公开(公告)号：US06511810B2

公开(公告)日：2003-01-28

申请号：US09898323

申请日：2001-07-03

申请人： Wanli Bi ,  Kenneth J. Livak ,  Will Bloch

发明人： Wanli Bi ,  Kenneth J. Livak ,  Will Bloch

IPC分类号： C12Q168

CPC分类号： C12Q1/6862 ,  C12Q2531/137 ,  C12Q2521/319

摘要： Disclosed are methods for detecting or quantifying one or more target polynucleotide sequences in a sample. In one aspect, a sample is contacted with first and second probe pair that are capable of hybridizing to a selected target sequence and a corresponding complementary sequence, respectively. Probe cleavage and ligation results in the formation of ligation products which can be generated in an exponential fashion when the target sequence and/or complement are present in the sample. In another embodiment, a single probe pair can be used to form ligation product in a linear fashion from a complementary template. Reagents and kits are also disclosed.

公开(公告)号：US20100276580A1

公开(公告)日：2010-11-04

申请号：US12611892

申请日：2009-11-03

申请人： Muhammad A. Sharaf ,  Wanli Bi ,  Kenneth J. Livak

发明人： Muhammad A. Sharaf ,  Wanli Bi ,  Kenneth J. Livak

IPC分类号： G12B13/00 ,  G01N21/64 ,  G01N21/25

CPC分类号： G01N21/274 ,  G01N21/278 ,  G01N21/6428 ,  G01N21/6452

摘要： Aspects of the present invention provide a method and apparatus of generating a calibration matrix for a spectral detector instrument. A calibration plate contains one or more dye mixtures in each well of the calibration plate at known absolute concentration. From the calibration plate, aspects of the present invention are used to prepare a concentration matrix based on the dyes used in the assay and the different dye mixtures used in the calibration plate. An excitation source exposes the calibration plate causing the spectral species in each of the wells to fluoresce. The emission spectra for the different dye mixtures of dyes as gathered by the spectral detector instrument at different points in the range of spectra is used to generate a spectral matrix. Bilinear calibration is performed on the concentration matrix and the spectral matrix as to determine a calibration matrix relating spectra directly to absolute concentrations.

摘要翻译： 本发明的方面提供了一种生成用于光谱检测器仪器的校准矩阵的方法和装置。 校准板在已知的绝对浓度下在校准板的每个孔中含有一种或多种染料混合物。 根据校准板，本发明的各方面用于制备基于测定中使用的染料和校准板中使用的不同染料混合物的浓度基质。 激发源暴露校准板，导致每个孔中的光谱物质发荧光。 由光谱检测仪器在光谱范围内的不同点收集的不同染料染料混合物的发射光谱用于产生光谱矩阵。 对浓度矩阵和光谱矩阵进行双线性校准，以确定直接将光谱与绝对浓度相关的校准矩阵。

公开(公告)号：US20080001099A1

公开(公告)日：2008-01-03

申请号：US11428385

申请日：2006-07-01

申请人： MUHAMMAD A. SHARAF ,  Wanli Bi ,  Kenneth J. Livak

发明人： MUHAMMAD A. SHARAF ,  Wanli Bi ,  Kenneth J. Livak

IPC分类号： G01N21/64

CPC分类号： G01N21/274 ,  G01N21/278 ,  G01N21/6428 ,  G01N21/6452

摘要： Aspects of the present invention provide a method and apparatus of generating a calibration matrix for a spectral detector instrument. A calibration plate contains one or more dye mixtures in each well of the calibration plate at known absolute concentration. From the calibration plate, aspects of the present invention are used to prepare a concentration matrix based on the dyes used in the assay and the different dye mixtures used in the calibration plate. An excitation source exposes the calibration plate causing the spectral species in each of the wells to fluoresce. The emission spectra for the different dye mixtures of dyes as gathered by the spectral detector instrument at different points in the range of spectra is used to generate a spectral matrix. Bilinear calibration is performed on the concentration matrix and the spectral matrix as to determine a calibration matrix relating spectra directly to absolute concentrations.

摘要翻译： 本发明的方面提供了一种生成用于光谱检测器仪器的校准矩阵的方法和装置。 校准板在已知的绝对浓度下在校准板的每个孔中含有一种或多种染料混合物。 根据校准板，本发明的各方面用于制备基于测定中使用的染料和校准板中使用的不同染料混合物的浓度基质。 激发源暴露校准板，导致每个孔中的光谱物质发荧光。 由光谱检测仪器在光谱范围内的不同点收集的不同染料染料混合物的发射光谱用于产生光谱矩阵。 对浓度矩阵和光谱矩阵进行双线性校准，以确定直接将光谱与绝对浓度相关的校准矩阵。

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。

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。

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

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。

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。

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。