Exonuclease sequencing: Combining a protein nanopore and processive enzyme for the sequential identification of DNA bases as they pass through the pore.
In the exonuclease method of DNA sequencing, a protein nanopore is coupled with a processive enzyme, an exonuclease. The enzyme cleaves individual DNA bases from a DNA strand. These bases enter the nanopore and undergo a binding event before passing through the pore. During this binding event, they cause characteristic disruption in current that can be used to identify the DNA bases in sequence.
Identification of individual DNA bases using a protein nanopore
"Wild type" (naturally occurring) α-hemolysin nanopore alone is not capable of differentiating DNA bases. Oxford Nanopore uses protein engineering techniques to adapt the nanopore for the detection of DNA bases.
In 2008, an Oxford Nanopore paper in Nature Nanotechnology demonstrated chemical modifications to the nanopore such as covalent attachment of cyclodextrin to the internal aperture of the nanopore. This acts as a binding site for individual DNA bases and allows accurate measurement of their passage through the nanopore binding site. This allows the identification of individual nucleoside 5’-monophosphate molecules (DNA bases) to a standard commensurate with a high resolution DNA sequencing technology. In addition the paper demonstrated the direct identification of the modified base methylated cytosine.
The following figure is an electronic trace showing DNA bases in solution entering the nanopore. The bases individually, transiently bind to the cyclodextrin adapter. Each time a base passes through the pore there is a disruption in the current. The diagram shows four different magnitudes of disruption which can be classified as C, G, A or T.
These individual current disruption events can be displayed as data points on a histogram, shown below. This demonstrates the different levels of current disruption shown by each type of DNA base as it passes through the pore. The histogram also illustrates how the level of confidence in the identity of a base may be assessed. For example in this diagram below, if a single event resulted in a residual current of 20pA then there would be a very high level of confidence that a dGMP had translocated the nanopore. In cases where there may be some ambiguity, analysis will show a two-way base ambiguity rather than four-way. So for example, an event that may be G or T would not be A or C.
The following histogram shows the addition of the modified base methylated cytosine, which can be directly distinguished from the four standard bases by using different experimental conditions.
