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    System for real time detection of nucleic acid amplification products
    1.
    发明授权
    System for real time detection of nucleic acid amplification products 失效
    核酸扩增产物实时检测系统

    公开(公告)号：US5928907A

    公开(公告)日：1999-07-27

    申请号：US752973

    申请日：1996-12-02

    申请人： Timothy M. Woudenberg ,  Kevin S. Bodner ,  Charles R. Connell ,  Alan M. Ganz ,  Lincoln J. McBride ,  Paul G. Saviano ,  John Shigeura ,  David H. Tracy ,  Eugene F. Young ,  Linda G. Lee

    发明人： Timothy M. Woudenberg ,  Kevin S. Bodner ,  Charles R. Connell ,  Alan M. Ganz ,  Lincoln J. McBride ,  Paul G. Saviano ,  John Shigeura ,  David H. Tracy ,  Eugene F. Young ,  Linda G. Lee

    IPC分类号： C12N15/09 ,  C12P19/34 ,  C12Q1/68 ,  G01N21/03 ,  G01N21/64 ,  G01N21/75 ,  G01N21/78 ,  G01N33/50 ,  G01N33/58

    CPC分类号： G01N21/6428 ,  C12Q1/6818 ,  C12Q1/6851 ,  C12Q1/686 ,  G01N21/0303 ,  G01N2021/6484

    摘要： A system is provided for carrying out real time fluorescence-based measurements of nucleic acid amplification products. In a preferred embodiment of the invention, an excitation beam is focused into a reaction mixture through a surface, the reaction mixture containing (i) a first fluorescent indicator capable of generating a first fluorescent signal whose intensity is proportional to the amount of an amplification product in the volume of the reaction mixture illuminated by the excitation beam and (ii) a second fluorescent indicator homogeneously distributed throughout the reaction mixture capable of generating a second fluorescent signal proportional to the volume of reaction mixture illuminated by the excitation beam. Preferably, the excitation beam is focused into the reaction mixture by a lens through a portion of a wall of a closed reaction chamber containing the reaction mixture. The same lens is used to collect the first and second fluorescent signals generated by the first and second fluorescent indicators, respectively, in response to the excitation beam. The ratio of the fluorescent intensities of the first and second fluorescent signals provides a stable quantitative indicator of the amount of amplification product synthesized in the course of the amplification reaction.

    摘要翻译： 提供了一种用于进行核酸扩增产物的实时基于荧光的测量的系统。 在本发明的优选实施方案中，激发光束通过表面聚焦到反应混合物中，反应混合物含有（i）第一荧光指示剂，其能够产生第一荧光信号，其强度与扩增产物的量成比例 在由激发束照射的反应混合物的体积中和（ii）均匀分布在整个反应混合物中的第二荧光指示器，其能够产生与由激发束照射的反应混合物的体积成比例的第二荧光信号。 优选地，激发光束通过透镜通过包含反应混合物的封闭反应室的壁的一部分聚焦到反应混合物中。 相同的透镜被用于响应于激发光束分别收集由第一和第二荧光指示器产生的第一和第二荧光信号。 第一和第二荧光信号的荧光强度比提供了在扩增反应过程中合成的扩增产物的量的稳定的定量指标。

    Passive internal references for the detection of nucleic acid amplification products
    3.
    发明授权
    Passive internal references for the detection of nucleic acid amplification products 失效
    被动内部参考用于检测核酸扩增产物

