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.

QuicklyreducedCost

1.Denatureadouble-strandedDNAtosingle-stranded byincreasingtemperature.2.Radioactively label one 5'endoftheDNAfragment tobesequenced byakinase reaction using gamma-32P-ATP.3.CleaveDNAstrand at specific positionsusing chemical reactions.- Reaction 1:Guanines (andtosomeextent theadenines)aremethylatedbydimethyl sulfate- Reaction 2:Purines (A+G)aredepurinated using formicacid,- Reaction 3:Pyrimidines (C+T)arehydrolysed usinghydrazine.- Reaction 4:Hydrazine+salt (sodiumchloride)inhibits thereaction of

thyminefortheC-only reaction.

- àNOTE:concentration ofchemicals is chosen toonly cause1modification inamolecule ofinterest

- à ThemodifiedDNAsmay thenbecleavedbyhotpiperidine

- Now infour reaction tubes,we will haveseveral differently sized DNAstrands that carry 32Pat 5’end

- Fragments areelectrophoresed inhigh-resolutionacrylamidegels forsize separation.

- These gels areplaced underX-ray film,which then yields aseries ofdarkbandswhich showthelocationofradiolabeled DNAmolecules.Thefragments areordered bysizeandsowe candeducethesequence oftheDNAmolecule.

1.Maxam-GilbertMethodchemicalsequencing

32P

32P

Reaction 1:Guanines aremethylated bydimethyl sulfate- Setupreaction toinduceonly onemodification perDNAmolecule- Cutwithpiperidineatmodified site- Releaseofspecfic 5’labelled DNAfragmetns

G G G GG G G G32P

32P32P32P32P32P32P32P32P

1.Maxam-GilbertMethodchemicalsequencing

ProsMaxam-Gilbertsequencing was at one point morepopular than theSanger method.Purified DNAcould beused directly,while theSangermethod required that each read startbecloned forproductionofsingle-stranded DNA.

ConsCons included difficulties scaling up,andthehandling ofX-rays andradiolabeling,which were harmful totechnicians.

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

- Primer oligonucleotidesIdentical dsDNA molcules with

known primer target site

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 Sanger 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??

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??

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

γ β α

PNK

DNApolymerase

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

LeduebasilaritecnicheelettroforeticheperlaseparazionediframmentidiDNA(ediRNA).

• Elettroforesisugeldipoliacrilammide (PAGE):• ilgelèottenutoperpolimerizzazione insoluzioneacquosatamponatadiacrilammide conunapiccolapercentualedibisacrilammide traduevetriconintercapedinedi0,5–2mmmantenutiverticalmente.Ilgelècostituitodaunaretetridimensionalecovalentedelpolimeroedèsostanzialmenteungelirreversibile.

PolyAcrylamide Gel Electrophoresis

PAGE

ilgelèottenutoperpolimerizzazioneinsoluzioneacquosatamponatadiacrilammide conunapiccolapercentualedibisacrilammide traduevetriconintercapedinedi0,5–2mmmantenutiverticalmente.Ilgelècostituitodaunaretetridimensionalecovalentedelpolimeroedèsostanzialmenteungelirreversibile.

Standardinlabuntil ca.1995

Readlength:500– 700nucleotdi

Cleavage frequency : 4nEnzyme that recognizes 4 bases -> 44 = 256

6 bases -> 46 = 40968 bases -> 48 = 65536

Primer is radioactively labelled!!All fragments produced by DNA Polymerasecan be visualized by autoradiography

ddNTPs

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

dd CTGA dd CTGA

Template sequence

2.1. Automated sequencingbased on Sanger technique

Dye-terminator Sanger sequencing

Classicradioactive

Dye-terminatorSanger sequencing

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

Dye-terminator ddNTPs

- 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

1%

99%

1%

99%

1%

99%

1%

99%

1%

99%

Primer

Labeling of each dideoxy-type enables performing sequencing of 4 nucleotide types in only one lane

3. Massive parallel seqeuncing

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)

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

Flowcellcontainssurfacewithmillionsofwells

àEachwellcontainsbeadsmountedwith2speciesofoligonucleotidesthathybridizewithadaptoroligosofDNAlibrary

àDNAlibrarywillbeloadedontotheflowcellinadeterminedconcentration:

ONLYONEMOLECULEOFDNAWILLBEPROCESSEDFORSEQUENCINGINASINGLE

WELL

CLUSTERAMPLIFICATION:

-makingDNAlibrary(~300bpfragments)-ligationofadaptersAandB tothefragments

- complementaryprimersareligatedtothesurface- pairingwithChiP ed ssDNA atrandomposition inthewelloftheflowcell

CLUSTERAMPLIFICATION:

1wellinaflow-cellwithbillionsofwells

1well,coveredwithmillionsof2typesofoligos

Bridgeamplification:takesplaceonsurfaceofbeads(eachbeadismountedwith2species ofoligos;eacholigo canhybridizetoaDNAlibraryfragment):initiation

GeneCore

Onthesurface:complementaryoligos

CLUSTERAMPLIFICATION:

DNApolymerase

New filament covalentlylinked to surface

EMBLGeneCore

CLUSTERAMPLIFICATION:

REVERSIBLECHAINTERMINATORS:

Instead of promoting irreversible primer extension like the Sangerdye terminator method, the reversible chain terminators methoduses a cyclic method that consists of nucleotide incorporation,fluorescence imaging and cleavage.The figure shows a modified nucleotide with a cleavable dye andreversible blocking group. Once the blocking group is removed, a3’OH is formedand anew nucleotidemay come in.

NOTE: no classic dNTPs are used for sequencing!!!!

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.

sequencingbysynthesiswith“3’blockedreversibleterminator”+cleavablefluorescentlylabel (foreachnucleotide)

illumina.com

1. Startofsynthesisusingprimer=incorporationoffluorescent3’blockedreversibleterminator:synthesisblocked

2. Scanningoffluorescentsignalsofallwellsofflow-cellwithlaser(image)3. Dyecleavage+eliminationofreversibleblockinggroup4. washstep1. Repeatsteps1-4ca.150xREADLENGTH:ca:150ntfromeachprimer(2x150nt=300nt)

Well 1

Well 2

Well 3

Well 4

Illumina:massiveparallelsequencing:

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

Illumina:pairedendsequencing increasesinformationcontent

https://www.youtube.com/watch?v=9YxExTSwgPM

1° strand sequencing bySP1

2° strand sequencing bySP2

ClusterregenerationSP2primer

Sequencederivedfromoneamplifiedcluster

Readlength:50–max.300ntReaddoesnotnecessarilycoverentirelibraryDNAfragment

Identifiedsequence

Identifiedsequence

Dataanalysis:obtainedsequencereadsarealignedalonggenomicDNAsequenceà highnumberofreadsnecessarytoobtain

fullsequencecoverage

Max.output:0.5- 35giga-bases=3.5*1010=10xhumangenome

PILE– UPALIGNEMENTACROSSTHEREFERENCEGENOME