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    Determination of genotype of an amplification product at multiple allelic sites
    23.
    发明申请
    Determination of genotype of an amplification product at multiple allelic sites 审中-公开
    确定多个等位基因位点扩增产物的基因型

    公开(公告)号：US20080187913A1

    公开(公告)日：2008-08-07

    申请号：US11592716

    申请日：2006-11-03

    申请人： Kenneth J. Livak ,  Federico Goodsaid

    发明人： Kenneth J. Livak ,  Federico Goodsaid

    IPC分类号： C12Q1/68

    CPC分类号： C12Q1/6858 ,  C12Q1/6818 ,  C12Q2561/101 ,  C12Q2537/143 ,  C12Q2563/107 ,  C12Q2531/119

    摘要： A method is provided for genotyping a target sequence at least two allelic sites by a 5′ nuclease amplification reaction. In one embodiment, the method includes performing a nucleic acid amplification on a target sequence having at least two different allelic sites using a nucleic acid polymerase having 5′-3′ nuclease activity and a primer capable of hybridizing to the target sequence in the presence of two or more sets of allelic oligonucleotide probes wherein: each set of allelic oligonucleotide probes is for detecting a different allelic site of the target sequence, each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5′ relative to a sequence to which the primer hybridizes to the target sequence, and at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer; detecting a fluorescence spectrum of the amplification; calculating a fluorescence contribution of each fluorescer to the fluorescence spectrum; and determining a presence or absence of the different allelic variants at the two or more different allelic sites based on the fluorescence contribution of each fluorescer to the combined fluorescence spectrum.

    摘要翻译： 提供了一种通过5'核酸酶扩增反应将靶序列至少两个等位基因位点进行基因分型的方法。 在一个实施方案中，该方法包括使用具有5'-3'核酸酶活性的核酸聚合酶和能够与靶序列杂交的引物在具有至少两个不同等位基因位点的靶序列上进行核酸扩增 两组或更多组等位基因寡核苷酸探针，其中：每组等位基因寡核苷酸探针用于检测靶序列的不同等位基因位点，每组等位基因寡核苷酸探针包括与等位基因位点上的不同等位基因变体互补的两个或多个探针 被该组探针检测到，等位基因位点相对于引物与靶序列杂交的序列为5'，并且至少所有等位基因寡核苷酸探针中的所有等位基因寡核苷酸探针包括与其它探针不同的荧光剂和猝灭剂 位于探针上以淬灭荧光剂的荧光; 检测扩增的荧光光谱; 计算每个荧光剂对荧光光谱的荧光贡献; 并且基于每个荧光剂对组合的荧光光谱的荧光贡献，确定两个或更多个不同等位基因位点处的不同等位基因变体的存在或不存在。

    Binary probe and clamp composition
    24.
    发明授权
    Binary probe and clamp composition 有权
    二进制探针和夹具组成

    公开(公告)号：US07057025B2

    公开(公告)日：2006-06-06

    申请号：US10112677

    申请日：2002-03-28

    申请人： Kenneth J. Livak ,  Michael Egholm ,  Michael W. Hunkapiller

    发明人： Kenneth J. Livak ,  Michael Egholm ,  Michael W. Hunkapiller

    IPC分类号： C07H21/02 ,  C07H21/04 ,  C12Q1/68 ,  C12P19/34

    CPC分类号： C12Q1/6816 ,  C12Q1/6818 ,  C12Q2549/126 ,  C12Q2537/119 ,  C12Q2525/179

    摘要： Binary probe and clamp compositions conduct methods for target hybridization detection. Where the probe is a substrate for exonuclease cleavage, the composition provides quantitation and detection of PCR products, by real-time and end-point measurements. Where the probe is an amplification primer, the composition provides an improved method for labelling and detection of PCR products. Probes and clamps may be labelled with fluorescent dyes, quenchers, hybridization-stabilizing moieties, chemiluminescent dyes, and affinity ligands. Clamps may be nucleic acid analogs, such as 2-aminoethylglycine PNA.

    摘要翻译： 二进制探针和夹具组合物进行靶杂交检测的方法。 当探针是外切核酸酶切割的底物时，组合物通过实时和终点测量提供PCR产物的定量和检测。 当探针是扩增引物时，组合物提供了PCR产物的标记和检测的改进方法。 探针和夹具可以用荧光染料，猝灭剂，杂交稳定部分，化学发光染料和亲和配体标记。 夹具可以是核酸类似物，例如2-氨基乙基甘氨酸PNA。

    Binary probe and clamp composition and methods for a target hybridization detection
    26.
    发明授权
    Binary probe and clamp composition and methods for a target hybridization detection 有权
    用于靶杂交检测的二进制探针和夹钳组成及方法

    公开(公告)号：US06432642B1

    公开(公告)日：2002-08-13

    申请号：US09232000

    申请日：1999-01-15

    申请人： Kenneth J. Livak ,  Michael Egholm ,  Michael W. Hunkapiller

    发明人： Kenneth J. Livak ,  Michael Egholm ,  Michael W. Hunkapiller

    IPC分类号： C12Q168

    CPC分类号： C12Q1/6816 ,  C12Q1/6818 ,  C12Q2549/126 ,  C12Q2537/119 ,  C12Q2525/179

    摘要： Binary probe and clamp compositions conduct methods for target hybridization detection. Where the probe is a substrate for exonuclease cleavage, the composition provides quantitation and detection of PCR products, by real-time and end-point measurements. Where the probe is an amplification primer, the composition provides an improved method for labelling and detection of PCR products. Probes and clamps may be labelled with fluorescent dyes, quenchers, hybridization-stabilizing moieties, chemiluminescent dyes, and affinity ligands. Clamps may be nucleic acid analogs, such as 2-aminoethylglycine PNA.

