公开(公告)号：US06790605B1

公开(公告)日：2004-09-14

申请号：US09375605

申请日：1999-08-17

申请人： Jay M. Short

发明人： Jay M. Short

IPC分类号： C12Q100

CPC分类号： C12N9/00 ,  C12N9/16 ,  C12N9/2445 ,  C12N15/102 ,  C12N15/1034 ,  C12Y301/03001 ,  C12Y302/01021

摘要： Disclosed is a process for obtaining an enzyme having a specified enzyme activity derived from a heterogeneous DNA population by screening, for the specified enzyme activity, a library of clones containing DNA from the heterogeneous DNA population which have been exposed to directed mutagenesis towards production of the specified enzyme activity. Also disclosed is a process for obtaining an enzyme having a specified enzyme activity by screening, for the specified enzyme activity, a library of clones containing DNA from a pool of DNA populations which have been exposed to directed mutagenesis in an attempt to produce in the library of clones DNA encoding an enzyme having one or more desired characteristics which can be the same or different from the specified enzyme activity.

摘要翻译： 公开了一种获得具有来自异质DNA群体的特定酶活性的酶的方法，对于规定的酶活性，筛选含有已经暴露于定向诱变的异源DNA群体的DNA的克隆的文库， 指定的酶活性。 还公开了获得具有特定酶活性的酶的方法，通过筛选已经暴露于定向诱变的DNA群体中含有DNA的克隆的特定酶活性文库，试图在文库中产生 的克隆DNA，其编码具有一个或多个可以与指定酶活性相同或不同的所需特征的酶。

公开(公告)号：US06713282B2

公开(公告)日：2004-03-30

申请号：US10099816

申请日：2002-03-14

申请人： Jay M. Short ,  Gerhard Johann Frey

发明人： Jay M. Short ,  Gerhard Johann Frey

IPC分类号： C12P2106

CPC分类号： C07K14/445 ,  A61K39/00 ,  A61K2039/53 ,  C12N9/00 ,  C12N9/14 ,  C12N9/16 ,  C12N9/88 ,  C12N15/102 ,  C12N15/1027 ,  C12N15/1034 ,  C12Q1/6811 ,  C12Y301/11002

摘要： This invention provides methods of obtaining novel polynucleotides and encoded polypeptides by the use of non-stochastic methods of directed evolution (DirectEvolution™). A particular advantage of end-selection-based methods is the ability to recover full-length polynucleotides from a library of progeny molecules generated by mutagenesis methods. These methods include non-stochastic polynucleotide site-saturation mutagenesis (Gene Site Saturation Mutagenesis™) and non-stochastic polynucleotide reassembly (GeneReassembly™). This invention provides methods of obtaining novel enzymes that have optimized physical &/or biological properties. Through use of the claimed methods, genetic vaccines, enzymes, small molecules, and other desirable molecules can be evolved towards desirable properties. For example, vaccine vectors can be obtained that exhibit increased efficacy for use as genetic vaccines. Vectors obtained by using the methods can have, for example, enhanced antigen expression, increased uptake into a cell, increased stability in a cell, ability to tailor an immune response, and the like. Furthermore, this invention provides methods of obtaining a variety of novel biologically active molecules, in the fields of antibiotics, pharmacotherapeutics, and transgenic traits.

公开(公告)号：US06635449B2

公开(公告)日：2003-10-21

申请号：US10108077

申请日：2002-03-26

申请人： Jay M. Short

发明人： Jay M. Short

IPC分类号： C12P2106

CPC分类号： C07K14/445 ,  A61K39/00 ,  A61K2039/53 ,  C12N9/00 ,  C12N9/16 ,  C12N15/102 ,  C12N15/1027 ,  C12N15/1034 ,  C12Q1/6811 ,  C12Y301/11002

