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1. 发明申请

WO2004072297A2 TERMINAL-PHOSPHATE-LABELED NUCLEOTIDES AND METHODS OF USE 审中-公开
标题翻译：终端磷酸酯标记的核苷酸和使用方法
公开(公告)号：WO2004072297A2
公开(公告)日：2004-08-26
申请号：PCT/US2004/002785
申请日：2004-01-30
申请人： AMERSHAM BIOSCIENCES CORP , FULLER, Carl , KUMAR, Shiv , SOOD, Anup , NELSON, John
发明人： FULLER, Carl , KUMAR, Shiv , SOOD, Anup , NELSON, John
IPC分类号： C12Q
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 substrate 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记。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记物的可检测的变化。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。

2. 发明申请

WO2004072238A2 TERMINAL-PHOSPHATE-LABELED NUCLEOTIDES WITH NEW LINKERS 审中-公开
标题翻译：带有新连接器的终端磷酸酯标记的核苷酸
公开(公告)号：WO2004072238A2
公开(公告)日：2004-08-26
申请号：PCT/US2004/003284
申请日：2004-02-05
申请人： AMERSHAM BIOSCIENCES CORP , KUMAR, Shiv , MCDOUGALL, Mark , SOOD, Anup , NELSON, John , FULLER, Carl , MACKLIN, John , MITSIS, Paul
发明人： KUMAR, Shiv , MCDOUGALL, Mark , SOOD, Anup , NELSON, John , FULLER, Carl , MACKLIN, John , MITSIS, Paul
IPC分类号： C12N
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。

3. 发明申请

WO2004071155A3 SOLID PHASE SEQUENCING 审中-公开
公开(公告)号：WO2004071155A3
公开(公告)日：2004-08-26
申请号：PCT/US2004/003283
申请日：2004-02-05
申请人： AMERSHAM BIOSCIENCES CORP , SOOD, Anup , KUMAR, Shiv , NELSON, John , FULLER, Carl
发明人： SOOD, Anup , KUMAR, Shiv , NELSON, John , FULLER, Carl
IPC分类号： C12Q1/68
摘要： The present invention describes methods of sequencing 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. In some instances the labeled polyphosphate may be detected directly via the label and provide information on the nucleic acid. 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 characterization of a target nucleic acid.

4. 发明申请

WO2005108994A1 FLUORESCENCE RESONANCE ENERGY TRANSFER ENZYME SUBSTRATES 审中-公开
标题翻译：荧光共振能量转移酶基质
公开(公告)号：WO2005108994A1
公开(公告)日：2005-11-17
申请号：PCT/US2005/013141
申请日：2005-04-18
申请人： AMERSHAM BIOSCIENCES CORP , KUMAR, Shiv , SOOD, Anup
发明人： KUMAR, Shiv , SOOD, Anup
IPC分类号： G01N33/542
CPC分类号： G01N33/542 , C12Q1/34
摘要： Disclosed are compounds of formula (I) wherein D 1 is a first dye moiety whose fluorescence properties may be modulated so as to be suitable as a donor or an acceptor in an energy transfer arrangement; D 2 is a second dye moiety suitable as an acceptor or a donor in an energy transfer arrangement with said first dye; L is a linking group comprises 2-200 linked atoms, wherein said linking group optionally includes an enzyme cleavage site; and M is an enzyme cleavable group chosen to modulate the fluorescence properties of D 1 . The compounds of formula (I) may be used as reporter molecules for detecting biochemical cleavage events in assays that employ fluorescence resonance energy transfer.
摘要翻译：公开了式（I）的化合物，其中D 1是第一染料部分，其荧光性质可以被调节以适合作为能量转移装置中的给体或受体; D 2是与所述第一种染料在能量转移布置中适合作为受体或供体的第二染料部分; L是包含2-200个连接原子的连接基团，其中所述连接基团任选地包括酶切割位点; M是选择调节D 1荧光性质的酶切割基团。式（I）的化合物可以用作用于检测采用荧光共振能量转移的测定中生化裂解事件的报道分子。

