公开(公告)号：US5888739A

公开(公告)日：1999-03-30

申请号：US926054

申请日：1997-09-09

申请人： J. Bruce Pitner ,  James G. Nadeau ,  Glenn P. Vonk

发明人： J. Bruce Pitner ,  James G. Nadeau ,  Glenn P. Vonk

IPC分类号： G01N33/58 ,  C07H21/04 ,  C12N15/09 ,  C12Q1/68 ,  G01N21/78

CPC分类号： C12Q1/6818 ,  Y10S436/80 ,  Y10S436/805 ,  Y10T436/143333

摘要： G-quartet structures have been found to be useful in fluorescence assays to detect a selected nucleic acid sequence. When oligonucleotides containing these structures are labeled with a donor fluorophore and an acceptor dye, the folding or interaction of the oligonucleotides in the G-quartet structure brings the donor-acceptor pair into close proximity, allowing an interaction between the two labels which results in quenching of donor fluorescence or a change in other fluorescence properties which are the result of the interaction of two dyes in close proximity. The G-quartet structure unfolds or is otherwise disrupted upon hybridization to its complementary sequence, increasing the distance between the two dye labels. This results in decreased donor quenching or a change in another proximity-related fluorescence parameter. The associated increase in donor fluorescence intensity or the change in another fluorescence parameter may be monitored as an indication of the presence of a selected nucleic acid sequence. Alternatively, in some cases a decrease in acceptor fluorescence may be monitored as an indication of the presence of the selected nucleic acid sequence when the acceptor is also a fluorophore. Related structures, such as the i-tetraplex, may also be useful in similar methods for detection of a selected nucleic acid sequence.

摘要翻译： 已经发现G-四重结构在荧光测定中可用于检测选定的核酸序列。 当含有这些结构的寡核苷酸用供体荧光团和受体染料标记时，G-四重结构中寡核苷酸的折叠或相互作用使供体 - 受体对紧密接近，允许两个标记之间的相互作用导致淬灭 的供体荧光或其他荧光性质的变化，这两种染料是两种染料相互作用的结果。 G-四重结构在与其互补序列杂交时展开或以其它方式被破坏，增加了两个染料标记之间的距离。 这导致供体猝灭减少或另一邻近相关荧光参数的变化。 可以监测供体荧光强度的相关增加或另一荧光参数的变化作为选择的核酸序列的存在的指示。 或者，在一些情况下，当受体也是荧光团时，可以监测受体荧光的降低作为选择的核酸序列的存在的指示。 相关结构，例如i-tetraplex，也可用于检测所选择的核酸序列的相似方法中。

公开(公告)号：US5846726A

公开(公告)日：1998-12-08

申请号：US855085

申请日：1997-05-13

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.

公开(公告)号：US5811269A

公开(公告)日：1998-09-22

申请号：US640378

申请日：1996-04-30

申请人： James G. Nadeau ,  Cheryl H. Dean ,  James L. Schram ,  Deborah R. Howard ,  Margaret S. Dey ,  David J. Wright

发明人： James G. Nadeau ,  Cheryl H. Dean ,  James L. Schram ,  Deborah R. Howard ,  Margaret S. Dey ,  David J. Wright

IPC分类号： C12N15/09 ,  C07H21/04 ,  C12Q1/04 ,  C12Q1/68 ,  C12R1/32 ,  C12R1/325 ,  C12R1/33 ,  C12P19/34

CPC分类号： C12Q1/689 ,  C12Q2600/16

摘要： Primers and methods for adapter-mediated multiplex amplification of the IS6110 insertion element of the Mycobacterium tuberculosis (M.tb) complex and a 16S rDNA target common to essentially all mycobacteria are described. In certain embodiments, the primers are optimized for efficient multiplex amplification in thermophilic SDA. The multiplex Strand Displacement Amplification methods of the invention are capable, in a single amplification reaction, of simultaneously identifying M. tuberculosis and providing a screen for substantially all of the clinically relevant species of mycobacteria. Also disclosed are internal control sequences designed for coamplification with the two targets, allowing assessment of amplification efficiency and/or quantitation of the targets.

摘要翻译： 描述了针对结核分枝杆菌（M.tb）复合物的IS6110插入元件和基本上所有分枝杆菌共同的16S rDNA靶的衔接子介导的多重扩增的引物和方法。 在某些实施方案中，针对嗜热SDA中的有效多重扩增优化引物。 本发明的多重链位移扩增方法能够在单个扩增反应中同时鉴定结核分枝杆菌并提供基本上所有临床相关分枝杆菌物种的筛选。 还公开了设计用于与两个靶标共扩增的内部控制序列，允许评估靶标的扩增效率和/或定量。

公开(公告)号：US5658738A

公开(公告)日：1997-08-19

申请号：US539516

申请日：1995-12-11

申请人： James G. Nadeau ,  Mary Lee Ciolkowski ,  Erwin A. Vogler

发明人： James G. Nadeau ,  Mary Lee Ciolkowski ,  Erwin A. Vogler

IPC分类号： A61K38/00 ,  C07H21/00 ,  C12N15/113 ,  C12Q1/68 ,  A01N43/72 ,  C07H21/04

CPC分类号： C12N15/1137 ,  C07H21/00 ,  C12Q1/6811 ,  A61K38/00 ,  C12N2310/13 ,  C12N2310/317 ,  C12N2310/3183

摘要： The present invention relates to bi-directional nucleic acid ligand compounds wherein at least two oligonucleotides of opposite sequence polarity are linked to a connecting compound at their same respective terminii; either the 5' terminii or the 3' terminii. These compounds are useful for binding protein or small molecule targets and thus may be used as diagnostic or therapeutic agents.

