Nanopore Mechanism Allows Single Strand DNA Sequencing

The study, which was published in in the March 25, 2012, online edition of the journal Nature Biotechnology, was based on a nanopore structure that had been created by genetic engineering of a protein pore from Mycobacterium smegmatis. The diameter of the pore was such that only a single strand of DNA could pass through. A molecule of DNA polymerase was attached to the nanopore and acted to pull the DNA strand through the pore. The nanopore structure was placed in a membrane surrounded by potassium-chloride solution, with a small voltage applied to create an ion current flowing through the nanopore.

Details presented in the study revealed that the electrical signature produced by the pore changed depending on the type of nucleotide traveling through it. Each type of DNA nucleotide - cytosine, guanine, adenine, and thymine – produced a distinctive signature. The nanopore complex was tested with six different strands of DNA, and results corresponded to the already known DNA sequence of the strands, which had readable regions 42 to 53 nucleotides long.

“There is a clear path to a workable, easily produced sequencing platform,” said senior author Dr. Jens Gundlach, professor of physics at the University of Washington (Seattle, USA). “We augmented a protein nanopore we developed for this purpose with a molecular motor that moves a DNA strand through the pore a nucleotide at a time. The motor pulls the strand through the pore at a manageable speed of tens of milliseconds per nucleotide, which is slow enough to be able to read the current signal.”

“Epigenetic modifications are rather important for things like cancer," said Dr. Gundlach. “Being able to provide DNA sequencing that can identify epigenetic changes is one of the charms of the nanopore sequencing method. With techniques like this, it might get down to a 10-dollar or 15-minute genome project. It is moving fast.”

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