5. 发明申请

WO2004085539A2 CYANINE DYE LABELLING REAGENTS WITH MESO-SUBSTITUTION 审中-公开
标题翻译：具有MESO替代品的氰基染料标签试剂
公开(公告)号：WO2004085539A2
公开(公告)日：2004-10-07
申请号：PCT/US2004/007833
申请日：2004-03-12
申请人： AMERSHAM BIOSCIENCES CORP , CHEN, Chung-Yuan , KUMAR, Shiv
发明人： CHEN, Chung-Yuan , KUMAR, Shiv
IPC分类号： C09B
CPC分类号： C09B23/164 , C07D209/24 , C09B23/0058 , C09B23/02 , G01N33/582
摘要： Disclosed are cyanine dyes having meso-substituents in the polymethine chain linking the heterocyclic ring systems. The dyes are of formula (I) in which groups R 3 and R 4 are attached to the Z 1 ring structure and groups R 5 and R 6 are attached to the Z 2 ring structure, and n = 1, 2 or 3; Z 1 and Z 2 independently represent the atoms necessary to complete one ring, or two fused ring aromatic or heteroaromatic systems, each ring having five or six atoms selected from carbon atoms and optionally no more than two atoms selected from oxygen, nitrogen and sulphur; X and Y are the same or different and are selected from oxygen, sulphur, -CH=CH- and the group (II) at least one of groups R 1 , R 2 , R 3 , R 4 , R 5 , R 6 (and R 8 and R 9 if present) is the group -E-F where E is a spacer group and F is a target bonding group; one of groups R 7 is selected from -CN, -Cl, -F, -CF 3 and -C(O)R 10 wherein R 10 is selected from H, C 1 -C 6 alkyl and aryl. Preferred group R 7 is meso-substituted -CN, which confers an unexpected hypsochromic shift of approximately 40nm in the emission spectrum, when compared with the corresponding unsubstituted analogue. The dyes are useful for labelling and detecting a variety of target molecules.
摘要翻译：公开了在连接杂环体系的聚甲炔链中具有内消旋取代基的花青染料。染料具有式（I），其中R 3和R 4基团连接到Z 1环结构，R 5和R 6基团连接到Z 2环上结构，n = 1,2或3; Z 1和Z 2独立地表示完成一个环或两个稠环芳族或杂芳族体系所必需的原子，每个环具有五个或六个选自碳原子和任选不超过两个选自氧，氮和硫; X和Y相同或不同，选自氧，硫，-CH = CH-，基团（II）R 1，R 2，R 3，R 4中的至少一个 R 5，R 6（和R 8和R 9如果存在）是基团-EF，其中E是间隔基团，F是目标键合基团; R 7中的一个选自-CN，-Cl，-F，-CF 3和-C（O）R 10，其中R 10选自H，C 1 -C 6烷基和芳基。优选的基团R 7是内消旋 - 取代的-CN，当与相应的未取代的类似物相比时，其在发射光谱中赋予约40nm的意想不到的变色位移。染料可用于标记和检测各种靶分子。

