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1. 发明授权

US5723296A Amplification and detection of mycobacterial DNA K nucleic acids 失效
标题翻译：分枝杆菌DNA K核酸的扩增和检测
公开(公告)号：US5723296A
公开(公告)日：1998-03-03
申请号：US644729
申请日：1996-05-10
申请人： Colleen Marie Nycz , James G. Nadeau , Patricia Brinkley Scott , Daryl Dee Shank , Patricia Anne Spears
发明人： Colleen Marie Nycz , James G. Nadeau , Patricia Brinkley Scott , Daryl Dee Shank , Patricia Anne Spears
IPC分类号： C12N15/09 , C07H21/04 , C12N15/00 , C12Q1/68 , C12R1/32 , C12R1/325 , C12R1/33 , C12R1/34 , C12P19/34 , C12Q1/70
CPC分类号： C12Q1/689
摘要： Methods for identification or detection of a species of an organism or a group of related species of an organism by species non-specific amplification of a target sequence followed by species- or group-specific detection of the amplification products. Also provided are a target sequence which is amplifiable in multiple species of mycobacteria using a single pair of amplification primers and species- and group-specific detector probes for hybridization to the assay regioin of the amplified target. Blocking oligonucleotides are employed to allow discrimination among species in which the amplified target sequences are sufficiently similar that they cross-hybridize to an assay probe.
摘要翻译：通过物种非特异性扩增靶序列，然后进行扩增产物的物种或组特异性检测来鉴定或检测生物体或一组相关物种的物种的方法。还提供了使用单对扩增引物和用于与扩增靶的测定区域杂交的物种和组特异性检测器探针在多种分枝杆菌中可扩增的靶序列。使用阻断寡核苷酸以允许在其中扩增的靶序列足够相似以使其与测定探针杂交的物种之间进行区分。

2. 发明授权

US5736365A Multiplex nucleic acid amplification 失效
标题翻译：多重核酸扩增
公开(公告)号：US5736365A
公开(公告)日：1998-04-07
申请号：US705225
申请日：1996-08-29
申请人： George Terrance Walker , James G. Nadeau , Patricia Anne Spears , Colleen M. Nycz , Daryl Dee Shank , James L. Schram , Stewart Russel Jurgensen
发明人： George Terrance Walker , James G. Nadeau , Patricia Anne Spears , Colleen M. Nycz , Daryl Dee Shank , James L. Schram , Stewart Russel Jurgensen
IPC分类号： C12N15/09 , C12Q1/04 , C12Q1/68 , C12R1/32 , C12R1/33 , C12P19/34 , C07H21/04 , C12Q1/70
CPC分类号： C12Q1/689 , C12Q1/6851 , C12Q2600/16
摘要： Primers and methods for adapter-mediated multiplex amplification of the IS6110 insertion element of Mycobacterium tuberculosis (M.tb) and the 16S ribosomal gene of Mycobacterium tuberculosis, useful for simultaneously detecting and/or identifying species of the M. tuberculosis complex and other clinically relevant Mycobacterium species. Multiplex Strand Displacement Amplification (SDA) is used in a single amplification reaction which is capable of simultaneously identifying M. tuberculosis and providing a screen for substantially all of the clinically relevant species of Mycobacteria. Also disclosed are methods for adapter-mediated multiplex amplification of multiple target sequences and a single internal control sequence for determination of sample efficacy or quantitation of the targets. In a preferred embodiment, an internal control sequence is included in the amplification reaction and coamplified with the IS6110 and 16S target sequences as an indication of sample amplification activity or to quantitate the initial amount of target sequences in the sample.
摘要翻译：针对结核分枝杆菌（M.tb）的IS6110插入元件和结核分枝杆菌的16S核糖体基因的适配器介导的多重扩增的引物和方法，可用于同时检测和/或鉴定结核分枝杆菌复合物和其他临床相关的物种分枝杆菌种。多重链位移扩增（SDA）用于能够同时鉴定结核分枝杆菌并为基本上所有临床相关分枝杆菌物种提供筛选的单一扩增反应。还公开了用于多重靶序列的衔接子介导的多重扩增的方法和用于确定靶的样品功效或定量的单个内部控制序列的方法。在优选的实施方案中，内部对照序列包括在扩增反应中并与IS6110和16S靶序列共扩增，作为样品扩增活性的指示或定量样品中靶序列的初始量。

