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公开(公告)号：US08323929B2
公开(公告)日：2012-12-04
申请号：US12419737
申请日：2009-04-07
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。
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公开(公告)号：US20090131647A1
公开(公告)日：2009-05-21
申请号：US11724180
申请日：2007-03-15
申请人： James G. Nadeau , Tobin J. Hellyer
发明人： James G. Nadeau , Tobin J. Hellyer
IPC分类号： C07H21/04
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。
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公开(公告)号：USRE39885E1
公开(公告)日：2007-10-16
申请号：US09082247
申请日：1998-05-20
申请人： James G. Nadeau , George T. Walker
发明人： James G. Nadeau , George T. Walker
CPC分类号： C12Q1/6844 , C12Q2565/525 , C12Q2561/113 , C12Q2531/119
摘要： Methods for detecting, immobilizing or localizing primer extension products of a Strand Displacement Amplification reaction which are coupled to, and an indication of, amplification of the target sequence. The primer extension products are secondary, target-specific DNA products generated concurrently with SDA of the target sequence and can therefore be used to detect and/or measure target sequence amplification in real-time. In general, the secondary amplification products are not amplifiable and remain inert in the SDA reaction after they are formed without interfering with amplification of the target sequence. The secondary amplification products may be designed or modified to contain special features to facilitate their detection, immobilization or localization.
摘要翻译： 用于检测，固定或定位连接于链序列扩增反应的引物延伸产物的方法，以及扩增靶序列的指示。 引物延伸产物是与靶序列的SDA同时产生的次要靶特异性DNA产物，因此可用于实时检测和/或测量靶序列扩增。 通常，二次扩增产物在其形成后不能扩增并在SDA反应中保持惰性，而不干扰靶序列的扩增。 二次扩增产物可以被设计或修改以包含特殊特征以促进其检测，固定或定位。
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公开(公告)号：US07223536B2
公开(公告)日：2007-05-29
申请号：US09778168
申请日：2001-02-07
CPC分类号： C12Q1/6827 , C12Q2565/1025 , C12Q2535/125 , C12Q2531/119 , C12Q2531/101
摘要： The present invention provides methods for detecting and identifying sequence variations in a nucleic acid sequence of interest using a detector primer. It has been found that the reduced efficiency of primer extension by DNA polymerases when the 3′ end of a primer does not hybridize perfectly with the target can be adapted for use as a means for distinguishing or identifying the nucleotide in the target which is at the site where the diagnostic mismatch between the detector primer and the target occurs. The detector primer hybridizes to the sequence of interest and is extended with polymerase. The efficiency of detector primer extension is detected as an indication of the presence and/or identity of the sequence variation in the target. The inventive methods make use of nucleotide mismatches at or near the 3′ end of the detector primer to discriminate between the nucleotide sequence of interest and a second nucleotide sequence which may occur at that same site in the target. The methods are particularly well suited for detecting and identifying single nucleotide differences between a target sequence of interest (e.g., a mutant allele of a gene) and a second nucleic acid sequence (e.g., a wild type allele for the same gene).
摘要翻译： 本发明提供使用检测器引物检测和鉴定感兴趣的核酸序列中的序列变异的方法。 已经发现，当引物的3'端没有与靶完全杂交时，通过DNA聚合酶的引物延伸的效率降低可以适用于区分或鉴定靶中的核苷酸的手段 发现检测器引物与靶标之间的诊断不匹配的位置。 检测引物与感兴趣的序列杂交，并用聚合酶扩增。 检测器引物延伸的效率被检测为目标中序列变异的存在和/或身份的指示。 本发明的方法利用在检测器引物的3'末端附近或其附近的核苷酸错配来区分目的核苷酸序列和可能发生在靶的相同位点的第二核苷酸序列。 所述方法特别适用于检测和鉴定靶标目标序列（例如，基因的突变等位基因）和第二核酸序列（例如，相同基因的野生型等位基因）之间的单核苷酸差异。
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公开(公告)号：US5935791A
公开(公告)日：1999-08-10
申请号：US933749
申请日：1997-09-23
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.
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公开(公告)号：US5928869A
公开(公告)日：1999-07-27
申请号：US865675
申请日：1997-05-30
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.
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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
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。
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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
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.
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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
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。
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公开(公告)号：US5658738A
公开(公告)日：1997-08-19
申请号：US539516
申请日：1995-12-11
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。
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