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1.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：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：US06653107B2
公开(公告)日：2003-11-25
申请号：US09818182
申请日：2001-03-27
申请人： James M. Dunn , Jean-Michel Lacroix
发明人： James M. Dunn , Jean-Michel Lacroix
IPC分类号： C12P1934
CPC分类号： C12Q1/6881 , C12Q1/6818 , C12Q1/6851 , C12Q1/6858 , C12Q1/6869 , C12Q1/703 , C12Q2600/156
摘要： A method for obtaining information about the allelic type of a sample of genetic material derived from an HIV-infected sample relies on the observation that using a second nested set of sequencing primers ensures the maximum potential for sequencing a particular sample. To perform the method, reagents suitable for performing the tests are suitably packaged as a kit.
摘要翻译： 获得关于衍生自HIV感染样品的遗传物质样本的等位基因类型的信息的方法依赖于使用第二嵌套测序引物组确定了对特定样品进行测序的最大潜力的观察。 为了执行该方法，适合于进行测试的试剂被适当地包装成试剂盒。
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公开(公告)号：US6007983A
公开(公告)日：1999-12-28
申请号：US938641
申请日：1997-09-26
申请人： James M. Dunn , Jean-Michel Lacroix
发明人： James M. Dunn , Jean-Michel Lacroix
CPC分类号： C12Q1/703
摘要： A streamlined, hierarchical method for obtaining information about the allelic type of a sample of genetic material derived from an HIV-infected sample relies on the observation that 93-95% of the known mutational variants of the reverse transcriptase and protease genes of HIV-1 can be determined by evaluating the positions of the A and T nucleotides within the sample. Thus, a substantial fraction of all mutational variations can be unequivocally identified by performing two initial sequencing reactions on the sample in which only ddA and ddT are used as chain terminators. For the small fraction of samples which are not identifiable based on the positions of these two bases, a second test is performed in which the sequence is determined in the 3'-direction for all four bases. This test will identify substantially all of the remaining samples. For those for which an ambiguity remains, however, a final test in which the sequence of the sample is determined in both the 3' and 5-direction for all four bases is performed. To perform the method, reagents suitable for performing the three tests within the hierarchy are suitably packaged as a kit containing two or more sub-kits. The first sub-kit contains reagents for performing A and T sequencing. The additional sub-kits contains reagents for performing a four-base sequence determination on one or both strands of the target DNA.
摘要翻译： 用于获得关于衍生自HIV感染样品的遗传物质样品的等位基因类型的信息的流线型分层方法依赖于93-95％已知的逆转录酶突变变体和HIV-1的蛋白酶基因 可以通过评估样品中A和T核苷酸的位置来确定。 因此，通过对其中仅使用ddA和ddT作为链终止剂的样品进行两个初始测序反应，可以明确地鉴定所有突变变异的相当大部分。 对于基于这两个碱基的位置不可识别的小部分样品，进行第二次测试，其中所有四个碱基在3'方向上确定序列。 该测试将基本上识别所有剩余的样品。 然而，对于那些仍然存在歧义的那些，执行最终测试，其中对于所有四个碱基在3'和5方向上确定样品的序列。 为了执行该方法，适合于在层次结构内执行三个测试的试剂适当地包装为包含两个或更多个子试剂盒的试剂盒。 第一个试剂盒包含用于进行A和T测序的试剂。 另外的子试剂盒包含用于在目标DNA的一条或两条链上进行四碱基序列测定的试剂。
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公开(公告)号：US06265152B1
公开(公告)日：2001-07-24
申请号：US09418720
申请日：1999-10-15
申请人： James M. Dunn , Jean-Michel Lacroix
发明人： James M. Dunn , Jean-Michel Lacroix
IPC分类号： C12Q170
CPC分类号： C12Q1/6881 , C12Q1/6818 , C12Q1/6851 , C12Q1/6858 , C12Q1/6869 , C12Q1/703 , C12Q2600/156
摘要： A method for obtaining information about the allelic type of a sample of genetic material derived from an HIV-infected sample relies on the observation that using a second nested set of sequencing primers ensures the maximum potential for sequencing a particular sample. To perform the method, reagents suitable for performing the tests are suitably packaged as a kit.
