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    Hairpin-mediated amplification method
    2.
    发明授权
    Hairpin-mediated amplification method 有权

    公开(公告)号：US10697006B2

    公开(公告)日：2020-06-30

    申请号：US15692215

    申请日：2017-08-31

    Applicant: Agilent Technologies, Inc.

    Inventor： Brian Jon Peter ,  Robert A. Ach

    IPC: C12P19/34 ,  C12Q1/6853 ,  C12Q1/6806

    Abstract: In some embodiments, the amplification method may comprise producing a reaction mix comprising: a nucleic acid sample, a polymerase, nucleotides, a forward primer that hybridizes to a sequence in the bottom strand of a fragment in the sample, and a reverse primer. The reverse primer has a hairpin structure comprising a loop, a stem and a 3′ overhang of at least 8 nucleotides, wherein the 3′ overhang hybridizes to a sequence in the top strand of the fragment. Subjecting the reaction mix at least two rounds of denaturation, renaturation and primer extension conditions results in extension the forward and reverse primers to produce an amplification product that contains: a double stranded region comprising a nick adjacent to the 5′ end of the reverse primer, and the loop of the first hairpin primer. Primer sets and kits for performing the methods are also provided.

    Method for Fragmenting Genomic DNA Using CAS9
    3.
    发明申请
    Method for Fragmenting Genomic DNA Using CAS9 审中-公开

    公开(公告)号：US20200172959A1

    公开(公告)日：2020-06-04

    申请号：US16748267

    申请日：2020-01-21

    Applicant: AGILENT TECHNOLOGIES, INC.

    Inventor： Brian Jon Peter ,  Robert A. Ach ,  Michael Walter ,  Bram Herman

    IPC: C12Q1/6806 ,  C12N9/22 ,  C12Q1/6869 ,  C12N15/10

    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.

    Method for fragmenting genomic DNA using CAS9
    4.
    发明授权
    Method for fragmenting genomic DNA using CAS9 有权

    公开(公告)号：US10577644B2

    公开(公告)日：2020-03-03

    申请号：US15339510

    申请日：2016-10-31

    Applicant: Agilent Technologies, Inc.

    Inventor： Brian Jon Peter ,  Robert A. Ach ,  Michael Walter ,  Bram Herman

    IPC: C12Q1/6806 ,  C12N15/11 ,  C12Q1/68 ,  C12P19/34 ,  C12N15/10 ,  C12Q1/6869 ,  C12N9/22

    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.

    METHOD FOR FRAGMENTING GENOMIC DNA USING CAS9
    7.
    发明申请
    METHOD FOR FRAGMENTING GENOMIC DNA USING CAS9 审中-公开
    使用CAS9分离基因组DNA的方法

    公开(公告)号：US20170044592A1

    公开(公告)日：2017-02-16

    申请号：US15339510

    申请日：2016-10-31

    Applicant: Agilent Technologies, Inc.

    Inventor： Brian Jon Peter ,  Robert A. Ach ,  Michael Walter ,  Bram Herman

    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。

    Polymerase idling method for single molecule DNA sequencing
    8.
    发明授权
    Polymerase idling method for single molecule DNA sequencing 有权

    公开(公告)号：US10590479B2

    公开(公告)日：2020-03-17

    申请号：US15353592

    申请日：2016-11-16

    Applicant: Agilent Technologies, Inc.

    Inventor： Brian Jon Peter ,  Joel Myerson

    IPC: C12Q1/68 ,  C12Q1/6869 ,  C12Q1/6816 ,  C12Q1/6834

    Abstract: A method for sequencing a nucleic acid is provided. In certain embodiments the method comprises obtaining a duplex comprising a nucleic acid and a primer, wherein the primer has a nuclease resistant 3′ end, combining the duplex with a chain terminator nucleotide and a proof-reading polymerase to produce a reaction in which the polymerase idles on the added chain terminator nucleotide, identifying the chain terminator nucleotide added to the end of the primer; and adding a nuclease-resistant nucleotide to the end of the primer after the polymerase has idled on and removed the added chain terminator nucleotide, thereby producing a duplex comprising the template and an extended primer that has a nuclease resistant 3′ end.

    NUCLEIC ACID ENRICHMENT USING CAS9
    10.
    发明申请
    NUCLEIC ACID ENRICHMENT USING CAS9 审中-公开

    公开(公告)号：US20170107560A1

    公开(公告)日：2017-04-20

    申请号：US15335139

    申请日：2016-10-26

    Applicant: Agilent Technologies, Inc.

    Inventor： Brian Jon Peter ,  Robert A. Ach

    IPC: C12Q1/68 ,  C12N9/22

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

    Abstract: A method of enriching for a fragment of a genome, as well as corresponding compositions and kits, are provided. In certain embodiments, the method comprises: (a) contacting a sample comprising fragmented DNA with a Cas9-gRNA complex comprising mutant Cas9 protein that has inactivated nuclease activity and a Cas9-associated guide RNA that is complementary to a site in the DNA, to produce a Cas9-fragment complex that comprises a fragment of the fragmented DNA; and (b) isolating the complex. In addition, other methods and compositions for Cas9/CRISPR-mediated nucleic acid manipulation are also provided.

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