FLUORESCENCE CORRELATION SPECTROSCOPY IN CELLS W/ TWO PHOTON EXCITATION

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In view of the utilization of human genome data the capability of sequencing the genetic information of single cells without prior amplification would potentiate speed and sensitivity of gene function recognition. We propose to carry out single molecule sequencing by following the stepwise addition of base pairs during production of the complementary DNA strand of a given (unknown) template strand by the enzyme DNA polymerase. The incorporation of each base at the polymerase active site is to be recognized by detection of a specific fluorescent marker. Preliminary studies on DNA synthesis on immobilized template strands utilized the fluorescent dye PicoGreen (Molecular Probes Inc.), and it is shown that individual molecules of newly synthesized DNA can be detected. Next, we attempt to detect the separate binding events of PicoGreen molecules to the newly generated DNA with a fluorescence correlation spectroscopy setup. The DNA sequencing approach will also utilize nanofabricated compon ents for DNA manipulation and possible fluid flow.