DNAsequencing:Importance

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

Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res 2001

PyrosequencingSolidPhase

Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res 2001

Pyrogram

Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res 2001

454LifeSciencesSequencer

454LifeSciencesSequencer
Advantages
Fast,accurate
Greatforsmall,simplegenomes,
Disadvantages
Readsonly~100200bp
Crappyforlargecomplexgenomes(likeours)
Homopolymerstretches(8+)aredifficulttoread

AlternativestoDyeTerminatorSequencing
Others:
Microarraysequencingakasequencingby
hybridization

AlternativestoDyeTerminatorSequencing
Others:
Nanoporesequencing