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公开(公告)号：US5273879A
公开(公告)日：1993-12-28
申请号：US614180
申请日：1990-11-13
申请人： Thomas C. Goodman , Martin Becker , Edwin F. Ullman , Samuel Rose
发明人： Thomas C. Goodman , Martin Becker , Edwin F. Ullman , Samuel Rose
CPC分类号： C12Q1/701 , C12Q1/6813 , C12Q1/683 , C12Q1/6844 , C12Q1/6853 , C12Q1/6858
摘要： A Kit is disclosed for a method for producing multiple copies of a primary polynucleotide sequence located at the 3' terminus of a polynucleotide. The method comprises (a) forming in the presence of nucleoside triphosphates and template-dependent polynucleotide polymerase an extension of a primary polynucleotide sequence hybridized with a template sequence of a single stranded pattern polynucleotide comprising two or more template sequences each containing one or more site specific cleavage sequences, (b) cleaving into fragments said extension at cleavable polynucleotide sequences in the presence of means for specifically cleaving said cleavable polynucleotide sequences when said extension is hybridized with said site specific cleavage sequences, (c) dissociating said fragments, (d) hybridizing said fragments with single stranded pattern polynucleotide, and repeating steps (a)-(d). Steps (a)-(d) may be conducted simultaneously or wholly or partially sequentially. The method may be applied in the detection of a polynucleotide analyte in a sample suspected of containing such analyte to facilitate such detection. Also disclosed are compositions for conducting the method of the invention.
摘要翻译： 公开了用于产生位于多核苷酸的3'末端的多核苷酸序列的多个拷贝的方法的试剂盒。 该方法包括（a）在核苷三磷酸和模板依赖性多聚核苷酸聚合酶的存在下形成与单链图案多核苷酸的模板序列杂交的主要多核苷酸序列的延伸，所述单链图案多核苷酸包含两个或更多个模板序列，每个模板序列含有一个或多个位点特异性 裂解序列，（b）当所述延伸与所述位点特异性切割序列杂交时，在存在用于特异性切割所述可切割多核苷酸序列的手段的存在下，在可切割多核苷酸序列处切割成片段，（c）解离所述片段，（d）杂交 所述具有单链图案多核苷酸的片段，以及重复步骤（a） - （d）。 步骤（a） - （d）可以同时或全部或部分顺序进行。 该方法可以应用于怀疑含有此类分析物的样品中的多核苷酸分析物的检测以促进这种检测。 还公开了用于进行本发明方法的组合物。
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公开(公告)号：US6124090A
公开(公告)日：2000-09-26
申请号：US438149
申请日：1995-05-09
CPC分类号： C12Q1/682 , C12Q1/6844 , C12Q1/6853 , C12Q1/6862 , C12Q1/689
摘要： A method is disclosed for determining the presence of a polynucleotide analyte in a sample suspected of containing the analyte. The method comprises (a) forming as a result of the presence of an analyte a single stranded polynucleotide comprising a target polynucleotide binding sequence flanked by first and second polynucleotide sequences that differ from the sequence of the analyte or a sequence complementary to the analyte sequence, (b) forming multiple copies of the single stranded polynucleotide, and (c) detecting the single stranded polynucleotide. Also disclosed is a method of producing at least one copy of a single stranded polynucleotide. The method comprises (a) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase an extension of a polynucleotide primer at least the 3'-end of which has at least a 10 base sequence hybridizable with a second sequence flanking the 3'-end of the single stranded polynucleotide, the second sequence being partially or fully complementary with at least a 10 base first sequence flanking the 5' end of the single stranded polynucleotide, (b) dissociating the extended polynucleotide primer and the single stranded polynucleotide, (c) repeating step a and (d) dissociating the extended polynucleotide primer and the copy of the single stranded polynucleotide.
