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51. 发明授权

US06372497B1 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination 失效
公开(公告)号：US06372497B1
公开(公告)日：2002-04-16
申请号：US09590774
申请日：2000-06-08
申请人： Willem P. C. Stemmer
发明人： Willem P. C. Stemmer
IPC分类号： C12N1500
CPC分类号： C40B40/02 , C07K14/43595 , C07K14/545 , C07K16/00 , C07K2317/565 , C07K2317/622 , C12N9/2471 , C12N9/86 , C12N15/1027 , C12N15/1034 , C12N15/1037 , C12N15/1058 , C12N15/52 , C12N15/64 , C12Q1/68 , C12Q1/6811 , C12Y302/01023 , C12Y305/02006 , H01L39/228 , C12Q2525/179
摘要： A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.

52. 发明授权

US06297053B1 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination 有权
公开(公告)号：US06297053B1
公开(公告)日：2001-10-02
申请号：US09501698
申请日：2000-02-10
申请人： Willem P. C. Stemmer
发明人： Willem P. C. Stemmer
IPC分类号： C12N1500
CPC分类号： C40B40/02 , C07K14/43595 , C07K14/545 , C07K16/00 , C07K2317/565 , C07K2317/622 , C12N9/2471 , C12N9/86 , C12N15/1027 , C12N15/1034 , C12N15/1037 , C12Q1/68 , C12Q1/6811 , C12Y302/01023 , C12Y305/02006 , C40B40/08 , C40B50/06
摘要： A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.

53. 发明授权

US06287861B1 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination 有权
标题翻译：通过迭代选择和重组产生具有所需特征的多核苷酸的方法
公开(公告)号：US06287861B1
公开(公告)日：2001-09-11
申请号：US09133508
申请日：1998-08-12
申请人： Willem P. C. Stemmer , Andreas Crameri
发明人： Willem P. C. Stemmer , Andreas Crameri
IPC分类号： C12N1500
CPC分类号： C40B40/02 , C07K14/43595 , C07K14/545 , C07K16/00 , C07K2317/565 , C07K2317/622 , C12N9/2471 , C12N9/86 , C12N15/1027 , C12N15/1034 , C12N15/1037 , C12Q1/68 , C12Q1/6811 , C12Y302/01023 , C12Y305/02006 , C40B40/08 , C40B50/06
摘要： A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.
摘要翻译：描述了随机断裂后DNA重组的方法及其通过体外或体内重组诱变核酸序列的应用。特别地，描述了用于生产编码突变蛋白的核酸片段或多核苷酸的方法。本发明还涉及一种反复循环诱变，洗牌和选择的方法，其允许蛋白质的体外或体内的定向分子进化。

54. 发明授权

US06277638B1 Methods for generating polynucleotides having desired characteristics by iterative selection and recombination 有权
公开(公告)号：US06277638B1
公开(公告)日：2001-08-21
申请号：US09232863
申请日：1999-01-15
申请人： Willem P. C. Stemmer
发明人： Willem P. C. Stemmer
IPC分类号： C12N1500
CPC分类号： C40B40/02 , C07K14/43595 , C07K14/545 , C07K16/00 , C07K2317/565 , C07K2317/622 , C12N9/2471 , C12N9/86 , C12N15/1027 , C12N15/1034 , C12N15/1037 , C12Q1/68 , C12Q1/6811 , C12Y302/01023 , C12Y305/02006 , C40B40/08 , C40B50/06
摘要： A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.

55. 发明授权

US5811238A Methods for generating polynucleotides having desired characteristics by iterative selection and recombination 失效
公开(公告)号：US5811238A
公开(公告)日：1998-09-22
申请号：US564955
申请日：1995-11-30
申请人： Willem P. C. Stemmer , Andreas Crameri
发明人： Willem P. C. Stemmer , Andreas Crameri
IPC分类号： C12N15/09 , A61K38/00 , A61K39/395 , C07K14/435 , C07K14/545 , C07K16/00 , C12N9/38 , C12N9/86 , C12N15/00 , C12N15/10 , C12P21/08 , C12Q1/68 , C40B40/02 , G01N33/53 , G01N33/566
CPC分类号： C40B40/02 , C07K14/43595 , C07K14/545 , C07K16/00 , C12N15/1027 , C12N15/1034 , C12N15/1037 , C12N9/2471 , C12N9/86 , C12Q1/68 , C12Q1/6811 , C12Y302/01023 , C12Y305/02006 , C40B40/08 , C40B50/06 , C07K2317/565 , C07K2317/622
摘要： A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.

