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    Encoding and decoding reactions for determining target molecules
    1.
    发明授权
    Encoding and decoding reactions for determining target molecules 失效
    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）。

    Multiplex Amplification for the Detection of Nucleic Acid Variations
    3.
    发明申请
    Multiplex Amplification for the Detection of Nucleic Acid Variations 审中-公开
    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。

    Multiplexed Amplification of Short Nucleic Acids
    4.
    发明申请
    Multiplexed Amplification of Short Nucleic Acids 审中-公开
    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。

    Systems and Methods for Isolating Nucleic Acids
    7.
    发明申请
    Systems and Methods for Isolating Nucleic Acids 失效
    用于分离核酸的系统和方法

    公开(公告)号：US20090071830A1

    公开(公告)日：2009-03-19

    申请号：US12179455

    申请日：2008-07-24

    申请人： Charles S. Vann ,  Maxim G. Brevnov ,  David W. Ruff ,  Kenneth J. Livak

    发明人： Charles S. Vann ,  Maxim G. Brevnov ,  David W. Ruff ,  Kenneth J. Livak

    IPC分类号： B01D57/02 ,  B01D61/42 ,  C25B9/08

    CPC分类号： C12N15/101 ,  G01N27/44782

    摘要： A system for collecting target nucleic acids from a sample can include at least one sample chamber configured to receive a sample containing target nucleic acids and other material, at least one collection chamber removably mountable relative to the at least one sample chamber and configured to collect target nucleic acids separated from the other material, a filter removably mountable relative to the at least one sample chamber and configured to be disposed between the at least one sample chamber and the at least one collection chamber when the at least one collection chamber is mounted relative to the at least one sample chamber. The system may further include a pair of electrodes configured to generate an electric field sufficient to cause target nucleic acids in the at least one sample chamber to migrate via electrophoresis from the at least one sample chamber through the filter into the at least one collection chamber, wherein the filter may be configured to permit passage of target nucleic acids and to block passage of material of a size larger than the target nucleic acids.

    摘要翻译： 用于从样品收集目标核酸的系统可以包括至少一个样品室，其被配置为接收含有靶核酸和其它材料的样品，至少一个收集室，其相对于至少一个样品室可拆卸地安装并且被配置成收集目标 与其他材料分离的核酸，相对于至少一个样品室可拆卸地安装的过滤器，并且被配置为当所述至少一个收集室相对于所述至少一个收集室安装时，被设置在所述至少一个样品室和所述至少一个收集室之间 所述至少一个样品室。 该系统可以进一步包括一对电极，其被配置为产生足以使所述至少一个样品室中的靶核酸经由电泳从所述至少一个样品室通过所述过滤器迁移到所述至少一个收集室中的电场， 其中所述过滤器可以被配置为允许靶核酸的通过并阻止尺寸大于所述靶核酸的材料的通过。

    Length determination of nucleic acid repeat sequences by discontinuous primer extension
    8.
    发明授权
    Length determination of nucleic acid repeat sequences by discontinuous primer extension 有权
    通过不连续引物延伸确定核酸重复序列的长度

    公开(公告)号：US07294464B2

    公开(公告)日：2007-11-13

    申请号：US10892403

    申请日：2004-07-14

    申请人： Kenneth J. Livak ,  Adam L. Lowe ,  Andrew J. Blasband

    发明人： Kenneth J. Livak ,  Adam L. Lowe ,  Andrew J. Blasband

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

    CPC分类号： C12Q1/6827 ,  C12Q1/6837 ,  C12Q1/6858 ,  C12Q1/6874 ,  C12Q2565/514 ,  C12Q2533/101 ,  C12Q2525/151 ,  C12Q2525/131 ,  C12Q2565/515 ,  C12Q2525/161 ,  C12Q2535/125 ,  C12Q2525/197 ,  C12Q2563/107

    摘要： Disclosed is a method for determining the number of repeat units in a repeat region of a target nucleic acid. In a first aspect, the method of the invention includes the steps of annealing a primer to a target nucleic acid; performing a first primer extension reaction using a first primer extension reagent; separating the target-primer hybrid and unreacted first primer extension reagent; performing a second primer extension reaction using a second primer extension reagent, wherein at least one of the first or second primer extension reagents includes an extendible nucleotide having a label attached thereto; separating the target-primer hybrid from unreacted second primer extension reagent; measuring a signal produced by the label; treating the label so as to render the label undetectable; and repeating the above steps until the signal is substantially less than a signal detected in a previous cycle.

    摘要翻译： 公开了确定靶核酸重复区域中重复单元数目的方法。 在第一方面，本发明的方法包括将引物退火至靶核酸的步骤; 使用第一引物延伸试剂进行第一引物延伸反应; 分离靶 - 引物杂交体和未反应的第一引物延伸试剂; 使用第二引物延伸试剂进行第二引物延伸反应，其中第一或第二引物延伸试剂中的至少一个包括具有附着标记的标记的可延伸核苷酸; 将靶引物杂交体与未反应的第二引物延伸试剂分离; 测量标签产生的信号; 处理标签以使标签不可检测; 并重复上述步骤，直到信号基本上小于在先前循环中检测到的信号。

    Computer logic for fluorescence genotyping at multiple allelic sites
    9.
    发明授权
    Computer logic for fluorescence genotyping at multiple allelic sites 有权
    用于多个等位基因位点荧光基因分型的计算机逻辑

    公开(公告)号：US6154707A

    公开(公告)日：2000-11-28

    申请号：US324709

    申请日：1999-06-03

    申请人： Kenneth J. Livak ,  Federico Goodsaid

    发明人： Kenneth J. Livak ,  Federico Goodsaid

    IPC分类号： G01N21/64 ,  C12M1/00 ,  C12Q1/68 ,  G01N33/53 ,  G01N33/566 ,  G06G7/58

    CPC分类号： C12Q1/6858 ,  C12Q1/6818

    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.

    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在目标序列中与靶序列杂交的引物对具有至少两个不同等位基因位点的靶序列进行核酸扩增 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上的不同等位基因变体互补的两个或多个探针 通过该组探针检测位点，所述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。

    Probe Based Nucleic Acid Detection
    10.
    发明申请
    Probe Based Nucleic Acid Detection 有权
    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。