摘要翻译： 获得关于衍生自HIV感染样品的遗传物质样本的等位基因类型的信息的方法依赖于使用第二嵌套测序引物组确定了对特定样品进行测序的最大潜力的观察。 为了执行该方法，适合于进行测试的试剂被适当地包装成试剂盒。
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8.发明授权Method and kit for quantitation and nucleic acid sequencing of nucleic acid analytes in a sample 失效用于样品中核酸分析物的定量和核酸测序的方法和试剂盒
公开(公告)号：US5795722A
公开(公告)日：1998-08-18
申请号：US819912
申请日：1997-03-18
申请人： Jean-Michel Lacroix , James M. Dunn
发明人： Jean-Michel Lacroix , James M. Dunn
CPC分类号： C12Q1/6851 , C12Q1/703
摘要： A method for quantitative and qualitative analysis of a nucleic acid analyte in a sample suspected to contain the nucleic acid analyte first combines the sample with a control nucleic acid, and two primer pairs, a first primer pair effective to amplify a conserved region of the nucleic acid analyte if present in the sample to produce a conserved fragment having a first length and to amplify the control nucleic acid to produce a control fragment having a second length different from the first length, and a second primer pair effective to amplify a second region of the nucleic acid analyte to produce a sequencing fragment. One member of the first primer pair is labeled with a detectable label, and one member of the second primer pair may be labeled with a label such as biotin effective to permit capture of the primer. The combined mixture is then processed to amplify the sample and control nucleic acid using the first and second primer pairs to produce an amplification product mixture containing conserved fragments, sequencing fragments and control fragments when the nucleic acid analyte is present in the sample, and only control fragment when the nucleic acid analyte is not present in the sample. This product mixture is analyzed to determine the relative amounts of conserved fragments and control fragments in the amplification product mixture to quantify the amount of nucleic acid analyte in the sample and used as a starting point for a reaction to determine the sequence of the sequencing fragment.
摘要翻译： 用于定量和定性分析怀疑含有核酸分析物的样品中的核酸分析物的方法首先将样品与对照核酸组合，并且将两个引物对，有效扩增核酸保守区的第一引物对 如果存在于样品中以产生具有第一长度的保守片段并扩增对照核酸以产生具有不同于第一长度的第二长度的对照片段的第二引物对，以及有效扩增第二区域的第二引物对 核酸分析物以产生测序片段。 第一引物对的一个成员用可检测的标记物标记，第二引物对的一个成员可以用有效的标记，例如生物素来标记以允许捕获引物。 然后，当核酸分析物存在于样品中时，将合并的混合物用第一和第二引物对进行处理以扩增样品和对照核酸，以产生含有保守片段，测序片段和对照片段的扩增产物混合物，并且仅控制 片段，当核酸分析物不存在于样品中。 分析该产物混合物以确定扩增产物混合物中保守片段和对照片段的相对量，以量化样品中核酸分析物的量并用作确定测序片段序列的反应的起始点。
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9.发明授权Method and kit for quantitation and nucleic acid sequencing of nucleic acid analytes in a sample 失效用于样品中核酸分析物的定量和核酸测序的方法和试剂盒公开(公告)号：US06830887B2
公开(公告)日：2004-12-14
申请号：US10082546
申请日：2002-02-25
申请人： Jean-Michel Lacroix
发明人： Jean-Michel Lacroix
IPC分类号： C12Q168
CPC分类号： C12Q1/6818 , C12Q1/6851 , C12Q1/6869 , C12Q1/703 , C12Q2600/156
摘要： Quantitative and qualitative analysis of a nucleic acid analyte in a sample suspected to contain the nucleic acid analyte if achieved by first preparing a reaction mixture containing the sample and a known amount of an internal quantitation standard. At least a first aliquot of the reaction mixture is combined with a set of amplification reagents effective to amplify nucleic acids in the reaction mixture. The set of reagents includes at least one primer pair which is effective to amplify a first region of the nucleic acid analyte if present in the sample to produce a first amplified sample fragment and to amplify at least a portion of the internal quantitation standard to produce a control fragment. Amplification results in the formation of an amplification product mixture containing first amplified sample fragments and control fragments when the nucleic acid analyte is present in the sample, and only control fragments when the nucleic acid analyte is not present in the sample. The relative amounts of first amplified sample fragments and control fragments are analyzed to quantify the amount of nucleic acid analyte in the sample, and the sequence of the first amplified sample fragments is determined to assess the qualitative characteristics of any nucleic acid analyte. The internal quantification fragment is derived from the analyte nucleic acid by the incorporation of a plurality of sequence variations. These sequence variations include at least a first sequence variation effective to render the internal quantitation standard distinguishable from the first amplified sample fragment, and a second sequence variation effective to substantially eliminate the production of sequencing products from interaction of the internal quantitation standard and the first sequencing primer.
摘要翻译： 如果通过首先制备含有样品的反应混合物和已知量的内部定量标准物来实现，怀疑含有核酸分析物的样品中的核酸分析物的定量和定性分析。 将至少第一等份的反应混合物与一组有效扩增反应混合物中的核酸的扩增试剂组合。 该组试剂包括至少一个引物对，其有效地扩增核酸分析物的第一区域，如果存在于样品中以产生第一扩增的样品片段并扩增至少一部分内部定量标准物以产生 控制片段。 当核酸分析物存在于样品中时，扩增导致形成含有第一扩增样品片段和对照片段的扩增产物混合物，并且当核酸分析物不存在于样品中时，仅控制片段。 分析第一扩增样品片段和对照片段的相对量，以量化样品中核酸分析物的量，并确定第一扩增样品片段的序列以评估任何核酸分析物的定性特征。 内部定量片段通过并入多个序列变体从分析物核酸得到。 这些序列变异包括至少第一序列变异，其有效地使内部定量标准与第一扩增样品片段可区分，以及有效地基本上消除测序产物从内部定量标准与第一次测序的相互作用产生的第二序列变异 底漆。
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