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A NANOPORE FOR DNA SEQUENCING

Guided and Checked By: Dr. Prakash C. Jha Sir

http://www.upenn.edu/pennnews/sites/default/files/imagecache/large_news_photo/news/images/JR.jpg

Presented BY:Shreya M .Modi

M.Phil. in Nano scienceCentral University of Gujarat.

shreyamodi20@gmail.com

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OUTLINE

IntroductionTypes & ApplicationDNA SequencingDifferent methods

-Using α-Hemolysin

-Using MspA

-Using Graphene etc……Conclusion References

http://www.ks.uiuc.edu/Research/graphenepores/FIG/system.png

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INTRODUCTION

• Nanoporous materials consist of a regular organic or inorganic framework supporting a regular, porous structure.

• The size of the pores is generally 100 nanometers or smaller.

• Classified as Bulk• Examples – Zeolites, A.Carbon, etc…

https://engineering.purdue.edu/ChE/Research/Areas/Nanotech/Images/Nanotech-02.jpg

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Types

Microporous materials

0.2-2.0 nm

Mesoporous materials

2-50 nm

Macroporous materials50-1000 nm

http://www.rsc.org/images/b814508c-400-FOR-TRIDION_tcm18-139492.jpg

http://nfm.kaust.edu.sa/PublishingImages/Nanoporous%20materials-2s.jpg

http://www.nature.com/nmat/journal/v11/n11/carousel/nmat3404-f4.jpg

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APPLICATIONS

http://rryoo.kaist.ac.kr/image/sub_3.jpg

6http://www.rsc.org/ejga/CE/2010/b909643b-ga.gif

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DNA SEQUENCING

• DNA sequencing is the process of determining the precise order of Nucleotides within a DNA molecule.

• DNA sequencing may be used to determine the sequence of individual genes, larger genetic regions, full chromosomes or entire genomes.

http://upload.wikimedia.org/wikipedia/commons/thumb/3/3d/Radioactive_Fluorescent_Seq.jpg/220px Radioactive_Fluorescent_Seq.jpg

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DNA SEQUENCING GENERATIONS

•Low throughput•High cost•Accurate•Broad user base

•Optical•Amplification needed•Highly parallel •Improved cost andThroughput•More centralisedusers

•Optical•Single-molecule•Highly parallel•Cost similar•New applications

•Or electronic,clonal

•Direct electrical (no optics)•Single-molecule, highly parallel•Transformation of workflow•Designed to broaden user base, deliver step change in cost, power•New applications

Now + anticipated

1st GenSanger

2nd Gen-parallised

2nd Gen-single mol or electronic

SangerGAII (Solexa/Illumina)

SOLiD (Agencourt/LIFE)FLX (454/Roche)

HelicosPacific Biosciences

Ion Torrent(LIFE Starlight)

Nanopores

AnticipatedNowThen + Now

$70M $200k --- $50k ---- $20k --- 15k--- ?$5k - $?

Next-single mol AND electronic

Estimated cost of a human genome using these technologies

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De NOVO

• DNA from individual bacterial clones is sequenced and the sequence is assembled by using overlapping DNA regions.

http://upload.wikimedia.org/wikipedia/en/thumb/6/60/DNA_Sequencing_gDNA_libraries.jpg/220px DNA_Sequencing_gDNA_libraries.jpg

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SANGER SEQUENCING (CE)

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454: Pyrosequencing

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WHAT IS A NANOPORE?Nanopore = ‘very small hole’Electrical current flows through the holeIntroduce analyte of interest into the hole identify “analyte” by the disruption or block to the electrical currentCurrent

flow

curr

ent

in

out

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APPLICATION OF NANOPORE

Sensor array chip: many nanopores in parallel

DNA Sequencing Proteins Polymers Small Molecules

Adaptable protein nanopore:

Appl

icati

on

Spec

ific

Gen

eric

Pla

tfor

m

Electronic read-out system

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NANO PORE FOR DNA SEQUENCING

• A nanopore is simply a small hole, of the order of 1 nanometer in internal diameter.

• Certain porous transmembrane cellular proteins act as nanopores, and nanopores have also been made by etching a somewhat larger hole

• α-Hemolysin Nanopore• MspA Nanopore• Graphene Nanopore

https://pubs.acs.org/cen/_img/87/i10/8710sci2_DNA1.jpg

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α-HEMOLYSIN

• TOXIN

• DNA or RNA strands can translocate through the pore of alpha-hemolysin, producing the ionic current blockades that reflect the chemical structure of individual strands

http://upload.wikimedia.org/wikipedia/en/thumb/8/82/The_process_of_hemolysis.png/450px-The_process_of_hemolysis.png

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ALPHA HEMOLYSIN

• A single nucleotide resolution has been demonstrated for DNA hairpins raising the prospect of creating a nanopore sensor capable of reading the nucleotide

http://www.ks.uiuc.edu/~alek/HEMOLYSIN/stochasticSensor.jpg

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DNA SEQUENCING BY MSPA

http://ars.els-cdn.com/content/image/1-s2.0-S1368764612000180-gr1.jpg

Mycobacterium smegmatis

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Conti….