摘要翻译： 本发明涉及双向核酸配体化合物，其中相对序列极性的至少两个寡核苷酸在相同的末端与连接化合物连接; 5'末端或3'末端。 这些化合物可用于结合蛋白质或小分子靶标，因此可用作诊断或治疗剂。

公开(公告)号：US09404160B2

公开(公告)日：2016-08-02

申请号：US13518211

申请日：2010-12-21

申请人： James G. Nadeau ,  Bernard J. H. Verwer

发明人： James G. Nadeau ,  Bernard J. H. Verwer

IPC分类号： C12Q1/68

CPC分类号： C12Q1/689 ,  C12Q2600/16

摘要： Presented herein are methods for the detection of the presence or absence of one or more microorganisms in a sample. The method deploys a plurality of probe sets to detect a plurality of microorganisms. The probes in the probe set are detectably labeled. At least one probe set has probes labeled with a combination of detectable labels. The number of detectable labels used in the plurality of probe sets numbers less than the number of microorganisms being detected by the probe set.

摘要翻译： 本文提出的是用于检测样品中一种或多种微生物的存在或不存在的方法。 该方法部署多个探针组以检测多个微生物。 探针组中的探针可以被可检测地标记。 至少一个探针组具有用可检测标记的组合标记的探针。 在多个探针组中使用的可检测标记的数量小于由探针组检测到的微生物数量。

公开(公告)号：US20120329050A1

公开(公告)日：2012-12-27

申请号：US13518211

申请日：2010-12-21

申请人： James G. Nadeau ,  Bernard J.H. Verwer

发明人： James G. Nadeau ,  Bernard J.H. Verwer

IPC分类号： C12Q1/68 ,  G01N21/76 ,  C12M1/34 ,  G01N21/64

CPC分类号： C12Q1/689 ,  C12Q2600/16

摘要： Presented herein are methods for the detection of the presence or absence of one or more microorganisms in a sample. The method deploys a plurality of probe sets to detect a plurality of microorganisms. The probes in the probe set are detectably labeled. At least one probe set has probes labeled with a combination of detectable labels. The number of detectable labels used in the plurality of probe sets numbers less than the number of microorganisms being detected by the probe set.

摘要翻译： 本文提出的是用于检测样品中一种或多种微生物的存在或不存在的方法。 该方法部署多个探针组以检测多个微生物。 探针组中的探针可以被可检测地标记。 至少一个探针组具有用可检测标记的组合标记的探针。 在多个探针组中使用的可检测标记的数量小于由探针组检测到的微生物数量。

公开(公告)号：US20120276538A1

公开(公告)日：2012-11-01

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 ,  G01N21/65 ,  G01N21/64

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。

公开(公告)号：US06379888B1

公开(公告)日：2002-04-30

申请号：US09406074

申请日：1999-09-27

申请人： James G. Nadeau ,  C. Preston Linn ,  J. Bruce Pitner ,  Cheryl H. Dean ,  G. Terrance Walker

发明人： James G. Nadeau ,  C. Preston Linn ,  J. Bruce Pitner ,  Cheryl H. Dean ,  G. Terrance Walker

IPC分类号： C12Q168

CPC分类号： C12Q1/6818 ,  Y10T436/143333 ,  C12Q2563/137 ,  C12Q2537/1373

摘要： Signal primers are employed for detection of nucleic acid target sequences by fluorescence quenching mechanisms. The signal primer comprises a first and a second oligonucleotide and is partially single-stranded and partially double-stranded. In the presence of target, the second oligonucleotide of the signal primer is displaced from the first and a conformational change in a reporter probe occurs which changes the distance between the members of a donor/quencher dye pair linked to the reporter probe. The change in proximity between the dyes causes an increase or a decrease in fluorescence quenching, which is detected as an indication of the presence of the target sequence.

