公开(公告)号：US07560254B2

公开(公告)日：2009-07-14

申请号：US10651558

申请日：2003-08-29

申请人： Anup Sood ,  Shiv Kumar ,  Carl Fuller ,  John Nelson ,  John Macklin

发明人： Anup Sood ,  Shiv Kumar ,  Carl Fuller ,  John Nelson ,  John Macklin

IPC分类号： C12P19/34 ,  C12Q1/68 ,  C07H21/02 ,  C07H21/04 ,  C07H21/00

CPC分类号： C12Q1/6853 ,  C12Q1/6858 ,  C12Q2525/125 ,  C12Q2521/525 ,  C12Q2521/319

摘要： A method of characterizing a nucleic acid sample is provided that includes the steps of: (a) conducting a DNA polymerase reaction that includes the reaction of a template, an allele specific primer, at least one terminal phosphate-labeled nucleotide, DNA polymerase, and optionally an enzyme having 3′→5′ exonuclease activity when the primer is non-hydrolyzable, which reaction results in the production of labeled polyphosphate; (b) permitting the labeled polyphosphate to react with a phosphatase to produce a detectable species; (c) detecting the detectable species; and (d) characterizing the nucleic acid sample based on such detection.

摘要翻译： 提供了表征核酸样品的方法，其包括以下步骤：（a）进行DNA聚合酶反应，其包括模板，等位基因特异性引物，至少一个末端磷酸酯标记的核苷酸，DNA聚合酶和 任选地当引物不可水解时具有3'→5'核酸外切酶活性的酶，该反应导致标记的多磷酸盐的产生; （b）允许标记的多磷酸酯与磷酸酶反应以产生可检测的物质; （c）检测可检测物种; 和（d）基于这种检测来表征核酸样品。

公开(公告)号：US07393640B2

公开(公告)日：2008-07-01

申请号：US10772996

申请日：2004-02-05

申请人： Shiv Kumar ,  Mark McDougall ,  Anup Sood ,  John Nelson ,  Carl Fuller ,  John Macklin ,  Paul Mitsis

发明人： Shiv Kumar ,  Mark McDougall ,  Anup Sood ,  John Nelson ,  Carl Fuller ,  John Macklin ,  Paul Mitsis

IPC分类号： C12Q1/68 ,  C12P19/34

CPC分类号： C07H19/10 ,  C07H19/20 ,  C07H21/00 ,  C12P19/34

摘要： The present invention describes methods of using terminal-phosphate-labeled nucleotides in the presence of a manganese salt to enhance their substrate properties towards various enzymes. Particularly described are methods of detecting a nucleic acid in a sample, based on the use of terminal-phosphate-labeled nucleotides as substrates for nucleic acid polymerases, in the presence of a manganese salt. Further provided are manganese complexes of terminal-phosphate-labeled nucleotides as well as terminal-phosphate-labeled nucleotides with new linkers with enhanced substrate properties.

摘要翻译： 本发明描述了在锰盐存在下使用末端磷酸酯标记的核苷酸以增强其对各种酶的底物性质的方法。 特别描述的是在锰盐存在的情况下，基于使用末端磷酸酯标记的核苷酸作为核酸聚合酶的底物，检测样品中的核酸的方法。 还提供了末端磷酸酯标记的核苷酸的锰络合物以及具有增强的底物性质的新接头的末端磷酸酯标记的核苷酸。

公开(公告)号：US07033762B2

公开(公告)日：2006-04-25

申请号：US10113025

申请日：2002-04-01

申请人： John Nelson ,  Carl Fuller ,  Anup Sood ,  Shiv Kumar

发明人： John Nelson ,  Carl Fuller ,  Anup Sood ,  Shiv Kumar

IPC分类号： C12Q1/68

CPC分类号： C12Q1/6823 ,  C12Q2545/114 ,  C12Q2521/525 ,  C12Q2521/101

摘要： A method of characterizing a nucleic acid sample is provided that includes the steps of: (a) conducting a DNA polymerase reaction that includes the reaction of a template, a non-hydrolyzable primer, at least one terminal phosphate-labeled nucleotide, DNA polymerase, and an enzyme having 3′→5′ exonuclease activity which reaction results in the production of labeled polyphosphate; (b) permitting the labeled polyphosphate to react with a phosphatase to produce a detectable species characteristic of the sample; (c) detecting the detectable species; and (d) characterizing the nucleic acid sample based on the detection.

