lezione 7 dna seqeuncing - moodle2.units.it€¦ · 1. maxam e gilbert:firstmethod 2. sanger...
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DNASEQUENCING
Techniques:
1.Maxam eGilbert:first method
2.Sanger Sequencing:basis forall seqeuncing tecniques
3.MassiveParallel Seqeuncing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA.
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QuicklyreducedCost
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1.Denatureadouble-strandedDNAtosingle-stranded byincreasingtemperature.2.Radioactively label one 5'endoftheDNAfragment tobesequenced byakinasereaction using gamma-32P-ATP.3.CleaveDNAstrand at specific positionsusing chemical reactions.- Reactino 1:Guanines (andtosomeextent theadenines)aremethylated bydimethyl sulfate- Reaction 2:Purines (A+G)aredepurinated using formicacid,- Reaction 3:Pyrimidines (C+T)arehydrolysed usinghydrazine.- Reaction 4:Hydrazine+salt (sodiumchloride)inhibits the
reaction ofthyminefortheC-only reaction.
- àNOTE:concentration ofchemicals is chosen toonly cause1,odification inamolecule ofinterest
- à ThemodifiedDNAsmay thenbecleavedbyhotpiperidine
- Now infour reaction tubes,we will haveseveral differently sizedDNAstrands that carry 32Pat 5’end
- Fragments areelectrophoresed inhigh-resolutionacrylamidegelsforsizeseparation.
- These gels areplaced underX-ray film,which then yields aseries ofdarkbandswhich showthelocationofradiolabeled DNAmolecules.Thefragments areordered bysizeandsowe candeducethesequence oftheDNAmolecule.
1.Maxam-GilbertMethodchemicalsequencing
32P
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1.Maxam-GilbertMethodchemicalsequencing
ProsMaxam-Gilbertsequencingwas at onepointmorepopular than theSanger method.Purified DNAcould beused directly,while theSanger method required thateach readstartbecloned forproductionofsingle-strandedDNA.
ConsCons included difficulties scaling up,andthehandlingofX-rays andradiolabeling,whichwere harmful totechnicians.
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2. Sequenziamento di DNA mediante il metodo di Sanger
Sequenziamento con il metodo dei dideossinucleotidi
F. Sanger 13 agosto 1918 – 19 novembre 2013
Due premi Nobel. Uno per iI sequenziamentodell’insulina ed uno per il sequenziamento del genomadel fago φ−X174
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Mix primer oligonucleotides andMany identical dsDNA molcules
Polymerase elongates template DNA
Primer
Primer
Primer
Primer
DNA Polymerase
Denature DNA (95C)Anneal primer (ca 60C)
go to 37C
add dATP, dTTP, dCTP, dGTPand DNA polymerase
dATPdCTPdTTPdGTPDNA Pol
General concept in a sequencing reaction:The synthesis of a new strand of DNA from a ss template DNA
PROBLEM: HOW CAN WE READ THE NEWLY SYNTHEZISED DNA SEQUENCE??
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A great trick: using di-deoxyribonucleoside triphosphates to terminatethe synthesis of DNA molecules
di-deoxyadenine triphosphate
deoxyadenine triphosphate
Concept: mixing a low amount of ddATPs into a high amount of dATP (ca 1:100):A pool of DNA molecules will be generated in which DNA molecules terminate
at all possible A sites.
ddNTP enable me to terminate sequencing at a definedposition in the newly synthesized DNA molecule
PROBLEM: How can detect sequencing products??
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32
γ-32PATP
Primer
Primer
Primer
Denature DNA (95C)Anneal primer (ca 60C)
go to 37C
dATPdCTPdTTPdGTP+ ddATPDNA Pol
+32P-gammaATP+ Polynucleotide kinase
Primer32P
radioactively labeled primerà SEQUENCE SPECIFICà PRIMER BINDING SITE IN
DNA MUST BE KNOWN32P 32P
32P32P
32P
32P
Primer ddATP
Primer ddATP
Primer ddATP
Denaturing Polyacrylamidegel electrophoresis
autoradiography
Radioactive labelingof sequencing primer
General concept in a sequencing reaction:The synthesis of a new strand of DNA from a ss template DNA
A
A
AXXX
XXX
Size ofDNAnewlysynthesized DNAfragment
32P
32P
32P
32P
32P
32P
T
T
T
ddATP:dATP=1:100
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Classic Sanger sequencing of a DNA fragment requires 4 parallelsequencing reactions
ddATP
A
A
A
Tube
1: d
NTP
mix
with
ddG
TPTu
be2:
dNT
P m
ix w
ith d
dATP
Tube3: dNTP mix with ddCTPTube4: dNTP mix with ddTTP
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LeduebasilaritecnicheelettroforeticheperlaseparazionediframmentidiDNA(ediRNA).
