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。

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。

公开(公告)号：US4486533A

公开(公告)日：1984-12-04

申请号：US460684

申请日：1983-01-24

申请人： Alan M. Lambowitz

发明人： Alan M. Lambowitz

IPC分类号： C12N15/00 ,  C12N1/00 ,  C12N1/14 ,  C12P19/34 ,  C12P21/00 ,  C12P21/02

CPC分类号： C12N15/00

摘要： A functional extrachromosomal element capable of replication in filamentous fungi is provided. The extrachromosomal element employs (1) a mitochondrial replicating element or (2) a lower organism replication sequence recognized by the fungus, in combination with foreign DNA to provide replication, transcription, and translation of foreign regulatory elements and genes. The extrachromosomal element is exemplified by a mitochondrial replicating system from Neurospora.The cell strain E. coli HB101 containing the plasmid pALS-1-1 has been deposited at the A.T.C.C. on July 13, 1982, for patent purposes and given the designation ATCC 39157.The cell strain E. coli HB101 containing the plasmid pALS-2 has been deposited at the A.T.C.C. on July 13, 1982, for patent purposes and given the designation ATCC 39158.

摘要翻译： 提供了能够在丝状真菌中复制的功能性染色体外元件。 染色体外元件采用（1）线粒体复制元件或（2）由真菌识别的下生物复制序列，与外来DNA结合，提供外源调节元件和基因的复制，转录和翻译。 染色体外元件的例子是来自神经孢子虫的线粒体复制体系。 含有质粒pALS-1-1的细胞株大肠杆菌HB101已经在A.T.C.C. 1982年7月13日授权专利，并命名为ATCC 39157.含有质粒pALS-2的细胞株大肠杆菌HB101已经保藏在A.T.C.C. 1982年7月13日，专利申请并授予ATCC 39158。

公开(公告)号：US07592161B2

公开(公告)日：2009-09-22

申请号：US10277643

申请日：2002-10-22

申请人： Alan M. Lambowitz ,  Huatao Guo ,  Michael Karberg

发明人： Alan M. Lambowitz ,  Huatao Guo ,  Michael Karberg

IPC分类号： C12P19/34 ,  C12P12/06

CPC分类号： C12N15/1058 ,  C12N9/22 ,  C12N15/102 ,  C12N15/1082 ,  C12N15/11 ,  C12N15/63 ,  C12N15/90

摘要： The present invention provides a system and methods for analyzing the function of nucleotide integrases and modified group II introns. The system comprises a donor plasmid comprising a wild-type or modified group II intron, a recipient plasmid comprising a DNA recognition site and a promoterless reporter gene downstream of the DNA target site, and a host cell. The method comprises the steps of transforming a host cell with the donor and recipient plasmids, assaying for expression of the reporter gene, isolating plasmid DNA from the cotransformed cells, and analyzing the plasmid DNA to confirm that the group II intron has been inserted into the target sequence. The present invention also provides a method for simultaneously analyzing the activity of two or more modified nucleotide integrases. The present invention also relates to methods of preparing a library of donor plasmids containing a plurality of diverse modified group II intron DNA sequences.

摘要翻译： 本发明提供了用于分析核苷酸整合酶和修饰的II组内含子的功能的系统和方法。 该系统包含包含野生型或修饰的II族内含子的供体质粒，包含DNA靶位点下游的DNA识别位点和无启动子报告基因的受体质粒和宿主细胞。 该方法包括以下步骤：用供体和受体质粒转化宿主细胞，测定报道基因的表达，从共转化的细胞中分离质粒DNA，并分析质粒DNA以证实第II组内含子已插入 目标序列。 本发明还提供了一种同时分析两种或更多种修饰的核苷酸整合酶的活性的方法。 本发明还涉及制备含有多个不同修饰的II组内含子DNA序列的供体质粒文库的方法。

公开(公告)号：US10113156B2

公开(公告)日：2018-10-30

申请号：US13254223

申请日：2010-03-04

申请人： Alan M. Lambowitz ,  Sabine Mohr ,  Georg Mohr ,  Eman Ghanem

发明人： Alan M. Lambowitz ,  Sabine Mohr ,  Georg Mohr ,  Eman Ghanem

IPC分类号： C12N9/12 ,  C12P19/34

摘要： Stabilized reverse transcriptase fusion proteins including a thermostable reverse transcriptase connected to a stabilizer protein are described. Attaching the stabilizer protein to the thermostable reverse transcriptase stabilizes the fusion protein and can aid in its purification, provide increased solubility, allow for longer storage, or allow the fusion protein to be used under more rigorous conditions such as higher temperature. The stabilized reverse transcriptase fusion protein can also include a linker between the stabilizer protein and the thermostable reverse transcriptase. The stabilized reverse transcriptase fusion proteins are suitable for use in nucleic acid amplification methods such as the reverse transcription polymerase chain reaction and other applications involving cDNA synthesis.

