公开(公告)号：US20180237818A1

公开(公告)日：2018-08-23

申请号：US15953784

申请日：2018-04-16

Applicant: Agilent Technologies, Inc.

Inventor： Jeffrey Robert Sampson ,  Derek Lee Lindstrom

IPC: C12P19/34 ,  C12N15/66 ,  C12N15/10 ,  C12N15/64

CPC classification number: C12P19/34 ,  C12N15/10 ,  C12N15/64 ,  C12N15/66

Abstract: Methods for making a synthetic nucleic acid which comprise: (a) identifying a conflicting nucleotide sequence in a target sequence; (b) inserting a masking sequence into the conflicting sequence to produce a disrupted target sequence, wherein: (i) the masking sequence comprises recognition sites for one or more Type IIS restriction endonucleases; and (ii) digestion of said disrupted target sequence by said one or more Type IIS restriction endonucleases followed by re-ligation reconstitutes the target sequence; (c) synthesizing a polynucleotide comprising the disrupted target sequence using polymerase chain assembly; and (d) removing the masking sequence from said polynucleotide by digesting said polynucleotide with said one or more Type IIS restriction endonucleases followed by re-ligation of the digestion product, thereby producing a polynucleotide comprising said target sequence.

公开(公告)号：US10689680B2

公开(公告)日：2020-06-23

申请号：US15953784

申请日：2018-04-16

Applicant: Agilent Technologies, Inc.

Inventor： Jeffrey Robert Sampson ,  Derek Lee Lindstrom

IPC: C12P19/34 ,  C12N15/10 ,  C12N15/64 ,  C12N15/66

Abstract: Methods for making a synthetic nucleic acid which comprise: (a) identifying a conflicting nucleotide sequence in a target sequence; (b) inserting a masking sequence into the conflicting sequence to produce a disrupted target sequence, wherein: (i) the masking sequence comprises recognition sites for one or more Type IIS restriction endonucleases; and (ii) digestion of said disrupted target sequence by said one or more Type IIS restriction endonucleases followed by re-ligation reconstitutes the target sequence; (c) synthesizing a polynucleotide comprising the disrupted target sequence using polymerase chain assembly; and (d) removing the masking sequence from said polynucleotide by digesting said polynucleotide with said one or more Type IIS restriction endonucleases followed by re-ligation of the digestion product, thereby producing a polynucleotide comprising said target sequence.

公开(公告)号：US20150010953A1

公开(公告)日：2015-01-08

申请号：US13935201

申请日：2013-07-03

Applicant: AGILENT TECHNOLOGIES, INC.

Inventor： Derek Lee Lindstrom ,  Jeffrey R. Sampson ,  Daniel E. Ryan

IPC: C12N15/10 ,  C12P19/34

CPC classification number: C12N15/1006 ,  C12N15/1003 ,  C12P19/34 ,  C12Q2521/514 ,  C12Q2525/301

Abstract: Provided herein is a method for producing a population of oligonucleotides that has reduced synthesis errors. In certain embodiments, the method comprises: a) obtaining an initial population of hairpin oligonucleotide molecules that each comprise a double-stranded stem region and a loop region; b) contacting the double-stranded region of the hairpin oligonucleotide molecules with a mismatch binding protein; and c) eliminating any molecules that bind to the mismatch binding protein, thereby producing a population of oligonucleotides that has reduced synthesis errors. A kit and a composition for performing the method are also provided.

Abstract translation: 本文提供了一种减少合成错误的寡核苷酸群体的制备方法。 在某些实施方案中，所述方法包括：a）获得每个包含双链茎区和环区的发夹寡核苷酸分子的初始群; b）使发夹寡核苷酸分子的双链区域与错配结合蛋白接触; 和c）消除与错配结合蛋白结合的任何分子，从而产生具有降低的合成错误的寡核苷酸群。 还提供了用于执行该方法的试剂盒和组合物。

公开(公告)号：US20150344927A1

公开(公告)日：2015-12-03

申请号：US14649111

申请日：2013-11-05

Applicant: AGILENT TECHNOLOGIES, INC.

