An extremely high throughput can, potentially, be achieved since the conversion (which is performed in bulk), allows parallel processing of millions of different DNA fragments, and the single-molecule nanopore readout can readily employ thousands of nanopores probed simultaneously using the high speed EMCCD camera.
In recent feasibility studies, Meller has been able to show, for the first time, that ~5 nm solid-state nanopores can be used to unzip, and optically read the identity of the four converted nucleotides with a high signal to background ratio. Moreover, because the readout method employs optical imaging, it can image multiple pores simultaneously, creating the first multi-pore readout.
Use of TIRF is vital as it permits high spatiotemporal resolution and allows wide-field optical detection of individual DNA molecules as they translocate through multiple nanopores. In addition, TIRF greatly reduces background fluorescence that may be created when the 640nm laser beam is focused to an off-axis point at the back of a high numerical aperture objective.
Using a dichroic mirror, the fluorescence emissions are split into two separate optical paths. The resultant images are projected side by side onto the Andor camera working at maximum gain and a 1ms integration time.
This new opto-electrical technique has major implications for future approaches to DNA sequencing.
With a sequencing rate of between 50-250 bases per second, this already generates a DNA readout speed that is faster than other single molecule methods. However, Meller believes there is scope to push this up to greater than 500 bases per second by adapting the technique for 4-color analysis. Using one fluorophore for each base would instantly halve the length of the converted DNA and thus double the detection speed, as well as increasing the accuracy of the base calling. It would also reduce any potential errors created as a result of frame shifting in a two-color approach.
There is also scope to increase readout speeds by optimizing the reagents.
In addition, since the readout process does not involve an enzymatic step, the speed per nanopore will solely be determined by the limits of detection offered by state of the art CCD or CMOS technologies. Furthermore, the process is readily controlled by the voltage applied on the SiN membrane.
This means that as soon as progress is made in raising imaging speeds, it will translate immediately into further increases in readout rates.
Use of a highly sensitive and ultra-fast EMCCD is central to this new sequencing method, as it relies on fast multicolor optical readout, from many nanopores simultaneously.
The Andor iXon DU-860 offers an optimal format for this sequencing technology since its 24μm size pixels allow an efficient light collection combined with the > 500 fps readout speed for full frame, or higher rates for subimages. It also has low readout noise and a very high EM gain, which are key features for this approach to DNA sequencing.
Back illumination version provides extremely high quantum yield, which is extremely beneficial for high-speed single molecule detection
This new wide-field optical detection technique has an inherent advantage in that numerous pores can be probed simultaneously. This makes it both fully scalable and more cost-effective on a cost-per-base basis, than other ‘new’ approaches. It also means Optipore could be the ideal basis for future, ‘fourth generation’, sequencing systems, especially those directed at routine clinical diagnostics, where price-per-base will be a key factor in expediting their adoption.
- McNally, B., A. Singer, Z. Yu, Y. Sun, Z. Weng, and A. Meller. 2010. Optical Recognition of Converted DNA Nucleotides for Single-Molecule DNA Sequencing Using Nanopore Arrays. Nano Letters 10, 2237-2244.
- Soni, V. G., A. Singer, Z. Yu, Y. Sun, B. McNally, and A. Meller. 2010. Synchronous optical and electrical detection of bio-molecules traversing through solid-state nanopores. Rev. Sci. Instru. 81, 014301-307.