DNA sequencing

DNA sequencing Maxam and Gilbert, Sanger, Automated, Nextgen sequencing

1. DNA Sequencing
Dr.Manikandan Kathirvel M.Sc., Ph.D., (NET)
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
(Reaccredited with "A" Grade by NAAC)
Affiliated to BengaluruNorthUniversity,
K. Narayanapura, Kothanur(PO)
Bengaluru 560077

2. DNASequencing:
DNAsequencingreferstomethodsfordeterminingtheorderofthenucleotidesbasesadenine,guanine,cytosineand
thymineinamoleculeofDNA.
ThefirstDNAsequencewasobtainedbyacademicresearchers,usinglaboratoriesmethodsbasedon2-dimensional
chromatographyintheearly1970s.
Bythedevelopmentofdyebasedsequencingmethodwithautomatedanalysis,DNAsequencinghasbecomeeasierand
faster.

3. MethodsofDNAsequencing:
Twomainmethodsarewidelyknowntobeusedtosequence
DNA:
1.TheChemicalMethod(alsocalledtheMaxam–Gilbert
methodafteritsinventors).Byusingthismethod,theyhad
sequenced24nucleotidesonly.However,theirmethod
waspublishedaftertwoyearsofSanger’smethod.
2.TheChainTerminationMethod(alsoknownasthe
Sangerdideoxymethodafteritsinventor).
Maxam–Gilberttechniquedependsontherelativechemical
liabilityofdifferentnucleotidebonds,whereastheSanger
methodinterruptselongationofDNAsequencesby
incorporatingdideoxynucleotidesintothesequences.
Thechainterminationmethodisthemethodmoreusually
usedbecauseofitsspeedandsimplicity.
Sequencing of an Oligonucleotide by Maxam-Gilbert method

4. 1. Chemical Cleavage Method (Maxam–Gilbert Method):
MaxamGilbertsequencingisamethodofDNAsequencingdevelopedby
AllanMaxamandWalterGilbertin1976–1977.Thismethodisbasedon
nucleobase-specificpartialchemicalmodificationofDNAandsubsequent
cleavageatspecificbasesoftheDNAbackboneatsitesadjacenttothe
modifiednucleotides.
Themethodrequiresradioactivelabellingatoneendandpurificationofthe
DNAfragmenttobesequenced.
Chemicaltreatmentgeneratesbreaksatsmallproportionsofoneortwo
ofthefournucleotidebasedineachoffourreactions(G,A+G,C,T+
C).
Thusaseriesoflabelledfragmentsisgenerated,fromtheradiolabelledend
tothefirst‘cut’siteineachmolecule.
Thefragmentsinthefourreactionsarearrangedsidebysideingel
electrophoresisforsizeseparation.
Tovisualizethefragments,thegelisexposedtoX-rayfilmfor
autoradiography,yieldingaseriesofdarkbandseachcorrespondingtoa
radiolabelledDNAfragment,fromwhichthesequencemaybeinferred.
Features:
Base-specific cleavage of DNA by certain chemicals
Four different chemicals, one for each base
A set of DNA fragments of different sizes
DNA fragments contain up to 500 nucleotides
Hydrazine:T+C
HydrazineNaCl:C
Dimethylsulfate:A+G
Piperidine:G
Sequencing of an Oligonucleotide by Maxam-Gilbert method