3. 发明授权

US09315863B2 Sequence-specific methods for homogeneous, real-time detection of lamp products 有权
标题翻译：灯具产品均匀，实时检测的序列特异性方法
公开(公告)号：US09315863B2
公开(公告)日：2016-04-19
申请号：US13505598
申请日：2010-11-04
申请人： James G. Nadeau
发明人： James G. Nadeau
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6865 , C12Q2525/307
摘要： Presented herein are methods and compositions for generating sequence-specific, secondary amplification products during Loop-mediated Isothermal Amplification (LAMP). Conventional LAMP produces a preponderance of high molecular weight DNA structures concatenated into self-complementary hairpins, which are not amenable to detection by routine probe-based hybridization methods, making multiplex detection of two or more targets or sequence variants in closed-tube formats extremely difficult. Provided herein, for example, are methods for generating secondary LAMP products bearing a fragment of the original target sequence embedded within low-molecular weight products that are devoid of competitive hairpin structures, the lack of which enhances probe-based detection of target sequences. These secondary products can, for example, be produced in real-time, during the LAMP process, and can provide the option of detecting multiple target sequences within a single tube using, e.g., a homogenous, real-time fluorescence format.
摘要翻译：本文提出了在环介导的等温扩增（LAMP）期间产生序列特异性二级扩增产物的方法和组合物。常规的LAMP产生连接到自身互补发夹中的高分子量DNA结构的优势，其不适于通过常规基于探针的杂交方法的检测，使得封闭管形式中的两个或更多个目标或序列变体的多重检测非常困难。例如，本文提供的是产生具有嵌入低分子量产物中的原始靶序列的片段的次级LAMP产物的方法，所述片段不含有竞争力的发夹结构，其缺乏可增强靶序列的基于探针的检测。这些次要产物可以例如在LAMP过程期间实时产生，并且可以提供使用例如均匀的实时荧光格式在单个管内检测多个靶序列的选项。

4. 发明授权

US5919630A Detection of nucleic acids by fluorescence quenching 失效
公开(公告)号：US5919630A
公开(公告)日：1999-07-06
申请号：US186030
申请日：1998-11-04
申请人： James G. Nadeau , J. Bruce Pitner , James L. Schram , C. Preston Linn , Glenn P. Vonk , G. Terrance Walker
发明人： James G. Nadeau , J. Bruce Pitner , James L. Schram , C. Preston Linn , Glenn P. Vonk , G. Terrance Walker
IPC分类号： G01N21/64 , C07H21/00 , C12N15/09 , C12Q1/68 , G01N33/58 , C07H21/04 , C12P19/34
CPC分类号： C12Q1/6818 , C12Q1/6825
摘要： Single-stranded signal primers are modified by linkage to two dyes which form a donor/acceptor dye pair. The two dyes are positioned in sufficiently close spatial proximity on the signal primer that the fluorescence of the first dye is quenched by the second dye. The signal primer may further comprise a restriction endonuclease recognition site (RERS) between the two dyes. As the signal primer is initially single-stranded and remains single-stranded in the absence of target, the restriction endonuclease recognition site is not cleavable or nickable by the restriction endonuclease. In the presence of target, however, signal primer and the restriction endonuclease recognition site are rendered double-stranded and cleavable or nickable by the restriction endonuclease. Cleavage or nicking separates the two dyes and a change in fluorescence due to decreased quenching is detected as an indication of the presence of the target sequence or of target sequence amplification.

5. 发明授权

US5840487A Internal controls for isothermal nucleic acid amplification reactions 失效
公开(公告)号：US5840487A
公开(公告)日：1998-11-24
申请号：US801542
申请日：1997-02-18
申请人： James G. Nadeau , Michael C. Little
发明人： James G. Nadeau , Michael C. Little
IPC分类号： C12Q1/68 , C12P19/34
CPC分类号： C12Q1/6865 , C12Q1/6851 , C12Q1/689
摘要： Methods employing internal oligonucleotide standards in isothermal nucleic acid amplification reactions to determine the efficacy of the amplification reaction and to quantify pre-amplification target levels.