摘要翻译： 公开了一种用于确定疑似含有分析物的样品中多核苷酸分析物的存在的方法。 该方法包括（a）作为分析物存在的结果形成单链多核苷酸，所述单链多核苷酸包含不同于分析物序列或与分析物序列互补的序列的第一和第二多核苷酸序列侧翼的靶多核苷酸结合序列， （b）形成多重拷贝的单链多核苷酸，和（c）检测单链多核苷酸。 还公开了生产单链多核苷酸的至少一个拷贝的方法。 该方法包括（a）在核苷三磷酸和模板依赖性多核苷酸聚合酶存在下形成多核苷酸引物的延伸，所述多核苷酸引物的至少3'末端具有至少10个碱基序列，可与3'- 单链多核苷酸的末端，第二个序列与单链多核苷酸的5'端侧翼的至少10个碱基第一序列部分或完全互补，（b）解离延伸的多核苷酸引物和单链多核苷酸，（c ）重复步骤a和（d）解离延伸的多核苷酸引物和单链多核苷酸的拷贝。
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公开(公告)号：US5827649A
公开(公告)日：1998-10-27
申请号：US242931
申请日：1994-05-16
CPC分类号： C12Q1/682 , C12Q1/6844 , C12Q1/6853 , C12Q1/6862 , C12Q1/689
摘要： A method is disclosed for determining the presence of a polynucleotide analyte in a sample suspected of containing the analyte. The method comprises (a) forming as a result of the presence of an analyte a single stranded polynucleotide comprising a target polynucleotide binding sequence flanked by first and second polynucleotide sequences that differ from the sequence of the analyte or a sequence complementary to the analyte sequence, (b) forming multiple copies of the single stranded polynucleotide, and (c) detecting the single stranded polynucleotide. Also disclosed is a method of producing at least one copy of a single stranded polynucleotide. The method comprises (a) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase an extension of a polynucleotide primer at least the 3'-end of which has at least a 10 base sequence hybridizable with a second sequence flanking the 3'-end of the single stranded polynucleotide, the second sequence being partially or fully complementary with at least a 10 base first sequence flanking the 5' end of the single stranded polynucleotide, (b) dissociating the extended polynucleotide primer and the single stranded polynucleotide, (c) repeating step a and (d) dissociating the extended polynucleotide primer and the copy of the single stranded polynucleotide.
摘要翻译： 公开了一种用于确定疑似含有分析物的样品中多核苷酸分析物的存在的方法。 该方法包括（a）作为分析物存在的结果形成单链多核苷酸，所述单链多核苷酸包含不同于分析物序列或与分析物序列互补的序列的第一和第二多核苷酸序列侧翼的靶多核苷酸结合序列， （b）形成多重拷贝的单链多核苷酸，和（c）检测单链多核苷酸。 还公开了生产单链多核苷酸的至少一个拷贝的方法。 该方法包括（a）在核苷三磷酸和模板依赖性多核苷酸聚合酶存在下形成多核苷酸引物的延伸，所述多核苷酸引物的至少3'末端具有至少10个碱基序列，可与3'- 单链多核苷酸的末端，第二个序列与单链多核苷酸的5'端侧翼的至少10个碱基第一序列部分或完全互补，（b）解离延伸的多核苷酸引物和单链多核苷酸，（c ）重复步骤a和（d）解离延伸的多核苷酸引物和单链多核苷酸的拷贝。
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公开(公告)号：US5397698A
公开(公告)日：1995-03-14
申请号：US146297
申请日：1993-11-02
申请人： Thomas C. Goodman , Martin Becker , Edwin F. Ullman , Samuel Rose
发明人： Thomas C. Goodman , Martin Becker , Edwin F. Ullman , Samuel Rose
CPC分类号： C12Q1/701 , C12Q1/6813 , C12Q1/683 , C12Q1/6844 , C12Q1/6853 , C12Q1/6858
摘要： A method is disclosed for producing multiple copies of a primary polynucleotide sequence located at the 3' terminus of a polynucleotide. The method comprises (a) forming in the presence of nucleoside triphosphates and template-dependent polynucleotide polymerase an extension of a primary polynucleotide sequence hybridized with a template sequence of a single stranded pattern polynucleotide comprising two or more template sequences each containing one or more site specific cleavage sequences, (b) cleaving into fragments said extension at cleavable polynucleotide sequences in the presence of means for specifically cleaving said cleavable polynucleotide sequences when said extension is hybridized with said site specific cleavage sequences, (c) dissociating said fragments, (d) hybridizing said fragments with single stranded pattern polynucleotide, and repeating steps (a)-(d). Steps (a)-(d) may be conducted simultaneously or wholly or partially sequentially. The method may be applied in the detection of a polynucleotide analyte in a sample suspected of containing such analyte to facilitate such detection. Also disclosed are compositions for conducting the method of the invention.
