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公开(公告)号：US5789168A
公开(公告)日：1998-08-04
申请号：US640672
申请日：1996-05-01
申请人： James Leushner , May Hui , James M. Dunn , Marina T. Larson
发明人： James Leushner , May Hui , James M. Dunn , Marina T. Larson
CPC分类号： B01L7/525 , B01L7/52 , C12Q1/6841 , C12Q1/6881 , C12Q1/689 , C12Q1/703 , C12Q2600/156 , C12Q2600/16 , G01N2035/00237
摘要： Amplification and sequencing of a selected region of a target nucleic acid polymer are be performed in a single vessel. The sample is added to an amplification mixture containing a thermally stable polymerase and nucleoside feedstocks. Chain terminating dideoxynucleosides are added either at the beginning of the amplification reaction or during the course of the amplification. A thermally stable polymerase which incorporates dideoxynucleotides into an extending oligonucleotide at a rate which is no less than about 0.4 times the rate of incorporation of deoxynucleosides can be used in the amplification mixture or added with the chain terminating nucleoside.
摘要翻译： 靶核酸聚合物的选定区域的扩增和测序可以在单个容器中进行。 将样品加入到含有热稳定聚合酶和核苷原料的扩增混合物中。 在扩增反应开始时或扩增过程中加入链终止双脱氧核苷。 将扩增寡核苷酸以不低于脱氧核苷的并入速率的约0.4倍的速率掺入延伸寡核苷酸的热稳定聚合酶可以用于扩增混合物中或加入链终止核苷。
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2.发明授权Method, compositions and kit for detection and identification of microorganisms 失效用于微生物检测和鉴定的方法，组合物和试剂盒
公开(公告)号：US5888736A
公开(公告)日：1999-03-30
申请号：US807138
申请日：1997-02-27
CPC分类号： B01L7/525 , B01L7/52 , C12Q1/6841 , C12Q1/6858 , C12Q1/6869 , C12Q1/6881 , C12Q1/689 , C12Q1/703 , C12Q2600/156 , G01N2035/00237
摘要： Evaluation of a sample for the presence and qualitative nature of a microorganism can be performed in a single vessel by combining a natural abundance DNA sample with a sequencing mixture containing a primer pair, a thermally stable polymerase such as ThermoSequenase.TM. which incorporates dideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than about 0.4 times the rate of incorporation of deoxynucleotides, nucleotide triphosphate feedstocks, and a chain terminating nucleotide triphosphate. The mixture is processed through multiple thermal cycles for annealing, extension and denaturation to produce a product mixture which is analyzed by electrophoresis.
摘要翻译： 通过将天然丰度DNA样品与含有引物对的测序混合物，热稳定聚合酶如ThermoSequenase TM组合，可以在单个容器中评估样品的存在和定性性质，其将双脱氧核苷酸掺入延伸的 核酸聚合物的速率不低于脱氧核苷酸，核苷酸三磷酸酯原料和链终止核苷酸三磷酸的引入速率的约0.4倍。 通过多个热循环处理混合物进行退火，延伸和变性以产生通过电泳分析的产物混合物。
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公开(公告)号：US5830657A
公开(公告)日：1998-11-03
申请号：US684498
申请日：1996-07-19
申请人： James Leushner , May Hui , James M. Dunn , Marina T. Larson
发明人： James Leushner , May Hui , James M. Dunn , Marina T. Larson
CPC分类号： B01L7/52 , B01L7/525 , C12Q1/6841 , C12Q1/6881 , C12Q1/689 , C12Q1/703 , C12Q2600/156 , C12Q2600/16 , G01N2035/00237
摘要： Sequencing of a selected region of a target nucleic acid polymer in a genomic DNA sample can be performed in a single vessel by combining the sample with a sequencing mixture containing a primer pair, a thermally stable polymerase such as Thermo Sequenase.TM. which incorporates dideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than about 0.4 times the rate of incorporation of deoxynucleotides, nucleotide feedstocks, and a chain terminating nucleotide. The mixture is processed through multiple thermal cycles for annealing, extension and denaturation to produce a product mixture which is analyzed by electrophoresis.