56. 发明授权

US5514568A Enzymatic inverse polymerase chain reaction 失效
标题翻译：酶反向聚合酶链反应
公开(公告)号：US5514568A
公开(公告)日：1996-05-07
申请号：US184751
申请日：1994-01-19
申请人： Willem P. C. Stemmer
发明人： Willem P. C. Stemmer
IPC分类号： C07H21/04 , C12P19/34 , C12Q1/68 , C12Q1/70
CPC分类号： C07H21/04 , C12P19/34 , C12Q1/68 , C12Q1/70
摘要： The invention is directed to a method of introducing at least one predetermined change in a nucleic acid sequence of a double-strand DNA. The method includes: (a) providing a first primer and a second primer capable of directing said predetermined change in said nucleic acid sequence, said first and second primers comprising a nucleic acid sequence substantially complementary to said double-stranded DNA so as to allow hybridization, a class IIS restriction enzyme recognition sequence and cleavage sites; (b) hybridizing said first and second primers to opposite strands of said double-stranded DNA to form a first pair of primer-templates oriented in opposite directions; (c) extending said first pair of primer-templates to create double-stranded molecules; (d) hybridizing said first and second primers at least once to said double-stranded molecules to form a second pair of primer-templates; (e) extending said second pair of primer-templates to produce double-stranded linear molecules terminating with class IIS restriction enzyme recognition sequences; and (f) restricting said double-stranded linear molecules with a class IIS restriction enzyme to form restricted linear molecules containing said change in said nucleic acid sequence. Also provided is a method of producing at least two changes located at one or more positions within a nucleic acid sequence. Synthetic primers for producing such changes are provided as well.
摘要翻译：本发明涉及在双链DNA的核酸序列中引入至少一个预定变化的方法。该方法包括：（a）提供能够引导所述核酸序列中所述预定变化的第一引物和第二引物，所述第一引物和第二引物包含与所述双链DNA基本上互补的核酸序列，以便允许杂交一类IIS限制酶识别序列和切割位点; （b）将所述第一和第二引物与所述双链DNA的相反链杂交以形成沿相反方向取向的第一对引物模板; （c）延伸所述第一对引物 - 模板以产生双链分子; （d）将所述第一和第二引物与所述双链分子杂交至少一次以形成第二对引物模板; （e）延伸所述第二对引物模板以产生以IIS限制酶识别序列终止的双链线性分子; 和（f）用IIS限制酶限制所述双链线性分子以形成包含所述核酸序列中所述变化的限制性线性分子。还提供了产生位于核酸序列内的一个或多个位置处的至少两个改变的方法。还提供了用于产生这种变化的合成引物。

57. 发明授权

US5498530A Peptide library and screening method 失效
标题翻译：肽库和筛选方法
公开(公告)号：US5498530A
公开(公告)日：1996-03-12
申请号：US290641
申请日：1994-08-15
申请人： Peter J. Schatz , Millard G. Cull , Jeff F. Miller , Willem P. C. Stemmer
发明人： Peter J. Schatz , Millard G. Cull , Jeff F. Miller , Willem P. C. Stemmer
IPC分类号： C12N1/21 , C12N15/10 , C12Q1/68 , G01N33/68 , C12P21/02 , C12N15/72 , C12N15/74 , C12N15/79
CPC分类号： G01N33/68 , C12N15/1075
摘要： A random peptide library constructed by transforming host cells with a collection of recombinant vectors that encode a fusion protein comprised of a DNA binding protein and a random peptide and also contain a binding site for the DNA binding protein can be used to screen for novel ligands. The screening method results in the formation of a complex comprising the fusion protein bound to a receptor through the random peptide ligand and to the recombinant DNA vector through the DNA binding protein.
摘要翻译：通过用编码由DNA结合蛋白和随机肽组成的融合蛋白的重组载体的集合转化宿主细胞构建的随机肽文库，并且还含有DNA结合蛋白的结合位点，可用于筛选新配体。筛选方法导致通过DNA结合蛋白形成包含通过随机肽配体与受体结合的融合蛋白和重组DNA载体的复合物。

58. 发明授权

US5491074A Association peptides 失效
标题翻译：关联肽
公开(公告)号：US5491074A
公开(公告)日：1996-02-13
申请号：US67387
申请日：1993-05-24
申请人： Lois Aldwin , Mark Madden , Willem P. C. Stemmer
发明人： Lois Aldwin , Mark Madden , Willem P. C. Stemmer
IPC分类号： C07K5/117 , C07K7/06 , C07K14/47 , C12N15/62 , C12Q1/00 , C12Q1/68 , G01N33/531 , C12P21/02
CPC分类号： C07K7/06 , C07K14/47 , C07K5/1024 , C12N15/62 , C12Q1/00 , C12Q1/68 , G01N33/531 , C07K2319/00 , C07K2319/23 , C07K2319/24 , C07K2319/42
摘要： Peptides that form lightly associated homodimers can be used to form dimers and multimers of other molecules and molecular motifs of interest. These association peptides can dimerize regardless of whether motifs are added to the amino-terminus of the peptide, or the carboxy terminus of the peptide, although additions to the carboxy-terminus of the association peptides require the presence of certain acidic residues.
摘要翻译：形成轻度相关同源二聚体的肽可用于形成其他分子的二聚体和多聚体以及感兴趣的分子基序。无论是将肽添加到肽的氨基末端还是肽的羧基末端，这些缔合肽都可以二聚化，尽管缔合肽的羧基末端的添加需要存在某些酸性残基。