Inventor： Jeffrey Robert Sampson ,  Derek Lee Lindstrom

IPC: C12P19/34 ,  C12N15/64

CPC classification number: C12P19/34 ,  C12N15/10 ,  C12N15/64 ,  C12N15/66

Abstract: Provided herein is a method comprising: producing a first complex by annealing: a first nucleic acid comprising, in order, a first unique sequence, a first central sequence and a second unique sequence; and a second nucleic acid comprising, in order, said second unique sequence, a second central sequence and a third unique sequence; wherein the first, second and third unique sequences do not hybridize with each other; subjecting said first complex to multiple rounds of primer extension to extend the first and second nucleic acids using each other as a template, thereby producing a first product molecule that contains, in order, the first unique sequence, the first central sequence, the second unique sequence, the second central sequence and the third unique sequence; and circularizing said first product molecule by intramolecularly ligating the ends of said product molecule together. Kits and compositions relating to the method are also provided.

Abstract translation: 本文提供的方法包括：通过退火产生第一配合物：第一核酸，依次包含第一唯一序列，第一中心序列和第二唯一序列; 和第二核酸，依次包含所述第二唯一序列，第二中心序列和第三唯一序列; 其中所述第一，第二和第三独特序列彼此不杂交; 使所述第一复合物经受多轮引物延伸以使第一和第二核酸彼此作为模板，从而产生第一产物分子，依次包含第一独特序列，第一中心序列，第二独特序列 序列，第二中心序列和第三个唯一序列; 并通过将所述产物分子的末端分子内连接在一起使所述第一产物分子环化。 还提供了与该方法相关的试剂盒和组合物。

公开(公告)号：US20140357523A1

公开(公告)日：2014-12-04

申请号：US14290896

申请日：2014-05-29

Applicant: Agilent Technologies, Inc.

Inventor： Gusti Zeiner ,  Derek Lee Lindstrom ,  Brian Jon Peter ,  Robert A. Ach

IPC: C12Q1/68

CPC classification number: C12Q1/6806 ,  C12N9/22 ,  C12N15/1003 ,  C12N15/1034 ,  C12Q1/6869 ,  C12Y301/00 ,  Y10T436/143333

Abstract: A method for fragmenting a genome is provided. In certain embodiments, the method comprises: (a) combining a genomic sample containing genomic DNA with a plurality of Cas9-gRNA complexes, wherein the Cas9-gRNA complexes comprise a Cas9 protein and a set of at least 10 Cas9-associated guide RNAs that are complementary to different, pre-defined, sites in a genome, to produce a reaction mixture; and (b) incubating the reaction mixture to produce at least 5 fragments of the genomic DNA. Also provided is a composition comprising at least 100 Cas9-associated guide RNAs that are each complementary to a different, pre-defined, site in a genome. Kits for performing the method are also provided. In addition, other methods, compositions and kits for manipulating nucleic acids are also provided.

Abstract translation: 提供了一种分割基因组的方法。 在某些实施方案中，所述方法包括：（a）将含有基因组DNA的基因组样品与多个Cas9-gRNA复合物组合，其中所述Cas9-gRNA复合物包含Cas9蛋白和至少10个Cas9相关导向RNA的集合，其中 与基因组中不同的，预定义的位点互补，以产生反应混合物; 和（b）孵育反应混合物以产生至少5个基因组DNA片段。 还提供了包含至少100种与基因组中不同的，预定义的位点互补的Cas9相关导向RNA的组合物。 还提供了用于执行该方法的套件。 此外，还提供用于操作核酸的其它方法，组合物和试剂盒。

公开(公告)号：US10421957B2

公开(公告)日：2019-09-24

申请号：US14320431

申请日：2014-06-30

Applicant: Agilent Technologies, Inc.

Inventor： Derek Lee Lindstrom

IPC: C12Q1/68 ,  C12N15/10

Abstract: This disclosure provides, among other things, a method of combining nucleic acid fragments, comprising: (a) providing two double-stranded DNA molecules with a common sequence, wherein the common sequence is at the end of each molecule; (b) nicking one strand in the common sequence of both molecules at a respective nicked site; (c) moderately denaturing both molecules to remove a single-stranded fragment from the nicked site to one end of each molecule, wherein the single-stranded fragment includes the common sequence in part or in whole, resulting in an overhanging sequence in each molecule, and the overhanging sequences in both molecules are complementary to each other; (d) allowing the overhanging sequences of both molecules to anneal to each other, and ligating the molecules. Alternative ways for performing the method are also provided.

