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1. 发明授权

US5536649A Decontamination of nucleic acid amplification reactions using uracil-N-glycosylase (UDG) 失效
标题翻译：使用尿嘧啶-N-糖基化酶（UDG）进行核酸扩增反应的去污
公开(公告)号：US5536649A
公开(公告)日：1996-07-16
申请号：US283117
申请日：1994-07-29
申请人： Melinda S. Fraiser , George T. Walker , James L. Schram
发明人： Melinda S. Fraiser , George T. Walker , James L. Schram
IPC分类号： C12N15/09 , C12Q1/68 , C12P19/34
CPC分类号： C12Q1/6844 , C12Q1/6848
摘要： Methods for inactivating contaminating amplicons in isothermal nucleic acid amplification reactions such as SDA. Uracil is incorporated into the amplicons produced by amplification in the place of thymine (T) using novel SDA reaction conditions. If these amplicons contaminate a subsequent amplification reaction, they may be inactivated as templates (i.e., rendered unamplifiable) by treatment with UDG. As isothermal amplification does not involve elevated temperatures, the UDG may be inactivated during the subsequent amplification of specific target sequences by inclusion of the UDG inhibitor protein Ugi. Incorporation of dU has unexpectedly been found to enhance the amplification power of SDA as compared to conventional SDA reactions. The methods may also be used to detect UDG activity in reagents or samples.
摘要翻译：在等温核酸扩增反应（如SDA）中灭活污染扩增子的方法。尿嘧啶通过使用新的SDA反应条件通过扩增代替胸腺嘧啶（T）而产生的扩增子。如果这些扩增子污染随后的扩增反应，则它们可以通过用UDG处理作为模板灭活（即，使其不能扩增）。由于等温扩增不包括升高的温度，所以UDG可以在随后通过包含UDG抑制剂蛋白Ugi扩增特异性靶序列期间失活。与传统的SDA反应相比，意外地发现dU的引入增强了SDA的放大功率。该方法也可用于检测试剂或样品中的UDG活性。

2. 发明授权

US5561044A Detection of mycobacteria by multiplex strand displacement nucleic acid amplification 失效
标题翻译：通过多重链置换核酸扩增检测分枝杆菌
公开(公告)号：US5561044A
公开(公告)日：1996-10-01
申请号：US398305
申请日：1995-03-03
申请人： George T. Walker , James G. Nadeau , Patricia A. Spears , Colleen M. Nycz , Daryl D. Shank , James L. Schram , Stewart R. Jurgensen
发明人： George T. Walker , James G. Nadeau , Patricia A. Spears , Colleen M. Nycz , Daryl D. Shank , James L. Schram , Stewart R. Jurgensen
IPC分类号： C12N15/09 , C12Q1/04 , C12Q1/68 , C12R1/32 , C12R1/33 , C12P19/34
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

US5470723A Detection of mycobacteria by multiplex nucleic acid amplification 失效
标题翻译：通过多重核酸扩增检测分枝杆菌
公开(公告)号：US5470723A
公开(公告)日：1995-11-28
申请号：US111076
申请日：1993-08-24
申请人： George T. Walker , James G. Nadeau , Patricia A. Spears , Colleen M. Nycz , Daryl D. Shank , James L. Schram , Stewart R. Jurgensen
发明人： George T. Walker , James G. Nadeau , Patricia A. Spears , Colleen M. Nycz , Daryl D. Shank , James L. Schram , Stewart R. Jurgensen
IPC分类号： C12N15/09 , C12Q1/04 , C12Q1/68 , C12R1/32 , C12R1/33 , C07H15/12 , C07H17/00 , C12P19/34
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 ant 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，扩增反应中包含内部控制序列并与IS6110蚂蚁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。

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

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。

6. 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。

7. 发明授权

US09823253B2 Assays using surface-enhanced raman spectroscopy (SERS)-active particles 有权
公开(公告)号：US09823253B2
公开(公告)日：2017-11-21
申请号：US12531508
申请日：2008-03-20
申请人： Kristin Weidemaier , Christian Sandmann , W. Shannon Dillmore , James L. Schram , W. William Stewart , Robert E. Pearson , Helen Hsieh , Steven Keith , Rajendra R. Bhat , Andrea Liebmann-Vinson , Adam Craig Curry , Alexander G. Lastovich
发明人： Kristin Weidemaier , Christian Sandmann , W. Shannon Dillmore , James L. Schram , W. William Stewart , Robert E. Pearson , Helen Hsieh , Steven Keith , Rajendra R. Bhat , Andrea Liebmann-Vinson , Adam Craig Curry , Alexander G. Lastovich
IPC分类号： C12Q1/68 , G01N33/53 , G01N33/58 , G01N21/65 , G01N33/543
CPC分类号： G01N33/587 , G01N21/658 , G01N33/54333
摘要： Disclosed herein are diagnostic assays using surface enhanced Raman spectroscopy (SERS)-active particles, including liquid-based assays; magnetic capture assays; microparticle-nanoparticle satellite structures for signal amplification in an assay; composite SERS-active particles useful for enhanced detection of targets; and sample tubes and processes for using the same.

8. 发明授权

US5928869A Detection of nucleic acids by fluorescence quenching 失效
公开(公告)号：US5928869A
公开(公告)日：1999-07-27
申请号：US865675
申请日：1997-05-30
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 , C07H21/00 , 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.

9. 发明授权

US5846726A Detection of nucleic acids by fluorescence quenching 失效
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.

10. 发明授权

US5811269A Detection of mycobacteria by multiplex nucleic acid amplification 失效
标题翻译：通过多重核酸扩增检测分枝杆菌
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。

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