1. A NANOPORE FOR DNA SEQUENCING
Guided and Checked By:
Dr. Prakash C. Jha Sir
Presented BY:
Shreya M .Modi
M.Phil. in Nano science
Central University of Gujarat.
shreyamodi20@gmail.com
1
http://www.upenn.edu/pennnews/sites/default/files/imagecache/large_news_photo/news/images/JR.jpg

2. OUTLINE
 Introduction
 Types & Application
 DNA Sequencing
 Different methods
-Using α-Hemolysin
-Using MspA
-Using Graphene etc……
 Conclusion
 References
http://www.ks.uiuc.edu/Research/graphenepores/FIG/system.png 2

3. 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 3

4. Types
Microporous Mesoporous Macroporous
materials materials materials
0.2-2.0 nm 2-50 nm 50-1000 nm
http://nfm.kaust.edu.sa/Publishing http://www.nature.com/nma
http://www.rsc.org/images/b814508c Images/Nanoporous%20materials- t/journal/v11/n11/carousel/n
4
-400-FOR-TRIDION_tcm18-139492.jpg 2s.jpg mat3404-f4.jpg

5. 5
http://rryoo.kaist.ac.kr/image/sub_3.jpg

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

7. 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 7
Radioactive_Fluorescent_Seq.jpg

8. DNA SEQUENCING GENERATIONS
Then + Now Now Now + anticipated Anticipated
1st Gen 2nd Gen 2nd Gen Next
Sanger -parallised -single mol or electronic -single mol AND electronic
•Optical
•Optical
•Low •Single-molecule •Direct electrical (no optics)
•Amplification needed
throughput •Highly parallel •Single-molecule, highly parallel
•Highly parallel
•High cost •Cost similar •Transformation of workflow
•Improved cost and
•Accurate •New applications •Designed to broaden user base,
Throughput
•Broad user deliver step change in cost, power
•More centralised
base •Or electronic, •New applications
users
clonal
Helicos
GAII (Solexa/Illumina)
Pacific Biosciences
Sanger SOLiD (Agencourt/LIFE) Nanopores
Ion Torrent
FLX (454/Roche)
(LIFE Starlight)
Estimated cost of a human genome using these technologies
$70M $200k --- $50k ---- $20k --- 15k--- ?$5k - $?
8

9. De NOVO
http://upload.wikimedia.org/wi
kipedia/en/thumb/6/60/DNA_S
equencing_gDNA_libraries.jpg/
220px
DNA_Sequencing_gDNA_librari
es.jpg
• DNA from individual bacterial clones is sequenced and the
sequence is assembled by using overlapping DNA regions. 9

10. SANGER SEQUENCING (CE)
10

11. 454: Pyrosequencing
11

12. WHAT IS A NANOPORE?
Nanopore = „very small hole‟
Electrical current flows through the hole
Introduce analyte of interest into the hole 
identify “analyte” by the disruption or block to
Current electrical current
the
flow
12

13. APPLICATION OF NANOPORE
Adaptable protein nanopore:
Application
Specific
DNA Sequencing Proteins Polymers Small Molecules
Sensor array chip: many nanopores in parallel
Generic Platform
Electronic read-out system
13

14. 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
14
https://pubs.acs.org/cen/_img/87/i10/8710sci2_DNA1.jpg

15. 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-
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The_process_of_hemolysis.png

16. 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
16
http://www.ks.uiuc.edu/~alek/HEMOLYSIN/stochasticSensor.jpg

17. DNA SEQUENCING BY MSPA
Mycobacterium smegmatis
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http://ars.els-cdn.com/content/image/1-s2.0-S1368764612000180-gr1.jpg

18. 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.
18
http://www.pnas.org/content/107/37/16060/F1.small.gif

19. • (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.
19
http://www.pnas.org/content/107/37/16060/F2.medium.gif

20. 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 20

21. ADVANTAGES
• Direct
• Fast
• Inexpensive
Disadvantage
- Reduced Conduction
- Smaller Coupling
21

22. 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 22

23. 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.
23
http://www.ks.uiuc.edu/Research/graphenepores/FIG/ATGC.png

24. 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.
24
http://www.ks.uiuc.edu/Research/graphenepores/FIG/pot.png

25. ULTIMATELY: WILL WE SEQUENCE
EVERY SPECIES?
1995
2002 2005
2000
2002 2009
25
http://seedmagazine.com/interactive/genome/

26. CONCLUSION
• According to advancement and applications of
nanopores, it indicates that DNA sequencing
carried out by Nanopores are best Methods
than others.
26

27. 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
27

28. 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– 28
7707.

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