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    Detection of halogenated precursors incorporated into DNA
    6.
    发明授权
    Detection of halogenated precursors incorporated into DNA 失效
    检测掺入DNA的卤代前体

    公开(公告)号：US5747258A

    公开(公告)日：1998-05-05

    申请号：US667294

    申请日：1996-06-20

    申请人： Zbigniew D. Darzynkiewicz ,  Xun Li ,  Frank N. Traganos ,  Myron R. Melamed

    发明人： Zbigniew D. Darzynkiewicz ,  Xun Li ,  Frank N. Traganos ,  Myron R. Melamed

    IPC分类号： C12Q1/68 ,  C12Q1/02 ,  G01N21/75 ,  G01N21/76

    CPC分类号： C12Q1/6841 ,  C12Q1/68 ,  Y10T436/19

    摘要： A method for detecting halogenated precursors incorporated into DNA is presented. The method is based on the selective photolysis of DNA by ultraviolet (UV) light at the sites of an incorporated halogenated precursor, such as the thymidine base analogs 5-bromo-2-deoxyuridine (BrdUrd), 5-iodo-2-deoxyuridine (IdUrd), 5-fluoro-2-deoxyuridine (FdUrd), or 5-chloro-2-deoxyuridine (CldUrd). The 3'-hydroxyl termini of the DNA single strand breaks generated during photolysis may be marked directly or indirectly with a fluorescent label. The DNA termini are directly labeled with fluorochrome-conjugated deoxyuridine triphosphate (dUTP) catalyzed by exogenous terminal deoxynucleotidyl transferase or DNA polymerase (nick translation system). The DNA termini are indirectly labeled with either biotin- or digoxygenin-conjugated dUTP; the incorporated biotin or digoxygenin is then detected following binding of fluorochrome-conjugated avidin or anti-digoxygenin antibody, respectively. The labeled DNA may be analyzed in situ by flow cytometry or fluorescence microscopy. Alternatively, the DNA may be isolated and analyzed by conventional methods, including gel electrophoresis and blotting assays, prior to marking with a flourescent label. The method does not require denaturation of the DNA and may be used with cells in suspension, thin tissue sections, bacteria, and viruses. The method has application in the analysis of cell proliferation and genotoxicity tests.

    摘要翻译： 提出了一种检测掺入DNA中的卤代前体的方法。 该方法基于在掺入的卤化前体如胸苷碱基类似物5-溴-2-脱氧尿苷（BrdUrd），5-碘-2-脱氧尿苷（BrdUrd）的位置处的紫外线（UV）光的DNA的选择性光解 IdUrd），5-氟-2-脱氧尿苷（FdUrd）或5-氯-2-脱氧尿苷（CldUrd）。 在光解过程中产生的DNA单链断裂的3'-羟基末端可以用荧光标记直接或间接标记。 DNA末端用外源末端脱氧核苷酸转移酶或DNA聚合酶（切口翻译系统）催化的荧光染料缀合的脱氧尿苷三磷酸（dUTP）直接标记。 DNA末端用生物素或地加氧联素缀合的dUTP间接标记; 然后在分别与荧光染料缀合的抗生物素蛋白或抗 - 地高辛素抗体结合后检测掺入的生物素或地加氧原素。 标记的DNA可以通过流式细胞术或荧光显微镜原位分析。 或者，可以在用荧光标记标记之前通过常规方法（包括凝胶电泳和印迹测定）分离和分析DNA。 该方法不需要DNA的变性，并且可以与悬浮液，薄组织切片，细菌和病毒中的细胞一起使用。 该方法在细胞增殖和基因毒性试验分析中具有应用前景。