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1. 发明授权

US06306596B1 Methods for cleaving single-stranded and double-stranded DNA substrates with nucleotide integrase 失效
公开(公告)号：US06306596B1
公开(公告)日：2001-10-23
申请号：US09257770
申请日：1999-02-25
申请人： Allen M. Lambowitz , Steven Zimmerly , Huatao Guo , Georg Mohr , Clifford James Beall
发明人： Allen M. Lambowitz , Steven Zimmerly , Huatao Guo , Georg Mohr , Clifford James Beall
IPC分类号： C12Q168
CPC分类号： C12N15/10 , C12Q1/68 , C12Q1/6813 , C12Q1/6823 , C12Q2563/119 , C12Q2525/185 , C12Q2521/313
摘要： Methods, employing a nucleotide integrase, for cleaving single-stranded RNA substrates, single-stranded DNA substrates, and double-stranded DNA substrates at specific sites and for inserting a nucleic acid molecule into the cleaved substrate are provided. One method uses a nucleotide integrase to cleave one strand of a double-stranded DNA substrate. The method comprises the steps of: providing an isolated nucleotide integrase comprising a group II intron RNA having two hybridizing sequences for hybridizing with two intron RNA binding sequences on the top strand of the DNA substrate, and a group II-intron encoded protein which binds to a first sequence element of the substrate; and reacting the nucleotide integrase with the double-stranded DNA substrate to permit the nucleotide integrase to cleave the top strand of the DNA substrate and to insert the group II intron RNA into the cleavage site. The method of cleaving both strands of a double-stranded DNA substrate comprises the steps of: providing a nucleotide integrase comprising a group II intron RNA having two hybridizing sequences for hybridizing with two intron RNA binding sequences on one strand of the substrate, and a group II-intron encoded protein that interacts with a first sequence element and a second sequence element in the recognition site of the substrate; and reacting the nucleotide integrase with the double-stranded DNA substrate such that the nucleotide integrase cleaves both strands of the DNA substrate and inserts the group II intron RNA into the cleavage site of the one strand. The method for cleaving a single-stranded nucleic acid substrate comprises the steps of: providing a nucleotide integrase having two hybridizing sequences for hybridizing with two intron RNA-binding sequences on the single-stranded substrate, and a group II intron encoded protein; and reacting the nucleotide integrase with the single stranded nucleic acid substrate to allow the nucleotide integrase to cleave the substrate and to attach the group II intron RNA molecule thereto.

2. 发明授权

US6027895A Methods for cleaving DNA with nucleotide integrases 失效
公开(公告)号：US6027895A
公开(公告)日：2000-02-22
申请号：US31897
申请日：1998-02-27
申请人： Allen M. Lambowitz , Steven Zimmerly , Huatao Gau , Georg Mohr , Clifford James Beall
发明人： Allen M. Lambowitz , Steven Zimmerly , Huatao Gau , Georg Mohr , Clifford James Beall
IPC分类号： A01N43/04 , C12P19/34 , C12Q1/68
CPC分类号： A01N43/04 , C12P19/34 , C12Q1/68
摘要： The present invention provides new methods, employing a nucleotide integrase, for cleaving single-stranded RNA substrates, single-stranded DNA substrates, and double- stranded DNA substrates at specific sites and for inserting a nucleic acid molecule into the cleaved substrate. One method uses a nucleotide integrase to cleave one strand of a double-stranded DNA substrate. The method comprises the steps of: providing a nucleotide integrase comprising a group II intron RNA having two hybridizing sequences that are capable of hybridizing with two intron RNA binding sequences on the one strand of the DNA substrate, and a group II-intron encoded protein which binds to a first sequence element of the substrate; and reacting the nucleotide integrase with the double-stranded DNA substrate under conditions that permit the nucleotide integrase to cleave the one strand of the DNA substrate and to insert the group II intron RNA into the cleavage site. The method of cleaving both strands of a double-stranded DNA substrate comprises the steps of: providing a nucleotide integrase comprising a group II intron RNA having two hybridizing sequences that are capable of hybridizing with two intron RNA binding sequences on one strand of the substrate, and a group II-intron encoded protein that is capable of binding to a first sequence element and to a second sequence element in the recognition site of the substrate; and reacting the nucleotide integrase with the double-stranded DNA substrate such that the nucleotide integrase cleaves both strands of the DNA substrate and inserts the group II intron RNA into the cleavage site of the one strand. The method for cleaving a single-stranded nucleic acid substrate comprises the steps of: providing a nucleotide integrase having two hybridizing sequences that are capable of hybridizing with two intron RNA-binding sequences on the single-stranded substrate, and a group II intron encoded protein; and reacting the nucleotide integrase with the single stranded nucleic acid substrate for a time and at a temperature sufficient to allow the nucleotide integrase to cleave the substrate and to attach the group II intron RNA molecule thereto.