5. Procedure:
1.DNAextractionistheveryfirststep.Afterthat,theDNAisdenaturedusing
theheatdenaturationmethodandsingle-strandedDNAisgenerated.
2.Thephosphate(5’P)endoftheDNAisremovedandlabelledbythe
radiolabeledP
32
.Theenzymenamedphosphataseremovesthephosphate
fromtheDNAandsimultaneously,thekinaseaddsthe
32
Ptothe5’endofit.
3.4differentchemicalsareusedtocleaveDNAatfourdifferentpositions;
hydrazineandhydrazineNaClareselectivelyattackpyrimidinenucleotides
whiledimethylsulfateandpiperidineattackpurinenucleotides.
Hydrazine:T+C
HydrazineNaCl:C
Dimethylsulfate:A+G
Piperidine:G
4.Anequalvolumeof4differentssDNAsamplesistakeninto4differenttubes
eachcontaining4differentchemicals.Thesamplesareincubatedfor
sometimesandelectrophoresedinpolyacrylamidegelelectrophoresis.The
resultsofthechemicalscleavageoffourdifferenttubesareshowninthe
figurebelow.
5.AutoradiographyisusedtovisualizetheseparationofDNAfragments.Due
totheradiolabelled
32
PendoftheDNA,theDNAbandsvisualizedthrough
autoradiography.
Sequencing of an Oligonucleotide by Maxam-Gilbert method

6. Procedure:
1.DNAextractionistheveryfirststep.Afterthat,theDNAisdenaturedusing
theheatdenaturationmethodandsingle-strandedDNAisgenerated.
2.Thephosphate(5’P)endoftheDNAisremovedandlabelledbythe
radiolabeledP
32
.Theenzymenamedphosphataseremovesthephosphate
fromtheDNAandsimultaneously,thekinaseaddsthe
32
Ptothe5’endofit.
3.4differentchemicalsareusedtocleaveDNAatfourdifferentpositions;
hydrazineandhydrazineNaClareselectivelyattackpyrimidinenucleotides
whiledimethylsulfateandpiperidineattackpurinenucleotides.
Hydrazine:C+T
HydrazineNaCl:C
Dimethylsulfate:A+G
Piperidine:G
4.Anequalvolumeof4differentssDNAsamplesistakeninto4differenttubes
eachcontaining4differentchemicals.Thesamplesareincubatedfor
sometimesandelectrophoresedinpolyacrylamidegelelectrophoresis.The
resultsofthechemicalscleavageoffourdifferenttubesareshowninthe
figurebelow.
5.AutoradiographyisusedtovisualizetheseparationofDNAfragments.Due
totheradiolabelled
32
PendoftheDNA,theDNAbandsvisualizedthrough
autoradiography.
Sequencing of an Oligonucleotide by Maxam-Gilbert method

7. Advantages:
PurifiedDNAcanbereaddirectly
HomopolymericDNArunsaresequencedasefficientlyas
heterogeneousDNAsequences
CanbeusedtoanalyzeDNAproteininteractions(i.e.
footprinting)
Canbeusedtoanalyzenucleicacidstructureandepigenetic
modificationstoDNA
Disadvantages:
Itrequiresextensiveuseofhazardouschemicals.
Ithasarelativelycomplexsetup/technicalcomplexity.
Itisdifficultto“scaleup”andcannotbeusedtoanalyze
morethan500basepairs.
Thereadlengthdecreasesfromincompletecleavage
reactions.

8. 2. Sanger Sequencing-DideoxyChain terminator
method
Sangersequencing,alsoknownasthe“chain
terminationmethod”,isamethodfordeterminingthe
nucleotidesequenceofDNA.
ThemethodwasdevelopedbytwotimeNobel
LaureateFrederickSangerandhiscolleaguesin
1977,hencethenametheSangerSequence.
Themethodisalsoknownasthefirst-generation
DNAsequencingmethod.
Sanger’s method of gene sequencing is also known as
dideoxychain termination method.