59. 发明授权

US5439829A Immobilization of biologically active molecules by changing the Oxidation state of a chelated transition metal ion 失效
标题翻译：通过改变螯合过渡金属离子的氧化态来固定生物活性分子
公开(公告)号：US5439829A
公开(公告)日：1995-08-08
申请号：US826928
申请日：1992-01-24
申请人： Leslie D. Anderson , James A. Cook , Gary S. David , Susan M. Hochschwender , Mary S. Kasher , Michele C. Smith , Willem P. C. Stemmer
发明人： Leslie D. Anderson , James A. Cook , Gary S. David , Susan M. Hochschwender , Mary S. Kasher , Michele C. Smith , Willem P. C. Stemmer
IPC分类号： C07K14/575 , C07K1/14 , C07K1/16 , C07K1/22 , C07K7/06 , C07K7/08 , C07K14/025 , C07K14/47 , C07K14/82 , C07K16/00 , C07K16/08 , C07K16/30 , C07K16/46 , C07K17/02 , C07K17/06 , C07K19/00 , C12N20060101 , C12N15/09 , C12N15/62 , C12P21/02 , C12R1/19 , G01N33/53 , G01N33/547 , G01N33/543 , C07K17/00 , C12N11/00 , G01N33/549
CPC分类号： C07K7/06 , C07K1/16 , C07K14/005 , C07K14/4736 , C07K14/82 , C07K16/00 , C07K16/084 , C07K16/3007 , C07K16/461 , C07K17/06 , C07K7/08 , C12N15/62 , C07K2317/24 , C07K2319/00 , C07K2319/20 , C07K2319/40 , C12N2710/20022 , C12N2710/22022 , Y10S436/824 , Y10S530/81 , Y10S530/812 , Y10S530/813 , Y10S530/816
摘要： A chelating agent is covalently bonded to a biologically active molecule such as an enzyme or antibody, the biologically active molecule is contacted with a support containing a bound transition metal ion whereby the metal ion is chelated by the chelating agent and the oxidation state of the metal ion is changed by treatment with an oxidizing or a reducing agent to provide a kinetically inert: oxidation state to immobilize the biologically active molecule on the support. The transition metal ion is preferably Co(II), Cr(II) or Ru(III) and the oxidation state of the metal ion is changed to Co(III), Cr(III) or Ru(II), respectively. The chelating agent can be iminodiacetic acid, nitrilotriacetic acid, terpyridine, bipyridine, triethylenetetraamine, biethylenetriamine, 1,4,7-triazacyclonane or a chelating peptide. Certain chelating agents can immobilize more than one biologically active molecule at a metal ion site on the support. The immobilized biologically active molecules can be used in affinity chromatography or in assay systems.
摘要翻译：螯合剂共价结合到生物活性分子如酶或抗体上，使生物活性分子与含有结合的过渡金属离子的载体接触，由此金属离子被螯合剂螯合，并且金属的氧化态通过用氧化还原剂处理来改变离子以提供动态惰性：氧化态以将生物活性分子固定在载体上。过渡金属离子优选为Co（II），Cr（II）或Ru（III），金属离子的氧化态分别变为Co（III），Cr（III）或Ru（II）。螯合剂可以是亚氨基二乙酸，次氮基三乙酸，三联吡啶，联吡啶，三亚乙基四胺，二亚乙基三胺，1,4,7-三氮杂环或螯合肽。某些螯合剂可以在载体上的金属离子位置固定多于一种生物活性分子。固定化的生物活性分子可用于亲和色谱法或测定系统中。

60. 发明授权

US08589085B2 Methods of populating data structures for use in evolutionary simulations 有权
标题翻译：填充用于进化模拟的数据结构的方法
公开(公告)号：US08589085B2
公开(公告)日：2013-11-19
申请号：US13434261
申请日：2012-03-29
申请人： Sergey A. Selifonov , Willem P. C. Stemmer
发明人： Sergey A. Selifonov , Willem P. C. Stemmer
IPC分类号： G01N31/00 , G01N33/48
CPC分类号： G06N3/126 , G06F19/14 , G06F19/18 , G06F19/22 , G06F19/24
摘要： In particular, this invention provides novel methods of populating data structures for use in evolutionary modeling. In particular, this invention provides methods of populating a data structure with a plurality of character strings. The methods involve encoding two or more biological molecules into character strings to provide a collection of two or more different initial character strings; selecting at least two substrings from the pool of character strings; concatenating the substrings to form one or more product strings about the same length as one or more of the initial character strings; adding the product strings to a collection of strings; and optionally repeating this process using one or more of the product strings as an initial string in the collection of initial character strings.
摘要翻译：特别地，本发明提供了用于进化建模的填充数据结构的新颖方法。特别地，本发明提供了用多个字符串填充数据结构的方法。所述方法包括将两个或更多个生物分子编码成字符串以提供两个或多个不同的初始字符串的集合; 从字符串池中选择至少两个子字符串; 连接子串以形成与一个或多个初始字符串相同长度的一个或多个产品串; 将产品字符串添加到字符串集合中; 并且可选地使用一个或多个产品串将该过程重复为初始字符串的集合中的初始字符串。

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