Single-Molecule Fluorescence Detection of Leukemia

描述(由申请人提供)：本提案的目标是开发一种基于单分子荧光的分析方法，用于定量检测RNA或基因组DNA样本中未扩增的白血病特异性序列。单分子荧光方法提供了检测包含特定靶序列的单个核酸片段的方法。最先进的双色单分子荧光流式细胞术允许在亚毫微摩尔浓度下检测目标片段。这种极端的灵敏度消除了在检测之前放大目标的需要。因此，基于单分子荧光的靶标检测不受聚合酶链式反应(PCR)扩增的限制，这些限制包括：靶标长度限制，由于放大杂质和非特异性扩增而导致的假阳性，以及由于PCR扩增过程的性质而导致的定量靶标检测困难。单分子荧光检测的成功开发将为白血病的早期诊断和最小残留疾病检测提供强大的分析工具。然而，单分子荧光方法的发展受到两个因素的阻碍：在低的亚皮摩尔目标浓度下对未扩增的基因组靶进行低效的探针杂交/标记，以及过量的未结合的探针贡献的高荧光背景。为了消除这些障碍，我们将开发有效的靶标记法和由于未结合的探针而减少荧光背景的方法。最后，我们将通过检测从具有良好特性的人类细胞系获得的样本中未扩增的白血病RNA和DNA靶标，来证明双色单分子荧光方法用于白血病诊断的可行性。

DESCRIPTION (provided by applicant): 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.