next generation dna sequencing technology

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

next generation dna sequencing technology

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