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    Gene site saturation mutagenesis
    92.
    发明申请
    Gene site saturation mutagenesis 审中-公开
    基因位点饱和诱变

    公开(公告)号：US20090130718A1

    公开(公告)日：2009-05-21

    申请号：US11285302

    申请日：2005-11-22

    申请人： Jay M. Short

    发明人： Jay M. Short

    IPC分类号： C12P19/34

    CPC分类号： C07K14/445 ,  A61K39/00 ,  A61K2039/53 ,  C07K16/00 ,  C07K16/005 ,  C07K2317/565 ,  C12N9/00 ,  C12N9/14 ,  C12N9/16 ,  C12N15/102 ,  C12N15/1027 ,  C12N15/1034

    摘要： A method for producing progeny polynucleotides and polypeptides by Gene Site Saturation Mutagenesis (GSSM). The method provides a set of degenerate primers corresponding to codons of a template polynucleotide, and performs polymerase elongation to produce progeny polynucleotides, which contain sequences corresponding to the degenerate primers. The progeny polynucleotides can be expressed and screened for directed evolution.

    摘要翻译： 通过基因位点饱和诱变（GSSM）产生后代多核苷酸和多肽的方法。 该方法提供了一组对应于模板多核苷酸的密码子的简并引物，并进行聚合酶延伸以产生含有对应于简并引物的序列的后代多核苷酸。 可以表达和筛选后代多核苷酸用于定向进化。

    Combinatorial screening of mixed populations of organisms
    98.
    发明授权
    Combinatorial screening of mixed populations of organisms 失效

    公开(公告)号：US07018793B1

    公开(公告)日：2006-03-28

    申请号：US09663620

    申请日：2000-09-15

    申请人： Jay M. Short

    发明人： Jay M. Short

    IPC分类号： C12Q1/68

    CPC分类号： C12N9/00 ,  C12N9/16 ,  C12N9/2445 ,  C12N15/102 ,  C12N15/1027 ,  C12N15/1034 ,  C12N15/1093 ,  C12Y302/01021

    摘要： Provided is a method of screening gene libraries derived from a mixed population of organisms for a bioactivity or biomolecule of interest. The mixed population of organisms can be a cultured population or an uncultured population from, for example, the environment. Also provided are methods of screening isolates or enriched populations of organisms, which isolates include a population that is spatially, temporally, or hierarchical, for example, of a particular species, genus, family, or class of organisms. Identified clones containing a biomolecule or bioactivity of interest can be further variegated or the DNA contained in the clone can be variegated to create novel biomolecules or bioactivities of interest.

    Saturation mutageneis in directed evolution
    99.
    发明授权
    Saturation mutageneis in directed evolution 有权
    定向进化中的饱和诱变

    公开(公告)号：US06764835B2

    公开(公告)日：2004-07-20

    申请号：US10309587

    申请日：2002-12-04

    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 ,  C12N15/1058

    摘要： Disclosed is a rapid and facilitated method of producing from a parentlal template polynucleotide, a set of mutagenized progeny polynculeotides whereby at each original codon position there is produced at least one substitute codon encoding each of the 20 naturally encoded amino acids. Accordingly, there is also provided a method of producing from a parental template polypeptide, a set of mutagenized progeny polypeptides wherein each of the 20 naturally encoded amino acids is represented at each original amino acid position. The method provided is termed site-saturation mutagenesis, or simply saturation mutagenesis, and can be used in combination with other mutagenization processes, such as, for example, a process wherein two or more related polynucleotides are introduced into a suitable host cell such that a hybrid polynucleotide is generated by recombination and reductive reassortment. Also provided are vector and expression vehicles incuding such polynucleotides, polypeptides expressed by the hybrid polynucleotides and a method for screening for hybrid polypeptides.

    摘要翻译： 公开了一种由亲本模板多核苷酸，一组诱变的后代多核苷酸产生的快速和便利的方法，其中在每个原始密码子位置产生编码20个天然编码氨基酸中的每一个的至少一个替代密码子。 因此，还提供了从亲本模板多肽，一组诱变的后代多肽生产其中每个原始氨基酸位置上表示20个天然编码氨基酸的方法。 所提供的方法称为位点饱和诱变或简单的饱和诱变，并且可以与其它诱变过程组合使用，例如其中将两个或更多个相关多核苷酸引入合适的宿主细胞中的方法，使得 通过重组和还原重配产生杂交多核苷酸。 还提供了包含这种多核苷酸的载体和表达载体，由杂交多核苷酸表达的多肽和杂交多肽的筛选方法。

    End selection in directed evolution
    100.
    发明授权
    End selection in directed evolution 有权
    定向演化中的终极选择

    公开(公告)号：US06696275B2

    公开(公告)日：2004-02-24

    申请号：US09867262

    申请日：2001-05-29

    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。