6. 发明申请

WO2004029579A2 FLUORESCENT LABELING REAGENTS WITH MULTIPLE DONORS AND ACCEPTORS 审中-公开
标题翻译：荧光标签试剂与多个捐助者和接受者
公开(公告)号：WO2004029579A2
公开(公告)日：2004-04-08
申请号：PCT/US2003/030361
申请日：2003-09-25
申请人： AMERSHAM BIOSCIENCES CORP , KUMAR, Shiv , CHEN, Chung-Yuan
发明人： KUMAR, Shiv , CHEN, Chung-Yuan
IPC分类号： G01N
CPC分类号： C09B11/08 , C07D311/82 , C07D405/14 , C07D487/04 , C09B11/04 , C09B11/24 , C12Q1/6869 , C12Q2527/125
摘要： Disclosed is a novel class of fluorescent resonance energy transfer (FRET) labelling reagents, based on and synthesised from easily prepared dye building blocks. The labelling reagents are in the form of "cassettes" which enable their attachment to a wide variety of biological and other materials. A labelling reagent comprises at least two fluorescent dye moieties covalently linked via a linker group and optionally having a target bonding group for attaching the reagent to a target. The energy transfer labelling reagents may be bound to target materials through covalent or non-covalent attachment. The dyes are selected so that the emission spectrum of a first (or donor) dye overlaps the absorption spectrum of a second dye, thereby allowing energy transfer to occur between the dyes. The dye building blocks are 4', 5'-bis-aminomethyl-fluorescein and/or its 5(6)-carboxylic acid and having the structure (I). In addition to the embodiment of the invention which includes a single donor and a single acceptor fluorochrome, the fluorescent energy transfer labelling reagents according to the invention may further comprise one or more third fluorochromes each having third absorption and emission spectra covalently attached to said first or second fluorochromes.
摘要翻译：公开了一种新型的荧光共振能量转移（FRET）标记试剂，基于并由易于制备的染料积木合成。标记试剂是“盒”的形式，使其能够连接到各种各样的生物和其他材料上。标记试剂包含至少两个通过接头基团共价连接的荧光染料部分，并且任选地具有用于将试剂连接到靶上的目标结合基团。能量转移标记试剂可以通过共价或非共价键连接到目标材料上。选择染料使得第一（或供体）染料的发射光谱与第二染料的吸收光谱重叠，从而允许在染料之间发生能量转移。染料结构单元是4'，5'-双氨基甲基 - 荧光素和/或其5（6） - 羧酸并具有结构（I）。除了包括单一供体和单受体荧光染料的本发明的实施方案之外，根据本发明的荧光能量转移标记试剂还可以包含一种或多种第三荧光染料，每个第三荧光染料具有共价连接到所述第一或第第二荧光染料。

7. 发明申请

WO2004029578A2 ENERGY TRANSFER DYES, TERMINATORS AND USE THEREOF 审中-公开
标题翻译：能量转运染色体，终端及其使用
公开(公告)号：WO2004029578A2
公开(公告)日：2004-04-08
申请号：PCT/US2003/030360
申请日：2003-09-25
申请人： AMERSHAM BIOSCIENCES CORP , KUMAR, Shiv , CHEN, Chung-Yuan , RAO, Sudhakar
发明人： KUMAR, Shiv , CHEN, Chung-Yuan , RAO, Sudhakar
IPC分类号： G01N
CPC分类号： C09B11/08 , C07D311/82 , C07D405/14 , C07D487/04 , C09B11/04 , C09B11/24 , C12Q1/6869 , C12Q2527/125
摘要： The present invention relates to a novel set of four fluorescently labeled dye terminators. Two of them are single dye labeled terminators and the other two dye terminators are based on fluorescent resonance energy transfer (FRET). The FRET dye terminators are generated from the 4', 5'-bis-aminomethylfluorescein. Of the two amino groups of the donor dye, 4', 5'-bis-aminomethylfluorescein, one amino group is used to attach the acceptor dye, and the other amino group is used to attach the dideoxynucleoside-5'-triphosphate. Their preparation, energy transfer efficiency, and use as labels, specifically, in DNA sequencing reactions is disclosed.
摘要翻译：本发明涉及一组四种荧光标记的染料终止剂。其中两个是单染色标记终止子，另外两个染料终止子是基于荧光共振能量转移（FRET）。 FRET染料终止子由4'，5'-双氨基甲基荧光素生成。在供体染料的两个氨基中，4'，5'-双氨基甲基荧光素，1个氨基用于连接受体染料，而另一个氨基用于连接双脱氧核苷-5'-三磷酸。公开了它们的制备，能量转移效率和用作标记，特别是在DNA测序反应中。

