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1. 发明申请

US20130045881A1 Probe Based Nucleic Acid Detection 有权
标题翻译：基于探针的核酸检测
公开(公告)号：US20130045881A1
公开(公告)日：2013-02-21
申请号：US13467933
申请日：2012-05-09
申请人： Kenneth J. Livak , Stacey N. Myers , Jun Wang , Xiaohui Wang
发明人： Kenneth J. Livak , Stacey N. Myers , Jun Wang , Xiaohui Wang
IPC分类号： G01N21/64 , C40B30/04
CPC分类号： C12Q1/6818 , C12Q2525/151
摘要： The invention provides a method for detecting a target nucleotide sequence by tagging the nucleotide sequence with a nucleotide tag, providing a probe oligonucleotide with a melting temperature Tm1, comprising a regulatory sequence and a nucleotide tag recognition sequence; incorporating the probe oligonucleotide into the tagged polynucleotide in a polynucleotide amplification reaction, providing a regulatory oligonucleotide with a melting temperature Tm2, comprising a sequence segment that is at least partially complementary to the regulatory sequence, amplifying the tagged target nucleic acid sequence in a PCR amplification reaction using the probe oligonucleotide as a primer, and detecting the amplification product; wherein Tm1 and Tm2 are higher than the annealing temperature associated with the polynucleotide amplification reaction.
摘要翻译：本发明提供了通过用核苷酸标签标记核苷酸序列来检测靶核苷酸序列的方法，提供了具有调节序列和核苷酸标签识别序列的融解温度Tm1的探针寡核苷酸; 在多核苷酸扩增反应中将探针寡核苷酸掺入标记的多核苷酸中，提供具有解链温度Tm2的调节性寡核苷酸，其包含与调节序列至少部分互补的序列片段，在PCR扩增中扩增标记的靶核酸序列使用探针寡核苷酸作为引物的反应，检测扩增产物; 其中Tm1和Tm2高于与多核苷酸扩增反应相关的退火温度。

2. 发明申请

US20120288857A1 MULTIFUNCTIONAL PROBE-PRIMERS 审中-公开
标题翻译：多功能探测器
公开(公告)号：US20120288857A1
公开(公告)日：2012-11-15
申请号：US13366178
申请日：2012-02-03
申请人： Kenneth J. Livak
发明人： Kenneth J. Livak
IPC分类号： C07H21/00 , G01N21/64 , C12Q1/68
CPC分类号： C12Q1/6876 , C12Q1/6818 , C12Q1/6823 , C12Q2525/161 , C12Q2525/197 , C12Q2565/1015
摘要： Methods and reagents for detection and analysis of nucleic acids are provided. Certain methods involves an encoding amplification in which a target sequence is associated with probe-binding sequences and optionally with indexing sequences, (2) an optional distribution step in which the product of the encoding amplification is split into multiple aliquots, and (3) a decoding and detection step in which the presence, absence, quantity, or relative amount of the target sequence in the aliquots is determined. The detection step makes use of a multifunctional “self-digesting” molecular probe comprising a primer polynucleotide and a probe oligonucleotide, linked in a 5′-5′ orientation.
摘要翻译：提供了用于检测和分析核酸的方法和试剂。某些方法涉及编码扩增，其中靶序列与探针结合序列和任选的索引序列相关，（2）任选的分布步骤，其中编码扩增的产物被分成多个等分试样，和（3）a 解码和检测步骤，其中确定等分试样中靶序列的存在，不存在，数量或相对量。检测步骤使用包含以5'-5'方向连接的引物多核苷酸和探针寡核苷酸的多功能自消化分子探针。