摘要： This invention provides methods of obtaining novel polynucleotides and encoded polypeptides by the use of non-stochastic methods of directed evolution (DirectEvolution™). A particular advantage of exonuclease-mediated reassembly methods is the ability to reassemble nucleic acid strands that would otherwise be problematic to chimerize. Exonuclease-mediated reassembly methods can be used in combination with other mutagenesis methods provided herein. These methods include non-stochastic polynucleotide site-saturation mutagenesis (Gene Site Saturation Mutagenesis™) and non-stochastic polynucleotide reassembly (GeneReassembly™). This invention provides methods of obtaining novel enzymes that have optimized physical &/or biological properties. Through use of the claimed methods, genetic vaccines, enzymes, small molecules, and other desirable molecules can be evolved towards desirable properties. For example, vaccine vectors can be obtained that exhibit increased efficacy for use as genetic vaccines. Vectors obtained by using the methods can have, for example, enhanced antigen expression, increased uptake into a cell, increased stability in a cell, ability to tailor an immune response, and the like. Furthermore, this invention provides methods of obtaining a variety of novel biologically active molecules, in the fields of antibiotics, pharmacotherapeutics, and transgenic traits.

公开(公告)号：US06579704B2

公开(公告)日：2003-06-17

申请号：US09851542

申请日：2001-05-08

申请人： Jay M. Short

发明人： Jay M. Short

IPC分类号： C12P1934

CPC分类号： C12Q1/6869 ,  C12Q2527/125

摘要： The present invention relates to a method for sequencing nucleic acids. In particular, such method includes incorporating certain modified nucleotides referred to as Simtides into nucleic acid strands. These Simtides are complexes comprising a nucleotide base (or analog thereof), a linker, and a label capable of generating a detectable signal for sequencing. A Simtide may be incorporated into a sequencing fragment as a primer component, as a chain elongator, or as a chain terminator.

公开(公告)号：US06489145B1

公开(公告)日：2002-12-03

申请号：US08962504

申请日：1997-10-31

申请人： Jay M. Short

发明人： Jay M. Short

IPC分类号： C12P1934

CPC分类号： C12Q1/6811 ,  C12N15/1027

摘要： Disclosed is a method of producing random polynucleotides by introducing two or more related polynucleotides into a suitable host cell such that a hybrid polynucleotide is generated by recombination and reductive reassortment. Also provided are vector and expression vehicles including such polynucleotides, polypeptides expressed by the hybrid polynucleotides and a method for screening for hybrid polypeptides.

摘要翻译： 公开了通过将两个或更多个相关多核苷酸引入合适的宿主细胞中产生随机多核苷酸的方法，使得通过重组和还原重配产生杂交多核苷酸。 还提供了载体和表达载体，包括这样的多核苷酸，由杂交多核苷酸表达的多肽和杂交多肽的筛选方法。

公开(公告)号：US06444426B1

公开(公告)日：2002-09-03

申请号：US09437905

申请日：1999-11-09

申请人： Jay M. Short ,  Eric J. Mathur

发明人： Jay M. Short ,  Eric J. Mathur

IPC分类号： C12Q170

CPC分类号： C12N15/1093 ,  C12N15/1096 ,  C12Q1/6811

摘要： Disclosed is a process for forming a normalized genomic DNA library from an environmental sample by (a) isolating a genomic DNA population from the environmental sample; (b) at least one of (i) amplifying the copy number of the DNA population so isolated and (ii) recovering a fraction of the isolated genomic DNA having a desired characteristic; and (c) normalizing the representation of various DNAs within the genomic DNA population so as to form a normalized library of genomic DNA from the environmental sample. Also disclosed is a normalized genomic DNA library formed from an environmental sample by the process.

摘要翻译： 公开了一种通过（a）从环境样品中分离基因组DNA群体从环境样品形成归一化基因组DNA文库的方法; （b）至少一个（i）扩增如此分离的DNA群体的拷贝数，和（ii）回收具有所需特征的分离的基因组DNA的一部分; 和（c）使基因组DNA群体内的各种DNA的表达正常化，从而形成来自环境样品的基因组DNA的归一化文库。 还公开了通过该方法由环境样品形成的归一化的基因组DNA文库。

公开(公告)号：US06361974B1

公开(公告)日：2002-03-26

申请号：US09535754

申请日：2000-03-27

申请人： Jay M. Short ,  Tsotne David Djavakhishvili ,  Gerhard Johann Frey

发明人： Jay M. Short ,  Tsotne David Djavakhishvili ,  Gerhard Johann Frey

IPC分类号： C12P2106

CPC分类号： C07K14/445 ,  A61K39/00 ,  A61K2039/53 ,  C12N9/00 ,  C12N9/16 ,  C12N15/102 ,  C12N15/1027 ,  C12N15/1034 ,  C12Q1/6811 ,  C12Y301/11002

