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1.发明申请Thermostable enzyme promoting the fidelity of thermostable DNA polymerases-for improvement of nucleic acid synthesis and amplification in vitro 有权促进耐热DNA聚合酶保真度的热稳定酶 - 用于体外改进核酸合成和扩增公开(公告)号：US20060078928A1
公开(公告)日：2006-04-13
申请号：US11241116
申请日：2005-09-29
Applicant: Waltraud Ankenbauer , Frank Laue , Harald Sobek , Michael Greif
Inventor： Waltraud Ankenbauer , Frank Laue , Harald Sobek , Michael Greif
CPC classification number: C12N15/10 , C12N9/1252 , C12N9/22 , C12Q1/686 , C12Q2521/101
Abstract: A purified thermostable enzyme is derived form the thermophilic archaebacterium Archaeoglobus fulgidus. The enzyme can be native or recombinant, is stable under PCR conditions and exhibits double strand specific exonuclease activity. It is a 3′-5′ exonuclease and cleaves to produce 5′-mononucleotides. Thermostable exonucleases are useful in many recombinant DNA techniques, in combination with a thermostable DNA polymerase like Taq especially for nucleic acid amplification by the polymerase chain reaction (PCR).
Abstract translation: 纯化的热稳定性酶源自嗜热古细菌大肠球菌（Archaeoglobus fulgidus）。 酶可以是天然的或重组的，在PCR条件下是稳定的并且表现出双链特异性外切核酸酶活性。 它是一个3'-5'核酸外切酶，并切割产生5'-单核苷酸。 热稳定的核酸外切酶可用于许多重组DNA技术，与热稳定DNA聚合酶如Taq组合，特别是通过聚合酶链反应（PCR）进行核酸扩增。
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公开(公告)号：US20110250598A1
公开(公告)日：2011-10-13
申请号：US13042628
申请日：2011-03-08
Applicant: Ulrike Fischer , Michael Greif , Harald Sobek , Johann-Peter Thalhofer
Inventor： Ulrike Fischer , Michael Greif , Harald Sobek , Johann-Peter Thalhofer
CPC classification number: C12Q1/686 , C12N9/1252 , C12Q2527/137 , C12Q2521/101
Abstract: The present invention relates to a formulation of a thermostable DNA polymerase which is completely free of detergents and its particular use in real time polymerase chain reaction (PCR). Such a formulation may be obtained if the selected purification method does not require the addition of a detergent at any purification step.
Abstract translation: 本发明涉及完全不含洗涤剂的热稳定性DNA聚合酶的制剂及其在实时聚合酶链式反应（PCR）中的特别用途。 如果所选择的纯化方法不需要在任何纯化步骤中添加洗涤剂，则可以获得这样的制剂。
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3.发明授权Thermostable enzyme promoting the fidelity of thermostable DNA polymerases—for improvement of nucleic acid synthesis and amplification in vitro 有权促进耐热DNA聚合酶保真度的热稳定酶 - 用于体外改进核酸合成和扩增公开(公告)号：US07410782B2
公开(公告)日：2008-08-12
申请号：US11241116
申请日：2005-09-29
Applicant: Waltraud Ankenbauer , Frank Laue , Harald Sobek , Michael Greif
Inventor： Waltraud Ankenbauer , Frank Laue , Harald Sobek , Michael Greif
CPC classification number: C12N15/10 , C12N9/1252 , C12N9/22 , C12Q1/686 , C12Q2521/101
Abstract: A purified thermostable enzyme is derived form the thermophilic archaebacterium Archaeoglobus fulgidus. The enzyme can be native or recombinant, is stable under PCR conditions and exhibits double strand specific exonuclease activity. It is a 3′-5′ exonuclease and cleaves to produce 5′-mononucleotides. Thermostable exonucleases are useful in many recombinant DNA techniques, in combination with a thermostable DNA polymerase like Taq especially for nucleic acid amplification by the polymerase chain reaction (PCR).