摘要翻译： 基因组DNA样品中靶核酸聚合物的选定区域的测序可以通过将样品与含有引物对的测序混合物，热稳定聚合酶如Thermo Sequenase TM组合而在单个容器中进行，其将二脱氧核苷酸掺入到 以不低于脱氧核苷酸，核苷酸原料和链终止核苷酸并入速率的约0.4倍的速率延伸核酸聚合物。 通过多个热循环处理混合物进行退火，延伸和变性以产生通过电泳分析的产物混合物。
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公开(公告)号：US06413718B1
公开(公告)日：2002-07-02
申请号：US09065748
申请日：1998-04-24
IPC分类号： C12Q168
CPC分类号： C12Q1/6869 , B01L7/52 , B01L7/525 , G01N2035/00237
摘要： Sequencing of a selected region of a target nucleic acid polymer in a natural abundance DNA sample can be performed in a single vessel by combining the sample with a sequencing mixture containing a primer pair, a thermally stable polymerase such as Thermo Sequenase™ which incorporates dideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than about 0.4 times the rate of incorporation of deoxynucleotides, nucleotide feedstocks, and a chain terminating nucleotide. The reaction mixture also includes an unconventional nucleotide and an appropriate enzyme for degradation of nucleic acid polymers containing the unconventional nucleotide. The mixture is processed through multiple thermal cycles for annealing, extension and denaturation to produce a product mixture which is analyzed by electrophoresis.
摘要翻译： 天然丰度DNA样品中靶核酸聚合物的选定区域的测序可以通过将样品与含有引物对的测序混合物，热稳定聚合酶如Thermo Sequenase TM组合而在单个容器中进行，其结合 双脱氧核苷酸以不低于脱氧核苷酸，核苷酸原料和链终止核苷酸并入速率的约0.4倍的速率转化成延伸的核酸聚合物。 反应混合物还包括用于降解含有非常规核苷酸的核酸聚合物的非常规核苷酸和合适的酶。 通过多个热循环处理混合物进行退火，延伸和变性以产生通过电泳分析的产物混合物。
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公开(公告)号：US06274315B1
公开(公告)日：2001-08-14
申请号：US09065748
申请日：1998-04-24
IPC分类号： C12Q168
CPC分类号： C12Q1/6869 , B01L7/52 , B01L7/525 , G01N2035/00237 , C12Q2535/101 , C12Q2525/117 , C12Q2521/513 , C12Q2521/101
摘要： Sequencing of a selected region of a target nucleic acid polymer in a natural abundance DNA sample can be performed in a single vessel by combining the sample with a sequencing mixture containing a primer pair, a thermally stable polymerase such as Thermo Sequenase™ which incorporates dideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than about 0.4 times the rate of incorporation of deoxynucleotides, nucleotide feedstocks, and a chain terminating nucleotide. The reaction mixture also includes an unconventional nucleotide and an appropriate enzyme for degradation of nucleic acid polymers containing the unconventional nucleotide. The mixture is processed through multiple thermal cycles for annealing, extension and denaturation to produce a product mixture which is analyzed by electrophoresis.
摘要翻译： 天然丰度DNA样品中靶核酸聚合物的选定区域的测序可以通过将样品与含有引物对的测序混合物，热稳定聚合酶如Thermo Sequenase TM组合而在单个容器中进行，其结合 双脱氧核苷酸以不低于脱氧核苷酸，核苷酸原料和链终止核苷酸并入速率的约0.4倍的速率转化成延伸的核酸聚合物。 反应混合物还包括用于降解含有非常规核苷酸的核酸聚合物的非常规核苷酸和合适的酶。 通过多个热循环处理混合物进行退火，延伸和变性以产生通过电泳分析的产物混合物。
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公开(公告)号：US6083699A
公开(公告)日：2000-07-04
申请号：US9483
申请日：1998-01-20
申请人： James Leushner , May Hui , James M. Dunn , Marina T. Larson , Jean-Michel Lacroix , Robert Shipman
发明人： James Leushner , May Hui , James M. Dunn , Marina T. Larson , Jean-Michel Lacroix , Robert Shipman
CPC分类号： C12Q1/703 , B01L7/52 , B01L7/525 , C12Q1/6841 , C12Q1/6869 , C12Q1/6881 , C12Q1/689 , G01N2035/00237
摘要： A method is provided for simultaneously determining the positions of a selected nucleotide base in a target region of both strands of a denatured duplex nucleic acid polymer. The nucleic acid polymer is combined with a reactant mixture comprising first and second oligonucleotide primers, said primers binding to the sense and antisense strands, respectively, of the nucleic acid polymer at a location flanking the target region; a thermostable DNA polymerase; a chain-terminating nucleotide triphosphate complementary to the selected nucleotide base; and other reagents for synthesis of chain extension products to form a reaction mixture. This mixture is processed through a plurality of thermal cycles, each including at least a chain extension phase and a denaturation phase to produce chain extension products. These chain extension products are evaluated to determine the positions of the selected bases. The method of the invention differs from the prior art, because the first and second oligonucleotide primers are each labeled with different, spectroscopically-distinguishable fluorescent labels. The method therefore obtains information about both DNA strands simultaneously while providing improved sensitivity as a result of the non-linear increase in the amount of DNA which results from the production of additional templates molecules from unterminated fragments.