    公开(公告)号：US5736333A

    公开(公告)日：1998-04-07

    申请号：US657989

    申请日：1996-06-04

    申请人： Kenneth J. Livak ,  Lincoln J. McBride

    发明人： Kenneth J. Livak ,  Lincoln J. McBride

    IPC分类号： C12N15/09 ,  C12Q1/68 ,  C07H21/04 ,  C12P19/34

    CPC分类号： C12Q1/6851

    摘要： The invention relates to passive internal references for use in quantitating the formation of amplification products in a nucleic amplification reaction. The internal amplification reference molecules of the invention comprise a first and second fluorophore joined together through a backbone connector. The first and second fluorophores are joined on the backbone in a configuration that permits the energy transfer from the first fluorophore to the second fluorophore. The backbone connector is selected so as not to bind to the target nucleic acid sequence under nucleic acid amplification conditions. Preferably, the backbone connector is a polynucleotide. Another aspect of the invention is to provide passive internal reference molecule containing reagent compositions for use in nucleic acid amplification reactions. The compositions comprise the internal amplification reference molecule of the invention and a nucleic acid amplification reaction buffer. The reagent compositions, optionally, include additional components required for nucleic acid amplification reactions. The invention also provides improved methods of measuring the amount of amplification product in nucleic acid amplification reactions employing fluorescer-quencher probe assays, including methods for the real-time measurement of amplification product formation. The methods comprise the step of adding the internal reference molecule of the invention to the amplification reaction mixture. Fluorescence of the second fluorophore on the internal reference may then be measured and used to calculate changes in fluorescence of the fluorophore on a fluorescer-quencher probe.

    摘要翻译： 本发明涉及用于量化核酸扩增反应中扩增产物形成的无源内参考。 本发明的内部扩增参考分子包含通过骨架连接器连接在一起的第一和第二荧光团。 第一和第二荧光团以支持能量从第一荧光团转移到第二荧光团的构型在主链上连接。 选择骨架连接器，以便在核酸扩增条件下不与靶核酸序列结合。 优选地，骨架连接器是多核苷酸。 本发明的另一方面是提供含有用于核酸扩增反应的试剂组合物的被动内参照分子。 组合物包含本发明的内部扩增参考分子和核酸扩增反应缓冲液。 试剂组合物任选地包括核酸扩增反应所需的另外的组分。 本发明还提供了使用荧光猝灭剂探针测定法测量核酸扩增反应中扩增产物量的改进方法，包括用于实时测量扩增产物形成的方法。 所述方法包括将本发明的内部参考分子加入到扩增反应混合物中的步骤。 然后可以测量第二荧光团在内部参考物上的荧光，并用于计算荧光团在荧光猝灭剂探针上的荧光团的荧光变化。

    Nucleic acid detection with separation
    5.
    发明授权
    Nucleic acid detection with separation 失效
    分离核酸检测

    公开(公告)号：US6054266A

    公开(公告)日：2000-04-25

    申请号：US276139

    申请日：1988-11-23

    申请人： Mel N. Kronick ,  Douglas H. Keith ,  Lincoln J. McBride ,  Norman M. Whiteley ,  Michael W. Hunkapiller

    发明人： Mel N. Kronick ,  Douglas H. Keith ,  Lincoln J. McBride ,  Norman M. Whiteley ,  Michael W. Hunkapiller

    IPC分类号： G01N33/50 ,  C12Q1/68 ,  G01N30/88 ,  G01N33/58 ,  C07H21/04

    CPC分类号： C12Q1/6813 ,  C12Q1/6804 ,  C12Q1/6823

    摘要： Nucleic acid sequences are detected by a multi-step process, involving labeling sample nucleic acid sequences, duplexing the labeled sample with a probe having a coupling element, immobilizing all of the duplexed probe and target sequence and unduplexed probe, separating specifically immobilized nucleic acid from free and non-specifically immobilized nucleic acid, releasing specifically immobilized nucleic acid, and detecting the presence of the sequence of interest by means of the label. The labeled sequence may be characterized by sizing, e.g. electrophoresis. The method provides for a sensitive and rapid means for accurate detection of sequences of interest in a wide variety of situations.

    摘要翻译： 通过多步法检测核酸序列，包括标记样品核酸序列，用具有偶联元件的探针双链化标记的样品，固定所有双链体探针和靶序列和未折射的探针，将特异性固定的核酸与 游离和非特异性固定化的核酸，释放特异性固定化的核酸，并通过标记物检测感兴趣的序列的存在。 标记序列的特征可以是尺寸，例如 电泳。 该方法提供用于在各种情况下精确检测感兴趣序列的敏感且快速的方法。