Prof. David Deamer - UCSC Biomedical Research Faculty

Dave Deamer is the founder of the UCSC Nanopore Project, which has pioneered the use of ion channels ("nanopores") for the analysis of single RNA and DNA molecules. The proect is now co-directed by Deamer and Professor Mark Akeson and involves faculty collaborators from UCSC and other institutions, as well as numerous postdoctoral fellows, graduate and undergraduate students.

The research group uses a prototype ion channel for its detector, which is formed by alpha-hemolysin, a 33 kD protein secreted by Staphylococcus aureus. The heptamer assembly is about 10 nm in total length, with a relatively narrow beta-barrel segment that is believed to span the membrane barrier, and a broader segment (the 'mushroom cap') that extends into one of the aqueous compartments. The mouth of the channel is about 2.6 nm in diameter -- large enough to accommodate B-form duplex DNA. The pore then widens into a vestibule that abruptly narrows to a limiting aperture of 1.5 nm at about the level of membrane-solution interface. This limiting aperture is slightly larger in diameter than an extended single strand of DNA.

In their experimental set-up, single hemolysin channels are embedded in bilayers formed across a 20 micron aperture at the end of a Teflon patch tube that was developed in the laboratory. This horizontal bilayer configuration permits sampling of nucleic acids in very small volumes (tens of microliter) . This is ideally configured for complementary technologies such as fluorescence microscopy and atomic force microscopy. In principle, the patch tube coupled with a durable thin film could also be moveable and miniaturized to the size of an intracellular glass electrode.

In early proof of principle experiments, the Deamer laboratory, along with colleagues at NIST and Harvard, used reduction of this otherwise unimpeded ionic current to detect individual single-stranded DNA and RNA molecules as they traversed the a-hemolysin pore [Kasianowicz et al., 1996]. As predicted by the crystallographic model, ssDNA (but not dsDNA) caused ionic current blockades characteristic of translocation events and DNA in the trans compartment could be amplified by PCR. The polynucleotide blockade durations were strand-length dependent, additional evidence that DNA traverses the pore as an extended single strand.

Since the original publication in 1996, the Santa Cruz nanopore group has made significant progress on real-time analysis of single RNA and single DNA molecules. A dozen other laboratories worldwide are also investigating nanopores as analytical devices. Deamer, Akeson, and their research associates use two operating modes to analyze DNA. In the first, a constant applied voltage threads single-stranded DNA or RNA across the pore in a linear manner. This permits discriminattion among single polymer molecules based on their nucleotide composition and to read along RNA or DNA block copolymers at roughly 20-nucleotide precision. The second approach is to capture a single duplex DNA molecule in the channel vestibule without translocation. Over a period of tens-to-hundreds of milliseconds, the dynamics of the base-pairs in the vestibule can be examined. Once the analysis is complete, the voltage is reversed, ejecting the DNA duplex back into the cis compartment. The cycle can be repeated many times, analyzing several thousand duplex molecules in minutes. In this voltage-pulse or 'tasting' mode, single base-pair precision is achieved, suggesting that single nucleotide polymorphism (SNP) or point mutation analysis on individual molecules is feasible. The fact that the hemolysin nanopore can achieve single base pair resolution is encouraging, and with colleagues at Harvard Deamer and Akeson are now developing a synthetic nanopore device that may ultimately be applicable in high throughput DNA sequencing. This research is supported by a grant from NHGRI, the division of NIH that funds research on the human genome.

The Nanopore Project would like to acknowledge the generous support of the National Human Genome Research Institute of the NIH