Albany 2013: Book of Abstracts
June 11-15 2013
©Adenine Press (2012)
Nanopore Immobilization of DNA Polymerase Enhances Single-Molecule Sequencing
A zero-mode waveguide (ZMW) is a nano-scale optical waveguide driven at a frequency below its cutoff. In this mode, the electric field, instead of travelling down the axis of the conducting cavity, decays exponentially. By fabricating waveguides with sub-wavelength diameters and illuminating them with laser light, the electric field in the waveguide is confined enough to enable single-molecule optical detection at micromolar concentration (Levene et al. 2003) Immobilizing single DNA polymerases in ZMW’s and using special phosphate-fluorescently-labeled dNTP’s forms the basis for single-molecule real-time DNA sequencing, one of the most promising next generation sequencing platforms (Eid, et al., 2009). In this method, the polymerase replicates the sample DNA, and as it incorporates new bases into the product strand, the labeled dNTP’s emit a burst of light before the phosphate is cleaved off. The sequence of colors corresponds to the DNA sequence (see Figure 1 below from Eid, et al., 2009). Because the ZMW aperture’s diameter is sub-diffraction-limit, it is impossible to optically distinguish one polymerase in a ZMW from two. Having only one polymerase in each waveguide is critical to sequencing accuracy. In its present state, experimenters use diffusion to fill ZMW’s with polymerases, resulting in a Poisson distribution for filling ZMW’s, and consequently a theoretical limit of 36.8% of ZMW’s having only one polymerase (Eid, et al., 2009). We achieve full polymerase occupancy of ZMW’s by fabricating the structures on an ultrathin silicon nitride membrane and drilling a nanopore at the base of each waveguide with an ion beam. A short DNA fragment with biotin on either end is conjugated to a streptavidin and then drawn into the nanopore with a voltage bias. There is then a free biotin at the base of the ZMW. A polymerase-streptavidin complex can diffuse into the ZMW and bind to the exposed biotin. Because the nanopore is too small to fit more than one molecule, only one ZMW will bind to a biotin in the nanopore. Upon flushing the ZMW chamber, the biotin-bound polymerase will remain trapped in the pore, and only a single polymerase will remain at the base of each waveguide.
Figure 1. Present SMRT sequencing configuration (from Eid, et al. 2009)
Figure 2. Proposed SMRT scheme with nanopore. Not to scale.
Eid, J., et al., Real-Time DNA Sequencing from Single Polymerase Molecules. Science, 2009. 323(5910): p. 133-138.