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公开(公告)号：US08158388B2
公开(公告)日：2012-04-17
申请号：US11401826
申请日：2006-04-11
申请人： Thomas C. Evans , Lixin Chen , Chudi Guan , Rebecca Kucera , Barton Slatko , Romualdas Vaisvila
发明人： Thomas C. Evans , Lixin Chen , Chudi Guan , Rebecca Kucera , Barton Slatko , Romualdas Vaisvila
IPC分类号： C12P19/34
CPC分类号： C12Q1/6844 , C12P19/34 , C12Q1/6848 , C12Q1/686 , C12Q2521/101 , C12Q2521/501 , C12Q2521/331 , C12Q2521/301 , C12Q2521/514
摘要： Methods and compositions are provided for repairing a polynucleotide so that it can be copied with improved fidelity and/or yield in, for example, an amplification reaction. This involves the use of a reaction mixture that includes a ligase and a cofactor selected from NAD+ or ATP and incubating the polynucleotide with the reaction mixture in the absence of Endonuclease VI.The reaction mixture may further contain an AP endonuclease and a polymerase. If used, these enzymes may be selected according to their ability to withstand high temperatures. For example, the reaction mixture may be used prior to a polynucleotide synthesis reaction in which case enzymes that are not thermophilic may be used. The repair reaction is not time sensitive with respect to seconds, minutes or hours of incubation in the enzyme mixture in as much as the repair is effected rapidly and prolonged incubation is not generally adverse.
摘要翻译： 提供了用于修复多核苷酸的方法和组合物，使得其可以在例如扩增反应中以改进的保真度和/或产率复制。 这包括使用包含连接酶和选自NAD +或ATP的辅因子并在不存在核酸内切酶VI的情况下将多核苷酸与反应混合物一起温育的反应混合物。 反应混合物还可以含有AP内切核酸酶和聚合酶。 如果使用，这些酶可以根据其耐受高温的能力来选择。 例如，反应混合物可以在多核苷酸合成反应之前使用，在这种情况下可以使用不是嗜热的酶。 维修反应对于酶混合物中的几秒钟，几分钟或几小时的孵育时间并不敏感，只要修复迅速进行并且延长的孵育通常不是不利的。
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公开(公告)号：US5834285A
公开(公告)日：1998-11-10
申请号：US222715
申请日：1994-04-04
CPC分类号： C07K16/12 , C12N9/1252
摘要： Recombinant DNA polymerases from archaebacteria as well as isolated DNA coding for such polymerases are provided. The isolated DNA is obtained by use or DNA or antibody probes prepared from the DNA encoding T. litoralis DNA polymerase and the T. litoralis DNA polymerase respectively. Also provided are methods for producing recombinant archaebacteria thermostable DNA polymerase and methods for enhancing the expression of such polymerases by identifying, locating and removing introns from within the DNA coding for such DNA polymerases.
摘要翻译： 提供了来自古细菌的重组DNA聚合酶以及编码这种聚合酶的分离的DNA。 分离的DNA是通过使用DNA或抗体探针获得的，分别是DNA编码的DNA或抗体，分别由编码泰氏针茅DNA和聚合酶链反应的DNA聚合酶。 还提供了用于产生重组古细菌耐热性DNA聚合酶的方法和通过鉴定，定位和从编码这种DNA聚合酶的DNA内鉴定和去除内含子来增强这些聚合酶的表达的方法。
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公开(公告)号：US08697408B2
公开(公告)日：2014-04-15
申请号：US13464548
申请日：2012-05-04
CPC分类号： C12N15/10 , C12N9/93 , C12N15/66 , C12P19/34 , C12Q1/6832 , Y02P20/52 , C12Q2521/501 , C12Q2527/127 , C12Q2527/137
摘要： Compositions and methods are provided for enhancing enzymatic ligation between nucleic acid fragments that relies on one or more small molecule enhancers having a size of less than 1000 daltons. For example, enhancement of ligation efficiencies are observed for double-stranded nucleic acid fragments that are blunt-ended, have a single nucleotide overhang at the ligation end, or have staggered ends compared to ligation under similar conditions in the absence of the one or more small molecule ligation enhancer. The use of small molecule enhancers for ligating nucleic acids results in an increased number of transformed host cells after transformation with the ligated molecules. This enhancement can be observed with chemically transformed host cells and with host cells transformed by electroporation.