3. 发明申请

US20120115143A1 Universal Probe Assay Methods 有权
标题翻译：通用探针测定方法
公开(公告)号：US20120115143A1
公开(公告)日：2012-05-10
申请号：US13280214
申请日：2011-10-24
申请人： Kenneth J. Livak , Jason A. A. West , Robert C. Jones
发明人： Kenneth J. Livak , Jason A. A. West , Robert C. Jones
IPC分类号： C12Q1/68
CPC分类号： C12Q1/682 , C12Q1/6876 , C12Q2600/178 , C12Q2525/155 , C12Q2525/307 , C12Q2537/161
摘要： Reagents and methods are provided for detecting the presence of a target polynucleotide in a sample are disclosed. In one aspect, a method for producing a labeled amplification product by amplifying a target nucleic acid sequence to produce an amplification product comprising the target sequence, a first probe-binding sequence 5′ to the target sequence, and a second probe-binding sequence 3′ to the target sequence, thereby producing an amplification product; and hybridizing a first detection probe to the amplification product, said first detection probe comprising a first segment that hybridizes to the first probe-binding sequence and a second segment that hybridizes to the second probe-binding sequence, thereby producing a labeled amplification product is disclosed.
摘要翻译：提供了用于检测样品中靶多核苷酸的存在的试剂和方法。一方面，通过扩增靶核酸序列以产生包含靶序列的扩增产物，与靶序列的第一探针结合序列5'和第二探针结合序列3来产生标记的扩增产物的方法 '到靶序列，从而产生扩增产物; 并将第一检测探针与扩增产物杂交，所述第一检测探针包括与第一探针结合序列杂交的第一片段和与第二探针结合序列杂交的第二片段，从而产生标记的扩增产物。

4. 发明授权

US07601821B2 Encoding and decoding reactions for determining target molecules 失效
标题翻译：用于确定靶分子的编码和解码反应
公开(公告)号：US07601821B2
公开(公告)日：2009-10-13
申请号：US11090830
申请日：2005-03-24
申请人： Mark R. Andersen , Kenneth J. Livak , Adam Broomer , Caifu Chen
发明人： Mark R. Andersen , Kenneth J. Livak , Adam Broomer , Caifu Chen
IPC分类号： C07H21/04 , C12Q1/68
CPC分类号： C12Q1/6869 , C12Q1/6851 , C12Q2525/191 , C12Q2525/185 , C12Q2521/501 , C12Q2525/155 , C12Q2521/313 , C12Q2537/143 , C12Q2525/161
摘要： The present invention is directed to methods, reagents, and kits for detecting the presence or absence of (or quantifying) target polynucleotide sequences and proteins in at least one sample using encoding and decoding reactions. When a particular target polynucleotide is present in a sample for example, a reaction product is formed in the encoding reaction that includes addressable primer portions. At least one labeling probe and at least one address primer can be employed in the decoding amplification reaction thereby providing a detectable signal value depending upon whether a sequence is present or absent. In some embodiments, the encoding comprises a ligation reaction with linker probes, and single nucleotide polymorphisms (SNPs) are analyzed.
摘要翻译：本发明涉及用于使用编码和解码反应检测至少一个样品中靶多核苷酸序列和蛋白质的存在或不存在（或定量）的方法，试剂和试剂盒。当特定靶多核苷酸例如存在于样品中时，在包含可寻址引物部分的编码反应中形成反应产物。可以在解码扩增反应中使用至少一个标记探针和至少一个地址引物，从而根据序列是否存在提供可检测的信号值。在一些实施方案中，编码包含与接头探针的连接反应，并分析单核苷酸多态性（SNP）。

