-
1.发明授权
公开(公告)号：US5736333A
公开(公告)日：1998-04-07
申请号：US657989
申请日：1996-06-04
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。
-
公开(公告)号：US20130022973A1
公开(公告)日：2013-01-24
申请号：US13521400
申请日：2011-01-14
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。
-
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
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。
-
公开(公告)号：US20090239290A1
公开(公告)日：2009-09-24
申请号：US12404674
申请日：2009-03-16
申请人： Michael W. Hunkapiller , Dennis A. Gilbert , Kenneth J. Livak , Junko Stevens , Susan K. Eddins , Michael Lucero
发明人： Michael W. Hunkapiller , Dennis A. Gilbert , Kenneth J. Livak , Junko Stevens , Susan K. Eddins , Michael Lucero
IPC分类号： C12M1/00
CPC分类号： C12Q1/6858 , G16B50/00 , C12Q2547/101
摘要： An assay kit is provided that includes assay reagents stored in a single-tube container, and a data storage medium containing information about the contents of the container. Methods are provided for using the data provided with the kit to direct instruments and/or processes, for example, to control an instrument to perform amplification and/or sequencing reactions.
摘要翻译： 提供了包含存储在单管容器中的测定试剂和含有关于容器内容物的信息的数据存储介质的测定试剂盒。 提供了使用试剂盒提供的数据来指导仪器和/或过程的方法，例如控制仪器进行扩增和/或测序反应。
-
公开(公告)号：US20090239233A1
公开(公告)日：2009-09-24
申请号：US12408870
申请日：2009-03-23
申请人： Michael W. Hunkapiller , Dennis A. Gilbert , Kenneth J. Livak , Junko Stevens , Susan K. Eddins , Michael Lucero
发明人： Michael W. Hunkapiller , Dennis A. Gilbert , Kenneth J. Livak , Junko Stevens , Susan K. Eddins , Michael Lucero
IPC分类号： C12Q1/68
CPC分类号： G01N35/00732 , G01N35/1002
摘要： An assay kit is provided that includes assay reagents stored in a single-tube container, and a data storage medium containing information about the contents of the container. Methods are provided for using the data provided with the kit to direct instruments and/or processes, for example, to control an instrument to perform amplification and/or sequencing reactions.
-
公开(公告)号：US20090071830A1
公开(公告)日：2009-03-19
申请号：US12179455
申请日：2008-07-24
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。
-
7.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
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。
-
8.发明授权
公开(公告)号：US6154707A
公开(公告)日：2000-11-28
申请号：US324709
申请日：1999-06-03
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。
-
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
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。
-
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
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。
-
-
-
-
-
-
-
-
-
搜索结果
58 条记录 (耗时 0.037 秒)