DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

31. New Optical Methods for Sequencing Individual Molecules of DNA

Jonas Korlach, Michael Levene, Stephen W. Turner, Mathieu Foquet, Harold G. Craighead, and Watt W. Webb

Applied & Engineering Physics, Clark Hall, Cornell University, Ithaca, NY 14853

jk109@cornell.edu

A new method for determining the base pair sequence of a single molecule of DNA by following the dynamical stepwise activity of DNA polymerase synthesizing the complementary strand of a given template strand is under development. The technical challenges consist in the development of suitable enzymatic systems and in the recognition of individual sequential base additions. Replacing spatial resolution of bases in the DNA by temporal resolution of sequential nucleotide additions is made possible by using near-field and multiphoton laser optics for chromophore processing, and time-resolved photon counting for detection. Confinement of the excitation volume far below the diffraction limit by nanostructured devices permits an increase of substrate concentrations by about three orders of magnitude above the nanomolar range (required for the enzymatic systems under study), but still allowing sequential single molecule recordings and analysis.

The approach should enable the creation of a very fast sequencing protocol with long read lengths, and potentially highly parallel, integrated systems with large throughput. Each development step towards the sequencing goal appears fertile for the generation and improvement of analytic research systems capable of following biochemical and molecular biological processes (e.g., enzymatic activities) at the single molecule level. The optical tools will enable a characterization of these processes previously unattainable by conventional biochemical analysis. Specifically in respect to the sequencing proposal, this amounts to new knowledge of the photophysical and dynamical behavior of single DNA molecules, the generation and use of new fluorescent labels that can be incorporated into DNA in high densities, and the study of enzymes acting on DNA at the level of individual bases.