Moderator’s note: Make sure to read the forum intro and the recent Nature Biotech review linked there!

Accurate, single molecule DNA sequence determination using nanopores poses special challenges, which result from a cascade of effects, starting with thermal motion. A DNA molecule driven through a pore by electrostatic force alone will still undergo substantial thermal motion. To ensure that DNA bases pass through the pore one by one, never reversing direction, the electrostatic drift velocity must dominate thermal motion. The electrostatic potential energy gain of one nucleotide passing through the pore must be greater than the thermal energy. (As a reminder, the thermal energy divided by one unit charge is k _B T/e ~ 25mV.) Assuming some charge screening, ≥50mV is required across a pore to overcome thermal motion; usually ≥100mV is used. Typically, DNA passes through the pore at 0.05-1 Mb/s, unless other forces slow its motion. To detect individual bases “on the fly” thus requires sampling rates of 1 MHz or more. So far, the major approach to slowing down DNA translocation has been to use enzymes which naturally process along DNA, such as polymerases or exonucleases.

Questions to get us started: Let’s start with a gut check on our assumptions.

• What limits our ability to acquire meaningful information at these sampling rates?
• How can we slow/control translocation of DNA through a nanopore?
• How does this problem affect the various readout schemes that have been proposed?