Nanopore for dna sequencing by shreya
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Transcript of Nanopore for dna sequencing by shreya
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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.
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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
2002
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