摘要翻译： 公开了一种用于产生位于多核苷酸的3'末端的多核苷酸序列的多个拷贝的方法。 该方法包括（a）在核苷三磷酸和模板依赖性多聚核苷酸聚合酶的存在下形成与单链图案多核苷酸的模板序列杂交的主要多核苷酸序列的延伸，所述单链图案多核苷酸包含两个或更多个模板序列，每个模板序列含有一个或多个位点特异性 裂解序列，（b）当所述延伸与所述位点特异性切割序列杂交时，在存在用于特异性切割所述可切割多核苷酸序列的手段的存在下，在可切割多核苷酸序列处切割成片段，（c）解离所述片段，（d）杂交 所述具有单链图案多核苷酸的片段，以及重复步骤（a） - （d）。 步骤（a） - （d）可以同时或全部或部分顺序进行。 该方法可以应用于怀疑含有此类分析物的样品中的多核苷酸分析物的检测以促进这种检测。 还公开了用于进行本发明方法的组合物。
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公开(公告)号：US4994368A
公开(公告)日：1991-02-19
申请号：US76807
申请日：1987-07-23
申请人： Thomas C. Goodman , Martin Becker , Edwin F. Ullman , Samuel Rose
发明人： Thomas C. Goodman , Martin Becker , Edwin F. Ullman , Samuel Rose
CPC分类号： C12Q1/701 , C12Q1/6813 , C12Q1/683 , C12Q1/6844 , C12Q1/6853 , C12Q1/6858 , Y10T436/143333
摘要： A method is disclosed for producing multiple copies of a primary polynucleotide sequence located at the 3' terminus of a polynucleotide. The method comprises (a) forming in the presence of nucleoside triphosphates and template-dependent polynucleotide polymerase an extension of a primary polynucleotide sequence hybridized with a template sequence of a single stranded pattern polynucleotide comprising two or more template sequences each containing one or more site specific cleavage sequences, (b) cleaving into fragments said extension at cleavable polynucleotide sequences in the presence of means for specifically cleaving said cleavable polynucleotide sequences when said extension is hybridized with said site specific cleavage sequences, (c) dissociating said fragments, (d) hybridizing said fragments with single stranded pattern polynucleotide, and repeating steps (a)-(d). Steps (a)-(d) may be conducted simultaneously or wholly or partially sequentially. The method may be applied in the detection of a polynucleotide analyte in a sample suspected of containing such analyte to facilitate such detection. Also disclosed are compositions for conducting the method of the invention.
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公开(公告)号：US5508178A
公开(公告)日：1996-04-16
申请号：US194140
申请日：1994-02-09
CPC分类号： C12Q1/6858 , Y10S435/81
摘要： A method is disclosed for determining the presence of a polynucleotide analyte in a sample suspected of containing the analyte. The method comprises (a) forming as a result of the presence of an analyte a single stranded polynucleotide comprising a target polynucleotide binding sequence flanked by first and second polynucleotide sequences that differ from the sequence of the analyte or a sequence complementary to the analyte sequence, (b) forming multiple copies of the single stranded polynucleotide, and (c) detecting the single stranded polynucleotide. Also disclosed is a method of producing at least one copy of a single stranded polynucleotide. The method comprises (a) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase an extension of a polynucleotide primer at least the 3'-end of which has at least a 10 base sequence hybridizable with a second sequence flanking the 3'-end of the single stranded polynucleotide, the second sequence being partially or fully complementary with at least a 10 base first sequence flanking the 5' end of the single stranded polynucleotide, (b) dissociating the extended polynucleotide primer and the single stranded polynucleotide, (c) repeating step a and (d) dissociating the extended polynucleotide primer and the copy of the single stranded polynucleotide.