• Elettroforesisugeldipoliacrilammide (PAGE):• ilgelèottenutoperpolimerizzazione insoluzioneacquosatamponatadiacrilammide conunapiccolapercentualedibisacrilammide traduevetriconintercapedinedi0,5–2mmmantenutiverticalmente.Ilgelècostituitodaunaretetridimensionalecovalentedelpolimeroedèsostanzialmenteungelirreversibile.
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PolyAcrylamide Gel Electrophoresis
PAGE
ilgelèottenutoperpolimerizzazioneinsoluzioneacquosatamponatadiacrilammide conunapiccolapercentualedibisacrilammide traduevetriconintercapedinedi0,5–2mmmantenutiverticalmente.Ilgelècostituitodaunaretetridimensionalecovalentedelpolimeroedèsostanzialmenteungelirreversibile.
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Standardinlabuntil ca.1995
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Cleavage frequency : 4n
Enzyme that recognizes 4 bases -> 44 = 2566 bases -> 46 = 40968 bases -> 48 = 65536
Primer is radioactively labelled!!All fragments produced by DNA Polymerasecan be visualized by autoradiography
ddNTPs
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Use of Sequenase kit - Cabral et al., J. Biol Chem, 278:10006-10012
Can you read the DNA sequence?
NORMAL: GGT GCT CCT GGT GCT CCT GGT GCC CCT GGC CCC GTT GGC CCT GCT
MUTANT: GGT GCT CCT GGT GCT CCT GGT GCT CCT GGT GCC CCT GGC CCC GTT
AMMINO ACID SEQEUNCE: G A P G A P G A P G P V G P A
AMMINO ACID SEQEUNCE: G A P G A P G A P G A P G P V
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2.1. Automated sequencingbased on Sanger technique
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Dye-terminator Sanger sequencing
Classicradioactive
Dye-terminatorSanger sequencing
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Tre diversi modi di marcare iframmenti di Sanger:
1) I frammenti di Sanger sono resi radioattivi per incorporazione di α-dNTP marcato
Questo metodo richiede quattro reazioni di polimerizzazione separate e quattro corsie elettroforetiche.
2) Ciascun ddNTP è reso fluorescente con un fluoroforo diverso. Questo metodo consente anche di effettuare tutte le reazioni in un’unica provetta ed unica corsie elettroforeticha..
Dye-terminator Sanger sequencing
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Dye-terminator ddNTPs
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- ModifiedddNTPs areincorporated into theproducedDNA,however they alsostoptheextensionofthechain (hence they arecalled terminators).NOTE:ddNTP:dNTP=1:100).Theuseofthese terminating ddNTPscreatesaselectionofDNAfragments ofdifferingsize,eachofwhichends withaparticularnucleotidewhich is labeledwithadifferent coloureddye.
- Thefragments canthenbeseparatedaccordingtosize.Inconventional agarosegelelectrophoresis,asampleofDNAis loaded intoawell intheagaroseandanelectric current applied.Because theconditions within thegelgive theDNAanoverall negativecharge,theelectric field pushes theDNAthrough thegelfromthenegativeterminaltothepositiveterminal.Smaller fragmentsofDNAcanmovemorequickly through thegelthan larger fragments,andsotheDNAseparates outinto regions ofthegelwhich contain fragments ofasimilar size.Dye terminatorsequencing canbeperformedusingaconventional gel.Howeverinmoremodernautomated systems,theelectrophoresis is performed inathintubecalled acapillary.Nodye needs tobeincorporated into thegelas theDNAfragments arealready fluorescently labeledusingthedye terminators.
- As thesoup ofdifferently sizedDNAfragmentsseparatesoutinthecapillary,itproduces aseries ofcolouredbands.Eachbandrepresents fragmentsofDNAofaparticular size,andeach colour represents thebaseat which thefragmentterminates.Theshorter fragments,representing thebasesat thebeginningofthesequencewill move through thecapillary first.