3. 发明授权

US6001608A Methods of making an RNP particle having nucleotide integrase activity 失效
标题翻译：制备具有核苷酸整合酶活性的RNP颗粒的方法
公开(公告)号：US6001608A
公开(公告)日：1999-12-14
申请号：US85603
申请日：1998-05-27
申请人： Alan M. Lambowitz , Georg Mohr , Roland Saldanha , Manabu Matsuura , Clifford James Beall , Jiam Yang , Steven Zimmerly , Huatao Guo
发明人： Alan M. Lambowitz , Georg Mohr , Roland Saldanha , Manabu Matsuura , Clifford James Beall , Jiam Yang , Steven Zimmerly , Huatao Guo
IPC分类号： C12N9/22 , C12P19/34 , C07H21/04 , C12N9/12 , C12P21/06
CPC分类号： C12N9/22
摘要： Methods for preparing nucleotide integrases are provided. The nucleotide integrases are prepared by combining in vitro an excised, group II intron RNA, referred to hereinafter as "exogenous RNA", with a group II intron-encoded protein. The exogenous RNA is prepared by in vitro transcription of a DNA molecule which comprises a group II intron sequence. In one embodiment, the group II intron-encoded protein is made by introducing into a host cell a DNA molecule that comprises at least the open reading frame sequence of a group II intron and then expressing the open reading frame sequence in the host cell. The DNA molecule may comprise the open reading frame sequence operably linked to a promoter, preferably an inducible promoter. Thereafter, the cell is fractionated and the protein is recovered and combined in vitro with the exogenous RNA to provide RNP particles having nucleotide integrase activity. In another embodiment, the DNA molecule comprise a group II intron sequence that encodes both a group II intron RNA as well as a group II intron encoded protein. The DNA molecule is then expressed in the host cell to provide RNP particles that comprise the group II intron-encoded protein bound to the group II intron RNA. Thereafter, the RNP particles comprising the group II intron-encoded protein and the group II intron RNA are isolated from the cell and treated with a nuclease to remove the RNA and to provide the group II-intron encoded protein. The group II intron-encoded protein is then combined in vitro with the exogenous RNA to provide RNP particles having nucleotide integrase activity.
摘要翻译：提供了制备核苷酸整合酶的方法。核苷酸整合是通过将体外切割的II组内含子RNA（以下称为“外源RNA”）与II组内含子编码的蛋白质组合来制备的。通过体外转录包含II组内含子序列的DNA分子制备外源RNA。在一个实施方案中，II组内含子编码的蛋白质是通过向宿主细胞中引入至少包含第II组内含子的开放阅读框序列，然后在宿主细胞中表达开放阅读框序列的DNA分子来制备的。 DNA分子可以包含可操作地连接到启动子，优选诱导型启动子的开放阅读框序列。此后，分离细胞并回收蛋白质并将其与外源RNA组合以提供具有核苷酸整合酶活性的RNP颗粒。在另一个实施方案中，DNA分子包含编码II组内含子RNA以及II组内含子编码蛋白质的II组内含子序列。然后将DNA分子在宿主细胞中表达以提供包含与II组内含子RNA结合的II组内含子编码的蛋白质的RNP颗粒。此后，从细胞中分离包含II组内含子编码的蛋白质和II组内含子RNA的RNP颗粒，并用核酸酶处理以除去RNA并提供II组内含子编码的蛋白质。然后将II组内含子编码的蛋白质在体外与外源RNA组合以提供具有核苷酸整合酶活性的RNP颗粒。

