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Nanopore sequencing - limitations, pros and cons. Název:. Jméno: Jiří Kudr Datum : 12.6. 2015. Investice do rozvoje vzdělávání. Basics of nanopore analysis. Nanopore is a nano-scale pore ( very small hole ) .

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  1. Nanopore sequencing- limitations, pros and cons Název: Jméno: Jiří Kudr Datum : 12.6. 2015 Investice do rozvoje vzdělávání

  2. Basicsof nanopore analysis • Nanopore is a nano-scalepore (very small hole). • (1995) If a strand of DNA or RNA could be electrophoretically driven through • ananopore of suitable diameter, the nucleobases would similarly modulate the ionic • current through the nanopore. Patent awarded in 1998. • Same principle as Coultercounter (micro vs. nanoscale). • Nanopore analysis is an emerging technique that involves using a voltage to drive • molecules through a nanoscale pore in a membrane between two electrolytes, • and monitoring how the ionic current through the nanopore changes as single • molecules pass through it. • This approach allows charged polymers (including single-stranded DNA, double-stranded DNA and RNA) to be analysed with subnanometre resolution and without the need for labels or amplification. Principle of Coulter counter

  3. Advantages of nanopore seq. • It possess the potential to quickly and reliably sequence the entire human genome for less than $1000, and possibly for even less than $100. • Nanopore technology allows the investigation of native single molecules with high sampling bandwidth without the needfor labeling, chemical modifications, or surface immobilization = it‘s cheap. • Next advantage is long read lengths.

  4. Protein vs. solid-state nanopores • Protein nanopores (biopores) • Bioporesinclude sum ofpore-formingbacterialexotoxins • (α-hemolysin ofS. aureus, porin A ofMycobacteriumsmegmatis) • and otherpore-formingproteins (nanomotorofphage phi29). • Weak mechanical stability of a lipid membrane which serves as • supporting substrate for pore-forming proteins lowers lifetime of whole lipid-proteinassembly. • Lipid membraneis sensitive to pH, temperature and voltage. • Solid-state nanopores (pores in man-made solid membranes) • Solid membranespossessgood stability and canbeeasilymodified. • Additionaldetectionmechanismscanbeimplemented. • Poreswithvariousdiameter, charge and shapecanbefabricated. α-hemolysin

  5. Otherchallengesof nanopore analysis • Compared to ionic current measurement bandwidth, the translocationof DNAis too fast to reliably record ionic current levels of individual nucleotides. • Solutions: • to lower fluid temperatureYeh, L. H., Zhang, M. K., Joo, S. W., Qian, S. Z., Electrophoresis 2012, 33, 3458-3465 • poresurfacemodificationKrishnakumar, P., Gyarfas, B., Song, W. S., Sen, S., Zhang, P. M., Krstic, P., Lindsay, S., ACS Nano 2013, 7, 10319-10326 • use otherelectrolyte (LiCl) Kowalczyk, S. W., Wells, D. B., Aksimentiev, A., Dekker, C., NanoLett. 2012, 12, 1038-1044 • induceadapterswithinporeBanerjee, A., Mikhailova, E., Cheley, S., Gu, L. Q., Montoya, M., Nagaoka, • Y., Gouaux, E., Bayley, H., Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 8165-8170; Clarke, J., Wu, H. C., Jayasinghe, L., Patel, A., Reid, S., • Bayley, H., Nat. Nanotechnol. 2009, 4, 265-270. • OR use additionaldetectionmethod

  6. Detectionoftunnelingcurrent • Itisbased on creationofnanogapelectrodeswithinpore. • In case of nanopore platforms, tunneling refers to an electron transfer where the barrier between electrodes is nanometer sized gap. • If bias voltage is applied between nanogap electrodes, electrons • tend to move to positively charged electrode and tunneling • current which probe the translocated analyte can be detected. • The idea is that analytes have different electronic densities of • state due to their chemical composition and these properties • during electrodes-analyte junction affect tunneling current.

  7. Opticaldetectionoftranslocated analyte • Opticalapproach is immune tonoise pick-up or parasitic capacitance noisesources. Anderson, B. N., Assad, O. N., Gilboa, T., Squires, A. H., Bar, D., Meller, A., ACS Nano 2014.

  8. Oxford Nanopore Technologies • Average read length using the MinION is about 5.4 kb up to 10 kb. • J. Quick used MinIONs to read the genomes of Ebola viruses from 14 patients in as little as 48 hours. • NASA scientists even plan to send a MinION to the International Space Station, where astronauts would test it in microgravity. • Yet there is still plenty that the MinION cannot do (problems with large genomes, errors, long repertition). MinION (512 nanopores, £650 ) Voltrax – automated sample preparation. PromethION GridION

  9. Conclusion • Nanopore based sequencing has a POTENTIALto achieve direct reading of long sequences without need of sample preparation, labelling or amplification. • Nanoelectrodes divided by the area of nanopore, which uses current to probe the translocated analyte, serve as a nanopore additional detection mechanism. • Nowadays, however nanopore devices are not able to sequence DNA, they are unique single molecule tool for nanotechnology researchers. The nanopore platform with incorporated biorecognition elements can be used as a sensor. Further it is applicable in protein analysis, separations and other possibilities lie ahead.

  10. Acknowledgements Investice do rozvoje vzdělávání 11

  11. Thank you for your attention Investice do rozvoje vzdělávání

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