December 20, 2009

Faster, cheaper DNA sequencing method


Nature Nanotechnology: Electrostatic focusing of unlabelled DNA into nanoscale pores using a salt gradient

The technique uses electrical fields to feed long strands of DNA through four-nanometer-wide pores, much like threading a needle. The method uses sensitive electrical current measurements to detect single DNA molecules as they pass through the nanopores. The need for DNA amplification is reduced by 10,000 times.

Solid-state nanopores are sensors capable of analysing individual unlabelled DNA molecules in solution. Although the critical information obtained from nanopores (for example, DNA sequence) comes from the signal collected during DNA translocation, the throughput of the method is determined by the rate at which molecules arrive and thread into the pores. Here, we study the process of DNA capture into nanofabricated SiN pores of molecular dimensions. For fixed analyte concentrations we find an increase in capture rate as the DNA length increases from 800 to 8,000 base pairs, a length-independent capture rate for longer molecules, and increasing capture rates when ionic gradients are established across the pore. Furthermore, we show that application of a 20-fold salt gradient allows the detection of picomolar DNA concentrations at high throughput. The salt gradients enhance the electric field, focusing more molecules into the pore, thereby advancing the possibility of analysing unamplified DNA samples using nanopores





A team of researchers led by Boston University biomedical engineer Amit Meller is using electrical fields to efficiently draw long strands of DNA through nanopore sensors, drastically reducing the number of DNA copies required for a high throughput analysis.

Currently, genome sequencing utilizes DNA amplification to make billions of molecular copies in order to produce a sample large enough to be analyzed. In addition to the time and cost DNA amplification entails, some of the molecules – like photocopies of photocopies – come out less than perfect. Meller and his colleagues at BU, New York University and Bar-Ilan University in Israel have harnessed electrical fields surrounding the mouths of the nanopores to attract long, negatively charged strands of DNA and slide them through the nanopore where the DNA sequence can be detected. Since the DNA is drawn to the nanopores from a distance, far fewer copies of the molecule are needed.

Before creating this new method, the team had to develop an understanding of electro-physics at the nanoscale, where the rules that govern the larger world don't necessarily apply. They made a counterintuitive discovery: the longer the DNA strand, the more quickly it found the pore opening.

"That's really surprising," Meller said. "You'd expect that if you have a longer 'spaghetti,' then finding the end would be much harder. At the same time this discovery means that the nanopore system is optimized for the detection of long DNA strands -- tens of thousands basepairs, or even more. This could dramatically speed future genomic sequencing by allowing analysis of a long DNA strand in one swipe, rather than having to assemble results from many short snippets.

"DNA amplification technologies limit DNA molecule length to under a thousand basepairs," Meller added. "Because our method avoids amplification, it not only reduces the cost, time and error rate of DNA replication techniques, but also enables the analysis of very long strands of DNA, much longer than current limitations."

With this knowledge in hand, Meller and his team set out to optimize the effect. They used salt gradients to alter the electrical field around the pores, which increased the rate at which DNA molecules were captured and shortened the lag time between molecules, thus reducing the quantity of DNA needed for accurate measurements. Rather than floating around until they happened upon a nanopore, DNA strands were funneled into the openings.

By boosting capture rates by a few orders of magnitude, and reducing the volume of the sample chamber the researchers reduced the number of DNA molecules required by a factor of 10,000 – from about 1 billion sample molecules to 100,000.



13 page pdf supplemental information

SI-1 A theoretical Model for DNA capture rate into nanopores in the diffusion-limited and barrier limited regimes.
APPENDIX: Calculation of the electrical potential in the vicinity of a nanopore
SI-2 Finite-element numerical evaluation of the electrical potential near a nanopore in the absence and presence of salt gradients.
SI-3 Measurements of the DNA capture rate as a function of bulk DNA concentration.
SI-4 DNA translocation dynamics for varying salt concentration in the cis chamber.
SI-5 Table of the translocation current signal-to-noise ratios (S/N) with asymmetric salt.
SI-6 Offset voltage and IV measurements under asymmetric salt conditions.
SI-7 Determination of the EOF direction with uncharged polymers.

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