6. 发明授权

US5422252A Simultaneous amplification of multiple targets 失效
标题翻译：同时放大多个目标
公开(公告)号：US5422252A
公开(公告)日：1995-06-06
申请号：US73197
申请日：1993-06-04
申请人： George T. Walker , James G. Nadeau , Michael C. Little
发明人： George T. Walker , James G. Nadeau , Michael C. Little
IPC分类号： G01N33/566 , C07H21/04 , C12N15/09 , C12P19/34 , C12Q1/68 , G11B5/00 , G11B5/09 , G11B5/187 , G11B5/31 , G11B5/455 , G11B5/465 , G11B19/04 , C12Q1/70
CPC分类号： C12Q1/6853
摘要： Methods for multiplex amplification of target nucleic acid sequences using a single pair of primers. Defined sequences are appended to the ends of multiple target sequences as part of the amplification reaction so that no steps in addition to amplification are required. The target sequences with the appended defined sequences need not be isolated prior to amplification. In one embodiment for coamplification of two target sequences, a sequence corresponding to a terminal segment of the first target sequence is appended to one end of the second target sequence and a sequence corresponding to a terminal segment of the second target sequence is appended to one end of the first target sequence. Amplification of the two targets then requires only a single pair of primers. Alternatively, a single defined sequence may be appended to the 5' and 3' ends of any number of selected targets. All such modified target sequences may then be amplified using a single pair of primers which hybridize to the defined end-sequences.
摘要翻译：使用单对引物多重扩增靶核酸序列的方法。定义的序列作为扩增反应的一部分附加到多个靶序列的末端，因此不需要扩增步骤。具有附加定义序列的靶序列不需要在扩增前分离。在两个目标序列的共放大的一个实施例中，对应于第一目标序列的末端片段的序列被附加到第二目标序列的一端，并且与第二目标序列的末端片段相对应的序列被附加到一端的第一个靶序列。两个靶标的扩增只需要一对引物即可。或者，单个定义的序列可以附加到任意数目的所选目标的5'和3'端。然后可以使用与定义的末端序列杂交的单对引物扩增所有这些修饰的靶序列。

7. 发明申请

US20090246792A1 METHODS FOR DETECTING NUCLEIC ACID SEQUENCE VARIATIONS 有权
标题翻译：检测核酸序列变异的方法
公开(公告)号：US20090246792A1
公开(公告)日：2009-10-01
申请号：US12419737
申请日：2009-04-07
申请人： Sha-Sha WANG , Keith Thornton , James G. Nadeau , Tobin J. Hellyer
发明人： Sha-Sha WANG , Keith Thornton , James G. Nadeau , Tobin J. Hellyer
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6827 , C12Q2565/1025 , C12Q2535/125 , C12Q2531/119 , C12Q2531/101
摘要： The invention employs an unlabeled signal primer comprising a 5′ adapter sequence for detection of variations in nucleic acid target sequences. The detection system further comprises a reporter probe, the 3′ end of which hybridizes to the complement of the 5′ adapter sequence of the signal primer to produce a 5′ overhang. Polymerase is used to fill in the overhang and synthesize the complement of the 5′ overhang of the reporter probe. Synthesis of the reporter probe complement is detected, either directly or indirectly, as an indication of the presence of the target.
摘要翻译：本发明采用包含5'衔接子序列的未标记信号引物来检测核酸靶序列的变异。检测系统还包括报道探针，其3'末端与信号引物的5'衔接子序列的互补体杂交以产生5'突出端。聚合酶用于填充突出端并合成报告基因探针的5'突出端的互补体。直接或间接检测报道探针补体的合成，作为目标存在的指示。