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7.发明授权
公开(公告)号：US6090567A
公开(公告)日：2000-07-18
申请号：US445464
申请日：1995-05-22
申请人： Edward Benjamin Jakobovits , Joy L. Silen , Mark J. Levy , Thomas C. Goodman , Martin Becker , Edwin F. Ullman , Robert M. Caldwell , Richard R. Bott , Christopher Charles Barnett
发明人： Edward Benjamin Jakobovits , Joy L. Silen , Mark J. Levy , Thomas C. Goodman , Martin Becker , Edwin F. Ullman , Robert M. Caldwell , Richard R. Bott , Christopher Charles Barnett
IPC分类号： G01N33/542 , C12N9/04 , C12N15/09 , C12Q1/28 , C12Q1/32 , C12R1/19 , G01N33/535 , G01N33/569 , G01N33/58 , G01N33/78 , G01N33/94 , G01N33/53
CPC分类号： C12N9/0006 , C12Q1/28 , G01N33/535 , G01N33/56911 , G01N33/581 , G01N33/78 , G01N33/94 , G01N2333/904
摘要： Methods for immunoassay of analytes employing mutant glucose-6-phosphate dehydrogenase (G6PDH) enzymes as labels. In particular, the invention relates to the use of conjugates of an analyte or analyte analog and a mutant NAD.sup.+ dependent G6PDH differing from any precursor G6PDH by the deletion, substitution, or insertion, or any combination thereof of at least one amino acid per subunit. The invention also involves the construction of several mutations in precursor glucose-6-phosphate dehydrogenase (G6PDH) enzymes. Typically, the mutations involve deletion or substitution of one or more lysine residues, or introduction of one or more cysteine residues by insertion of cysteine to precursor G6PDH or substitution of precursor G6PDH amino acids residues with cysteine. The present invention also relates to conjugates of the subject enzymes and specific binding pair members, kits useful in performing the methods of the invention, cell lines producing the subject enzymes, DNA sequences encoding the subject enzymes, and vectors containing DNA encoding the subject enzymes and designed to allow a host cell to produce the subject enzymes.
摘要翻译： 使用突变型葡萄糖-6-磷酸脱氢酶（G6PDH）酶作为标记免疫测定分析物的方法。 特别地，本发明涉及通过每个亚基的至少一个氨基酸的缺失，取代或插入或其任何组合，使用不同于任何前体G6PDH的分析物或分析物类似物和突变体NAD +依赖性G6PDH的缀合物的用途。 本发明还涉及前体葡萄糖-6-磷酸脱氢酶（G6PDH）酶的几个突变的构建。 通常，突变涉及一个或多个赖氨酸残基的缺失或取代，或通过将半胱氨酸插入前体G6PDH或用半胱氨酸取代前体G6PDH氨基酸残基引入一个或多个半胱氨酸残基。 本发明还涉及本发明的酶和特异性结合对成员的缀合物，可用于实施本发明方法的试剂盒，产生本发明酶的细胞系，编码本发明酶的DNA序列，和含有编码本发明酶的DNA的载体和 旨在使宿主细胞产生目标酶。
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8.发明授权公开(公告)号：US06455288B1
公开(公告)日：2002-09-24
申请号：US08044857
申请日：1993-04-08
申请人： Edward Benjamin Jakobovits , Joy L. Silen , Mark J. Levy , Thomas C. Goodman , Martin Becker , Edwin F. Ullman , Robert M. Caldwell , Richard R. Bott , Christopher Charles Barnett
发明人： Edward Benjamin Jakobovits , Joy L. Silen , Mark J. Levy , Thomas C. Goodman , Martin Becker , Edwin F. Ullman , Robert M. Caldwell , Richard R. Bott , Christopher Charles Barnett
IPC分类号： C12N1552
CPC分类号： C12N9/0006 , C12Q1/28 , G01N33/535 , G01N33/56911 , G01N33/581 , G01N33/78 , G01N33/94 , G01N2333/904