9. Principle:
ThekeyprincipleoftheSangermethodwastheuseof
dideoxynucleotidetriphosphates(ddNTPs)asDNAchain
terminators.
ADNAprimerisattachedbyhybridizationtothetemplate
strandanddeoxynucleosidestriphosphates(dNTPPs)are
sequentiallyaddedtotheprimerstrandbyDNApolymerase.
TheM13primerisdesignedalongwiththeknown
sequencesat3’endofthetemplatestrand.
Thereactionmixturealsocontainsdideoxynucleoside
triphosphate(ddNTPs)alongwithusualdNTPs.
Ifduringreplication,ddNTPsisincorporatedinsteadof
usualdNTPsinthegrowingDNAstrandthenthe
replicationstopsatthatnucleotide.
The ddNTPsare analogue of dNTPs
ddNTPslackshydroxylgroup(-OH)atc3ofribosesugar,soitcannot
makephosphodiesterbondwithnestnucleotide,thusterminatesthe
nucleotidechain
RespectiveddNTPsofdNTPsterminateschainattheirrespectivesite.
ForexampleddATPterminatesatAsite.SimilarlyddCTP,ddGTPand
ddTTPterminatesatC,GandTsiterespectively.

10. Sanger Sequencing Steps
TherearethreemainstepstoSangersequencing.
1.Templatepreparation:DNASequenceforChain
TerminationPCR
2.Generationofnestedsetoflabelledfragments
3.SizeSeparationbyGelElectrophoresisandgelreading
4.GelAnalysis&DeterminationofDNASequence

11. 1.Templatepreparation:DNASequenceforChain
TerminationPCR
The DNA sequence of interest is used as a template for a special
type ofPCRcalled chain-termination PCR.
Steps:
1.Copies of template strand to be sequenced must be
prepared with short known sequences at 3’ end of the
template strand.
2.A DNA primer (M13 SEQUENCING PRIMER) is essential to
initiate replication of template, so primer preparation of
known sequences at 3’end is always required.

12. 2.Generationofnestedsetoflabelledfragments:
Steps:
Copiesofeachtemplateisdividedintofourbatchesand
eachbatchisusedfordifferentreplicationreaction.
Copiesofstandardprimer,normaldNTPsandDNA
polymeraseIareusedinallfourbatches.
TosynthesizefragmentsthatterminatesatA,
1.ddATP(canberadiolabelled)isaddedtothereaction
mixturetothebatchIalongwithdATP,dTTP,dCTPand
dGTP,
2.standardprimerand
3.DNApolymeraseI.
Similarly,togenerate,allfragmentsthatterminatesat
C,GandT,
therespectiveddNTPsi.e.ddCTP,ddGTPandddTTPareadded
respectivelytodifferentreactionmixtureondifferentbatch
alongwithusualdNTPs.

13. 3.SizeSeparationbyGelElectrophoresisandgelreading
Steps:
Thereactionmixturefromfourbatchesareloadedinto
fourdifferentwellonpolyacrylamidegeland
electrophoresed.
Theautoradiogramofthegelisreadtodeterminethe
orderofbasesofcomplementarystrandtothatof
templatestrand.
Thebandofshortestfragmentsisatthebottomof
autoradiogramsothatthesequencesof
complementarystrandarereadfrombottomtotop.
4.GelAnalysis&DeterminationofDNASequence
Thelaststepsimplyinvolvesreadingthegeltodeterminethe
sequenceoftheinputDNA.
InmanualSangersequencing,theuserreadsallfourlanesof
thegelatonce,movingbottomtotop,usingthelaneto
determinetheidentityoftheterminalddNTPforeachband.For
example,ifthebottombandisfoundinthecolumncorresponding
toddGTP,thenthesmallestPCRfragmentterminateswith
ddGTP,andthefirstnucleotidefromthe5’endoftheoriginal
sequencehasaguanine(G)base.

