Single-Molecule Fluorescence Detection of Leukemia

白血病的单分子荧光检测

• 批准号: 6709668
• 负责人: PETER Marvin GOODWIN
• 金额: $ 15.42万
• 依托单位: UNIVERSITY OF CALIF-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6709668
• 关键词: DNA; RNA; biomedical equipment development; biotechnology; cell line; diagnosis design /evaluation; diagnostic tests; early diagnosis; flow cytometry; fluorescence; fluorescent dye /probe; fusion gene; leukemia; messenger RNA; method development; neoplasm /cancer diagnosis; nucleic acid probes; nucleic acid quantitation /detection; oligonucleotides

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.

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测定方法，用于定量检测 RNA 或基因组 DNA 样品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可以检测亚飞摩尔浓度的目标片段。这种极高的灵敏度消除了在检测前放大目标的需要。因此，基于单分子荧光的目标检测不受聚合酶链式反应（PCR）扩增的限制，包括：目标长度限制、由于杂质扩增和非特异性扩增而导致的假阳性以及定量目标的困难由于 PCR 扩增过程的性质而导致的检测。单分子荧光检测的成功开发将为早期白血病诊断以及最小残留疾病检测提供强大的分析工具。然而，单分子荧光方法的进步受到两个因素的阻碍：低效的探针杂交/在亚皮摩尔目标浓度下对未扩增的基因组目标进行标记，以及过量的未结合探针造成的高荧光背景。为了消除这些障碍，我们将开发有效的目标标记和减少未结合探针引起的荧光背景的方法。最后，我们将通过检测从充分表征的人类细胞系获得的样本中未扩增的白血病 RNA 和 DNA 靶标，证明双色单分子荧光方法用于白血病诊断的可行性。