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A Synthetic Electronic
Nanopore for DNA
Sequencing
Mr. Aaron Choi, Computer Science, Sophomore
Mr. Davis Sneider, Biomedical Engineering, Sophomore
Mr. Saifuddin Aijaz, Chemical Engineering, Pre-Junior
Mentors:
Dr. David Wendell, Assistant Professor, Environmental Engineering
Dr. Vasile Nistor, Assistant Professor, Biomedical Engineering
Ms. Elizabeth Wurtzler, Graduate Student, Environmental
Engineering
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Introduction
• Background
• Goals & Tasks
• Time Schedule
– What we’ve done
• Inserting DNA
– What we’re looking for, what we’ve found
• Findings
• Conclusion
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DNA Sequencing Methods
• 454 pyrosequencing
– DNA is amplified inside water droplets (emulsion PCR) with
each drop containing a single DNA template attached to a
single primer-coated bead that forms a clonal colony.
– 700 bp read length
– 1 million reads per run
– ≈ $2500per run
• Ion Torrent
– dNTP is incorporated and is used by determining if a
hydrogen ion is released from the dNTP forming a bond
– Up to 400 bp read length
– Up to 80 million reads per run
– ≈ $750 per run.
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DNA Sequencing Methods
• Illumina Dye sequencing
– DNA and primers are put on
a slide, amplified with
polymerase so DNA
colonies can form. Then
nucleotides are added and
a camera takes images of
the nucleotides
– 50-300 bp per read
– Up to 3 billion reads per run
– ≈ $2000 per run
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Current Problem
• DNA sequencing can cost several
thousand dollars and take about a week
• Nanopore technology can save a lot of
money and reduce the time to one day
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Nanopores: What are they?
• They are extremely small holes.
• They have potential applications for
many kinds of developing technology
Oxford Nanopore Technologies
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Mycobacterium Smegmatis Porin A
(MspA)
• Hydrophilic inner
channel, hydrophobic
outer protein
membrane
• 1.4-2.8 nm in
diameter
• Dwell time of 810ms/ bp
Nature.com
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Alpha Hemolysin (αHL)
• Hydrophilic inner
channel, hydrophobic
outer protein
membrane
• 1.4-4.6 nm in diameter
• Dwell time of 0.0151
ms/bp
Nature.com
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Oxford Nanopore Technology
• “Oxford Nanopore Technologies® is
developing a new generation of nanoporebased electronic systems for analysis of
single molecules…”
• Use α-hemolysin nanopore
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Oxford Nanopore Technology
• Commercialize GridION™
systems
• Chip containing thousands
of microwells with
individual charges and a
single nanopore
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Hydraphile Nanopore
• A synthetic nanopore, created by Dr.
George Gokel at University of Missouri,
St. Louis
• Lariat Ethers
– Excellent cation selectivity
– Excellent binding and release kinetics
Royal Society of Chemistry
http://pubs.rsc.org/en/content/articlehtml/2000/cc/a903825f
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Why use it?
Synthetic
–More functional over broader
range
–Easier to store
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Applications
• We could detect cancer earlier and
much more efficiently
• DNA sequencing allows us to find many
genetic disorders
• Ability to detect viruses
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Our Goals
• To determine which buffer works best
• To test the hydraphile’s sequencing
ability
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Tasks
• Use Clampfit to analyze data from four
buffers
• Run items through nanopore:
– DNA
– Ion Solutions
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Time Schedule
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Conclusion from buffers tests
• Out of the four solutions used, it was
determined that KCl is the best choice to
use for nanopore sequencing as it gives
a more stable membrane.
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Potassium Buffer
• 1M KCl Buffer,
with 5mM HEPES
• pH 7.8
• Able to get data
with ease
• Analyzing Data
– Clampex
• 100< data points
Glogster.com
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Nanopore Insertion
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Pore Diameter Estimation
Use event data to:
• Find conductance of individual
events
• Estimate pore diameter by
comparing conductance to that of
other pores
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Detecting DNA Current Change
• Inserting DNA causes resistances in the
current across the membrane
– Negative charge across membrane
www.ks.uiuc.edu
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DNA Passing
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What We Measured
• 2 major measurements
– Blockage %
– Dwell Time (ms)
• DNA length
– 250 bp
– 500 bp
– 1,000 bp
– 2,500 bp
– 5,000 bp
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Results
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Results
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Results
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Results
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Results
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Results
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Results
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Results
• Found:
• blockage % for multiple lengths
of DNA
• dwell times for multiple lengths
of DNA
• Proved that DNA can pass
through the hydraphile nanopore
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What Does It Mean & What Is It
Useful For?
• Blockage %
– Tells us how much of the nanopore has been
blocked
– Helps us identify approximate width of DNA/RNA
strand
• Event Duration
– Tells us how long it took the DNA
segment to pass through the nanopore
– Helps us identify approximate length
of the DNA/RNA strand
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Conclusions
• Hydraphile:
– Can pass DNA
– Long dwell times are good for sequencing
• Requires more research
– More conformations suggested by
conductance data
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References
• Gokel, George. Hydraphiles: Design, Synthesis and Analysis of a
Family of Synthetic, Cation-conducting Channels. Tech. Royal
Society of Chemistry, 24 Dec. 1999. Web. 13 June 2014.
• "Towards the 15-minute Genome." The Economist. The
Economist Newspaper, 12 Mar. 2011. Web. 17 June 2014.
• Uddin A, Yemenicioglu S, Chen C-H, Corigliano E, Milaninia K
and Theogarajan L. Integration of solid-state nanopores in a
0.5Â um CMOS foundry process. Nanotechnology. IOPScience,
31 October 2013. Web. 2 July 2014.
• Wendell, D., Jing, P., Geng, J., Subramaniam, V., Lee, T. J.,
Montemagno, C., and Guo, P. (2009). "Translocation of
double-stranded DNA through membrane-adapted phi29
motor protein nanopores." Nat Nano, 4(11), 765-772.
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References (cont.)
• Butler, T. Z., Pavlenok, M., and Derrington, I. M. (2008). "Singlemolecule DNA detection with an engineered MspA protein
nanopore." Proc. Natl. Acad. Sciences, 105(52), 20647-20652.
• Niederweis, M. (2003). "Mycobacterial Porins - new channel
proteins in unique outer membranes." Molecular Microbiology,
49(5), 1167-1177.
• Shoseyov, O., and Levy, I. (2008). NanoBioTechnology
Bio Inspired Devices and Materials of the Future, Humana Press,
New Jersey.
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Thank You!
• We would like to thank NSF for funding
our research [Grant ID No.: DUE0756921 and EEC-1004623]
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Questions?