14. SignificanceofDNASequencing:
InformationobtainedbyDNAsequencingmakesitpossibletounderstandor
alterthefunctionofgenes.
DNAsequenceanalysisdemonstratesregulatoryregionsthatcontrolgene
expressionandgenetic“hotspots”particularlysusceptibletomutation.
ComparisonofDNAsequencesshowsevolutionaryrelationshipsthatprovide
aframeworkfordefiniteclassificationofmicroorganismsincludingviruses.
ComparisonofDNAsequencesfacilitatesidentificationofconservedregions,
whichareusefulfordevelopmentofspecifichybridizationprobestodetect
microorganismsincludingvirusesinclinicalsamples.
DNAsequencinghasbecomesufficientlyfastandinexpensivetoallow
laboratorydeterminationofmicrobialsequencesforidentificationofmicrobes.
Sequencingofthe16Sribosomalsubunitcanbeusedtoidentifyspecific
bacteria.Sequencingofvirusescanbeusedtoidentifythevirusand
distinguishdifferentstrains.
DNAsequencingshowsgenestructurethathelpsresearchworkerstofindout
thestructureofgeneproducts.

15. AutomatedDNAsequencing:
ThemanualSangermethodwastedious.However,recent
advancementintothesequencingmakesiteasyandrapidtouse.
Thesemi-automatedSangersequencingmethodisbasedonthe
principleofSanger’smethodwithsomeminorvariations.
Insteadofthe4differentreactions,theautomatedDNA
sequencingcarriedoutinthesingletubeandtheDNArunsina
singlelane.
Here,inthesemi-automatedDNAsequencing,thefluorescent-
labeledsetofprimersareused,insteadofddNTPs.Thusfour
differentprimersgivefourdifferentpeaks.
ThePAGEmethodisn’tcapableofseparatingallthefragments
inasinglereaction.Therefore,alternatively,thecapillarygel
electrophoresismethodispracticed.Thismethodseparateseach
andeverysinglefragmentprecisely.
ThecapillaryelectrophoresisusedtoseparateDNAmolecules
onthebasisofthesize,itispowerfulenoughtoseparatesingle
basepairfragment.Thechromatogramgeneratedthroughthe
C.Esenttheoutputasafluorescentpeak.
Theadvancedsemi-automatedSangersequencing
methodismoreaccurate,reliableandfasterthan
thetraditionalmethod.

16. Three Basic Steps of Automated Sanger Sequencing
ThereadcapacityoftheSangersequencingishigheras
comparedwiththechemicaldegradationmethod.Itcan
sequence700to800bpsequenceinasinglerun,therefore,
itismoresuitableforsequencingbacterialorother
prokaryoticgenomes.
Itismoreadvancedandautomated.Eventheerrorrateis
verylowascomparedwiththeconventionalchain
terminationmethod.Still,itistime-consumingandahigh-
costmethod.

17. AutomatedDNAsequencing

18. NextGenerationSequencing(NGS)
ImportantNextGenerationSequencingTechniques
Thenext-generationsequencingplatformisdifferentfromtheSangertechniqueorchain
terminationmethodofDNAsequencing.Broadly,itamplifiesmillionsofcopiesofa
particularfragmentinamassivelyparallelfashionandthe“reads”areanalyzedbythe
computationalprogram.
Pyrosequencing
Illumina(Solexa)sequencing
Lynxtherapeutics’massivelyparallelsignaturesequencing(MPSS)
Polonysequencing
SOLiDsequencing
DNAnanoballsequencing
Helioscopesinglemoleculesequencing
SinglemoleculeSMRTsequencing
Singlemoleculerealtime(RNAP)sequencing
NextGenerationSequencing(NGS)isapowerfulplatformthathasenabledthesequencingof
thousandstomillionsofDNAmoleculessimultaneously.

19. Thegenerationsofsequencing:
FirstGeneration
MaxamandGilbertDNAsequencingandSangerDNASequencing
SecondGenerationSequencing
Pyrosequencing
SequencingbyReversibleTerminatorChemistry
SequencingbyLigation
ThirdGenerationSequencing
SingleMoleculeFluorescentSequencing
SingleMoleculeRealTimeSequencing
SemiconductorSequencing
NanoporeSequencing
FourthGenerationSequencing
Aimsconductinggenomicanalysisdirectlyinthecell