This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.
| Frumkin, Jesse P; Patra, Biranchi N; Sevold, Anthony et al. (2016) The interplay between chromosome stability and cell cycle control explored through gene-gene interaction and computational simulation. Nucleic Acids Res 44:8073-85 |
| Johnson, Leah M; Gao, Lu; Shields IV, C Wyatt et al. (2013) Elastomeric microparticles for acoustic mediated bioseparations. J Nanobiotechnology 11:22 |
| Micheva-Viteva, Sofiya N; Shou, Yulin; Nowak-Lovato, Kristy L et al. (2013) c-KIT signaling is targeted by pathogenic Yersinia to suppress the host immune response. BMC Microbiol 13:249 |
| Ai, Ye; Sanders, Claire K; Marrone, Babetta L (2013) Separation of Escherichia coli bacteria from peripheral blood mononuclear cells using standing surface acoustic waves. Anal Chem 85:9126-34 |
| Sanders, Claire K; Mourant, Judith R (2013) Advantages of full spectrum flow cytometry. J Biomed Opt 18:037004 |
| Cushing, Kevin W; Piyasena, Menake E; Carroll, Nick J et al. (2013) Elastomeric negative acoustic contrast particles for affinity capture assays. Anal Chem 85:2208-15 |
| Piyasena, Menake E; Austin Suthanthiraraj, Pearlson P; Applegate Jr, Robert W et al. (2012) Multinode acoustic focusing for parallel flow cytometry. Anal Chem 84:1831-9 |
| Austin Suthanthiraraj, Pearlson P; Piyasena, Menake E; Woods, Travis A et al. (2012) One-dimensional acoustic standing waves in rectangular channels for flow cytometry. Methods 57:259-71 |
| Vuyisich, Momchilo; Sanders, Claire K; Graves, Steven W (2012) Binding and cell intoxication studies of anthrax lethal toxin. Mol Biol Rep 39:5897-903 |
| Chaudhary, Anu; Ganguly, Kumkum; Cabantous, Stephanie et al. (2012) The Brucella TIR-like protein TcpB interacts with the death domain of MyD88. Biochem Biophys Res Commun 417:299-304 |
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