- Anexcitation lasershines through thecapillaryandthelightemittedbythefluorescent dye is it returns toalower energy level is detectedbyadetectorsystem.As each colouredbandis detected,it creates asignal which is processedbythesequencer andpresentedas apeakonagraph.Eachpeak represents adifferent base
Dye-terminator Sanger sequencing
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Labeling of each dideoxy-type enables performing sequencing of 4 nucleotide types in only one lane
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3. Massive parallel seqeuncing
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UseDNAtogenerateDNAlibraries:à GenomicDNA(fragemented)à Otherlibraries(cDNA,ChIP,…)
Lecture3:Hallmarkdiscoveryandanalysisofhistonemodifications
NextgenerationsequencingofpoolsofDNAs
fusionofDNA
with linker oligo
Linkersserveasuniformprimer
bindingsites.Thisallowstheamplificationof
theentireDNAlibraryusing
only2typesofoligonucleotides
Amplifiedlibrary
READYFORMASSIVEPARALLELSEQEUNCING
BC:barcode.EachbiologicalsamplehascommonP7oligos(blueandyellow)andP5oligos(red/green);howeverforeachbiologicalsampleadefinedBCsequenceis
chosen.Thislinksthesequencingresulttothebiological
sampleàManysamplescanbesequencedatthesame
timeà (librariesareprepared
separately)
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Lecture3:Hallmarkdiscoveryandanalysisofhistonemodifications
ChIPseq:Analysisofepigeneticinformationonthesinglenucleotidelevelà GENERASTIONOFGENOMEWIDEEPIGENTICMAPS
IlluminaMassivelyParallelSequencing
Illumina offersthemostpotentmassivesequencinginstruments– leaderonthemarket
TheheartoftheIlluminaMassiveParallelSequenceristhe“FLOW-CELL”.AsurfacewithmillionsofsmallwellsthatallowthousandsofSanger-sequencingreactionInparallel=“massiveparallelsequencing”.IneachwellaSINGLEMOLECULEofDNAIsamplifiedandsequenced
https://www.illumina.com/company/video-hub/pfZp5Vgsbw0.html
https://www.youtube.com/watch?v=pfZp5Vgsbw0
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Flowcellcontainssurfacewithmillionsofwells
àEachwellcontainsbeadsmountedwith2speciesofoligonucleotidesthathybridizewithadaptoroligosofDNAlibrary
àDNAlibrarywillbeloadedontotheflowcellinadeterminedconcentration:
ONLYONEMOLECULEOFDNAWILLBEPROCESSEDFORSEQUENCINGINASINGLE
WELL
CLUSTERAMPLIFICATION:
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-makingDNAlibrary(~300bpfragments)-ligationofadaptersAandB tothefragments
- complementaryprimersareligatedtothesurface- pairingwithChiP ed ssDNA atrandomposition inthewelloftheflowcell
CLUSTERAMPLIFICATION:
1wellinaflow-cellwithbillionsofwells
1well,coveredwithmillionsof2typesofoligos
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Bridgeamplification:takesplaceonsurfaceofbeads(eachbeadismountedwith2species ofoligos;eacholigo canhybridizetoaDNAlibraryfragment):initiation
GeneCore
Onthesurface:complementaryoligos
CLUSTERAMPLIFICATION:
DNApolymerase
New filament covalentlylinked to surface
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EMBLGeneCore
CLUSTERAMPLIFICATION:
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REVERSIBLECHAINTERMINATORS:
Instead of promoting irreversible primer extension like the Sangermethod, the reversible chain terminators method uses a cyclicmethod that consists of nucleotide incorporation, fluorescenceimaging and cleavage. The figure below shows a modified nucleotidewith a cleavable dye and reversible blocking group. Once theblocking group is removed, a 3’OH is formed and a new nucleotidemay come in.
NOTE: no classic dNTPs are used for sequencing!!!!
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CLUSTERAMPLIFICATION:
Threedifferent3’-blockedreversible terminators were shownontheleft (A–C)andtwo3’-unblockedreversible terminatorswere shownontheright(D–E).Thechemical structures inred denote thereversible terminating groups.Arrows indicatethesiteofcleavage separating thefluorescent groups fromthenucleotide,andthechemical structures inbluedenote themolecular scars that areattached tothebase.
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sequencingbysynthesiswith“3’blockedreversibleterminator”+fluorescentlylabel (foreachnucleotide)
illumina.com
1. Synthesisusingprimer=incorporationoffluorescent3’blockedreversibleterminato:synthesisblocked
2. Scanningoffluorescentsignalsofallwellsofflow-cellwithlaser(image)3. Dyecleavage+eliminationofreversibleblockinggroup4. washstep1. Repeatsteps1-4ca.150xREADLENGTH:ca:150ntfromeachprimer(2x150nt=300nt)
Illumina:massiveparallelsequencing:
Well 1
Well 2
Well 3
Well 4
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DNA(0.1-1.0 ug)
Sample preparation Cluster growth
5’
5’3’
G
T
C
A
G
T
C
A
G
T
C
A
C
A
G
TC
A
T
C
A
C
C
TAG
CG
TA
GT
1 2 3 7 8 94 5 6
Image acquisition Base calling
T G C T A C G A T …
Sequencing
Illumina SequencingTechnologyRobust Reversible Terminator Chemistry Foundation
In each round of sequencing a fluorescently labelled ddNTP will be used for sequencing. ddATP carries different fluorphor than ddTTP, etc..
Sequencingfrom1end
Well 1
Well 2
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Illumina:pairedendsequencing increasesinformationcontent
https://www.youtube.com/watch?v=9YxExTSwgPM
1° strand sequencing bySP1
2° strand sequencing bySP2
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Sequencederivedfromoneamplifiedcluster
Readlength:50–max.300ntReaddoesnotnecessarilycoverentirelibraryDNAfragment
Identifiedsequence
Identifiedsequence
Dataanalysis:obtainedsequencereadsarealignedalonggenomicDNAsequenceà highnumberofreadsnecessarytoobtain
fullsequencecoverage
Max.output:0.5- 35giga-bases=3.5*1010=10xhumangenome
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