Meller Group : Single Molecule Biophysics & Nano-biotechnologyTechnion Logo

Opti-pore DNA Sequencing

The development of novel methods for extremely fast and low-cost whole human-genome sequencing remains to be a major goal for bio-engineering. With the introduction of second- and third-generation DNA sequencing approaches we observed a rapid decrease in DNA sequencing costs and a fast increase in the overall sequencing throughput. Currently, whole human genome sequencing is routinely preformed in large research centers around the world, providing unprecedented information on our personal genomic make-up and its disease-related alterations. Looking forward, it is widely accepted that the even cheaper and faster human genome sequencing technologies will be broadly applied for molecular diagnostics, replacing current methods.

With support from the National Human Genome Research Institiute at NIH, our group is developing a nanopore-based single-molecule sequencing method that involves optical readout of the nucleobases.

We are developing a novel single molecule DNA sequencing technique based on the optical readout of DNA molecule translocations through nanometer scale pores. 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 digital 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 the optical probing. Potentially, an extremely high throughput can be achieved, since the conversion (performed in bulk) allows both parallel processing of millions of various DNA fragments, while the single-molecule nanopore readout also readily employ thousands of nanopores probed simultaneously using a high speed camera.

 

Recently we have completed the feasibility 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. Moreover, because our readout method employs optical imaging, we can image multiple pores simultaneously, allowing us to perform the first multi-pore readout. Read more on this work in this paper.

To achieve extremely high throughput and enable human genome sequencing within a few hours a high degree of multiplexing is required.The optical readout permits a straightforward simultaneous detection from hundreds of nanopores fabricated in dense arrays (Figure 3). This is one of the prominent advantages of the opti-pore approach.

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.

 

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