公开(公告)号：US07125671B2

公开(公告)日：2006-10-24

申请号：US10651362

申请日：2003-08-29

申请人： Anup Sood ,  Shiv Kumar ,  John Nelson ,  Carl Fuller ,  Anuradha Sekher

发明人： Anup Sood ,  Shiv Kumar ,  John Nelson ,  Carl Fuller ,  Anuradha Sekher

IPC分类号： C12Q1/68 ,  C12P19/34

CPC分类号： C12Q1/6816 ,  C12Q1/6823 ,  C12Q1/6848 ,  C12Q1/686 ,  C12Q2525/101 ,  C12Q2521/525 ,  C12Q2521/101 ,  C12Q2565/301 ,  C12Q2561/113

摘要： The present invention relates generally to the use of terminal-phosphate-labeled nucleotides having three or more phosphates as substrates for nucleic acid polymerases and their use in DNA amplification. The labels employed are chemiluminescent, fluorescent, electrochemical and chromogenic moieties as well as mass tags and include those that are directly detectable, detectable after enzyme activation or feed into other processes to generate a different signal. The signal generated from the attached dyes may also be used to quantify the amount of amplification. Further provided are stabilizers that enhance the stability of terminal-phosphate labeled nucleoside polyphosphates in aqueous solutions and are useful for reducing non-enzymatic hydrolysis of these nucleotides, hence decrease background.

摘要翻译： 本发明一般涉及具有三种或更多种磷酸盐的末端磷酸酯标记的核苷酸作为核酸聚合酶的底物及其在DNA扩增中的应用。 使用的标记是化学发光，荧光，电化学和显色部分以及质量标签，并且包括直接可检测的标记，可在酶活化后进行或进入其它过程以产生不同信号。 从连接的染料产生的信号也可用于量化扩增量。 还提供了增强末端磷酸酯标记的核苷多磷酸酯在水溶液中的稳定性的稳定剂，并且可用于减少这些核苷酸的非酶水解，从而降低背景。

公开(公告)号：US07052839B2

公开(公告)日：2006-05-30

申请号：US10113030

申请日：2002-04-01

申请人： John Nelson ,  Carl Fuller ,  Anup Sood ,  Shiv Kumar

发明人： John Nelson ,  Carl Fuller ,  Anup Sood ,  Shiv Kumar

IPC分类号： C12Q1/68

CPC分类号： C07H19/10 ,  C07H19/20 ,  C07H21/00 ,  C12Q1/6816 ,  C12Q1/6851

摘要： The present invention describes methods of detecting a nucleic acid in a sample, based on the use of terminal-phosphate-labeled nucleotides as substrates for nucleic acid polymerases. The methods provided by this invention utilize a nucleoside polyphosphate, dideoxynucleoside polyphosphate, or deoxynucleoside polyphosphate analogue which has a colorimetric dye, chemiluminescent, or fluorescent moiety, a mass tag or an electrochemical tag attached to the terminal-phosphate. When a nucleic acid polymerase uses this analogue as a substrate, an enzyme-activatable label would be present on the inorganic polyphosphate by-product of phosphoryl transfer. Cleavage of the polyphosphate product of phosphoryl transfer via phosphatase leads to a detectable change in the label attached thereon. When the polymerase assay is performed in the presence of a phosphatase, there is provided a convenient method for real-time monitoring of DNA or RNA synthesis and detection of a target nucleic acid.

