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Jun 15, 2010 (Vol. 30, No. 12)

Next-Generation Sequencing Moves to Next-Next Level

As Technology Proves Its Mettle and Advances Expand Usage, Applications Are Proliferating

  • Electron Tunneling

    Click Image To Enlarge +
    As next-gen sequencing technology continues to be refined, scientists will gain a better understanding of inter-individual genomic variations as well as the complex interplay between genetic and epigenetic modifications. [Taraki/Fotolia]

    At the meeting, Stuart Lindsay, Ph.D., the Edward and Nadine Carson professor of physics and chemistry at Arizona State University, talked about recent advances that his group made in developing label-free sequencing by electron tunneling. It is known that, at moderate electrical fields, charged molecules such as DNA can translocate through single-wall nanotubes of approximately 2 nm in diameter, and the current generated by the nucleotide diffusion through the pore is sensitive to sequence, but the signal is averaged over several bases. An alternative is to measure the localized current owing to electron tunneling across the DNA.

    Electron tunneling, a quantum mechanical effect, can be confined to a space as small as a single base. This powerful technique presents several challenges such as the interference by contaminating molecules and fluctuations in the current that are caused by changes in atomic positions.

    To address these shortcomings, Dr. Lindsay and collaborators chemically functionalized one electrode with a reagent, 4-mercaptobenzoic acid, which binds the gold electrode through the sulphur atom to form monolayers, while its benzoic acid moiety is directed toward the solvent and forms hydrogen bonds with the passing nucleotides.

    The coupling that results is sufficiently weak to allow DNA to pass through the tunnel, but at the same time it is strong enough to restrict the molecular orientation of the nucleotides and generate a signal of increased selectivity with higher signal-to-noise ratio.

    As DNA enters the electron-gap tunnel, the current intensity indicates which nucleosides are passing and, as Dr. Lindsay and colleagues revealed, a distinctive signal can be generated for each of the four bases. “What we found is that we can generate distinct tunneling signatures for each of the four bases, and we can read the identity of a base with one read, which is pretty significant,” explained Dr. Lindsay.

  • Environmental Influences

    Next-generation sequencing provides a powerful tool to dissect the influences that environmental perturbations exert on the genome. In what became the first catalog of somatic mutations from a human cancer genome, Peter J. Campbell, Ph.D., group leader at the Wellcome Trust Sanger Institute’s Cancer Genome Project, and collaborators recently completed the sequencing of malignant melanoma and lymphoblastoid cell line genomes derived from the same person and, in a companion study, they examined the genomic mutational burden that accumulates in smokers who develop small-cell lung cancer.

    “We set out to use next-generation sequencing to examine genomic changes at a detail that was never looked at before, and we wanted to identify the vast majority or all mutations present in those cancers,” explained Dr. Campbell. The causes of these two cancers were relatively well characterized—melanoma is linked to sunlight, while lung cancer is caused by exposure to cigarette smoke.

    “We wanted to see if we are able to visualize the records of those exposures in the genome. And indeed that is what we found,” said Dr. Campbell. The investigators identified tens of thousands of mutations in both cancers, and the vast majority provided a hallmark that can be used to establish a direct link between exposure and mutations in the respective cells, in addition to signs of DNA repair processes that were functional in the respective cells.

    For example, over 22,000 somatic mutations, of which 134 were located within coding exons, were identified in small-cell lung cancer, providing a powerful signature that cigarette smoke leaves on the genome. In addition, the authors showed that over the lifetime of a cell clone that eventually becomes cancerous, one mutation would accumulate for approximately every 15 cigarettes smoked.

    Next-generation platforms allow genetic variation to be characterized in a high-throughput way, at a fraction of the cost that we were previously used to. “In the future, patients with cancer and other disorders can have their entire genome sequenced with the aim of understanding what is going on in that individual patient to cause that disease, and hopefully that will lead us to much better targeted therapeutic approaches,” added Dr. Campbell.

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