Faster, Less Expensive DNA Sequencing Method Developed

In a study published in the December 20, 2009, online edition of the journal Nature Nanotechnology, a team led by Boston University (BU; MA, USA) biomedical engineering associate professor Dr. Amit Meller detailed ground-breaking work in detecting DNA molecules as they pass through silicon nanopores. The method utilizes electrical fields to feed long strands of DNA through four-nanometer-wide pores, much like threading a needle. The technique uses sensitive electrical current measurements to detect single DNA molecules as they pass through the nanopores.

"The current study shows that we can detect a much smaller amount of DNA sample than previously reported,” said Dr. Meller. "When people start to implement genome sequencing or genome profiling using nanopores, they could use our nanopore capture approach to greatly reduce the number of copies used in those measurements.”

Currently, genome sequencing utilizes DNA amplification to make billions of molecular copies in order to generate a sample large enough to be studied. In addition to the time and cost DNA amplification entails, some of the molecules--similar to photocopies of photocopies--come out less than perfect. Dr. Meller and his colleagues at BU, New York University (New York, NY, USA) and Bar-Ilan University (Ramat-Gan, 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 researchers had to develop an understanding of electrophysics at the nanoscale, where the rules that govern the larger world do not necessarily apply. They made a counterintuitive finding: the longer the DNA strand, the more quickly it found the pore opening.

"That's really surprising,” Dr. Meller noted. "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 base pairs. 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.”

Utilizing this knowledge, Dr. Meller and his coworkers 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 precise measurements. Instead of floating around until they happened upon a nanopore, DNA strands were channeled into the openings.

By increasing 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 approximately 1 billion sample molecules to 100,000.


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