公开(公告)号：US09012183B2

公开(公告)日：2015-04-21

申请号：US14000513

申请日：2012-02-23

申请人： Alan M. Lambowitz ,  Sabine Mohr ,  Travis B. White ,  Scott Kuersten

发明人： Alan M. Lambowitz ,  Sabine Mohr ,  Travis B. White ,  Scott Kuersten

IPC分类号： C12P19/34 ,  C12N15/10 ,  C12Q1/68

CPC分类号： C12P19/34 ,  C12N15/1096 ,  C12Q1/6806 ,  C12Q2521/107 ,  C12Q2525/191 ,  C12Q2525/207

摘要： A method of preparing a DNA copy of a target polynucleotide using template switching is described. The method includes mixing a double stranded template/primer substrate made up of a DNA primer oligonucleotide associated with a complementary oligonucleotide template strand with a target polynucleotide in a reaction medium and adding a suitable amount of a non-retroviral reverse transcriptase to the reaction medium to extend the DNA primer oligonucleotide from its 3′ end to provide a DNA copy polynucleotide. The DNA copy polynucleotide includes a complementary target DNA polynucleotide that is synthesized using the target polynucleotide as a template. Methods of adding nucleotides to the double stranded template/primer substrate are also described. The method can be used to facilitate detection, PCR amplification, cloning, and determination of RNA and DNA sequences.

摘要翻译： 描述了使用模板切换来制备靶多核苷酸的DNA拷贝的方法。 该方法包括将由与互补寡核苷酸模板链相关的DNA引物寡核苷酸组成的双链模板/引物底物与靶多核苷酸混合在反应介质中，并向反应介质中加入适量的非逆转录病毒逆转录酶至 从其3'末端延伸DNA引物寡核苷酸以提供DNA拷贝多核苷酸。 DNA拷贝多核苷酸包括使用靶多核苷酸作为模板合成的互补靶DNA多核苷酸。 还描述了向双链模板/底物底物添加核苷酸的方法。 该方法可用于促进RNA和DNA序列的检测，PCR扩增，克隆和测定。

公开(公告)号：US20140004569A1

公开(公告)日：2014-01-02

申请号：US14000513

申请日：2012-02-23

申请人： Alan M. Lambowitz ,  Sabine Mohr ,  Travis B. White ,  Scott Kuersten

发明人： Alan M. Lambowitz ,  Sabine Mohr ,  Travis B. White ,  Scott Kuersten

IPC分类号： C12P19/34

CPC分类号： C12P19/34 ,  C12N15/1096 ,  C12Q1/6806 ,  C12Q2521/107 ,  C12Q2525/191 ,  C12Q2525/207

摘要： A method of preparing a DNA copy of a target polynucleotide using template switching is described. The method includes mixing a double stranded template/primer substrate made up of a DNA primer oligonucleotide associated with a complementary oligonucleotide template strand with a target polynucleotide in a reaction medium and adding a suitable amount of a non-retroviral reverse transcriptase to the reaction medium to extend the DNA primer oligonucleotide from its 3′ end to provide a DNA copy polynucleotide. The DNA copy polynucleotide includes a complementary target DNA polynucleotide that is synthesized using the target polynucleotide as a template. Methods of adding nucleotides to the double stranded template/primer substrate are also described. The method can be used to facilitate detection, PCR amplification, cloning, and determination of RNA and DNA sequences.

摘要翻译： 描述了使用模板切换来制备靶多核苷酸的DNA拷贝的方法。 该方法包括将由与互补寡核苷酸模板链相关的DNA引物寡核苷酸组成的双链模板/引物底物与靶多核苷酸混合在反应介质中，并向反应介质中加入适量的非逆转录病毒逆转录酶至 从其3'末端延伸DNA引物寡核苷酸以提供DNA拷贝多核苷酸。 DNA拷贝多核苷酸包括使用靶多核苷酸作为模板合成的互补靶DNA多核苷酸。 还描述了向双链模板/底物底物添加核苷酸的方法。 该方法可用于促进RNA和DNA序列的检测，PCR扩增，克隆和测定。

公开(公告)号：US20120009630A1

公开(公告)日：2012-01-12

申请号：US13254223

申请日：2010-03-04

申请人： Alan M. Lambowitz ,  Sabine Mohr ,  Georg Mohr ,  Eman Ghanem

发明人： Alan M. Lambowitz ,  Sabine Mohr ,  Georg Mohr ,  Eman Ghanem

IPC分类号： C12P19/34 ,  C12N15/63 ,  C12N9/12

CPC分类号： C12N9/1276 ,  C07K2319/00 ,  C07K2319/24 ,  C12P19/34 ,  C12Y207/07049

摘要： Stabilized reverse transcriptase fusion proteins including a thermostable reverse transcriptase connected to a stabilizer protein are described. Attaching the stabilizer protein to the thermostable reverse transcriptase stabilizes the fusion protein and can aid in its purification, provide increased solubility, allow for longer storage, or allow the fusion protein to be used under more rigorous conditions such as higher temperature. The stabilized reverse transcriptase fusion protein can also include a linker between the stabilizer protein and the thermostable reverse transcriptase. The stabilized reverse transcriptase fusion proteins are suitable for use in nucleic acid amplification methods such as the reverse transcription polymerase chain reaction and other applications involving cDNA synthesis.

摘要翻译： 描述了稳定的逆转录酶融合蛋白，包括与稳定蛋白连接的热稳定逆转录酶。 将稳定剂蛋白质连接到热稳定逆转录酶稳定融合蛋白并可以帮助其纯化，提供增加的溶解度，允许更长的储存，或允许融合蛋白在更严格的条件下使用，例如较高的温度。 稳定的逆转录酶融合蛋白还可以包括稳定蛋白和热稳定逆转录酶之间的接头。 稳定的逆转录酶融合蛋白适用于核酸扩增方法，如逆转录聚合酶链反应和涉及cDNA合成的其他应用。

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。