5. 发明授权

US6030787A Hybridization assay using self-quenching fluorescence probe 有权
标题翻译：使用自熄荧光探针的杂交测定
公开(公告)号：US6030787A
公开(公告)日：2000-02-29
申请号：US207170
申请日：1998-12-07
申请人： Kenneth J. Livak , Susan J. A. Flood , Jeffrey Mamoro , Khairuzzaman Bashar Mullah
发明人： Kenneth J. Livak , Susan J. A. Flood , Jeffrey Mamoro , Khairuzzaman Bashar Mullah
IPC分类号： C12N15/09 , C07H21/00 , C12N15/00 , C12Q1/68 , C07H19/04 , C07H21/04
CPC分类号： C12Q1/6818 , C12Q1/6834 , C12Q1/6837
摘要： A hybridization assay is provided which uses an oligonucleotide probe which includes a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule. The oligonucleotide probe is constructed such that the probe exists in at least one single-stranded conformation when unhybridized where the quencher molecule is near enough to the reporter molecule to quench the fluorescence of the reporter molecule. The oligonucleotide probe also exists in at least one conformation when hybridized to a target polynucleotide where the quencher molecule is not positioned close enough to the reporter molecule to quench the fluorescence of the reporter molecule. By adopting these hybridized and unhybridized conformations, the reporter molecule and quencher molecule on the probe exhibits different fluorescence signal intensities when the probe is hybridized and unhybridized. As a result, it is possible to determine whether the probe is hybridized or unhybridized based on a change in the fluorescence intensity of the reporter molecule, the quencher molecule, or a combination thereof. In addition, because the probe can be designed such that the quencher molecule quenches the reporter molecule when the probe is not hybridized, the probe can be designed such that the reporter molecule exhibits limited fluorescence until the probe is either hybridized or digested.
摘要翻译：提供了使用寡核苷酸探针的杂交测定法，其包括荧光报告分子和能够猝灭报告分子荧光的猝灭剂分子。构建寡核苷酸探针，使得探针在未杂交的情况下以至少一个单链构象存在，其中猝灭剂分子足够接近报道分子以淬灭报道分子的荧光。当寡核苷酸探针与靶多核苷酸杂交时，寡核苷酸探针也存在至少一个构象，其中猝灭剂分子不能与报道分子紧密接近以猝灭报道分子的荧光。通过采用这些杂交和非杂交构象，当探针杂交和未杂交时，探针上的报道分子和猝灭剂分子表现出不同的荧光信号强度。结果，可以基于报道分子，猝灭剂分子或其组合的荧光强度的变化来确定探针是杂交还是未杂交。此外，因为探针可以被设计成使得当探针不杂交时，猝灭剂分子淬灭报告分子，所以探针可被设计成使得报告分子在荧光探针杂交或消化之前显示有限的荧光。

6. 发明授权

US5962233A Determination of a genotype of an amplification product at multiple allelic sites 失效
公开(公告)号：US5962233A
公开(公告)日：1999-10-05
申请号：US18595
申请日：1998-02-04
申请人： Kenneth J. Livak , Federico Goodsaid
发明人： Kenneth J. Livak , Federico Goodsaid
摘要： A method is provided for genotyping a target sequence at 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'.fwdarw.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, andat 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; anddetermining 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.

7. 发明授权

US5538848A Method for detecting nucleic acid amplification using self-quenching fluorescence probe 失效
标题翻译：使用自熄荧光探针检测核酸扩增的方法
公开(公告)号：US5538848A
公开(公告)日：1996-07-23
申请号：US340558
申请日：1994-11-16
申请人： Kenneth J. Livak , Susan J. A. Flood , Jeffrey Marmaro
发明人： Kenneth J. Livak , Susan J. A. Flood , Jeffrey Marmaro
IPC分类号： C12N15/09 , C07H21/00 , C12N15/00 , C12Q1/68 , C07H21/04 , C12P19/34 , C12Q1/70
CPC分类号： C12Q1/6818 , C12Q1/6834 , C12Q1/6837
摘要： A method is provided for monitoring the progress of nucleic acid amplifications that rely on a nucleic acid polymerase having 5'.fwdarw.3' exonuclease activity. An important feature of the method is providing an oligonucleotide probe having a reporter molecule and a quencher molecule at either end such that the quencher molecule substantially quenches any fluorescence from the reporter whenever the oligonucleotide probe is in a single stranded state and such that the reporter is substantially unquenched whenever the oligonucleotide probe is in a double stranded state hybridized to a target polynucleotide.
摘要翻译：提供了一种监测依赖于具有5'→3'核酸外切酶活性的核酸聚合酶的核酸扩增进程的方法。该方法的一个重要特征是提供了在任一端具有报告分子和猝灭剂分子的寡核苷酸探针，使得猝灭剂分子在寡核苷酸探针处于单链状态时基本上使来自报道分子的任何荧光猝灭，每当寡核苷酸探针处于与靶多核苷酸杂交的双链状态时，基本上未被消除。

