Methods and compositions for detecting telomerase activity
    1.
    发明授权
    Methods and compositions for detecting telomerase activity 有权
    检测端粒酶活性的方法和组合物

    公开(公告)号:US07390621B2

    公开(公告)日:2008-06-24

    申请号:US10534978

    申请日:2003-11-12

    IPC分类号: C12Q1/68 C12P19/34

    摘要: A method for determining telomerase activity using primer extension followed with real time PCR quantification is disclosed. The method of the present invention provides a rapid, sensitive and accurate measurement for telomerase activity in a biological sample. In one embodiment, the method includes the steps of: adding the biological sample to a reaction tube containing a first reaction mixture having a first primer and nucleoside triphosphates, a second reaction mixture having a second primer and a DNA polymerase, and a wax layer that separates the first reaction mixture from the second reaction mixture; incubating the biological sample with the first reaction mixture; admixing the extension product with the second reaction mixture; amplifying and quantifying the extension product using real-time PCR and a control template. In another embodiment, the detection method includes an in situ primer extension step that allows the production of the extension product within an intact cell. In this embodiment, the extension product can be preserved under appropriate conditions for an extended time before the completion of the quantification step.

    摘要翻译: 公开了使用引物延伸测定端粒酶活性的方法,随后进行实时PCR定量。 本发明的方法提供对生物样品中端粒酶活性的快速,灵敏和准确的测量。 在一个实施方案中,该方法包括以下步骤:将生物样品加入到含有具有第一引物和核苷三磷酸的第一反应混合物的反应管中,具有第二引物和DNA聚合酶的第二反应混合物和蜡层, 将第一反应混合物与第二反应混合物分离; 用第一反应混合物孵育生物样品; 将延伸产物与第二反应混合物混合; 使用实时PCR扩增和定量扩增产物和对照模板。 在另一个实施方案中,检测方法包括原位引物延伸步骤,其允许在完整细胞内产生延伸产物。 在本实施例中,扩展产品可以在适当的条件下在量化步骤完成之前延长一段时间。