摘要： The present invention relates to methods for immunoassay of analytes employing mutant glucose-6-phosphate dehydrogenase (G6PDH) enzymes as labels. In particular, the invention relates-to the use of conjugates of an analyte or analyte analog and a mutant NAD+ dependent G6PDH differing from any precursor G6PDH by the deletion, substitution, or insertion, or any combination thereof of at least one amino acid per subunit. The invention also involves the construction of several mutations in precursor glucose-6-phosphate dehydrogenase (G6PDH) enzymes. Typically, the mutations involve deletion or substitution of one or more lysine residues, or introduction of one or more cysteine residues by insertion of cysteine to precursor G6PDH or substitution of precursor G6PDH amino acids residues with cysteine. The present invention also relates to conjugates of the subject enzymes and specific binding pair members, kits useful in performing the methods of the invention, cell lines producing the subject enzymes, DNA sequences encoding the subject enzymes, and vectors containing DNA encoding the subject enzymes and designed to allow a host cell to produce the subject enzymes.
摘要翻译： 本发明涉及使用突变型葡萄糖-6-磷酸脱氢酶（G6PDH）酶作为标记免疫测定分析物的方法。 特别地，本发明涉及分离物或分析物类似物和与任何前体G6PDH不同的突变体NAD +依赖性G6PDH通过每个亚基的缺失，取代或插入或其任何组合的至少一个氨基酸的缀合物的用途 。 本发明还涉及前体葡萄糖-6-磷酸脱氢酶（G6PDH）酶的几个突变的构建。 通常，突变涉及一个或多个赖氨酸残基的缺失或取代，或通过将半胱氨酸插入前体G6PDH或用半胱氨酸取代前体G6PDH氨基酸残基引入一个或多个半胱氨酸残基。 本发明还涉及本发明的酶和特异性结合对成员的缀合物，可用于实施本发明方法的试剂盒，产生本发明酶的细胞系，编码本发明酶的DNA序列，和含有编码本发明酶的DNA的载体和 旨在使宿主细胞产生目标酶。
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9.发明授权
公开(公告)号：US6033890A
公开(公告)日：2000-03-07
申请号：US445463
申请日：1995-05-22
申请人： Edward Benjamin Jakobovits , Joy L. Silen , Mark J. Levy , Thomas C. Goodman , Martin Becker , Edwin F. Ullman , Robert M. Caldwell , Richard R. Bott , Christopher Charles Barnett
发明人： Edward Benjamin Jakobovits , Joy L. Silen , Mark J. Levy , Thomas C. Goodman , Martin Becker , Edwin F. Ullman , Robert M. Caldwell , Richard R. Bott , Christopher Charles Barnett
IPC分类号： G01N33/542 , C12N9/04 , C12N15/09 , C12Q1/28 , C12Q1/32 , C12R1/19 , G01N33/535 , G01N33/569 , G01N33/58 , G01N33/78 , G01N33/94
CPC分类号： C12N9/0006 , C12Q1/28 , G01N33/535 , G01N33/56911 , G01N33/581 , G01N33/78 , G01N33/94 , G01N2333/904
摘要： The present invention relates to methods for immunoassay of analytes employing mutant glucose-6-phosphate dehydrogenase (G6PDH) enzymes as labels. In particular, the invention relates to the use of conjugates of an analyte or analyte analog and a mutant NAD.sup.+ dependent G6PDH differing from any precursor G6PDH by the deletion, substitution, or insertion, or any combination thereof of at least one amino acid per subunit. The invention also involves the construction of several mutations in precursor glucose-6-phosphate dehydrogenase (G6PDH) enzymes. Typically, the mutations involve deletion or substitution of one or more lysine residues, or introduction of one or more cysteine residues by insertion of cysteine to precursor G6PDH or substitution of precursor G6PDH amino acids residues with cysteine. The present invention also relates to conjugates of the subject enzymes and specific binding pair members, kits useful in performing the methods of the invention, cell lines producing the subject enzymes, DNA sequences encoding the subject enzymes, and vectors containing DNA encoding the subject enzymes and designed to allow a host cell to produce the subject enzymes.
