Sequencing

Comprehensive insight into sanger sequencing

1. Sequencing
•Sanger sequencing
•Chain termination sequencing
Sunil Bhandari
BalkumariCollege

2. Sequencing:
•ThefundamentalwayofanalyzingthestructureofDNA/protein,
whetheritisarecombinantplasmid,anaturalgene,orawhole
genomeorsynthesizedproteins,istodeterminethesequenceof
basesofwhichitiscomposed.
•Sequencingistheprocessofdeterminingtheactualsequenceof
nucleotides(As,Ts,Cs,andGspresentintheDNA)ordeterminingthe
actualaminoacidsequencepresentintheprotein.
•Today,withtherightequipmentandmaterials,sequencingashort
pieceofDNA/proteinisrelativelystraightforward.

3. •Sequencingistheprimarywayofcharacterizinga
macromolecules,whetheritbedeterminingtheorder
ofaminoacidsinaproteinorofbasesinanucleicacid
•Proteinsequencingwasaveryimportanttoolbefore
genescouldbeclonedandsequenced.
•However,withtheadventofrecombinantDNA
technology,sequencingofencodedproteinbeing
deducedfromthesequenceofthegene.
•InFeb2001,HumanGenomeProjectandCelera
publishedthehumangenomesequenceofnolessthan
threebillionbasepairs.(30,000genes,3billionsbases).

5. Principle of SangarSequencing
Principle
“ThedideoxychainterminationDNAsequencingtechnology(SangerSequencing)
utilizesthefactthatDNApolymeraseswillincorporateachainterminating2’,3’
dideoxynucleotidetriphosphateddNTPattheappropriatecomplementary
positionbutsynthesiswillbestoppedbytheincorporationoftheddNTPsat
the3endbecausethenextnucleotidetobeaddedlackstherequired3hydroxyl
groupfordNTPphosphodiesterbondformation.(Sangeretal1977)

6. What happens when
dNTP’Sare changed
into ddNTP’s?
•ddNTP’sdonothaveany
hydroxylgroupfor
furtherphosphodiester
bondformation,thusno
furtherbaseisadded
afterwards.Thisis
calledchaintermination.

7. Sangarsequencing/
chain termination reaction
•is a process of PCR with a few more bases on it
so,
•Part-I of process is: PCR
•Part-IIofprocessis:Chaindetection

8. Process
of
sanger
sequen-
cing

12. 5’-TAGCTGATACGAG-3’
OUTCOME OF GEL RADIOGRAPHY IS
THE COMPLIMETORY STRAND
COMPLIMENTOTY OF COMPLIMENT
STRAND IS TEMPLATE
THUS
OUR TEMPLATE:
3’-ATCGACTATGCTC-5’

15. Automation:
Sequencing Generation

16. 1st generation sequencing
•Protocolismuchmoresimilartosangersequencing.
•Sangersequencinghadbeenautomatedandalsoknownasfirstgeneration
sequencing
•Itutilizesallsameprotocoluptotemplatereplication(PCR)and
finalimagingstepwaschanged
•Thecomplicated(plustimeconsuming)andhazardousXray
exposureofpolyacrylamidegel(hadbeenreplacedbythe
additionofdetectorsthatdetectsthefluorescentlylabelled
ddNTPsbandswhentheyescapeoutofgelduring
electrophoresis
•Thendetectorsdetectionwasrecordedoncomputersandthe
wholesequenceisretrieveddirectlyfromcomputers

18. High throughput sequencing: NGS
Highthroughputsequencingmethods-fastandcheap
waystosequencigandeasytoanalyzelargegenomes.
A variety of different approaches are being used.
•TheygenerallyinvolvetheamplificationofDNAtemplatesbythe
polymerasechainreaction,andthephysicalbindingoftemplate
DNAtoasolidsurfaceortotinybeadscalledmicrobeads
•ThesetechniquesareoftenreferredtoasmassivelyparallelDNA
sequencing,becausethousandsormillionsofsequencing
reactionsarerunatoncetogreatlyspeeduptheprocess

19. NGS Example: Real time sequencing
Themethodofrealtimesequencinginvolvesimagingthe
continuousincorporationofdyelabellednucleotidesduringDNA
synthesis.
Pacific Biosciences platform:
•SingleDNApolymerasemoleculesareattachedtothebottomsurface
ofindividualzeromodewaveguidedetectors(Zmwdetectors).
•TheseZmwdetectorscanobtainsequenceinformationwhile
phospholinkednucleotidesarebeingincorporatedintothegrowing
primerstrand.
•Zmwdetectotrsendssignalstothecomputers.
•DNAsequenceeasilydetermined.

20. Fig: Pacific Biosciences platform: (Zmwdetectors)