Abstract translation: 纯化的热稳定性酶源自嗜热古细菌大肠球菌（Archaeoglobus fulgidus）。 酶可以是天然的或重组的，在PCR条件下是稳定的并且表现出双链特异性外切核酸酶活性。 它是一个3'-5'核酸外切酶，并切割产生5'-单核苷酸。 热稳定的核酸外切酶可用于许多重组DNA技术，与热稳定DNA聚合酶如Taq组合，特别是通过聚合酶链反应（PCR）进行核酸扩增。
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Patent Agency Ranking
4.发明授权Thermostable enzyme promoting the fidelity of thermostable DNA polymerases-for improvement of nucleic acid synthesis and amplification in vitro 有权促进耐热DNA聚合酶保真度的热稳定酶 - 用于体外改进核酸合成和扩增公开(公告)号：US07030220B1
公开(公告)日：2006-04-18
申请号：US09856850
申请日：2000-09-27
Applicant: Waltraud Ankenbauer , Frank Laue , Harald Sobek , Michael Greif
Inventor： Waltraud Ankenbauer , Frank Laue , Harald Sobek , Michael Greif
CPC classification number: C12N15/10 , C12N9/1252 , C12N9/22 , C12Q1/686 , C12Q2521/101
Abstract: A purified thermostable enzyme is derived from the thermophilic archaebacterium Archaeoglobus fulgidus. The enzyme can be native or recombinant, is stable under PCR conditions and exhibits double strand specific exonuclease activity. It is a 3′–5′ exonuclease and cleaves to produce 5′-mononucleotides. Thermostable exonucleases are useful in many recombinant DNA techniques, in combination with a thermostable DNA polymerase like Tag especially for nucleic acid amplification by the polymerase chain reaction (PCR).
Abstract translation: 纯化的热稳定性酶源自嗜热古细菌大肠球菌（Archaeoglobus fulgidus）。 酶可以是天然的或重组的，在PCR条件下是稳定的并且表现出双链特异性外切核酸酶活性。 它是一个3'-5'核酸外切酶，并切割产生5'-单核苷酸。 热稳定的核酸外切酶可用于许多重组DNA技术，与耐热性DNA聚合酶如Tag特异性用于通过聚合酶链反应（PCR）的核酸扩增。
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公开(公告)号：US09193959B2
公开(公告)日：2015-11-24
申请号：US13069514
申请日：2011-03-23
Applicant: Harald Sobek , Johann-Peter Thalhofer , Rainer Mueller , Manfred Schmidt , Michael Greif , Armin Ruf , Christian Rudolph
Inventor： Harald Sobek , Johann-Peter Thalhofer , Rainer Mueller , Manfred Schmidt , Michael Greif , Armin Ruf , Christian Rudolph
CPC classification number: C12N9/1247 , C12P19/34 , C12Y207/07006
Abstract: The present invention provides improved variants of T7 RNA polymerase by introducing novel mutations which lead to improved thermostability of the enzyme. According to the invention, amino acid substitutions at the positions Val426, Ser633, Val650, Thr654, Ala702, Val795, and combinations thereof are advantageous.
Abstract translation: 本发明通过引入导致酶的热稳定性的新突变来提供T7RNA聚合酶的改进变体。 根据本发明，位置Val426，Ser633，Val650，Thr654，Ala702，Val795及其组合的氨基酸取代是有利的。
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6.发明授权Reversibly modified thermostable enzymes for DNA synthesis and amplification in vitro 有权用于体外DNA合成和扩增的可逆修饰的热稳定酶公开(公告)号：US07378262B2
公开(公告)日：2008-05-27
申请号：US10317715
申请日：2002-12-11
Applicant: Harald Sobek , Michael Greif
Inventor： Harald Sobek , Michael Greif
CPC classification number: C12Q1/6848 , C12N9/1252 , C12N9/22 , C12N9/99 , C12Q1/686 , C12Q2549/101 , C12Q2521/319 , C12Q2521/101
Abstract: The invention relates to a composition comprising a first modified thermostable enzyme exhibiting 3′exonuclease activity but essentially no DNA polymerase activity and a second modified thermostable enzyme exhibiting DNA polymerase activity, whereas the fidelity of an amplification process is enhanced by the use of the composition in an amplification process in comparison to the use of the single second enzyme in an amplification process and, whereas said first and said second modified thermostable enzyme is reversibly modified by an inhibiting agent which results in essentially complete inactivation of enzyme activity, wherein incubation of said first and said second modified thermostable enzyme in an aqueous buffer at alkaline pH at a temperature less than 25° C. for 20 minutes results in no significant increase in the activity of said first and said second modified thermostable enzyme, wherein incubation at a temperature greater than 50° C. in an aqueous buffer at alkaline pH results in at least tow-fold increase in enzyme activity in less than 20 minutes which allow formation of primer extension products.