8. 发明申请

US20130022973A1 Multiplex Amplification for the Detection of Nucleic Acid Variations 审中-公开
标题翻译：用于检测核酸变异的多重扩增
公开(公告)号：US20130022973A1
公开(公告)日：2013-01-24
申请号：US13521400
申请日：2011-01-14
申请人： Carl L. G. Hansen , Oleh Petriv , Kevin Heyries , Kenneth J. Livak
发明人： Carl L. G. Hansen , Oleh Petriv , Kevin Heyries , Kenneth J. Livak
IPC分类号： C12Q1/68 , G01N21/64
CPC分类号： C12Q1/6851 , C12Q1/6883 , C12Q2600/156 , C12Q2600/16 , C12Q2537/143 , C12Q2537/165 , C12Q2563/159
摘要： Kits, primers, and methods are provided herein for detecting relative target source to reference source ratios in a biological sample, by distributing the biological sample into discrete subsamples, wherein the biological sample includes, a plurality of target molecules on a target source; and a plurality of reference molecules on a reference source; providing target primers directed to one or more of the plurality of target molecules and reference primers directed to one or more of the plurality of reference molecules; performing digital amplification with the target primers and the reference primers; and detecting the presence or absence of amplified target products with target probes and detecting the presence or absence of amplified reference products with reference probes, wherein the ratio of amplified target products to amplified reference products is indicative of a relative amount of target source to reference source in a biological sample.
摘要翻译：本文提供了试剂盒，引物和方法，用于通过将生物样品分配到离散的子样品中来检测生物样品中的相对目标来源参考源比例，其中生物样品包括目标源上的多个靶分子; 和参考源上的多个参考分子; 提供指向所述多个靶分子中的一个或多个的靶引物和指向所述多个参考分子中的一个或多个的参考引物; 用目标引物和参照引物进行数字扩增; 并用靶探针检测扩增的靶产物的存在或不存在，并用参考探针检测扩增的参考产物的存在或不存在，其中扩增的靶产物与扩增的参考产物的比率指示靶源与参考源的相对量在生物样品中。

9. 发明申请

US20120053069A1 Determination of a Genotype of an Amplification Product at Multiple Allelic Sites 审中-公开
标题翻译：确定多个等位基因位点扩增产物的基因型
公开(公告)号：US20120053069A1
公开(公告)日：2012-03-01
申请号：US12618686
申请日：2009-11-13
申请人： Kenneth J. LIVAK , Federico Goodsaid
发明人： Kenneth J. LIVAK , Federico Goodsaid
IPC分类号： C40B30/04 , C40B40/06 , 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 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 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 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'核酸酶扩增反应在至少两个等位基因位点对靶序列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多组等位基因寡核苷酸探针，其中等位基因寡核苷酸探针中的至少全部除一个之外包括与其它探针不同的荧光剂，以及位于探针上的猝灭剂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来确定不同等位基因变体的存在或不存在。

10. 发明申请

US20110251083A1 Multiplexed Amplification of Short Nucleic Acids 审中-公开
标题翻译：短核酸的多重扩增
公开(公告)号：US20110251083A1
公开(公告)日：2011-10-13
申请号：US12762272
申请日：2010-04-16
申请人： Kai Qin LAO , Kenneth J. Livak , Neil A. Straus
发明人： Kai Qin LAO , Kenneth J. Livak , Neil A. Straus
IPC分类号： C40B30/04 , C12P19/34 , C40B40/06 , C12Q1/68
CPC分类号： C12N15/1065 , C12Q1/6851 , C12Q1/686 , C12Q2525/301 , C12Q2525/161 , C12Q2563/179 , C12Q2537/143 , C12Q2525/207
摘要： The present teachings provide methods, compositions, and kits for reverse transcribing and amplifying small nucleic acids such as micro RNAs. High levels of multiplexing are provided by the use of a zip-coded stem-loop reverse transcription primer, along with a PCR-based pre-amplification reaction that comprises a zip-coded forward primer. Detector probes in downstream decoding PCRs can take advantage of the zip-code introduced by the stem-loop reverse transcription primer. In some embodiments, further amplification is achieved by cycling the reverse transcription reaction. The present teachings also provide compositions and kits useful for performing the reverse transcription and amplification reactions described herein.
摘要翻译：本教导提供用于逆转录和扩增小核酸如微RNA的方法，组合物和试剂盒。通过使用zip编码的茎 - 环逆转录引物以及包含经翻译的正向引物的基于PCR的预扩增反应来提供高水平的复用。下游解码PCR中的检测器探针可以利用茎环逆转录引物引入的zip码。在一些实施方案中，通过循环逆转录反应来实现进一步的扩增。本教导还提供可用于进行本文所述的逆转录和扩增反应的组合物和试剂盒。

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