摘要翻译： 本发明涉及使用突变型葡萄糖-6-磷酸脱氢酶（G6PDH）酶作为标记免疫测定分析物的方法。 特别地，本发明涉及通过每个亚基的至少一个氨基酸的缺失，取代或插入或其任何组合，使用不同于任何前体G6PDH的分析物或分析物类似物和突变体NAD +依赖性G6PDH的缀合物的用途。 本发明还涉及前体葡萄糖-6-磷酸脱氢酶（G6PDH）酶的几个突变的构建。 通常，突变涉及一个或多个赖氨酸残基的缺失或取代，或通过将半胱氨酸插入前体G6PDH或用半胱氨酸取代前体G6PDH氨基酸残基引入一个或多个半胱氨酸残基。 本发明还涉及本发明的酶和特异性结合对成员的缀合物，可用于实施本发明方法的试剂盒，产生本发明酶的细胞系，编码本发明酶的DNA序列，和含有编码本发明酶的DNA的载体和 旨在使宿主细胞产生目标酶。
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10.发明授权
公开(公告)号：US5595891A
公开(公告)日：1997-01-21
申请号：US555323
申请日：1990-07-19
申请人： Samuel Rose , Linda M. Western , Martin Becker , Edwin F. Ullman
发明人： Samuel Rose , Linda M. Western , Martin Becker , Edwin F. Ullman
CPC分类号： C12Q1/6853 , C12N15/10 , C12Q1/686
摘要： A method is disclosed for producing a single stranded polydeoxynucleotide having two segments that are non-contiguous and complementary with each other. The method comprises the steps of providing in combination (1) a polynucleotide having two non-contiguous, non-complementary nucleotide sequences S1 and S2 wherein S2 is 5' of S1 and is at least ten deoxynucleotides long and (2) an extender probe comprised of two deoxynucleotide sequences, wherein the sequence at the 3'-end of the extender probe is hybridizable with S1 and the other of the deoxynucleotide sequences is homologous to S2 and (b) extending the extender probe along the polynucleotide. The method can also comprise providing in the combination a polydoxynucleotide primer capable of hybridizing at least at its 3'-end with a nucleotide sequence complementary to S2 under conditions where (1) the extended extender probe is rendered single stranded, (2) the polydeoxynucleotide primer hybridizes with and is extended along the extended extender probe to form a duplex comprising extended primer, (3) the extended primer is dissociated from the duplex, and (4) the primer hybridizes with and is extended along the extended primer to form a duplex comprising extended primer, and repeating steps (3) and (4). The method finds particular application in the detection of polynucleotide analytes.
摘要翻译： 公开了一种用于生产具有彼此不连续和互补的两个区段的单链多脱氧核苷酸的方法。 该方法包括以下步骤：组合（1）具有两个非连续的非互补核苷酸序列S1和S2的多核苷酸，其中S2是S1的5'，长至少十个脱氧核苷酸，和（2） 的两个脱氧核苷酸序列，其中扩增物探针的3'末端的序列可与S1杂交，而另一个脱氧核苷酸序列与S2同源，并且（b）沿多核苷酸扩展扩增体探针。 该方法还可以包括提供能够在其3'末端与至少与S2互补的核苷酸序列的多核苷酸引物提供，其中（1）扩展扩增物探针是单链的，（2）多脱氧核苷酸 引物与扩展的扩增物探针杂交并延伸，以形成包含延伸引物的双链体，（3）延伸引物与双链体解离，和（4）引物与延伸引物杂交并延伸以形成双链体 包括延伸引物，并重复步骤（3）和（4）。 该方法在多核苷酸分析物的检测中具有特殊应用。
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