Abstract translation: 本发明涉及一种组合物，其包含显示出3'核酸酶活性但基本上不含DNA聚合酶活性的第一个修饰的热稳定酶，以及显示DNA聚合酶活性的第二个修饰的热稳定酶，而通过使用组合物来增强扩增过程的保真度 扩增过程与在扩增过程中单次第二酶的使用相比较，而所述第一和所述第二修饰的热稳定酶由抑制剂可逆地修饰，所述抑制剂导致酶活性基本完全失活，其中所述第一 并且所述第二修饰的热稳定酶在碱性pH在低于25℃的温度下在水性缓冲液中20分钟导致所述第一和所述第二修饰的热稳定酶的活性没有显着增加，其中在大于 50℃，在碱性pH溶液中的水性缓冲液中 在少于20分钟的时间内至少可以提高酶活性的三倍增加，从而形成引物延伸产物。
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公开(公告)号：US20110256589A1
公开(公告)日：2011-10-20
申请号：US13069514
申请日：2011-03-23
Applicant: Harald Sobek , Johann-Peter Thalhofer , Rainer Mueller , Manfred Schmidt , Michael Greif , Armin Ruf , Christian Rudolph
Inventor： Harald Sobek , Johann-Peter Thalhofer , Rainer Mueller , Manfred Schmidt , Michael Greif , Armin Ruf , Christian Rudolph
CPC classification number: C12N9/1247 , C12P19/34 , C12Y207/07006
Abstract: The present invention provides improved variants of T7 RNA polymerase by introducing novel mutations which lead to improved thermostability of the enzyme. According to the invention, amino acid substitutions at the positions Val426, Ser633, Val650, Thr654, Ala702, Val795, and combinations thereof are advantageous.
Abstract translation: 本发明通过引入导致酶的热稳定性的新突变来提供T7RNA聚合酶的改进变体。 根据本发明，位置Val426，Ser633，Val650，Thr654，Ala702，Val795及其组合的氨基酸取代是有利的。
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8.发明申请公开(公告)号：US20120252071A1
公开(公告)日：2012-10-04
申请号：US13436110
申请日：2012-03-30
Applicant: Michael Greif , Christian Rudolph , Manfred Schmidt , Harald Sobek , Johann-Peter Thalhofer
Inventor： Michael Greif , Christian Rudolph , Manfred Schmidt , Harald Sobek , Johann-Peter Thalhofer
CPC classification number: C12N9/1247 , C12Y207/07006
Abstract: The present disclosure provide novel variants of T7 RNA polymerase. Embodiments of T7 variants, according to the instant invention, include a Cysteine-Serine substitution on position 723 of the amino acid sequence of the T7 polypeptide. Embodiments of T7 variants according to the instant invention have a DNA-dependent RNA polymerase enzymatic activity and a reduced tendency to form intramolecular homodimers by way of oxidizing thiol groups. The amino acid substitutions within the T7 variants disclosed herein impact minimally, if at all, the RNA polymerase activity of the T7 polypeptide. Further, the mutations of the disclosed embodiments may optionally be combined with mutations which provide enhanced thermostability compared to the wild-type reference.
Abstract translation: 本公开提供了T7RNA聚合酶的新变体。 根据本发明的T7变体的实施方案包括T7多肽的氨基酸序列的位置723上的半胱氨酸 - 丝氨酸取代。 根据本发明的T7变体的实施方案具有DNA依赖性RNA聚合酶酶活性，并且通过氧化硫醇基形成分子内同型二聚体的趋势降低。 本文公开的T7变体内的氨基酸取代最小程度地影响T7多肽的RNA聚合酶活性。 此外，所公开的实施方案的突变可以任选地与与野生型参考物相比提供增强的热稳定性的突变组合。
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