DNA SEQUENCING Techniques: 1. Maxam e Gilbert:first method 2. Sanger Sequencing: basis for all seqeuncing tecniques 3. Massive Parallel 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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Lezione 7 DNA Seqeuncing - moodle2.units.it€¦ · 1. Maxam e Gilbert:firstmethod 2. Sanger Sequencing: basis for all seqeuncingtecniques 3. Massive Parallel Seqeuncing DNA sequencing
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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 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
- These gels areplaced underX-ray film,which then yields aseries ofdarkbandswhich showthelocationofradiolabeled DNAmolecules.Thefragments areordered bysizeandsowe candeducethesequence oftheDNAmolecule.
1.Maxam-GilbertMethodchemicalsequencing
32P
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.
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
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??
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
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
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
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
Labeling of each dideoxy-type enables performing sequencing of 4 nucleotide types in only one lane
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!!!!
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.