摘要翻译： 提供了一种用于同时测定变性双链体核酸聚合物的两条链的靶区域中所选核苷酸碱基的位置的方法。 核酸聚合物与包含第一和第二寡核苷酸引物的反应物混合物组合，所述引物分别与靶区域侧翼位置处的核酸聚合物的有义链和反义链结合; 热稳定DNA聚合酶; 与所选核苷酸碱基互补的链终止核苷酸三磷酸; 和其他用于合成链延伸产物的试剂形成反应混合物。 该混合物通过多个热循环进行处理，每个热循环至少包含延伸相和变性相以产生链延伸产物。 评估这些链延伸产物以确定所选碱基的位置。 本发明的方法与现有技术不同，因为第一和第二寡核苷酸引物各自用不同的光谱可区分的荧光标记进行标记。 因此，该方法同时获得关于两条DNA链的信息，同时由于从未终止的片段产生附加模板分子而导致的DNA量的非线性增加，提供了改善的灵敏度。
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公开(公告)号：US06214555B1
公开(公告)日：2001-04-10
申请号：US09311260
申请日：1999-05-13
申请人： James Leushner , May Hui , James M. Dunn , Jean-Michel LaCroix
发明人： James Leushner , May Hui , James M. Dunn , Jean-Michel LaCroix
IPC分类号： C12Q168
CPC分类号： C12Q1/689 , B01L7/52 , B01L7/525 , C12Q1/6841 , C12Q1/6869 , C12Q1/6881 , C12Q1/703 , G01N2035/00237 , C12Q2565/102 , C12Q2535/113
摘要： Evaluation of a sample for the presence and qualitative nature of a microorganism can be performed in a single vessel by combining a natural abundance DNA sample with a sequencing mixture containing a primer pair, a thermally stable polymerase such as ThermoSequenase™ which incorporates dideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than about 0.4 times the rate of incorporation of deoxynucleotides, nucleotide triphosphate feedstocks, and a chain terminating nucleotide triphosphate. The mixture is processed through multiple thermal cycles for annealing, extension and denaturation to produce a product mixture which is analyzed by electrophoresis.
摘要翻译： 通过将天然丰度DNA样品与含有引物对的测序混合物，热稳定聚合酶如ThermoSequenase TM（其将二脱氧核苷酸掺入到其中）合并，可以在单个容器中评估样品的存在和定性性质 延伸的核酸聚合物的速度不低于脱氧核苷酸，核苷酸三磷酸酯原料和链终止核苷酸三磷酸的引入速率的约0.4倍。 通过多个热循环处理混合物进行退火，延伸和变性以产生通过电泳分析的产物混合物。
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公开(公告)号：US06403303B1
公开(公告)日：2002-06-11
申请号：US08649950
申请日：1996-05-14
申请人： Robert Shipman , James Leushner , James M. Dunn
发明人： Robert Shipman , James Leushner , James M. Dunn
IPC分类号： C12Q168
CPC分类号： C12Q1/6827 , C12Q1/6886 , C12Q2600/156 , C12Q2600/16 , C12Q2537/143
摘要： Samples are tested for mutations in the BRCA1 gene using a hierarchical approach. First, each sample is amplified in one or more multiplex PCR amplification reactions. Each multiplex PCR reaction produces a mixture of amplified fragments. The sizes and amounts of these fragments are evaluated and compared to standard values reflecting the sizes and amounts of fragments produced when the same multiplex amplification is performed on the wild-type BRCA1 gene. Differences between the observed fragment sizes and/or amounts and those for the wild-type gene are indicative of a mutation with the BRCA1 gene of the sample. Next, one or more of the exons of the BRCA1 gene are sequenced, preferably only for those samples where no mutation was detected by analysis of the multiplex PCR fragments. The sequencing procedure can be performed by amplification and sequencing of the multiplex amplification mixture.
