The goal of this proposal is the development of a single-molecule fluorescence-based assay for quantitative detection of unamplified leukemia-specific sequences in RNA or genomic DNA samples. Single-molecule fluorescence methods provide the means to detect individual nucleic acid fragments that contain a specific target sequence. State of the art two-color single-molecule fluorescence flow cytometry permits detection of target fragments at sub-femtomolar concentrations. This extreme sensitivity eliminates the need to amplify the target prior to detection. As such, single-molecule fluorescence-based target detection is not subject to the limitations of polymerase chain reaction (PCR) amplification that include: target length limitations, false positives due to amplification of impurities and non-specific amplification, and difficulties with quantitative target detection due to the nature of the PCR amplification process. Successful development of a single molecule fluorescence-based assay will provide a powerful analytical tool for early leukemia diagnostics as well as for minimum residual disease detection. However, the advancement of single-molecule fluorescence methods has been hindered by two factors: inefficient probe hybridization/labeling of unamplified genomic targets at low, sub-picomolar target concentrations, and high fluorescence background contributed by excess, unbound probes. To eliminate these obstacles, we will develop methods for efficient target labeling and reduction of fluorescence background due to unbound probes. Finally, we will demonstrate the feasibility of the two-color single-molecule fluorescence approach for leukemia diagnostics by detection of unamplified leukemia RNA and DNA targets in samples obtained from well-characterized human cell lines.