摘要翻译： 提供了组合物和方法，用于增强依赖于一种或多种尺寸小于1000道尔顿的小分子增强剂的核酸片段之间的酶连接。 例如，对于平端的双链核酸片段，在连接末端具有单个核苷酸突出端，或者在不存在一个或多个相似条件的相似条件下与连接相比具有交错末端，观察到连接效率的增强 小分子连接增强子。 使用小分子增强子连接核酸导致用连接的分子转化后转化的宿主细胞数量增加。 可以通过化学转化的宿主细胞和通过电穿孔转化的宿主细胞来观察这种增强。
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公开(公告)号：US20100173364A1
公开(公告)日：2010-07-08
申请号：US12296827
申请日：2007-04-11
申请人： Thomas C. Evans, JR. , Barton Slatko , Lixin Chen , Romaldas Vaisvila , Chudi Guan , Rebecca Kucera
发明人： Thomas C. Evans, JR. , Barton Slatko , Lixin Chen , Romaldas Vaisvila , Chudi Guan , Rebecca Kucera
CPC分类号： C12Q1/686 , C12Q2521/514 , C12Q2521/501 , C12Q2521/301 , C12Q2525/119
摘要： Methods and compositions are provided for repairing a polynucleotide so that it can be copied with improved fidelity and/o yield in, for example, an amplification reaction. This involves the use of a reaction mixture that includes a DNA ligase and an effective amount of at least one endonuclease as well as a cofactor selected from NAD+ or ATP.
摘要翻译： 提供了用于修复多核苷酸的方法和组合物，使得其可以在例如扩增反应中以改进的保真度和/或产率复制。 这包括使用包括DNA连接酶和有效量的至少一种内切核酸酶以及选自NAD +或ATP的辅因子的反应混合物。
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公开(公告)号：US07851192B2
公开(公告)日：2010-12-14
申请号：US10585964
申请日：2004-11-22
申请人： Chudi Guan , Sanjay Kumar , Rebecca Kucera
发明人： Chudi Guan , Sanjay Kumar , Rebecca Kucera
CPC分类号： C12N9/22
摘要： Compositions and methods are provided that relate to a modified DNA cleaving enzyme having at least 35% amino acid sequence identity with T7 Endo I. The modified enzyme includes two catalytic centers separated by a β-bridge where the β-bridge contains at least one mutation having an effect of altering enzyme cleavage activity compared to the unmodified enzyme. Activities associated with the modified DNA cleaving enzyme that can be modulated in different reaction conditions include at least one of: (a) non-sequence specific nicking activity; (b) cleaving the second strand of a duplex DNA at a preexisting nick site to produce a linear duplex with a single strand overhang; (c) non-sequence specific DNA cleavage; (d) cleaving DNA flanking a mismatch; and (e) cleavage at a cruciform structure in a DNA duplex.
摘要翻译： 提供了与T7 Endo I具有至少35％的氨基酸序列同一性的修饰的DNA切割酶的组合物和方法。修饰的酶包括两个由“桥”分开的催化中心，其中“桥”至少包含 一个突变与未修饰的酶相比具有改变酶裂解活性的作用。 与可以在不同反应条件下调节的修饰的DNA切割酶相关的活性包括以下至少一个：（a）非序列特异性切口活性; （b）在预先存在的切口位点切割双链体DNA的第二链以产生具有单链突出端的线性双链体; （c）非序列特异性DNA切割; （d）切断位于不对称侧翼的DNA; 和（e）在DNA双链体中的十字形结构处的切割。
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6.发明申请公开(公告)号：US20070042379A1
公开(公告)日：2007-02-22
申请号：US10585964
申请日：2004-11-22
申请人： Chudi Guan , Sanjay Kumar , Rebecca Kucera
发明人： Chudi Guan , Sanjay Kumar , Rebecca Kucera
CPC分类号： C12N9/22
摘要： Compositions and methods are provided that relate to a modified DNA cleaving enzyme having at least 35% amino acid sequence identity with T7 Endo I. The modified enzyme includes two catalytic centers separated by a β-bridge where the β-bridge contains at least one mutation having an effect of altering enzyme cleavage activity compared to the unmodified enzyme. Activities associated with the modified DNA cleaving enzyme that can be modulated in different reaction conditions include at least one of: (a) non-sequence specific nicking activity; (b) cleaving the second strand of a duplex DNA at a preexisting nick site to produce a linear duplex with a single strand overhang; (c) non-sequence specific DNA cleavage; (d) cleaving DNA flanking a mismatch; and (e) cleavage at a cruciform structure in a DNA duplex.
摘要翻译： 提供了与T7 Endo I具有至少35％氨基酸序列同一性的修饰的DNA切割酶的组合物和方法。修饰的酶包括由β桥分开的两个催化中心，其中β-桥含有至少一个突变 与未修饰的酶相比具有改变酶裂解活性的作用。 与可以在不同反应条件下调节的修饰的DNA切割酶相关的活性包括以下至少一个：（a）非序列特异性切口活性; （b）在预先存在的切口位点切割双链体DNA的第二链以产生具有单链突出端的线性双链体; （c）非序列特异性DNA切割; （d）切断位于不对称侧翼的DNA; 和（e）在DNA双链体中的十字形结构处的切割。
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7.发明申请公开(公告)号：US20050158769A1
公开(公告)日：2005-07-21
申请号：US11018116
申请日：2004-12-21
IPC分类号： C12N15/09 , C12N1/15 , C12N1/19 , C12N1/21 , C12N5/10 , C12N9/10 , C12N9/22 , C12N15/54 , C12N15/55 , C12R1/265 , C12Q1/68 , C07H21/04 , C12N15/74
CPC分类号： C12N9/1007 , C12N9/22
摘要： A method for cloning restriction-modification system is provided whereby the target modification methylase is produced and confers full protection during all growth phases in which the cognate restriction enzyme is present. The method is employed in the cloning of the MseI restriction-modification system.