摘要翻译： 信号引物用于通过荧光猝灭机制检测核酸靶序列。 信号引物包含第一和第二寡核苷酸并且是部分单链和部分双链的。 在靶的存在下，信号引物的第二寡核苷酸从第一个位点移位，并且发生报告物探针中的构象变化，其改变与报道探针连接的供体/猝灭剂染料对的成员之间的距离。 染料之间接近度的变化导致荧光淬灭的增加或减少，其被检测为靶序列存在的指示。

公开(公告)号：US06261784B1

公开(公告)日：2001-07-17

申请号：US09599164

申请日：2000-06-22

申请人： James G. Nadeau ,  Helen V. Hsieh ,  J. Bruce Pitner ,  C. Preston Linn

发明人： James G. Nadeau ,  Helen V. Hsieh ,  J. Bruce Pitner ,  C. Preston Linn

IPC分类号： C12Q168

CPC分类号： C12Q1/6818 ,  C12Q2537/1373 ,  C12Q2531/143 ,  C12Q2531/119

摘要： Detector nucleic acids are employed for detection of nucleic acid target sequences by fluorescence quenching mechanisms. The detector nucleic acid comprises at least two oligonucleotides and is partially single-stranded and partially double-stranded. One of the two dyes of a donor/acceptor dye pair is linked to the first oligonucleotide and the other is linked to a second oligonucleotide such that they are in close spatial proximity when the first and second oligonucleotides are base-paired and donor fluorescence is quenched. A single second oligonucleotide may be hybridized to the first oligonucleotide or multiple second oligonucleotides may be hybridized to the first oligonucleotide and to each other, forming a junction structure comprising multiple donor/acceptor dye pairs. The detector oligonucleotide retains its partially single-stranded and partially double-stranded conformation in the absence of target. In the presence of target, however, the second oligonucleotide(s) of the detector nucleic acid is/are completely or partially displaced from the first, increasing the distance between the donor and acceptor dyes and causing a change in fluorescence which may be detected as an indication of the presence of the target sequence.

摘要翻译： 检测器核酸用于通过荧光猝灭机制检测核酸靶序列。 检测器核酸包含至少两个寡核苷酸并且是部分单链和部分双链的。 供体/受体染料对的两种染料之一与第一寡核苷酸连接，另一种与第二寡核苷酸连接，使得当第一和第二寡核苷酸是碱基配对并且供体荧光淬灭时它们处于紧密的空间接近 。 单个第二寡核苷酸可以与第一寡核苷酸杂交，或者多个第二寡核苷酸可以与第一寡核苷酸和彼此杂交，形成包含多个供体/受体染料对的连接结构。 检测器寡核苷酸在不存在目标时保留其部分单链和部分双链构象。 然而，在靶存在下，检测器核酸的第二寡核苷酸与第一寡核苷酸完全或部分位移，从而增加供体和受体染料之间的距离并引起荧光的变化，其可被检测为 指示靶序列的存在。

公开(公告)号：US6130047A

公开(公告)日：2000-10-10

申请号：US235583

申请日：1999-01-22

申请人： James G. Nadeau ,  Helen V. Hsieh ,  J. Bruce Pitner ,  C. Preston Linn

发明人： James G. Nadeau ,  Helen V. Hsieh ,  J. Bruce Pitner ,  C. Preston Linn

IPC分类号： G01N21/64 ,  C07H21/00 ,  C12N15/09 ,  C12Q1/68 ,  G01N33/542 ,  G01N33/566 ,  C07H21/04 ,  C12P19/34

CPC分类号： C12Q1/6818

摘要： Detector nucleic acids are employed for detection of nucleic acid target sequences by fluorescence quenching mechanisms. The detector nucleic acid comprises at least two oligonucleotides and is partially single-stranded and partially double-stranded. One of the two dyes of a donor/acceptor dye pair is linked to the first oligonucleotide and the other is linked to a second oligonucleotide such that they are in close spatial proximity when the first and second oligonucleotides are base-paired and donor fluorescence is quenched. A single second oligonucleotide may be hybridized to the first oligonucleotide or multiple second oligonucleotides may be hybridized to the first oligonucleotide and to each other, forming a junction structure comprising multiple donor/acceptor dye pairs. The detector oligonucleotide retains its partially single-stranded and partially double-stranded conformation in the absence of target. In the presence of target, however, the second oligonucleotide(s) of the detector nucleic acid is/are completely or partially displaced from the first, increasing the distance between the donor and acceptor dyes and causing a change in fluorescence which may be detected as an indication of the presence of the target sequence.

摘要翻译： 检测器核酸用于通过荧光猝灭机制检测核酸靶序列。 检测器核酸包含至少两个寡核苷酸并且是部分单链和部分双链的。 供体/受体染料对的两种染料之一与第一寡核苷酸连接，另一种与第二寡核苷酸连接，使得当第一和第二寡核苷酸是碱基配对并且供体荧光淬灭时它们处于紧密的空间接近 。 单个第二寡核苷酸可以与第一寡核苷酸杂交，或者多个第二寡核苷酸可以与第一寡核苷酸和彼此杂交，形成包含多个供体/受体染料对的连接结构。 检测器寡核苷酸在不存在目标时保留其部分单链和部分双链构象。 然而，在靶存在下，检测器核酸的第二寡核苷酸与第一寡核苷酸完全或部分位移，从而增加供体和受体染料之间的距离并引起荧光的变化，其可被检测为 指示靶序列的存在。