Boston University

Ultra Fast DNA Sequencing Using Nanopores and Optical Probes

In 2004 the National Human Genome Research Institute (NHGRI) at NIH launched a program for the development of revolutionary genome sequencing technologies - the "$1,000 Genome". The main objective of this project is to focus on applied research for the development of ultra fast and cheap DNA sequencing technologies. Although the "$1,000 per genome" should only be taken as a figure of merit, it sets a scale for the ultimate goal - between 4 and 6 orders of magnitude cheaper and faster than current state of the art technologies.

We are developing a novel single molecule DNA sequencing technique based on the optical readout of DNA molecule translocations through nanometer scale pores (see Ref #1). To increase the contrast between nucleotides we first convert the DNA to an expanded, digitized form by systematically substituting each and every base in the DNA sequence with a specific ordered pair of concatenated oligonucleotides (Figure 1). The converted DNA is hybridized with complementary molecular beacons of two colors. To detect the sequence, 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 a CCD camera (Figure 2). The unzipping process slows down the translocation of the DNA through the pore in a voltage-dependent manner, to a rate compatible with SM optical probing. Extremely high throughput potentially can be achieved since the conversion (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 a high speed CCD camera.

Recently we have completed the feasibilty studies of our DNA sequencing method. We showed, for the first time, that ~5 nm solid-state nanopores can be used to unzip, and optically read the identity of the 4 converted nucleotides with high signal to background ratio. Morover, beacuse our readout method employs optical imaging, we can image multiple pores simultaneuosly, allowing us to poerform the first multi-pore readout. Click here to down to download our Nano Letters paper.

To achieve the phenomenal throughput sets by the "$1,000 genome" project a high degree of multiplexing is required. Simultaneous optical detection from hundreds of nanopores in nanopore arrays will allow us to achieve this parallelism (Figure 3 ).

References:

1. D. Branton et. al. (2008) The potential and challenges of nanopore sequencing. Nature Biotech. 26, 1146-53.

2. Soni G. and A. Meller (2007) Progress Towards ultrafast DNA sequencing using solid state nanopores. Clin. Chem. 3, 1996-01.

3. Lee, J.W. and A. Meller. (2007). Rapid sequencing by direct nanoscale reading of nucleotide bases in individual DNA chains. In: "Perspective in Bioanalysis", Elsevier, Edt. K. Mitchelson.

4.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. 81:014301-014307.

5. 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 articles ASAP 05/12/10.

Support:

National Institute of Health

Figure 1

Figure 2
Figure 3