公开(公告)号：US07256019B2

公开(公告)日：2007-08-14

申请号：US10651355

申请日：2003-08-29

申请人： Anup Sood ,  Shiv Kumar ,  Carl Fuller ,  John Nelson

发明人： Anup Sood ,  Shiv Kumar ,  Carl Fuller ,  John Nelson

IPC分类号： C12Q1/68 ,  C12P19/34 ,  C07H21/04

CPC分类号： C07H19/20 ,  C07H19/10 ,  C07H21/00 ,  C12Q1/6816 ,  C12Q1/6851 ,  C12Q1/6869 ,  C12Q2525/186

摘要： The present invention describes terminal phosphate blocked nucleoside polyphosphates that are stable at high temperature and their use in nucleic acid amplification and analysis. Current invention further describes charge modified terminal phosphate blocked nucleoside polyphosphates for improved incorporation and direct loading of nucleic acid sequencing reactions onto separating media.

摘要翻译： 本发明描述了在高温下稳定的磷酸封端的磷酸核苷多磷酸，并且其用于核酸扩增和分析。 本发明进一步描述了电荷修饰的末端磷酸酯封端的核苷多磷酸酯，用于改进核酸测序反应在分离介质上的掺入和直接加载。

公开(公告)号：US07244566B2

公开(公告)日：2007-07-17

申请号：US10651582

申请日：2003-08-29

申请人： Anup Sood ,  Shiv Kumar ,  Carl Fuller ,  John Nelson

发明人： Anup Sood ,  Shiv Kumar ,  Carl Fuller ,  John Nelson

IPC分类号： C12Q1/68

CPC分类号： C12P19/34 ,  C12Q1/68

摘要： A method of characterizing an analyte sample is provided that includes the steps of: (a) anchoring the analyte to a nucleic acid template of known sequence; (b) conducting a DNA polymerase reaction that includes the reaction of a template, a non-hydrolyzable primer, at least one terminal phosphate-labeled nucleotide, DNA polymerase, and an enzyme having 3′→5′ exonuclease activity which reaction results in the production of labeled polyphosphate; (c) permitting the labeled polyphosphate to react with a phosphatase to produce a detectable species characteristic of the sample; (d) detecting the detectable species. The method may include the step of characterizing the nucleic acid sample based on the detection. Also provided are methods of analyzing multiple analytes in a sample, and kits for characterizing analyte samples.

摘要翻译： 提供表征分析物样品的方法，其包括以下步骤：（a）将分析物锚定到已知序列的核酸模板; （b）进行DNA聚合酶反应，其包括模板，不可水解的引物，至少一个末端磷酸酯标记的核苷酸，DNA聚合酶和具有3'→5'核酸外切酶活性的酶的反应，该反应导致 生产标记多磷酸盐; （c）允许标记的多磷酸酯与磷酸酶反应以产生样品的可检测物种特征; （d）检测可检测物种。 该方法可以包括基于检测来表征核酸样品的步骤。 还提供了分析样品中多种分析物的方法，以及用于表征分析物样品的试剂盒。

公开(公告)号：US07223541B2

公开(公告)日：2007-05-29

申请号：US10358818

申请日：2003-02-05

申请人： Carl Fuller ,  Shiv Kumar ,  Anup Sood ,  John Nelson

发明人： Carl Fuller ,  Shiv Kumar ,  Anup Sood ,  John Nelson

IPC分类号： C12Q1/68

CPC分类号： C12Q1/6823 ,  C07H19/10 ,  C07H19/20 ,  C07H21/00 ,  C12Q1/6816 ,  C12Q1/6851 ,  C12Q2521/525

摘要： The present invention relates to improved methods of detecting a target using a labeled substrate or substrate analog. The methods comprise reacting the substrate or substrate analog in an enzyme-catalyzed reaction which produces a labeled moiety with independently detectable signal only when such substrate or substrate analog reacts. The present invention, in particular, describes methods of detecting a nucleic acid in a sample, based on the use of terminal-phosphate-labeled nucleotides as substrates for nucleic acid polymerases. The methods provided by this invention utilize a nucleoside polyphosphate, dideoxynucleoside polyphosphate, or deoxynucleoside polyphosphate analogue which has a colorimetric dye, chemiluminescent, or fluorescent moiety, a mass tag or an electrochemical tag attached to the terminal-phosphate. When a nucleic acid polymerase uses this analogue as a substrate, an enzyme-activatable label would be present on the inorganic polyphosphate by-product of phosphoryl transfer. Cleavage of the polyphosphate product of phosphoryl transfer via phosphatase leads to a detectable change in the label attached thereon. When the polymerase assay is performed in the presence of a phosphatase, there is provided a convenient method for real-time monitoring of DNA or RNA synthesis and detection of a target nucleic acid.

