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：US07153658B2

公开(公告)日：2006-12-26

申请号：US10665671

申请日：2003-09-19

申请人： Mark R. Andersen ,  Michael W. Hunkapiller ,  Kenneth J. Livak ,  Eugene G. Spier ,  H. Michael Wenz

发明人： Mark R. Andersen ,  Michael W. Hunkapiller ,  Kenneth J. Livak ,  Eugene G. Spier ,  H. Michael Wenz

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

CPC分类号： C12Q1/6827 ,  C12Q2561/101 ,  C12Q2531/137 ,  C12Q2525/161

摘要： The present invention relates to methods and kits for detecting the presence or absence of (or quantitating) target nucleic acid sequences using ligation and amplification. The invention also relates to methods, reagents, and kits that employ addressable portions and labeled probes.

摘要翻译： 本发明涉及使用连接和扩增检测靶核酸序列的存在或不存在（或定量）的方法和试剂盒。 本发明还涉及采用可寻址部分和标记探针的方法，试剂和试剂盒。

公开(公告)号：US07132239B2

公开(公告)日：2006-11-07

申请号：US10455150

申请日：2003-06-04

申请人： Kenneth J. Livak ,  Federico Goodsaid

发明人： Kenneth J. Livak ,  Federico Goodsaid

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

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: 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; 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：US06773887B2

公开(公告)日：2004-08-10

申请号：US10038520

申请日：2001-10-22

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： C12Q168

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. In a second 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 from unreacted first primer extension reagent; performing a second primer extension reaction using a second primer extension reagent and with a primer termination reagent, the primer termination reagent including a nucleotide terminator having a label attached thereto; separating the target-primer hybrid from unreacted second primer extension reagent and unreacted primer termination reagent; measuring a signal produced by the label; and repeating the above steps until a signal is detected indicating incorporation of the nucleotide terminator. The invention further includes kits useful for practicing the above methods.

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。 在第二方面，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; 将靶引物杂交体与未反应的第一引物延伸试剂分离; 使用第二引物延伸试剂和引物终止试剂进行第二引物延伸反应，所述引物终止试剂包括具有附着标记的核苷酸终止子; 从未反应的第二引物延伸试剂和未反应的引物终止试剂中分离靶引物杂交体; 测量标签产生的信号; 并重复上述步骤直到检测到指示掺入核苷酸终止子的信号。 本发明还包括可用于实践上述方法的试剂盒。

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：US06358679B1

公开(公告)日：2002-03-19

申请号：US09645959

申请日：2000-08-24

申请人： Christian A. Heid ,  Kenneth J. Livak

发明人： Christian A. Heid ,  Kenneth J. Livak

IPC分类号： C12Q170

CPC分类号： C12Q1/686 ,  Y10S435/81 ,  C12Q2545/113 ,  C12Q2545/10

摘要： Methods of nucleic acid amplification with external controls are provided that verify the absence or presence of specific target sequences, and correct primers and probes. A single-stranded, external control polynucleotide is amplified with primers of the same sequence as target primers. Probes with detectable labels and sequences specific for target and external control polynucleotides allow for detection and measurement. The primers and the detectable probe are adjacent or substantially adjacent when hybridized to the external control polynucleotide. Target and control amplicons may be detected by increased fluorescence induced by polymerase-mediated 5′ nuclease cleavage or hybridization of a self-quenching probe complementary to both target and external control polynucleotides. A kit of PCR reagents can be dispensed into vessels for rapid and accurate nucleic acid amplification assay, with real-time or end-point measurements. The amplification control reagents, kits, and methods of the present invention provide positive and negative control tests which can be conducted concurrently with target amplification. Allelic differences at genetic loci can be detected, including single nucleotide polymorphisms (SNP).

摘要翻译： 提供使用外部对照的核酸扩增方法，其验证特定靶序列的不存在或存在，以及校正引物和探针。 使用与靶引物相同序列的引物扩增单链外部对照多核苷酸。 具有可检测标记和具有针对和外部对照多核苷酸序列的序列的探针允许检测和测量。 当与外部对照多核苷酸杂交时，引物和可检测的探针相邻或基本相邻。 可以通过聚合酶介导的5'核酸酶切割诱导的增加的荧光或与靶和外部对照多核苷酸互补的自猝灭探针的杂交来检测靶和对照扩增子。 PCR试剂盒可以分配到容器中，用于快速准确的核酸扩增测定，具有实时或终点测量。 本发明的扩增控制试剂，试剂盒和方法提供可以与靶扩增同时进行的阳性和阴性对照试验。 可以检测遗传基因座中的等位基因差异，包括单核苷酸多态性（SNP）。

公开(公告)号：US06258569B1

公开(公告)日：2001-07-10

申请号：US09436454

申请日：1999-11-08

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： C12N100

CPC分类号： C12Q1/6818 ,  C12Q1/6834 ,  C12Q1/6837 ,  C12Q2565/519 ,  C12Q2563/107 ,  C12Q2561/101 ,  C12Q2525/185 ,  C12Q2525/301 ,  C12Q2565/1015 ,  C12Q2565/107

摘要： Provided is a method of nucleic acid amplification. In one embodiment, the method comprises performing nucleic acid amplification on a target polynucleotide using a nucleic acid polymerase having 5′ to 3′ nuclease activity, a primer capable of hybridizing to the target polynucleotide, and an oligonucleotide probe under amplification conditions such that the probe hybridizes to the target polynucleotide 3′ relative to the primer and the probe does not hybridize with itself to form a hairpin structure. The oligonucleotide probe has at one end a fluorescent reporter and at the other end a quencher that quenches the fluorescence of the reporter molecule when both the fluorescent reporter and quencher are attached to the probe. Digestion of the oligonucleotide probe by the polymerase during amplification is effective to separate the reporter from the quencher, whereby a fluorescence signal of the reporter is increased.

摘要翻译： 提供了核酸扩增的方法。 在一个实施方案中，所述方法包括使用具有5'至3'核酸酶活性的核酸聚合酶在靶多核苷酸上进行核酸扩增，能够与靶多核苷酸杂交的引物和寡核苷酸探针在扩增条件下进行，使得探针 与靶多核苷酸3'相对于引物杂交，并且探针不与其自身杂交以形成发夹结构。 寡核苷酸探针在一端具有荧光报告基团，另一端是当荧光报道分子和猝灭剂连接到探针上时淬灭报道分子的荧光的猝灭剂。 在扩增期间通过聚合酶消化寡核苷酸探针有效地将报道分子从猝灭剂中分离出来，从而增加了报道分子的荧光信号。

公开(公告)号：US5876930A

公开(公告)日：1999-03-02

申请号：US558303

申请日：1995-11-15

申请人： Kenneth J. Livak ,  Susan J. A. Flood ,  Jeffrey Marmaro ,  Khairuzzaman Bashar Mullah

发明人： Kenneth J. Livak ,  Susan J. A. Flood ,  Jeffrey Marmaro ,  Khairuzzaman Bashar Mullah

IPC分类号： C12N15/09 ,  C07H21/00 ,  C12N15/00 ,  C12Q1/68 ,  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.