摘要翻译： 使用分层方法测试样品中BRCA1基因的突变。 首先，在一个或多个多重PCR扩增反应中扩增每个样品。 每个多重PCR反应产生扩增片段的混合物。 评估这些片段的大小和量，并将其与反映在对野生型BRCA1基因进行相同的多重扩增时产生的片段的大小和量的标准值进行比较。 观察到的片段大小和/或量与野生型基因的差异表明样品的BRCA1基因突变。 接下来，对BRCA1基因的一个或多个外显子进行测序，优选仅对通过多重PCR片段的分析检测未突变的那些样品进行测序。 测序程序可以通过多重扩增混合物的扩增和测序进行。
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9.发明授权Method and apparatus for thermal cycling and for automated sample preparation with thermal cycling 失效用于热循环的方法和设备以及用于热循环的自动化样品制备
公开(公告)号：US5897842A
公开(公告)日：1999-04-27
申请号：US703730
申请日：1996-08-27
申请人： James M. Dunn , James Leushner , John Renfrew , Paul Waterhouse , Alexandre M. Izmailov , Henryk Zaleski
发明人： James M. Dunn , James Leushner , John Renfrew , Paul Waterhouse , Alexandre M. Izmailov , Henryk Zaleski
CPC分类号： B01L7/52 , B01L7/525 , C12Q1/6841 , C12Q1/6881 , C12Q1/689 , C12Q1/703 , C12Q2600/156 , C12Q2600/16 , G01N2035/00237
摘要： An apparatus and method for thermally cycling a reaction mixture in a reaction vessel to expose the mixture to the varying temperatures necessary to, for example, achieve PCR amplification or the preparation of sequencing fragments using a cycle sequencing operation makes use of flow-through reaction vessels, such as capillary tubes, for the preparation and thermal cycling of reaction mixtures. In order to prevent loss of the reaction mixture from the vessels during heating, the thermal cycling apparatus of the invention provides means for sealing the proximal and distal end of each reaction vessel. The proximal ends can be sealed by coupling to a pump which permits movement of the samples within the reaction vessels. As to the distal ends, the reaction vessels can be sealed by pressing the distal end of each vessel against a sealing element with a conformable surface, or by immersing the distal end of each vessel in the reservoir of liquid, preferably of an oil, that is not miscible with the reaction mixture.
摘要翻译： 在反应容器中热循环反应混合物以将混合物暴露于例如实现PCR扩增所需的变化温度或使用循环测序操作制备测序片段的装置和方法使用流通反应容器 ，如毛细管，用于反应混合物的制备和热循环。 为了防止加热期间反应混合物从容器中的损失，本发明的热循环装置提供了用于密封每个反应容器的近端和远端的装置。 近端可以通过联接到允许样品在反应容器内移动的泵来密封。 对于远端，可以通过将每个容器的远端压靠具有适形表面的密封元件，或通过将每个容器的远端浸入液体储存器（优选油）中来密封反应容器， 与反应混合物不混溶。
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10.发明授权Method for evaluation of polymorphic genetic sequences, and the use thereof in identification of HLA types 失效多态遗传序列的评价方法及其在HLA类型鉴定中的应用
公开(公告)号：US5834189A
公开(公告)日：1998-11-10
申请号：US577858
申请日：1995-12-22
IPC分类号： C12N15/09 , C12Q1/68 , G01N27/447 , C12P19/34
CPC分类号： C12Q1/6858 , C12Q1/6869 , C12Q1/6881 , C12Q1/6883 , C12Q1/689 , G01N27/44721 , C12Q2600/156 , C12Q2600/16
摘要： The allelic type of a polymorphic genetic locus in a sample is identified by first combining the sample with a sequencing reaction mixture containing a polymerase, nucleotide feedstocks, one type of chain terminating nucleotide and a sequencing primer to form a plurality of oligonucleotide fragments of differing lengths, and then evaluating the length of the oligonucleotide fragments. As in a standard sequencing procedure, the lengths of the fragments indicate the positions of the type of base corresponding to the chain terminating nucleotide in the extended primer. Instead of performing and evaluating four concurrent reactions, one for each type of chain terminating nucleotide, however, the sample is concurrently combined with at most three, and preferably only one, sequencing reaction mixtures containing different types of chain terminating nucleotides. The information obtained from this test is evaluated prior to performing any additional tests on the sample. In many cases, evaluation of the positions of only a single base using one sequencing reaction will allow for allelic typing of the sample.
摘要翻译： 通过首先将样品与含有聚合酶，核苷酸原料，一种类型的终止核苷酸和测序引物的测序反应混合物合并，形成不同长度的多个寡核苷酸片段，来鉴定样品中多态性遗传位点的等位基因型 ，然后评估寡核苷酸片段的长度。 如在标准测序程序中，片段的长度表示对应于延伸引物中的链终止核苷酸的碱基类型的位置。 然而，代替执行和评估四种并发反应，每种类型的链终止核苷酸一个样品同时与至多三个，优选仅一个含有不同类型的链终止核苷酸的测序反应混合物组合。 在对样品进行任何额外的测试之前，先评估从该测试获得的信息。 在许多情况下，使用一个测序反应评估仅一个碱基的位置将允许样品的等位基因分型。
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