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

2. Principle of Coulter counter
Basics of nanopore analysis
Nanopore is a nano-scale pore (very small hole).
(1995) If a strand of DNA or RNA could be electrophoretically driven through
a nanopore of suitable diameter, the nucleobases would similarly modulate the ionic
current through the nanopore. Patent awarded in 1998.
Same principle as Coulter counter (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 need for labeling, chemical modifications, or surface immobilization = it‘s cheap.
Next advantage is long read lengths.

4. Protein vs. solid-state nanopores
Protein nanopores (biopores)
Biopores include sum of pore-forming bacterial exotoxins
(α-hemolysin of S. aureus, porin A of Mycobacterium smegmatis)
and other pore-forming proteins (nanomotor of phage phi29).
Weak mechanical stability of a lipid membrane which serves as
supporting substrate for pore-forming proteins lowers lifetime of whole lipid-protein assembly.
Lipid membrane is sensitive to pH, temperature and voltage.
Solid-state nanopores (pores in man-made solid membranes)
Solid membranes possess good stability and can be easily modified.
Additional detection mechanisms can be implemented.
Pores with various diameter, charge and shape can be fabricated.
α-hemolysin

5. Other challenges of nanopore analysis
Compared to ionic current measurement bandwidth, the translocation of DNA is too fast to reliably record ionic current levels of individual nucleotides.
Solutions:
to lower fluid temperature Yeh, L. H., Zhang, M. K., Joo, S. W., Qian, S. Z., Electrophoresis 2012, 33,
pore surface modification Krishnakumar, P., Gyarfas, B., Song, W. S., Sen, S., Zhang, P. M., Krstic, P., Lindsay, S., ACS Nano 2013, 7,
use other electrolyte (LiCl) Kowalczyk, S. W., Wells, D. B., Aksimentiev, A., Dekker, C., Nano Lett. 2012, 12,
induce adapters within pore Banerjee, 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, ; Clarke, J., Wu, H. C., Jayasinghe, L., Patel, A., Reid, S.,
Bayley, H., Nat. Nanotechnol. 2009, 4,
OR use additional detection method

6. Detection of tunneling current
It is based on creation of nanogap electrodes within pore.
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. Optical detection of translocated analyte
Optical approach is immune to noise pick-up or parasitic capacitance noise sources.
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 POTENTIAL to 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. Investice do rozvoje vzdělávání
Acknowledgements
Investice do rozvoje vzdělávání 11

11. Thank you for your attention
Investice do rozvoje vzdělávání