摘要翻译： 本发明涉及使用标记的底物或底物类似物检测靶的改进方法。 所述方法包括在酶催化的反应中使底物或底物类似物反应，其仅在这种底物或底物类似物反应时产生具有独立可检测信号的标记部分。 本发明特别地描述了基于使用末端磷酸酯标记的核苷酸作为核酸聚合酶的底物来检测样品中核酸的方法。 本发明提供的方法利用了具有比色染料，化学发光或荧光部分的核苷多磷酸，双脱氧核苷多聚磷酸酯或脱氧核苷多聚磷酸酯类似物，连接至末端磷酸酯的质量标签或电化学标签。 当核酸聚合酶使用该类似物作为底物时，酶活性标记将存在于磷酸转移的无机多磷酸盐副产物上。 通过磷酸酶切割磷酸转移的多磷酸盐产物导致附着在其上的标签的可检测的变化。 当在磷酸酶的存在下进行聚合酶测定时，提供了用于实时监测DNA或RNA合成和检测靶核酸的方便的方法。

公开(公告)号：US07109316B2

公开(公告)日：2006-09-19

申请号：US10226734

申请日：2002-08-23

申请人： Shiv Kumar ,  Anup Sood ,  Sudhakar Rao ,  John Nelson

发明人： Shiv Kumar ,  Anup Sood ,  Sudhakar Rao ,  John Nelson

IPC分类号： C07H21/02 ,  C07H21/04 ,  C07H19/04 ,  C12Q1/68

CPC分类号： C07H19/04

摘要： Oligonucleotide tagged nucleoside triphosphates, OTNTPs, which are substrates for polymerases and or terminal nucleotidyl transferases are provided as well as methods of making these OTNTPs. Further provided are OTNTPs with fluorescent dyes including energy transfer dyes, attached to the oligonucleotide chain, OTNTPs with unnatural bases incorporated in the oligonucleotide sequence and methods for incorporating these OTNTPs in DNA or RNA. Also provided are methods for using the oligonucleotides on OTNTPs for amplifying the oligo sequence on the OTNTP using an amplification method described above. Further provided are reactive bifunctional amidites, methods of making these compounds and methods for detecting single nucleotide polymorphisms using the above OTNTPs. Methods for detecting differential gene expression using the OTNTPs and methods of separating specifically modified DNA or RNA using the OTNTPs are also provided.

摘要翻译： 提供了寡聚核苷酸标记的核苷三磷酸酯，OTNTP，其是用于聚合酶和末端核苷酸转移酶的底物，以及制备这些OTNTP的方法。 还提供了含有荧光染料的OTNTP，其包括与寡核苷酸链连接的能量转移染料，掺入寡核苷酸序列中的非天然碱基的OTNTP以及将这些OTNTP引入DNA或RNA中的方法。 还提供了使用寡核苷酸在OTNTP上使用上述扩增方法在OTNTP上扩增寡核苷酸序列的方法。 还提供了反应性双官能脒，制备这些化合物的方法和使用上述OTNTP检测单核苷酸多态性的方法。 还提供了使用OTNTP检测差异基因表达的方法和使用OTNTP分离特异性修饰的DNA或RNA的方法。

公开(公告)号：US20180229323A1

公开(公告)日：2018-08-16

申请号：US15432237

申请日：2017-02-14

申请人： John Nelson

发明人： John Nelson

IPC分类号： B23K3/08 ,  B23K101/38

摘要： A soldering jig assembly for soldering conductors together includes a plate that may be positioned on a support surface. A pair of gripping units is provided. Each of the gripping units is coupled to the plate and each of the gripping units is selectively manipulated. Each of the gripping units engages an associated one of a pair of conductors.