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8/8/2019 Next Generation Dna Sequencing Technology
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Advances in sequencing
technology
byEugene Y. Chan
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DNA SEQUENCING
The term DNA sequencing refers tosequencing methods for determining
the order of the nucleotide bases- adenine,guanine, cytosine, and thymine- in amolecule of DNA
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TRADITIONAL SEQUENCING
METHODS
Maxam and Gilbert method
Sanger dideoxy method
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Maxam and Gilbert sequencing
method
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Sanger dideoxy sequencing
method
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NEXTGENERATIONDNA
SEQUEN
CING
TECHN
OLOG
IES
PYROSEQUENCING
NANOPORESEQUENCING
ZEROMODE WAVEGUIDE
EXONUCLEASESEQUENCING
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PYROSEQUE
NCING
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STEP-WISE PROCESS
Step 1A sequencing primer is hybridized to a single-stranded PCR
amplicon that serves as a template, and incubated with the
enzymes, DNA polymerase, ATP sulfurylase, luciferase, and
apyrase as well as the substrates, adenosine 5' phosphosulfate(APS), and luciferin.
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Step 2
The first deoxribonucleotide triphosphate (dNTP) is added to thereaction. DNA polymerase catalyzes the incorporation of the
deoxyribo-nucleotide triphosphate into the DNA strand, if it is
complementary to the base in the template strand. Each
incorporation event is accompanied by release of pyrophosphate
(PPi) in a quantity equimolar to the amount of incorporated
nucleotide.
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Step 3
ATP sulfurylase converts PPi to ATP in the presence of adenosine 5'
phosphosulfate (APS). This ATP drives the luciferase-mediatedconversion of luciferin to oxyluciferin that generates visible light in
amounts that are proportional to the amount of ATP. The light
produced in the luciferase-catalyzed reaction is detected by a charge
coupled device (CCD) chip and seen as a peak in the raw data output
(Pyrogram). The height of each peak (light signal) is proportional to
the number of nucleotides incorporated.
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Step 4
Apyrase, a nucleotide-degrading enzyme, continuously degrades
unincorporated nucleotides and ATP. When degradation is complete,another nucleotide is added
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Step 5
Addition of dNTPs is performed sequentially. It should be noted that
deoxyadenosine alfa-thio triphosphate (dATPS) is used as a substitutefor the natural deoxyadenosine triphosphate (dATP) since it is efficiently
used by the DNA polymerase, but not recognized by the luciferase. As
the process continues, the complementary DNA strand is built up and
the nucleotide sequence is determined from the signal peaks in the
Pyrogram trace
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NANOPORE SEQUENCING
Nanopore:- A nanopore is simply a small hole,
of the order of 1 nanometer in internal
diameter.
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THEORY BEHINDNANOPORE
SEQUENCING
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STEP-WISE PROCESS
STEP 1
Design and synthesis of modifiednucleotides
Site of modification
On 7-position of A (6-amino-hexylamino)
5-postion of T (Biotin)
T*>A*>G>C
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Whymodification is needed
To enhance discrimination between two purinesand two pyrimidines because these generate
similar electronic signature
STEP 2DNA extension reaction using modified
nucleotides.
Modified dATP and dTTP
and unmodified dCTP and dGTP
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STEP 3
Removing from resulting ds DNA, the ss DNA
containing modified nucleotides
STEP 4
ss DNA pass through a pore of suitable
diameter (nanopore)
STEP 5
Each type of nucleotide in the DNA has uniqueelectronic signature and this is used to
determine the sequence of nucleotides in ss
DNA
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ZERO-MODE WAVEGUIDESingle Molecule Real Time (SMRT)DNA
sequencing technology
ZMW is a cylindrical hole just a few tens of
nanometer in diameter, perforating a thin film
supported by a transparent substrate
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PHOSPHOLINKED NUCLEOTIDES
Step 1: Fluorescent phospholinked labeled nucleotidesare introduced into the ZMW.
Step 2: The base being incorporated is held in the
detection volume for tens of milliseconds, producing abright flash of light.
Step 3: The phosphate chain is cleaved, releasing theattached dye molecule.
Step 4-5: The process repeats.
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EXONUCLEASE SEQUENCING
This method of sequencing exploits 35
activity of exonuclease
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STEP-WISE PROCESS
STEP 1
labeling the nucleotides with base specific
tags suitable for fluorescence detection
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STEP 2
Selecting a desired single stranded fragment of
DNA and synthesizing its second strand with
the help of DNA polymerase
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STEP 3
Suspending the single DNA fragment in a
flowing sample stream
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STEPS 4 and 5
Sequentially cleaving labeled bases from the
free end of the DNA fragment using anexonuclease, and Detecting and identifying the
cleaved, labeled bases as they flow through a
focused laser beam
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APPLICATIONS OF NEXT
GEN
ERATION
DN
ASEQUENCINGTECHNOLOGY
Genomic medicine
Metagenomics
Comparative genomics
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REFERENCES
1) Clyde A. Hutchison(2007)DNA sequencing: bench to bedside
and beyond Nucleic Acids Research, Vol. 35, No. 18 62276237.
2) Eugene Y. Chan(2005) Advances in sequencing technologyMutation Research., 573, 1340
3)Healy Ken.,(2007)Nanopore-based single moleculeDNA
analysis Nanomedicine., 2(4), 459-481.
4)http://www.wipo.int/pctdb/en/wo.jsp?WO=2007146158
5)http://en.wikipedia.org/wiki/Pyrosequencing
6)http://www.pyrosequencing.com/DynPage.aspx?id=7454
7)http://www.nature.com/naturemethods
8) J.H. Jett, R.A. Keller, J.C. Martin, B.L. Marrone,R.K. Moyzis, R.L.
Ratliff, N.K. Seitzinger, E.B. Shera, and C.C. Stewart(1989)High-speed DNA sequencing: an approach based upon fluorescence
detection of single molecules Journal of Biomolecular Structure
& Dynamics.
9)) MostafaRonaghi, Mathias Uhln, andPl Nyrn (1998)DNA
SEQUENCING: A Sequencing Method Based onReal-Time
Pyrophosphate Science., 281, 363-365.
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10)Nava Whiteford, Niall Haslam, Gerald Weber, Adam Prgel-
Bennett1, JonathanW. Essex, Peter L. Roach, Mark Bradley2 and
Cameron Neylon*(2005) An analysis of the feasibility of short read
sequencing Nucleic Acids Research 33(19):e171;
doi:10.1093/nar/gni170 (ONLINE)
11)Neil Hall(2007) Advanced sequencing technologies and their
wider impact in microbiologyThe Journal of Experimental
Biology., 209, 1518-1525.
12)www.pacificbiosciences.com/.../pacbio_technology_backgroun
der.pdf
13)www.its.caltech.edu/.../Levene_zero_mode_waveguide_science
_2003.pdf