摘要： This invention provides methods of obtaining novel polynucleotides and encoded polypeptides by the use of non-stochastic methods of directed evolution (DirectEvolution™). A particular advantage of exonuclease-mediated reassembly methods is the ability to reassemble nucleic acid strands that would otherwise be problematic to chimerize. Exonuclease-mediated reassembly methods can be used in combination with other mutagenesis methods provided herein. These methods include non-stochastic polynucleotide site-saturation mutagenesis (Gene Site Saturation Mutagenesis™) and non-stochastic polynucleotide reassembly (GeneReassembly™). This invention provides methods of obtaining novel enzymes that have optimized physical &/or biological properties. Through use of the claimed methods, genetic vaccines, enzymes, small molecules, and other desirable molecules can be evolved towards desirable properties. For example, vaccine vectors can be obtained that exhibit increased efficacy for use as genetic vaccines. Vectors obtained by using the methods can have, for example, enhanced antigen expression, increased uptake into a cell, increased stability in a cell, ability to tailor an immune response, and the like. Furthermore, this invention provides methods of obtaining a variety of novel biologically active molecules, in the fields of antibiotics, pharmacotherapeutics, and transgenic traits.

摘要翻译： 本发明提供了通过使用定向进化（DirectEvolution TM）的非随机方法获得新的多核苷酸和编码的多肽的方法。 外切核酸酶介导的重组方法的一个特别优点是重新组装核酸链的能力，否则这些核酸链将成为嵌合的问题。 外切核酸酶介导的重组方法可以与本文提供的其它诱变方法组合使用。 这些方法包括非随机多核苷酸位点饱和诱变（Gene Site Saturation Mutagenesis TM）和非随机多核苷酸重组（GeneReassembly TM）。 本发明提供获得具有优化的物理和/或生物学特性的新型酶的方法。 通过使用所要求保护的方法，可以将遗传疫苗，酶，小分子和其它所需分子演变成期望的性质。 例如，可以获得表现出增加用作基因疫苗的功效的疫苗载体。 通过使用该方法获得的载体可具有例如增强的抗原表达，增加的细胞摄取，增加细胞的稳定性，定制免疫应答的能力等。 此外，本发明提供了在抗生素，药物治疗剂和转基因性状领域中获得各种新型生物活性分子的方法。

公开(公告)号：US06352842B1

公开(公告)日：2002-03-05

申请号：US09276860

申请日：1999-03-26

申请人： Jay M. Short ,  Gerhard J. Frey ,  Tsotne D. Djavakhishvili

发明人： Jay M. Short ,  Gerhard J. Frey ,  Tsotne D. Djavakhishvili

IPC分类号： C12P2106

CPC分类号： C07K14/445 ,  A61K39/00 ,  A61K2039/53 ,  C12N9/00 ,  C12N9/14 ,  C12N9/16 ,  C12N9/88 ,  C12N15/102 ,  C12N15/1027 ,  C12N15/1034 ,  C12Q1/6811 ,  C12Y301/11002

摘要： A directed evolution process comprising novel methods for generating improved progeny molecules having desirable properties, including, for example, a method for rapid and facilitated production from a parental polynucleotide template, of a set of mutagenized progeny polynucleotides wherein at least one codon encoding each of the 20 naturally encoded amino acids is represented at each original codon position. This method, termed site-saturation mutagenesis, or simply saturation mutagenesis, is preferably based on the use of the degenerate N,N,G/T sequence. Also, a method of producing from a parental polypeptide template, a set of mutagenized progeny polypeptides wherein each of the 20 naturally encoded amino acids is represented at each original amino acid position. Also, other mutagenization processes that can be used in combination with, or in lieu of, saturation mutagenesis, including, for example: (a) assembly and/or reassembly of polynucloetide building blocks (including sections of genes &/or of gene families) mediated by a source of exonuclease activity such as exonuclease III; and (b) introduction of two or more related polynucleotides into a suitable host cell such that a hybrid polynucleotide is generated by recombination and reductive reassortment. Also molecular property screening methods, including a preferred method, termed end selection, comprised of using an enzyme, such as a topoisomerase, a restriction endonuclease, &/or a nicking enzyme (such as N. BstNB I), to detect a specific terminal sequence in a working polynucleotide, to produce a ligatable end thereat, and to ligate and clone the working polynucleotide.

