Nanopore Sequencing
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The document discusses nanopore sequencing, a method for DNA sequencing involving the passage of single-stranded DNA through a nanoscopic pore. It explores the historical development, key figures, principles, advancements, and the pros and cons of the technique. Challenges include controlling the speed of DNA translocation and achieving high signal-to-noise ratios.
Nanopore Sequencing
- 1. NANOPORE SEQUENCING Somesh Shingane Kuldeep Sharma M.Tech Biological Engineering, IIT GANDHINAGAR.
- 2. Introduction Fourth Gen Sequencing Polynucleotide Sequencing Single Molecule Detection Constantly Evolving Nanometer Scale Pore Size
- 3. Background and Principle behind Nanopore Sequencing Around 1980s David Deamer had an implausible suggestion that it might be possible to sequence a single strand of DNA(ssDNA) by being drawn through a membrane’s nanoscopic pore by the process of electrophoresis. George Church’s interest in scaling up DNA sequencing for the HGP led him to propose that an electronically monitored embedded system across bilayer membrane might provide sequence information upon processing of DNA. Hagan Bayley’s interest in membrane proteins and the structure and assembly of oligomeric transmembrane protein pores, such as α-hemolysin, also led him and his colleagues to ask if pores could serve as biosensors of molecules and cation. With the collaboration of David Deamer ,John Kasianowicz together with Sergey Bezrukov at the US National Institutes of Health (NIH) studied the structure of α- hemolysin and compared with size of dsDNA(it was perfect fit!)
- 4. Pages from the book of David Deamer
- 5. Basics of Nanopore Sequencing
- 6. But… How do we know only ssDNA can pass through the nanaopore? How can we make sure that ssDNA is guided to the pore without any hindrances? How do we differentiate between the purines and pyrimidines just by the current signals when polynucleotide passes through the pores? How much time does it takes to identify the sequence and how many base pairs can it identify at a time or how much it should be? Is α-hemolysin the only nanopore for the sequencing purpose?
- 7. Timeline of Nanopore Sequencing
- 8. Minds Behind the Idea of Nanopore Sequencing
- 9. Construction Sensor Cell Electric reader unit Chambers separated by septum; Lipid bilayer; electrode o Transmembrane Protein subunit o Nucleic acid handling enzyme o Membrane unit
- 10. Transmembrane Protein Other Proteins : OmpF, OmpG, OmpA, NaLP, WZA
- 11. NA Handling Enzyme Phi29 DNA Polymerases RecD Helicases Source: PDB
- 15. Advancement Covalent linkage of transmembrane subunit to NA handling enzyme (Patent Application No. WO2010004265A1) SSB use to prevent secondary structure formation (Patent Application No. WO2014013259A1) Coupling the target polynucleotide to the membrane (Patent Application No. WO2012164270A1) Hairpin loop method for dsDNA sequencing (Patent Application No. WO2013014451A1)
- 16. Pros and Cons Nanopore Sequencing Pros Cons • Minimum amount of data processing and complex algorithm are required. • The DNA sequence analyzing system is yet to reach its maximum efficiency. • Can identify the genomic sequence at large scale ( upto gigabase pairs) at one go. • Handling the long strand of ssDNA is also a major concern i.e. to avoid self complimentary binding. • The size of pore is fixed and does not vary for large variation of environment or eperimental. • Though it can work in various experimental conditions yet it has some temperature limitations. • Real time sequencing at high rate of accuracy (upto 98%) • Controlling the speed of ssDNA passing through the pore is yet to be achieved at maximum efficiency. • Can sequence the entire human genome in less than $1000 • High signal to noise ratio is yet to obtain. • Translocation of polynucleotide through pores are in precise and control manner. • Number of run per pore membrane is also bit low.
- 17. THANK YOU