8. 发明授权

US06316200B1 Probes and methods for detection of nucleic acids 有权
标题翻译：用于检测核酸的探针和方法
公开(公告)号：US06316200B1
公开(公告)日：2001-11-13
申请号：US09590061
申请日：2000-06-08
申请人： James G. Nadeau , Tobin J. Hellyer
发明人： James G. Nadeau , Tobin J. Hellyer
IPC分类号： C12Q168
CPC分类号： C12Q1/6818 , C12Q1/6844 , C12Q1/6855 , C12Q2565/549 , C12Q2561/101 , C12Q2525/191
摘要： The invention employs an unlabeled signal primer comprising a 5′ adapter sequence for detection of nucleic acid target sequences. The detection system further comprises a reporter probe, the 3′ end of which hybridizes to the complement of the 5′ adapter sequence of the signal primer to produce a 5′ overhang. Polymerase is used to fill in the overhang and synthesize the complement of the 5′ overhang of the reporter probe. Synthesis of the reporter probe complement is detected, either directly or indirectly, as an indication of the presence of the target.
摘要翻译：本发明采用包含5'衔接子序列的未标记信号引物来检测核酸靶序列。检测系统还包括报道探针，其3'末端与信号引物的5'衔接子序列的互补体杂交以产生5'突出端。聚合酶用于填充突出端并合成报告基因探针的5'突出端的互补体。直接或间接检测报道探针补体的合成，作为目标存在的指示。

9. 发明授权

US5624825A Simultaneous amplification of multiple targets 失效
标题翻译：同时放大多个目标
公开(公告)号：US5624825A
公开(公告)日：1997-04-29
申请号：US451313
申请日：1995-05-26
申请人： George T. Walker , James G. Nadeau , Michael C. Little
发明人： George T. Walker , James G. Nadeau , Michael C. Little
IPC分类号： G01N33/566 , C07H21/04 , C12N15/09 , C12P19/34 , C12Q1/68 , G11B5/00 , G11B5/09 , G11B5/187 , G11B5/31 , G11B5/455 , G11B5/465 , G11B19/04
CPC分类号： C12Q1/6853
摘要： Methods for multiplex amplification of target nucleic acid sequences using a single pair of primers. Defined sequences are appended to the ends of multiple target sequences as part of the amplification reaction so that no steps in addition to amplification are required. The target sequences with the appended defined sequences need not be isolated prior to amplification. In one embodiment for coamplification of two target sequences, a sequence corresponding to a terminal segment of the first target sequence is appended to one end of the second target sequence and a sequence corresponding to a terminal segment of the second target sequence is appended to one end of the first target sequence. Amplification of the two targets then requires only a single pair of primers. Alternatively, a single defined sequence may be appended to the 5' and 3' ends of any number of selected targets. All such modified target sequences may then be amplified using a single pair of primers which hybridize to the defined end-sequences.
摘要翻译：使用单对引物多重扩增靶核酸序列的方法。定义的序列作为扩增反应的一部分附加到多个靶序列的末端，因此不需要扩增步骤。具有附加定义序列的靶序列不需要在扩增前分离。在两个目标序列的共放大的一个实施例中，对应于第一目标序列的末端片段的序列被附加到第二目标序列的一端，并且与第二目标序列的末端片段相对应的序列被附加到一端的第一个靶序列。两个靶标的扩增只需要一对引物即可。或者，单个定义的序列可以附加到任意数目的所选目标的5'和3'端。然后可以使用与定义的末端序列杂交的单对引物扩增所有这些修饰的靶序列。

10. 发明授权

US5270184A Nucleic acid target generation 失效
标题翻译：核酸靶产生
公开(公告)号：US5270184A
公开(公告)日：1993-12-14
申请号：US794399
申请日：1991-11-19
申请人： George T. Walker , Michael C. Little , James G. Nadeau
发明人： George T. Walker , Michael C. Little , James G. Nadeau
IPC分类号： C12N15/02 , C12N15/09 , C12Q1/68 , C12P19/34
CPC分类号： C12Q1/6846 , C12Q1/6853
摘要： The invention is a method for generating nucleic acid sequences ends which comprises;(a) hybridizing a primer to a nucleic acid sequence,(b) hybridizing a primer to the nucleic acid sequence in (a) located 5' to the primer in (a), and(c) polymerizing both primers so that the primer in (a) is displaced from the nucleic acid sequence.The invention provides a method for generating target nucleic acid sequences for subsequent amplification. The method is applicable to both DNA and RNA.

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