A Synthetic Electronic Nanopore for DNA Sequencing Mr. Aaron Choi, Computer Science, Sophomore Mr....
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Transcript of A Synthetic Electronic Nanopore for DNA Sequencing Mr. Aaron Choi, Computer Science, Sophomore Mr....
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
2
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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• 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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DNA Sequencing Methods
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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• Hydrophilic inner channel, hydrophobic outer protein membrane
• 1.4-2.8 nm in diameter
• Dwell time of 8-10ms/ bp
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Mycobacterium Smegmatis Porin A (MspA)
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 nanopore-based electronic systems for analysis of single molecules…”
• Use α-hemolysin nanopore
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• Commercialize GridION™ systems• Chip containing thousands
of microwells with individual charges and a single nanopore
Oxford Nanopore Technology
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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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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Tasks
• Use Clampfit to analyze data from four buffers
• Run items through nanopore:– DNA– Ion Solutions
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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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Pore Diameter Estimation
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Use event data to:• Find conductance of individual
events• Estimate pore diameter by
comparing conductance to that of other pores
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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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
• Found: • blockage % for multiple lengths
of DNA• dwell times for multiple lengths
of DNA• Proved that DNA can pass
through the hydraphile nanopore
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). "Single-molecule 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.: DUE-0756921 and EEC-1004623]
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