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Old 11-14-2012, 08:09 AM   #2
Location: San Diego

Join Date: Nov 2011
Posts: 25

No differentiation made between strand sequencing and exonuclease sequencing methods
Also, no distinction is made between biological and solid state pores

Major challenges of nanopore sequencing.

One of the biggest challenges is electronic noise. The electric current changes in the nanopore are in the range of 25 to 50 pA (25 - 50 x 10-9 A) and that is for the difference between having the nanopore empty or having DNA passing through it. Imagine how difficult it is to differentiate one base from the next!
Also, these are extremely small. Nanopores (natural and synthetic ones) can have diameters between 1.2nm (MspA pore) and 50nm (solid state). This presents a challenge for the interface, as the nanopores need to be connected to an outside circuit that processes the signals. If that wasn’t enough, the lipid bilayers on which biological nanopores are suspended are unstable and hard to manipulate. They are also very fragile and can break easily. As soon as that happens there is no more voltage between either side of the nanopores and thus translocation stops (i.e. the DNA does not move).
There are also uncertainties about whether translocation occurs at a constant speed and the complications of pore clogging.
Another issue is the speed at which the DNA strand moves through the nanopore: at a rate of 1 to 5μs per base. Some companies are trying to address this by controlling the speed of DNA strand by various protein engineering strategies, using phi29, for example.
One specific challenge of the exonuclease approach, where a processing enzyme chops individual bases of the DNA and feeds it to the nanopore is to make sure the released nucleotide goes into the nanopore. This is being addressed by attaching the enzyme to the nanopore, although this mechanism does not constitute a guarantee that the nucleotides will follow a particular path.
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