4. 发明授权

US5869634A Method of making an RNA particle for use in cleaving nucleic acid molecules and inserting a nucleic acid molecule into the cleavage site 失效
标题翻译：制备用于切割核酸分子并将核酸分子插入切割位点的RNA颗粒的方法
公开(公告)号：US5869634A
公开(公告)日：1999-02-09
申请号：US946617
申请日：1997-10-07
申请人： Alan M. Lambowitz , Steven Zimmerly , Jian Yang , Huatao Guo
发明人： Alan M. Lambowitz , Steven Zimmerly , Jian Yang , Huatao Guo
IPC分类号： C12N15/09 , C12N9/22 , C12N15/10 , C12Q1/68 , C07H21/02 , C07K14/00 , C12P19/34
CPC分类号： C12N15/10 , C12N9/22
摘要： The present invention provides new methods, employing a nucleotide integrase, for cleaving double-stranded and single stranded DNA substrates at specific sites and for attaching nucleic acid molecules to the cleaved DNA substrates. One method uses a nucleotide integrase to cleave one strand of a double-stranded DNA and to concomitantly attach a nucleic acid molecule to the cleaved strand. Another method uses a nucleotide integrase to cleave both strands of a double-stranded DNA substrate and to attach a nucleic acid molecule to one strand of the DNA substrate. Another method uses a nucleotide integrase to cleave both strands of a double-stranded DNA substrate and to attach an RNA molecule to one strand of the substrate and for attaching a cDNA to the other strand of the substrate. Another method cleaves single stranded DNA with the concomitant insertion of a nucleic acid molecule at the cleavage point. The nucleotide integrase comprises an RNP particle which comprises a group II intron RNA bound to a group II intron encoded protein. The present invention also relates to purified and reconstituted RNP particles and reconstituted RNP that cleave DNA substrates.
摘要翻译：本发明提供使用核苷酸整合酶在特定部位切割双链和单链DNA底物并将核酸分子附着于切割的DNA底物的新方法。一种方法使用核苷酸整合酶切割双链DNA的一条链，并将核酸分子同时连接到切割的链上。另一种方法使用核苷酸整合酶切割双链DNA底物的两条链，并将核酸分子连接到DNA底物的一条链上。另一种方法使用核苷酸整合酶切割双链DNA底物的两条链，并将RNA分子连接到底物的一条链上，并将cDNA连接到底物的另一条链上。另一种方法是在切割点处同时插入核酸分子来切割单链DNA。核苷酸整合酶包含RNP颗粒，其包含与II组内含子编码的蛋白质结合的II组内含子RNA。本发明还涉及纯化和重构的RNP颗粒和切割DNA底物的重组RNP。

5. 发明授权

US5698421A Ribonucleoprotein particles for cleaving double-stranded DNA and inserting an RNA/DNA molecule into the cleavage site 失效
标题翻译：用于切割双链DNA并将RNA / DNA分子插入切割位点的核糖核蛋白颗粒
公开(公告)号：US5698421A
公开(公告)日：1997-12-16
申请号：US526964
申请日：1995-09-12
申请人： Alan M. Lambowitz , Steven Zimmerly , Jian Yang , Huatao Guo
发明人： Alan M. Lambowitz , Steven Zimmerly , Jian Yang , Huatao Guo
IPC分类号： C12N15/09 , C12N9/22 , C12N15/10 , C12Q1/68 , C12P19/34 , C07H21/02 , C07H21/04
CPC分类号： C12N15/10 , C12N9/22
摘要： The present invention provides new methods, employing a nucleotide integrase, for cleaving double-stranded and single stranded DNA substrates at specific sites and for attaching nucleic acid molecules to the cleaved DNA substrates. One method uses a nucleotide integrase to cleave one strand of a double-stranded DNA and to concomitantly attach a nucleic acid molecule to the cleaved strand. Another method uses a nucleotide integrase to cleave both strands of a double-stranded DNA substrate and to attach a nucleic acid molecule to one strand of the DNA substrate. Another method uses a nucleotide integrase to cleave both strands of a double-stranded DNA substrate and to attach an RNA molecule to one strand of the substrate and for attaching a cDNA to the other strand of the substrate. Another method cleaves single stranded DNA with the concomitant insertion of a nucleic acid molecule at the cleavage point. The nucleotide integrase comprises an RNP particle which comprises a group II intron RNA bound to a group II intron encoded protein. The present invention also relates to purified and reconstituted RNP particles and reconstituted RNP that cleave DNA substrates.
摘要翻译：本发明提供了使用核苷酸整合酶在特定部位切割双链和单链DNA底物并将核酸分子附着于切割的DNA底物的新方法。一种方法使用核苷酸整合酶切割双链DNA的一条链，并将核酸分子同时连接到切割的链上。另一种方法使用核苷酸整合酶切割双链DNA底物的两条链，并将核酸分子连接到DNA底物的一条链上。另一种方法使用核苷酸整合酶切割双链DNA底物的两条链，并将RNA分子连接到底物的一条链上，并将cDNA连接到底物的另一条链上。另一种方法是在切割点处同时插入核酸分子来切割单链DNA。核苷酸整合酶包含RNP颗粒，其包含与II组内含子编码的蛋白质结合的II组内含子RNA。本发明还涉及纯化和重构的RNP颗粒和切割DNA底物的重组RNP。

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