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41. 发明授权

US6054279A Detection of nucleic acids by fluorescence quenching 失效
标题翻译：通过荧光猝灭检测核酸
公开(公告)号：US6054279A
公开(公告)日：2000-04-25
申请号：US120916
申请日：1998-07-20
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 , C21Q1/68
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.
摘要翻译：通过连接到形成供体/受体染料对的两种染料来修饰单链信号引物。这两种染料位于信号引物足够接近的空间上，第一种染料的荧光被第二种染料淬灭。信号引物还可以包含两种染料之间的限制性内切核酸酶识别位点（RERS）。由于信号引物最初是单链的，并且在不存在靶标的情况下保持单链，限制性内切核酸酶识别位点不受限制性内切核酸酶的切割或切割。然而，在靶的存在下，信号引物和限制性内切核酸酶识别位点被限制性内切核酸酶双链和切割或切割。切割或切口分离两种染料，并且由于猝灭降低引起的荧光变化被检测为目标序列或靶序列扩增的存在的指示。

42. 发明授权

US5958700A Detection of nucleic acids by fluorescence quenching 失效
标题翻译：通过荧光猝灭检测核酸
公开(公告)号：US5958700A
公开(公告)日：1999-09-28
申请号：US237510
申请日：1999-01-26
申请人： James G. Nadeau , J. Bruce Pitner , C. Preston Linn , James L. Schram
发明人： James G. Nadeau , J. Bruce Pitner , C. Preston Linn , James L. Schram
IPC分类号： G01N33/50 , C12N15/09 , C12Q1/68 , G01N33/566 , C07H21/04 , C12P19/34
CPC分类号： C12Q1/6827 , C12Q1/6818 , C12Q1/6858
摘要： A detector oligonucleotide having a sequence which forms an intramolecularly base-paired secondary structure is described for use in detecting nucleic acid target sequences and target sequence amplification. The detector oligonucleotide is further modified by linkage to two dyes which form a donor/acceptor dye pair. The two dyes are positioned on the detector oligonucleotide such that they are in close spatial proximity in the base-paired, folded secondary structure, thereby causing quenching of donor fluorescence. The detector oligonucleotide may optionally further comprise a restriction endonuclease recognition site (RERS) which remains partially or entirely single-stranded in the base-paired secondary structure. The RERS is flanked by the two dyes. In the presence of target, the base-paired secondary structure is unfolded or linearized, increasing the distance between the donor and acceptor dyes and causing a change in fluorescence of the donor and/or the acceptor. If an RERS is present, it is rendered double-stranded in the presence of target, allowing cleavage or nicking by a restriction endonuclease and separation of the two dyes onto separate nucleic acid fragments. This may further contribute to the magnitude of the change in fluorescence.
摘要翻译：描述了具有形成分子内碱基配对二级结构的序列的检测器寡核苷酸用于检测核酸靶序列和靶序列扩增。通过连接到形成供体/受体染料对的两种染料进一步修饰检测器寡核苷酸。两种染料位于检测器寡核苷酸上，使得它们在碱基配对的折叠的二级结构中处于紧密的空间接近处，由此引起供体荧光的猝灭。检测器寡核苷酸可以任选地进一步包含限制性内切核酸酶识别位点（RERS），所述限制性内切核酸酶识别位点在碱基配对二级结构中部分或全部保持单链。 RERS侧面有两种染料。在靶的存在下，碱基配对的二级结构被展开或线性化，增加供体和受体染料之间的距离并引起供体和/或受体的荧光变化。如果存在RERS，则在靶的存在下使其成为双链，允许通过限制性内切核酸酶切割或切割并将两种染料分离到单独的核酸片段上。这可能进一步有助于荧光变化的大小。

43. 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。

44. 发明授权

US5691145A Detection of nucleic acids using G-quartets 失效
标题翻译：使用G四重奏检测核酸
公开(公告)号：US5691145A
公开(公告)日：1997-11-25
申请号：US703755
申请日：1996-08-27
申请人： J. Bruce Pitner , Glenn P. Vonk , James G. Nadeau
发明人： J. Bruce Pitner , Glenn P. Vonk , James G. Nadeau
IPC分类号： G01N33/58 , C07H21/04 , C12N15/09 , C12Q1/68 , G01N21/78 , C07H21/02 , C12P19/34
CPC分类号： C12Q1/6818 , Y10S436/80 , Y10S436/805 , Y10T436/143333
摘要： Oligonucleotides which form G-quartet structures have been found to be useful in fluorescence assays to detect a selected nucleic acid sequence. When one end of the oligonucleotide is labeled with a donor fluorophore and the other end is labeled with an acceptor dye, the folding of the molecule 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 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.
摘要翻译：已经发现形成G-四重结构的寡核苷酸可用于荧光测定以检测所选择的核酸序列。当寡核苷酸的一端用供体荧光团标记，另一端用受体染料标记时，分子在G四重结构中的折叠使供体 - 受体对紧密接近，允许两者之间的相互作用导致供体荧光猝灭的标记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，增加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。

45. 发明授权

US5668265A Bi-directional oligonucleotides that bind thrombin 失效
标题翻译：结合凝血酶的双向寡核苷酸
公开(公告)号：US5668265A
公开(公告)日：1997-09-16
申请号：US614447
申请日：1996-03-12
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 , C07H21/04 , A61K35/00
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。

46. 发明授权

US5593867A Fluorerscence polarization detection of nucleic acid amplication 失效
标题翻译：荧光偏振检测核酸扩增
公开(公告)号：US5593867A
公开(公告)日：1997-01-14
申请号：US311474
申请日：1994-09-23
申请人： G. Terrance Walker , James G. Nadeau , C. Preston Linn
发明人： G. Terrance Walker , James G. Nadeau , C. Preston Linn
IPC分类号： C12N15/09 , C12Q1/68 , C12R1/32 , C12P19/34
CPC分类号： C12Q1/6844
摘要： Fluorescence polarization methods for detection of nucleic acid amplification. A fluorescently labelled oligodeoxynucleotide probe is converted from single- to double-stranded form in a target dependent manner during amplification of the target sequence. This conformational change is accompanied by an increase in fluorescence polarization values. The increase in fluorescence polarization can be measured on a transient-state fluorometer in real-time during the amplification reaction without any physical manipulation of the sample. The inventive methods therefore provide a closed, homogeneous system for both amplification of target sequences and detection of amplification. Alternatively, amplification may be detected in the fluorometer after the amplification reaction is completed.
摘要翻译：用于检测核酸扩增的荧光偏振方法。荧光标记的寡脱氧核苷酸探针在靶序列扩增期间以靶依赖的方式从单链形式转化为双链形式。这种构象变化伴随着荧光偏振值的增加。可以在扩增反应期间在瞬态荧光计上实时测量荧光偏振的增加，而不对样品进行任何物理操作。因此，本发明的方法提供用于靶序列扩增和扩增检测的封闭的均质系统。或者，扩增反应完成后，可以在荧光计中检测扩增。

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