Nanopore Sequencing - Challenges

Challenges

One challenge for the 'strand sequencing' method is in refining the method to improve its resolution to be able to detect single bases. In the early papers methods, a nucleotide needed to be repeated in a sequence about 100 times successively in order to produce a measurable characteristic change. This low resolution is because the DNA strand moves rapidly at the rate of 1 to 5μs per base through the nanopore. This makes recording difficult and prone to background noise, failing in obtaining single-nucleotide resolution. The problem is being tackled by either improving the recording technology or by controlling the speed of DNA strand by various protein engineering strategies. More recently effects of single bases due to secondary structure or released mononucleotides have been shown. Professor Hagan Bayley, founder of Oxford Nanopore, recently proposed that creating two recognition sites within an alpha hemolysin pore may confer advantages in base recognition.

One challenge for the 'exonuclease approach', where a processive enzyme feeds individual bases, in the correct order, into the nanopore, is to integrate the exonuclease and the nanopore detection systems. In particular, the problem is that when an exonuclease hydrolyzes the phosphodieseter bonds between nucleotides in DNA, the subsequentially released nucleotide is not necessarily guaranteed to directly move in to, say, a nearby alpha-hemolysin nanopore. One idea is to attach the exonuclease to the nanopore, perhaps through biotinylation to the beta barrel hemolsyin. The central pore of the protein may be lined with charged residues arranged so that the positive and negative charges appear on opposite sides of the pore. However, this mechanism is primarily discriminatory and does not constitute a mechanism to guide nucleotides down some particular path.