    摘要翻译： 二进制探针和夹具组合物进行靶杂交检测的方法。 当探针是外切核酸酶切割的底物时，组合物通过实时和终点测量提供PCR产物的定量和检测。 当探针是扩增引物时，组合物提供了PCR产物的标记和检测的改进方法。 探针和夹具可以用荧光染料，猝灭剂，杂交稳定部分，化学发光染料和亲和配体标记。 夹具可以是核酸类似物，例如2-氨基乙基甘氨酸PNA。

    Methods using exogenous, internal controls and analogue blocks during nucleic acid amplification
    27.
    发明授权
    Methods using exogenous, internal controls and analogue blocks during nucleic acid amplification 失效
    在核酸扩增过程中使用外源，内部对照和模拟嵌段的方法

    公开(公告)号：US5952202A

    公开(公告)日：1999-09-14

    申请号：US48880

    申请日：1998-03-26

    申请人： Kazuko Aoyagi ,  Kenneth J. Livak

    发明人： Kazuko Aoyagi ,  Kenneth J. Livak

    IPC分类号： C07H21/00 ,  C12Q1/68 ,  C12P19/34 ,  C07H21/04

    CPC分类号： C07H21/00 ,  C12Q1/6818

    摘要： Reporter-quencher probe assays of nucleic acid amplification, such as PCR, are rendered more meaningful by the addition of internal control reagents. An internal control polynucleotide is amplified with internal control primers and the product is measured by correlation with increased fluorescence by polymerase mediated-exonuclease cleavage or hybridization of the internal control probe. Probes specific for target and internal control polynucleotides are labelled with spectrally resolvable reporters, allowing for concurrent detection and measurement of target and control amplification. A kit of all PCR reagents can be dispensed into reaction chambers in a high-throughput system for rapid and accurate nucleic acid amplification assay, with real-time or end-point measurements. Fluorescent signals correlated to target and internal control levels are spectrally resolvable and measured concurrently. A non-extending oligonucleotide or nucleic analog "block", complementary to the internal control polynucleotide, is added to the amplification mixture to preclude amplification of the internal control polynucleotide and function as an internal negative control. The amplification control reagents, kits, and methods of the present invention provide positive and negative control tests occurring within, and measurable within, the reaction chamber.

    摘要翻译： 通过添加内部对照试剂使得核酸扩增（例如PCR）的报道 - 猝灭剂探针测定变得更有意义。 内部对照多核苷酸用内部对照引物扩增，通过聚合酶介导的外切核酸酶切割或内部对照探针的杂交与荧光增加相关来测量产物。 靶向和内部控制多核苷酸的特异性探针用光谱可分辨的报告物标记，允许同时检测和测量靶标和对照扩增。 所有PCR试剂的试剂盒可以在高通量系统中分配到反应室中，用于快速准确的核酸扩增测定，具有实时或终点测量。 与目标和内部控制水平相关的荧光信号可以光谱解析和并发测量。 将与内部对照多核苷酸互补的非延伸寡核苷酸或核苷酸类似物“嵌段”加入到扩增混合物中，以排除内部对照多核苷酸的扩增并作为内部阴性对照起作用。 本发明的扩增控制试剂，试剂盒和方法提供在反应室内部和内部可测量的阳性和阴性对照试验。

    Passive internal references for the detection of nucleic acid amplification products
    28.
    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。

    Differentiation of nucleic acid segments on the basis of nucleotide differences
    29.
    发明授权
    Differentiation of nucleic acid segments on the basis of nucleotide differences 失效
    基于核苷酸差异分化核酸片段

    公开(公告)号：US4879214A

    公开(公告)日：1989-11-07

    申请号：US272068

    申请日：1988-11-15

    申请人： John S. Kornher ,  Kenneth J. Livak

    发明人： John S. Kornher ,  Kenneth J. Livak

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

    CPC分类号： C12Q1/6858 ,  C12Q1/6816 ,  C12Q1/6876 ,  C12Q2600/156

    摘要： A process for distinguishing nucleic acid segments on the basis of nucleotide differences, thereby providing a rapid, convenient means for detecting mutation, is disclosed. The present process comprises synthesizing separately complementary nucleic acid strands on each of at least two target nucleic acid templates using a nucleic acid polymerase and nucleoside triphosphate substrates, wherein at least one of the natural nucleoside triphosphate substrates is replaced with a mobility-shifting analog; denaturing the synthesized strands from the templates if necessary; and comparing the mobility of the separately synthesized strands through a size-fractionation medium. A difference in mobility indicates that the target nucleic acid templates contain different numbers of total nucleotides and/or different numbers of nucleotides complementary to the mobility-shifting analogs. Thus, the process of the present invention can be used to distinguish homologous DNAs or RNAs differing by nucleotide substitutions that affect the number of mobility-shifting analog residues incorporated into synthesized strands, as well as to distinguish differences due to nucleotide insertion or deletion.