To decode the human genome and gain a greater understanding of diseases and their aetiology, advances need to be made in the rate at which the nucleotides that comprise DNA can be sequenced. Approaches to DNA sequencing have moved on considerably since the development of “chain-terminator” methods by Maxam and Gilbert, and by Sanger, in the1970s, and are now evolving beyond the so-called “next generation” toward today’s “third generation” systems.
The driving force behind these is a desire to:
- increase the read length and accelerate the read times, both of which were sacrificed in next generation systems in order to improve the throughput delivered by the traditional Sanger-based sequencers;
- reduce the price of the required instrumentation;
- cut the cost per sequenced DNA base.
Increasing the read length is vital since the longer the sequence, the more accurate the genomic map that is delivered.
The latest systems claim read lengths of up to 1,000 bases, increasing to 3,000 in certain instances.
Nanopores – A “Hole” New Approach
A single-molecule DNA detection using high-speed optical identification of individual converted DNA bases as they translocate through solid state nanopores with high temporal resolution (1000 frames per second), has been developed by Amit Meller, Associate Professor of Biomedical Engineering and Physics, Boston University1.
Translocation is a promising new approach since it can analyze extremely long DNA chains with a precision which is superior to current third generation systems, and competes very effectively with them on cost, speed, and accuracy. However, to be viable, it needs to be able to differentiate between the four nucleotides that comprise DNA on a single molecule level, and be capable of parallel readout.
The Technology Explained
Meller’s team is developing a novel single molecule DNA sequencing technique – called Optipore – based on the optical readout of DNA molecules as they translocate through nanometer scale pores.
A key to Meller’s approach to sequencing is the use of a custom total internal reflection fluorescence (TIRF) microscopy set-up, incorporating an ultra-sensitive Andor iXon 860 EMCCD camera to rapidly record fluorescence images from the nanopore membrane2.
To increase the contrast between the nucleotides, the DNA is first converted to an expanded, digitized form by systematically substituting each and every base in the DNA sequence with a specific ordered pair of concentrated oligonucleotides. The converted DNA is then hybridized with complementary molecular beacons labeled with two different colors. Nanopores are then used to sequentially ‘unzip’ the beacons. With each unzipping event a new fluorophore is un-quenched, giving rise to a series of photon flashes in two colors, which are recorded by the EMCCD camera. This unzipping process slows down the translocation of the DNA through the pore in a voltage-dependent manner, to a rate compatible with single molecule optical probing.
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.