It resolve single-nucleotides in single-stranded DNA when double-stranded DNA temporarily holds the nucleotides in the pore constriction.

Passing DNA with a series of double-stranded sections through MspA provides proof of principle of a simple DNA sequencing method using a nanopore.

http://www.pnas.org/content/107/37/16060/F1.small.gif

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• (A) The positive voltage attracts the negatively charged hairpin DNA into the pore.

• (B) The DNA threads through the pore until the wider hairpin duplex prevents further translocation.

• (C) After a few milliseconds the hairpin dissociates allowing for complete translocation.

http://www.pnas.org/content/107/37/16060/F2.medium.gif

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GRAPHENE FOR DNA SEQUENCING

Each nucleotide interacts with the nanopore to a varying degree, resulting in a characteristic electronic signal for each of the 4 nucleotides.

http://onlinelibrary.wiley.com/doi/10.1002/adfm.201002530/pdf

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ADVANTAGES

• Direct• Fast• Inexpensive

Disadvantage - Reduced Conduction - Smaller Coupling

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EDGE HYDROGENATION

When two H-bonds (dotted yellow lines) are formed simultaneously between the nitrogen atom of a DNA nucleobase and two H atoms attached to the graphene-edge. For the sake of clarity, only relevant atoms from the edge hydrogenated graphene electrodes and the DNA molecule have been visualized, omitting water molecules, counter ions, and the silicon-nitride membrane.

http://media.materialsviews.com/wp-content/uploads/2012/02/graphene-for-DNA-sequencing.gif

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DETECTING A-T AND G-C BASE PAIRS A-T base pairs to stretch more readily. It can also discriminate between A-T and G-C base

pairs which is the first step towards sequencing DNA.

http://www.ks.uiuc.edu/Research/graphenepores/FIG/ATGC.png

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Voltage-dependent Kinetics of DNA Transport

• The blocking is more effective at lower bias voltages. • At low bias hydrophobic interaction is strong so DNA stick to

graphene membrane.

http://www.ks.uiuc.edu/Research/graphenepores/FIG/pot.png

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ULTIMATELY: WILL WE SEQUENCE EVERY SPECIES?

http://seedmagazine.com/interactive/genome/

1995

2000

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2002

2005

2009

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CONCLUSION

• According to advancement and applications of nanopores, it indicates that DNA sequencing carried out by Nanopores are best Methods than others.

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REFERENCES

• http://www.ks.uiuc.edu/Research/graphenepores/

• https://pubs.acs.org/cen/science/87/8710sci2.html

• http://textbookofbacteriology.net/staph_3.html

• http://en.wikipedia.org/wiki/DNA_sequencing

• Faller M, et al. (2004) "The structure of a mycobacterial outer-membrane channel." Science.

• Butler TZ, Pavlenok M, Derrington I, Niederweis M, Gundlach J (2008). "Single-molecule DNA detection with an engineered MspA protein nanopore." Proc. Natl. Acad. Sci. 106 (9): 20647-20652.

• Purnell R, Mehta K, Schmidt J (2008). Nucleotide identification and orientation discrimination of DNA homopolymers immobilized in a protein nanopore. Nano Letters 8 (9): 3029-3034

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Conti…• Church, G.M.; Deamer, D.W., Branton, D., Baldarelli, R., Kasianowicz,

J. (1998) "US patent # 5,795,782 (filed March 1995) Characterization of individual polymer molecules based on monomer-interface interaction".

• Kasianowicz, JJ; Brandin E, Branton D, Deamer DW (1996-11-26). "Characterization of individual polynucleotide molecules using a membrane channel.". Proc Natl Acad Sci USA 93 (24): 13770–3. doi:10.1073/pnas.93.24.13770. PMC 19421. PMID 8943010.

• Manrao E, Derrington I, Pavlenok M, Niederweis M, Gundlach J (2011). "Nucleotide discrimination with DNA immobilized in the MspA nanopore." PLoS ONE 6

• Stoddart D, Heron A, Mikhailova E, Maglia G, Bayley H (2009). "Single-nucleotide discrimination in immobilized DNA oglionucleoties with a biological nanopore". Proc. Natl. Acad. Sci. USA 106: 7702–7707.

• .

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Thank You