Gene sequencing by scanning molecular excitation microscopy. Progress report, June 1990--June 1993

We propose to complete the development of scanning molecular excitation microscopy to rapidly sequence DNA. This new type of scanned-tip microscopy is specifically designed to map individual DNA bases in a non-destructive fashion. Base recognition is based on the external heavy atom effect between a heavy atom label on a specific DNA base and a fluorescent sensor molecule at the end of a scanned optical tip. As the tip is scanned very close to the heavy atom its emissions will change intensity and wavelength. For sequencing, heavy-atom labeled single-stranded DNA molecules will be synthesized and oriented on a flat substrate such as mica. An optical tag an one end of each DNA molecule will be rapidly located at low resolution using conventional fluorescence or scanning near-field optical microscopy. Scanning with the same optical tip, the low-resolution path of the DNA will be mapped using long-range interactions such as attractive van der Waals forces or Forester energy transfer. Subsequently, the labeled bases will be mapped at better than 5{Angstrom} resolution with the same tip using the external heavy atom effect, and the coordinates stored and processed by computer. The proposed microscope could be automated to quickly sequence intact lambda clones, YACK,more » or genomic Not I fragments, making genomic sequencing much more rapid and economical.« less