Murrafa Batoool 2012-ag-2600 Med-707.ppt

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1. NUCLEIC ACID SEQUENCING
Murrafa Batool
Mphil. CMS
University of Agriculture, Faisalabad

2. Outline
•Definition
•History
•Sequencing methods
•Applications
•Disadvantages
•Limitations

3. Nucleic Acid Sequencing
•Nucleic Acid Sequencing is the process of
determining of the precise order of nucleotides
within a DNA molecule. It includes any method
or technology that is used to determine the order
of four bases –adenine, guanine, cytosine and
thymine-in a strand of DNA.

4. History

5. Sequencing Methods
•Maxam/Gilbert chemical sequencing
•Sanger chain termination sequencing
Next-generation/Alternative methods:
•Massively parallel signature sequencing (MPSS)
•Polonysequencing
•454 pyrosequencing
•Illumina(Solexa) sequencing
•SOLiDsequencing
•Ion Torrent semiconductor sequencing
•DNA nanoballsequencing
•Single molecule real time (SMRT) sequencing
•Array sequencing

6. Maxam-Gilbert sequencing is performed by
chain breakageat specific nucleotides.
DMS
G
G
G
G
FA
G
A
G
G
A
G
A
A
H
C
T
T
C
T
C
C
T
H+S
C
C
C
C
Maxam-Gilbert Sequencing
DMS Dimethyl sulfate
FA Formic Acid
H Hydrazine
H+S Hydrazine+Sodiumchloride

7. Sequencing gels are read from bottom to top (5′to 3′).
G G+A T+C C
3′
A
A
G
C
A
A
C
G
T
G
C
A
G
5′
Longer fragments
Shortest fragments
G
A
Maxam-Gilbert Sequencing

9. Chain Termination (Sanger) Sequencing
•A modified DNA
replication reaction.
•Growing chains are
terminated by
dideoxynucleotides.

10. Chain terminates
at ddG
Chain Termination (Sanger) Sequencing
The 3′-OH group necessary for formation of the
phosphodiester bond is missing in ddNTPs.

11. Template area to be sequenced
-3′OH
TCGACGGGC…
5′OP-
Primer
Template
Chain Termination (Sanger) Sequencing
•Asequencingreactionmixincludeslabeled
primerandtemplate.
•Dideoxynucleotidesareaddedseparatelytoeach
ofthefourtubes.

12. ddATP + ddA
four dNTPs dAdGdCdTdGdCdCdCdG
ddCTP+ dAdGddC
four dNTPs dAdGdCdTdGddC
dAdGdCdTdGdCddC
dAdGdCdTdGdCdC ddC
ddGTP+ dAddG
four dNTPs dAdGdCdTddG
dAdGdCdTdGdCdCdC ddG
ddTTP + dAdGdCddT
four dNTPs dAdGdCdTdGdCdCdCdG
A
C
G
T
Chain Termination (Sanger) Sequencing

13. Chain Termination (Sanger) Sequencing
•Withadditionofenzyme(DNApolymerase),the
primerisextendeduntiladdNTPis
encountered.
•Thechainwillendwiththeincorporationofthe
ddNTP.
•WiththeproperdNTP:ddNTPratio,thechain
willterminatethroughoutthelengthofthe
template.
•AllterminatedchainswillendintheddNTP
addedtothatreaction.

14. Chain Termination (Sanger) Sequencing
•Thecollectionoffragmentsisasequencing
ladder.
•Theresultingterminatedchainsareresolvedby
electrophoresis.
•Fragmentsfromeachofthefourtubesare
placedinfourseparategellanes.

15. Cycle Sequencing
•Cyclesequencingischaintermination
sequencingperformedinathermalcycler.
•Cyclesequencingrequiresaheat-stableDNA
polymerase.

16. Fluorescent Dyes
•Fluorescentdyesaremulticyclicmoleculesthat
absorbandemitfluorescentlightatspecific
wavelengths.
•Examplesarefluoresceinandrhodamine
derivatives.
•Forsequencingapplications,thesemolecules
canbecovalentlyattachedtonucleotides.

17. Fluorescent Dyes
•Indyeprimersequencing,theprimercontains
fluorescentdye–conjugatednucleotides,labeling
thesequencingladderatthe5′endsofthe
chains.
•Indyeterminatorsequencing,thefluorescent
dyemoleculesarecovalentlyattachedtothe
dideoxynucleotides,labelingthesequencing
ladderatthe3′endsofthechains.
ddA
ddA

18. AC
GT
The fragments are
distinguished by size and
“color.”
Dye Terminator Sequencing
•Adistinctdyeor“color”isusedforeachofthe
fourddNTP.
•Sincetheterminatingnucleotidescanbe
distinguishedbycolor,allfourreactionscanbe
performedinasingletube.
A
T
G
T

19. Capillary
G
T
C
T
G
A
Slab gel
GA
TCG A T C
Dye Terminator Sequencing
The DNA ladder is resolved in one gel lane or in a
capillary.

20. Dye Terminator Sequencing
•The DNA ladder is read on an electropherogram.
CapillarySlab gel
5′AGTCTG
Electropherogram

21. 5′AGTCTG 5′AG(T/A)CTG 5′AGACTG
T/T T/A A/A
Automated Sequencing
•Dyeprimerordyeterminatorsequencingoncapillary
instruments.
•Sequenceanalysissoftwareprovidesanalyzed
sequenceintextandelectropherogramform.
•Peakpatternsreflectmutationsorsequencechanges.

22. Alternative Sequencing Methods:
Pyrosequencing
•Pyrosequencingisbasedonthegenerationof
lightsignalthroughreleaseofpyrophosphate
(PPi)onnucleotideaddition.
▫DNA
n+dNTPDNA
n+1+PP
I
•PPiisusedtogenerateATPfromadenosine
phosphosulfate(APS).
▫APS+PP
IATP
•ATPandluciferasegeneratelightby
conversionofluciferintooxyluciferin.

23. DNA sequence: A T C A GG CC T
Nucleotide added : A T C A G C T
Alternative Sequencing Methods:
Pyrosequencing
•Each nucleotide is added in turn.
•Only one of four will generate a light signal.
•The remaining nucleotides are removed enzymatically.
•The light signal is recorded on a pyrogram.

24. Applications
•Withitsstudywecanunderstandthefunctionofa
specificsequenceandthesequenceresponsiblefor
anydisease.
•WiththehelpofcomparativeDNAsequencestudy
wecandetectanymutation.
•DNAfingerprinting.
•Byknowingthewholegenomesequence,Human
genomeprojectgetcompleted.
•Forensics
•Agriculture
•Medicine

25. DISADVANTAGES
•OnekeydisadvantageofDNAanalysisisthepotential
forinvasionofindividualprivacy
•Becauseaperson'sDNArevealssomuchinformation
abouttheirphysicalstate,itissensitiveinformationthat
mustbecarefullyguarded
•Informationaboutanindividual'sethnicbackground
andparentagecouldbecomecausefordiscrimination
•Disadvantagesincludeincompletecoverage,whichcan
leadtofalsenormalresults,andtheabilitytotestonly
forunbalancedrearrangements(duplicationsand
deletions),andnotbalancedtranslocationsorinversions

26. Limitations
•Formanyoftheidentifiedabnormalities,the
clinicalsignificanceiscurrentlyunknown
•Next-generationsequencingalsorequires
sophisticatedbioinformaticssystems,fastdata
processingandlargedatastoragecapabilities,
whichcanbecostly
•Lackofcomputationalresourcesandstaffingto
analyzeandclinicallyinterpretthedata

27. THANK YOU