8. 发明申请

WO2020181129A1 HIGH ACCURACY NANOPORE-BASED SINGLE MOLECULE SEQUENCING BY SYNTHESIS WITH TAGGED NUCLEOTIDES 审中-公开
公开(公告)号：WO2020181129A1
公开(公告)日：2020-09-10
申请号：PCT/US2020/021253
申请日：2020-03-05
申请人： THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK , JU, Jingyue , FULLER, Carl, W. , KUMAR, Shiv , KALACHIKOV, Sergey , LI, Xiaoxu , MOROZOVA, Irina , RUSSO, James , JOCKUSCH, Steffen
发明人： JU, Jingyue , FULLER, Carl, W. , KUMAR, Shiv , KALACHIKOV, Sergey , LI, Xiaoxu , MOROZOVA, Irina , RUSSO, James , JOCKUSCH, Steffen
IPC分类号： G01N33/487 , C12Q1/6869 , C12Q1/6874 , C12N9/12 , C12M1/34 , C12P19/34 , C07H21/04
摘要： Disclosed herein are highly accurate approaches for single molecule electronic nanopore-based SBS.

9. 发明申请

WO2015148402A1 CHEMICAL METHODS FOR PRODUCING TAGGED NUCLEOTIDES 审中-公开
标题翻译：用于生产标记核酸的化学方法
公开(公告)号：WO2015148402A1
公开(公告)日：2015-10-01
申请号：PCT/US2015/022063
申请日：2015-03-23
申请人： THE TRUSTEES OF COLUMBIA UNIVERISTY IN THE CITY OF NEW YORK , GENIA TECHNOLOGIES, INC. , FULLER, Carl, W. , KUMAR, Shiv , JU, Jingyue , DAVIS, Randall , CHEN, Roger
发明人： FULLER, Carl, W. , KUMAR, Shiv , JU, Jingyue , DAVIS, Randall , CHEN, Roger
IPC分类号： C07H19/04
CPC分类号： C12Q1/6874 , C07H17/02 , C07H19/10 , C07H19/20 , C12Q1/6806
摘要： This disclosure provides systems and methods for attaching nano pore-detectable tags to nucleotides. The disclosure also provides methods for sequencing nucleic acids using the disdosed tagged nudeotides. Provided herein are nudeotides with attached tags and methods for attaching tags to nudeotides. The tags can be attached by chemical reactions, such as "dick chemistry". In an aspect, the present disdosure provides a tagged nudeotide, comprising: (a) a poly-phosphate moiety having a terminal phosphate; and (b) a tag covalently coupled to the terminal phosphate of the nudeotide by a triazole, a 1,2-diazine, a disulfide, a secondary amine, a hydrazone, a thio-acetamide, or a maleimide-thioadduct.
摘要翻译：本公开提供了将纳米孔可检测标签附着于核苷酸的系统和方法。本公开还提供了使用剔除的标记的核苷酸对核酸进行测序的方法。本文提供了具有附加标签的核苷酸和将标签附着到裸核苷酸的方法。标签可以通过化学反应来附着，例如“dick chemistry”。在一方面，本发明提供了一种标记的核苷酸，其包含：（a）具有末端磷酸酯的聚磷酸酯部分; 和（b）通过三唑，1,2-二嗪，二硫化物，仲胺，腙，硫代乙酰胺或马来酰亚胺 - 硫代加成酸共价偶联到核苷酸的末端磷酸酯上的标签。

10. 发明申请

WO2004020604A2 ALLELE SPECIFIC PRIMER EXTENSION 审中-公开
标题翻译： ALLELE专用主机扩展
公开(公告)号：WO2004020604A2
公开(公告)日：2004-03-11
申请号：PCT/US2003/027286
申请日：2003-08-29
申请人： AMERSHAM BIOSCIENCES CORP
发明人： SOOD, Anup , KUMAR, Shiv , FULLER, Carl , NELSON, John , MACKLIN, John
IPC分类号： C12N
CPC分类号： C12Q1/6869 , C12Q1/6823 , C12Q2600/156 , C12Q2535/125 , C12Q2525/125 , C12Q2521/125 , C12Q2565/301 , C12Q2525/101
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, 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，至少一个末端磷酸盐标记的核苷酸，DNA聚合酶和任选的具有3' 5'外切核酸酶活性，当该引物是不可水解的时，该反应导致标记的多磷酸盐的产生; （b）使标记的多磷酸酯与磷酸酯酶反应产生可检测的物质; （c）检测可检测物质; 和（d）基于这种检测表征核酸样品。

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