公开(公告)号：US09976162B2

公开(公告)日：2018-05-22

申请号：US14649111

申请日：2013-11-05

Applicant: Agilent Technologies, Inc.

Inventor： Jeffrey Robert Sampson ,  Derek Lee Lindstrom

IPC: C12P19/34 ,  C12N15/64 ,  C12N15/10 ,  C12N15/66

CPC classification number: C12P19/34 ,  C12N15/10 ,  C12N15/64 ,  C12N15/66

Abstract: Provided herein is a method comprising: producing a first complex by annealing: a first nucleic acid comprising, in order, a first unique sequence, a first central sequence and a second unique sequence; and a second nucleic acid comprising, in order, said second unique sequence, a second central sequence and a third unique sequence; wherein the first, second and third unique sequences do not hybridize with each other; subjecting said first complex to multiple rounds of primer extension to extend the first and second nucleic acids using each other as a template, thereby producing a first product molecule that contains, in order, the first unique sequence, the first central sequence, the second unique sequence, the second central sequence and the third unique sequence; and circularizing said first product molecule by intramolecularly ligating the ends of said product molecule together. Kits and compositions relating to the method are also provided.

公开(公告)号：US09873907B2

公开(公告)日：2018-01-23

申请号：US14290896

申请日：2014-05-29

Applicant: Agilent Technologies, Inc.

Inventor： Gusti Zeiner ,  Derek Lee Lindstrom ,  Brian Jon Peter ,  Robert A. Ach

IPC: C12Q1/68 ,  C12N15/10 ,  C12N9/22

CPC classification number: C12Q1/6806 ,  C12N9/22 ,  C12N15/1003 ,  C12N15/1034 ,  C12Q1/6869 ,  C12Y301/00 ,  Y10T436/143333

Abstract: A method for fragmenting a genome is provided. In certain embodiments, the method comprises: (a) combining a genomic sample containing genomic DNA with a plurality of Cas9-gRNA complexes, wherein the Cas9-gRNA complexes comprise a Cas9 protein and a set of at least 10 Cas9-associated guide RNAs that are complementary to different, pre-defined, sites in a genome, to produce a reaction mixture; and (b) incubating the reaction mixture to produce at least 5 fragments of the genomic DNA. Also provided is a composition comprising at least 100 Cas9-associated guide RNAs that are each complementary to a different, pre-defined, site in a genome. Kits for performing the method are also provided. In addition, other methods, compositions and kits for manipulating nucleic acids are also provided.

公开(公告)号：US20150031089A1

公开(公告)日：2015-01-29

申请号：US14320431

申请日：2014-06-30

Applicant: AGILENT TECHNOLOGIES, INC.

Inventor： Derek Lee Lindstrom

IPC: C12N15/10

Abstract: This disclosure provides, among other things, a method of combining nucleic acid fragments, comprising: (a) providing two double-stranded DNA molecules with a common sequence, wherein the common sequence is at the end of each molecule; (b) nicking one strand in the common sequence of both molecules at a respective nicked site; (c) moderately denaturing both molecules to remove a single-stranded fragment from the nicked site to one end of each molecule, wherein the single-stranded fragment includes the common sequence in part or in whole, resulting in an overhanging sequence in each molecule, and the overhanging sequences in both molecules are complementary to each other; (d) allowing the overhanging sequences of both molecules to anneal to each other, and ligating the molecules. Alternative ways for performing the method are also provided.

Abstract translation: 本公开尤其提供了组合核酸片段的方法，其包括：（a）提供具有共同序列的两条双链DNA分子，其中所述共同序列位于每个分子的末端; （b）在两个分子的共同序列中在相应的切口位点处切割一条链; （c）中和地变性两个分子以从每个分子的一端除去单链片段，其中单链片段部分或全部包括共同序列，导致每个分子中的突出序列， 两个分子中的突出序列互补; （d）允许两个分子的悬垂序列彼此退火并连接分子。 还提供了执行该方法的替代方法。