongtheway
Theykeytothisprocess:dideoxynucleotides
(ddNTPs)
Whattolabelforvisualization?
Primers?
Disadvantagesofprimerlabels:
fourreactions
tedious
limitedtocertainregions,customoligosor
limitedtoclonedinsertsbehinduniversalpriming
sites.
Advantages:itworks
Solution:
labeledterminatorsddNTPs
DNAAnalysis:DNASequencing
ddNTPsareanalagoustofaultyLEGOs,
NormalLEGOshave
littlepegsthatallow
themtostack
FaultyLEGOslackthe
littlepegsandnothingcan
stackonthemthus,
theyterminatethestack
10_07_1_enzym.dideoxy.jpg
10_07_2_enzym.dideoxy.jpg
Thisisgreatbut
Wouldntitbegreattoruneverythinginonelane?
Savespaceandtime,moreefficient
FluorescentlylabeltheddNTPssothatthey
eachappearadifferentcolor
07_02.jpg
07_02_2.jpg
07_03.jpg
DNAAnalysis:DNASequencing
http://www.dnai.org/b/index.html
virtualautoradrealtimeDNAsequenceoutputfromABI377
1. Tracefiles(dyesignals)areanalyzedand
basescalledtocreatechromatograms.
2. Chromatogramsfromoppositestrandsare
reconciledwithsoftwaretocreatedouble
strandedsequencedata.
AlternativestoDyeTerminatorSequencing
454Sequencingisamassivelyparallelsequencingbysynthesis(SBS)system
capableofsequencingroughly20megabases(20,000,000bp)ofrawDNAsequenceper4.5
hourrun
Comparetobestdyeterminatorsequencingrigtoday:ABI3730xl
(192capillariesx~1000bp)in5hrs(22.5hrruns)=196,000bp
454sequencingreliesonfixingnebulizedandadapterligatedDNAfragmentstosmallDNA
capturebeadsinawaterinoilemulsion.
DNAisfixedtothesebeadsisthenamplifiedbyPCR.
EachDNAboundbeadisplacedintoa~44mwellonaPicoTiterPlate,afiberopticchip.A
mixofenzymessuchaspolymerase,ATPsulfurylase,andluciferasearealsopackedintothe
well.
Thefournucleotides(TAGC)arewashedinseriesoverthePicoTiterPlate.
Ifanucleotidecomplementarytothetemplatestrandflowsintoawell,thepolymerase
extendstheexistingDNAstrandbyaddingnucleotide(s).
Additioninareactionthatgeneratesalightsignalthatisrecordedbyacamerainthe
instrument.
Pyrosequencing
PyrosequencingSolidPhase
Pyrogram
454LifeSciencesSequencer
454LifeSciencesSequencer
Advantages
Fast,accurate
Greatforsmall,simplegenomes,
Disadvantages
Readsonly~100200bp
Crappyforlargecomplexgenomes(likeours)
Homopolymerstretches(8+)aredifficulttoread
AlternativestoDyeTerminatorSequencing
Others:
Microarraysequencingakasequencingby
hybridization
AlternativestoDyeTerminatorSequencing
Others:
Nanoporesequencing