公开(公告)号：US06238884B1

公开(公告)日：2001-05-29

申请号：US09267118

申请日：1999-03-09

申请人： Jay M. Short ,  Gerhard Johann Frey

发明人： Jay M. Short ,  Gerhard Johann Frey

IPC分类号： C12P2106

CPC分类号： C12Q1/6811 ,  C12N15/102 ,  C12N15/1058

摘要： A directed evolution process comprising novel methods for generating improved progeny molecules having desirable properties, including, for example, a method for rapid and facilitated production from a parental polynucleotide template, of a set of mutagenized progeny polynucleotides wherein at least one codon encoding each of the 20 naturally encoded amino acids is represented at each original codon position. This method, termed site-saturation mutagenesis, or simply saturation mutagenesis, is preferably based on the use of the degenerate N,N,G/T sequence. Also, a method of producing from a parental polypeptide template, a set of mutagenized progeny polypeptides wherein each of the 20 naturally encoded amino acids is represented at each original amino acid position. Also, other mutagenization processes that can be used in combination with, or in lieu of, saturation mutagenesis, including, for example: (a) assembly and/or reassembly of polynucloetide building blocks, which building blocks can be sections of genes &/or of gene families; and (b) introduction of two or more related polynucleotides into a suitable host cell such that a hybrid polynucleotide is generated by recombination and reductive reassortment. Also, vector and expression vehicles including such polynucleotides and correspondingly expressed polypeptides. Also molecular property screening methods, including a preferred method, termed end selection, comprised of using an enzyme, such as a topoisomerase, a restriction endonuclease, &/or a nicking enzyme (such as N. BstNB I), to detect a specific terminal sequence in a working polynucleotide, to produce a ligatable end thereat, and to ligate and clone the working polynucleotide.

摘要翻译： 一种定向进化过程，其包括用于产生具有期望性质的改良后代分子的新方法，包括例如一组诱变后代多核苷酸的亲本多核苷酸模板的快速和便利生产的方法，其中至少一个编码 在每个原始密码子位置表示20个天然编码的氨基酸。 该方法，称为位点饱和诱变，或简单的饱和诱变，优选基于使用简并N，N，G / T序列。 另外，从亲本多肽模板产生一组诱变的后代多肽的方法，其中在每个原始氨基酸位置表示20个天然编码氨基酸中的每一个。 此外，可以与饱和诱变组合使用或代替饱和诱变使用的其它诱变过程，包括例如：（a）组合和/或重组多核苷酸结构单元，所述构建基团可以是基因的部分和/或 的基因家族; 和（b）将两种或更多种相关多核苷酸引入合适的宿主细胞中，使得通过重组和还原重配产生杂交多核苷酸。 而且，载体和表达载体包括这种多核苷酸和相应表达的多肽。 还包括称为末端选择的分子特性筛选方法，其包括使用酶，例如拓扑异构酶，限制性内切核酸酶和/或切酶（例如N.BstNB I），以检测特异性末端 序列，以在其中产生可连接的末端，并连接和克隆工作的多核苷酸。

公开(公告)号：US06183740B2

公开(公告)日：2001-02-06

申请号：US09318528

申请日：1999-05-25

申请人： Jay M. Short ,  Keith A. Kretz

发明人： Jay M. Short ,  Keith A. Kretz

IPC分类号： A61K3846

CPC分类号： C12N15/8242 ,  A01K2217/05 ,  A21D8/042 ,  A23K20/189 ,  A23L5/25 ,  A23L7/101 ,  A23L11/00 ,  A23L11/33 ,  A23L29/06 ,  A23L33/17 ,  A23L33/185 ,  A61K38/00 ,  C12C5/004 ,  C12G2200/15 ,  C12N9/16 ,  C12N15/8243 ,  C12N15/8245 ,  C12Y301/03026

摘要： A purified recombinant phytase enzyme derived from Escherichia coli B. The enzyme has a molecular weight of about 47.1 kilodaltons and has phytase activity (SEQ ID NO:2). The enzyme can be produced from native or recombinant host cells and can be used to aid in the digestion of phytate where desired. In particular, the phytase of the present invention can be used in foodstuffs to improve the feeding value of phytate rich ingredients.