Team:Vanderbilt/Project/Nanopore
Nanopore Sequencing
For the past several years, a third generation sequencing platform [1,2] has been under development that has its roots in biophysiologic studies of channel proteins. It utilizes a dually biotic and abiotic architecture to generate nucleic acid sequences of near limitless lengths from electrochemical data without the need for target sequence modification [1-11]. It is called nanopore sequencing, and it seeks to revolutionize the accessibility of genetic data through the use of molecular machines.
The history of nanopore sequencing is as brief as it is wildly promising. In the mid-1990’s, it was first proposed that a heterogeneous ssNA could be analyzed when passed through an interface by way of a nanoscale pore [3]. The Deamer group took inspiration for this idea from the electrophysiologic patch clamp technique, traditionally used to characterize membrane channel proteins, and reasoned that the procedure could be manipulated to quantify the apertures of ssNA protein channel candidates [4,5,9]. Shortly thereafter, they demonstrated that nucleotides passing through a channel could be distinguished given the reproducible number of ions predicted to be displaced by each of the bases3. Kasianowicz also showed that the translocation of nucleic acids through an alpha-hemolysin channel partitioning two chambers containing potassium chloride allowed characterization of polymer length via discernable blockage of ionic currents[5].
The foundational experiment leading up to today came when Deamer showed distinct patterns of pore current blockage were produced by chemically distinct oligonucleotides; pyrimidine oligos could be distinguished on the basis of blockade amplitude, while purine oligos could be distinguished on the basis of blockade duration[12].
A great number of refinements have since been made to this method, including the modulation of chamber environment, translocation speed (via rate-limiting motor activity [13]), and nanopore synthesis. One of the standout products of these efforts is Oxford Nanopore Technologies’ minION, a 500 channel nanopore sequencer that was first distributed to alpha testers in 2014. The device uses a channel / motor protein arrangement (likely MspA and phi-29 polymerase) to generate electrical data from transversing ssNA that is later translated into a sequence. Vanderbilt iGEM has had the incredible opportunity to conduct preliminary testing with the minION, and moving forward we hope to use nanopore sequencing to detect and quantify modified base signatures in mutagenized DNA samples.
References
- Kenneth Nelson, F. et al. in Current Protocols in Molecular Biology (John Wiley & Sons, Inc., 2001).
- Kling, J. Ultrafast DNA sequencing. Nat Biotech 21, 1425–1427 (2003).
- http://www.google.com/patents/EP1956367B1>Church, G., Deamer, D. W., Branton, D., Baldarelli, R. & Kasianowicz, J. Characterization of individual polymer molecules based on monomer-interface interactions. (2013).
- Cheeseman, P. C. Method for sequencing polynucleotides. (1994).
- Kasianowicz, J. J., Brandin, E., Branton, D. & Deamer, D. W. Characterization of individual polynucleotide molecules using a membrane channel. Proc Natl Acad Sci U S A 93, 13770–13773 (1996).
- Meller, A., Nivon, L. & Branton, D. Voltage-Driven DNA Translocations through a Nanopore. Phys. Rev. Lett. 86, 3435–3438 (2001).
- Liang, F. & Zhang, P. Nanopore DNA sequencing: Are we there yet? Sci. Bull. (2014).
- Scheicher, R. H., Grigoriev, A. & Α-hemolysinuja, R. DNA sequencing with nanopores from an ab initio perspective. J Mater Sci 47, 7439–7446 (2012).
- Branton, D. et al. The potential and challenges of nanopore sequencing. Nat Biotech 26, 1146–1153 (2008).
- Wang, H. Introducing polynucleotides and polypeptides into a device which includes a structure having an aperture therethrough; forming and analyzing the distribution pattern from the information obtained. (2004).
- Sampson, J. Synthesis and amplification of unstructured nucleic acids for rapid sequencing. (2002).
- Akeson, M., Branton, D., Kasianowicz, J. J., Brandin, E. & Deamer, D. W. Microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid, and polyuridylic acid as homopolymers or as segments within single RNA molecules. Biophys. J. 77, 3227–3233 (1999).
- Wendell, D. et al. Translocation of double-stranded DNA through membrane-adapted Φ29 motor protein nanopores. Nat Nano 4, 765–772 (2009).