摘要翻译： 提供克隆限制性修饰体系的方法，由此产生目标修饰甲基化酶，并在存在相关限制性内切酶的所有生长期赋予完全保护。 该方法用于克隆MseI限制性修饰系统。
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公开(公告)号：US5352778A
公开(公告)日：1994-10-04
申请号：US167238
申请日：1993-12-15
CPC分类号： C07K16/12 , C12N9/1252
摘要： Recombinant DNA polymeraset from archaebacteria as well as isolated DNA coding for such polymeraset are provided. The isolated DNA is obtained by use of DNA or antibody probet prepared from the DNA encoding T. litoralis DNA polymerate and the T. litoralis DNA polymerate respectively. Also provided are meshocs for producing recombinant archaebacteria thermostable DNA polymerase and methods for enhancing the expression of such polymeraset by identifying, locating and removing introns from within the DNA coding for such DNA polymerases.
摘要翻译： 提供了来自古细菌的重组DNA聚合酶以及编码这种聚合酶的分离的DNA。 分离的DNA通过使用分别由编码泰氏单胞菌DNA聚合物的DNA和泰氏单胞菌DNA聚合物制备的抗体探针获得。 还提供了用于产生重组古细菌耐热性DNA聚合酶的方法和通过从编码这种DNA聚合酶的DNA内鉴定，定位和去除内含子来增强这些聚合酶的表达的方法。
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9.发明授权
公开(公告)号：US5322785A
公开(公告)日：1994-06-21
申请号：US686340
申请日：1991-04-17
IPC分类号： C12N9/10 , C12N1/21 , C12N9/12 , C12N9/16 , C12N15/09 , C12N15/54 , C12N15/55 , C12R1/01 , C12R1/19 , C12N15/67 , C12N15/70 , C12N15/74
CPC分类号： C12N9/1252
摘要： There is provided an extremely thermostable enzyme obtainable from Thermococcus litoralis. The thermostable enzyme has a molecular weight of about 90,000-95,000 daltons, a half-life of about 60 minutes at 100.degree. C. in the absence of stabilizer, and a half-life of about 95 minutes at 100.degree. C. in the presence of stabilizer, such as octoxynol (TRITON X-100) or bovine serum albumin. The thermostable enzyme possesses a 3'-5' proofreading exonuclease activity. The thermostable enzyme may be native or recombinant and may be used for second-strand cDNA synthesis in cDNA cloning, DNA sequencing, and DNA amplification.
摘要翻译： 提供了可从Thermococcus litoralis获得的极热稳定的酶。 耐热酶的分子量约为90,000-95,000道尔顿，在不存在稳定剂的情况下，在100℃下半衰期约为60分钟，而在100℃存在时，半衰期约为95分钟 的稳定剂如辛苯醇（TRITON X-100）或牛血清白蛋白。 耐热酶具有3'-5'校对核酸外切酶活性。 耐热酶可以是天然的或重组的，并且可以用于cDNA克隆，DNA测序和DNA扩增中的第二链cDNA合成。
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公开(公告)号：US20120283144A1
公开(公告)日：2012-11-08
申请号：US13464548
申请日：2012-05-04
CPC分类号： C12N15/10 , C12N9/93 , C12N15/66 , C12P19/34 , C12Q1/6832 , Y02P20/52 , C12Q2521/501 , C12Q2527/127 , C12Q2527/137
摘要： Compositions and methods are provided for enhancing enzymatic ligation between nucleic acid fragments that relies on one or more small molecule enhancers having a size of less than 1000 daltons. For example, enhancement of ligation efficiencies are observed for double-stranded nucleic acid fragments that are blunt-ended, have a single nucleotide overhang at the ligation end, or have staggered ends compared to ligation under similar conditions in the absence of the one or more small molecule ligation enhancer. The use of small molecule enhancers for ligating nucleic acids results in an increased number of transformed host cells after transformation with the ligated molecules. This enhancement can be observed with chemically transformed host cells and with host cells transformed by electroporation.
摘要翻译： 提供了组合物和方法，用于增强依赖于一种或多种尺寸小于1000道尔顿的小分子增强剂的核酸片段之间的酶连接。 例如，对于平端的双链核酸片段，在连接末端具有单个核苷酸突出端，或者在不存在一个或多个相似条件的相似条件下与连接相比，具有交错末端，观察到连接效率的增强 小分子连接增强子。 使用小分子增强子连接核酸导致用连接的分子转化后转化的宿主细胞数量增加。 可以通过化学转化的宿主细胞和通过电穿孔转化的宿主细胞来观察这种增强。
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