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    Circularized templates for sequencing
    3.
    发明授权
    Circularized templates for sequencing 有权

    公开(公告)号：US10752944B2

    公开(公告)日：2020-08-25

    申请号：US14342725

    申请日：2012-09-06

    申请人： Steven T. Brentano ,  Dmitry Lyakhov ,  Matthew C. Friedenberg ,  Anne-Laure Shapiro

    发明人： Steven T. Brentano ,  Dmitry Lyakhov ,  Matthew C. Friedenberg ,  Anne-Laure Shapiro

    IPC分类号： C12Q1/68 ,  C12Q1/6869 ,  C12Q1/6855 ,  C12Q1/6844

    摘要： The invention provides methods of forming a circular template for sequencing a target nucleic acid. The circular template is generated by amplification of a segment of the target nucleic acid with chimeric primers with complementary 5′ ends. The circular template has a single nick or gap providing a site for initiation of template-directed extension for sequence analysis. Sequencing of a single template generates reads of alternating segments of the same strand of the target nucleic spaced by primer segments. The different reads of the same strand of the target nucleic acid can be compiled to generate a consensus sequence. Because only one strand of the target nucleic acid is sequenced per reaction, the present method avoids errors introduced by unwittingly combining sequences of both strands of a heteroduplex PCR product. Because only one strand of the target nucleic acid is sequenced per reaction, the present method avoids errors introduced by unwittingly combining sequences of both strands of a heteroduplex PCR product.

    CIRCULARIZED TEMPLATES FOR SEQUENCING
    4.
    发明申请
    CIRCULARIZED TEMPLATES FOR SEQUENCING 审中-公开
    用于测序的循环模板

    公开(公告)号：US20140378318A1

    公开(公告)日：2014-12-25

    申请号：US14342725

    申请日：2012-09-06

    申请人： Steven T. Brentano ,  Dmitry Lyakhov ,  Matthew C. Friedenberg ,  Anne-Laure Shapiro

    发明人： Steven T. Brentano ,  Dmitry Lyakhov ,  Matthew C. Friedenberg ,  Anne-Laure Shapiro

    IPC分类号： C12Q1/68

    摘要： The invention provides methods of forming a circular template for sequencing a target nucleic acid. The circular template is generated by amplification of a segment of the target nucleic acid with chimeric primers with complementary 5′ ends. The circular template has a single nick or gap providing a site for initiation of template-directed extension for sequence analysis. Sequencing of a single template generates reads of alternating segments of the same strand of the target nucleic spaced by primer segments. The different reads of the same strand of the target nucleic acid can be compiled to generate a consensus sequence. Because only one strand of the target nucleic acid is sequenced per reaction, the present method avoids errors introduced by unwittingly combining sequences of both strands of a heteroduplex PCR product. Because only one strand of the target nucleic acid is sequenced per reaction, the present method avoids errors introduced by unwittingly combining sequences of both strands of a heteroduplex PCR product.

    摘要翻译： 本发明提供形成用于测序靶核酸的圆形模板的方法。 通过用具有互补的5'末端的嵌合引物扩增靶核酸的片段来产生圆形模板。 圆形模板具有单个切口或间隙，提供用于启动用于序列分析的模板指导扩展的位点。 单个模板的测序产生与引物区间隔的靶核心的相同链的交替片段的读数。 可以编译目标核酸的相同链的不同读数以产生共有序列。 因为每个反应只对靶核酸的一条链进行测序，所以本方法避免了通过不经意地组合异源双链PCR产物的两条链的序列引入的错误。 因为每个反应只对靶核酸的一条链进行测序，所以本方法避免了通过不经意地组合异源双链PCR产物的两条链的序列引入的错误。

    Methods and compositions for nucleic acid amplification
    5.
    发明授权
    Methods and compositions for nucleic acid amplification 有权
    用于核酸扩增的方法和组合物

    公开(公告)号：US08198027B2

    公开(公告)日：2012-06-12

    申请号：US11962072

    申请日：2007-12-20

    申请人： Steven T. Brentano ,  Dmitry Lyakhov ,  James D. Carlson ,  Norman C. Nelson ,  Lyle J. Arnold, Jr.

    发明人： Steven T. Brentano ,  Dmitry Lyakhov ,  James D. Carlson ,  Norman C. Nelson ,  Lyle J. Arnold, Jr.

    IPC分类号： C12Q1/68 ,  C12P19/34 ,  C07H21/04

    CPC分类号： C12Q1/6881 ,  C12Q1/6834 ,  C12Q1/6844 ,  C12Q1/6865 ,  C12Q2525/155 ,  C12Q2525/186 ,  C12Q2525/197 ,  C12Q2531/143 ,  C12Q2565/543

    摘要： Compositions that are used in nucleic acid amplification in vitro are disclosed, which include a target specific universal (TSU) promoter primer or promoter provider oligonucleotide that includes a target specific (TS) sequence that hybridizes specifically to a target sequence that is amplified and a universal (U) sequence that is introduced into the sequence that is amplified, by using a primer for the universal sequence. Methods of nucleic acid amplification in vitro are disclosed that use one or more TSU oligonucleotides to attached a U sequence to a target nucleic acid in a target capture step and then use a primer for a U sequence in subsequent amplification steps performed in substantially isothermal conditions to make amplification products that contain a U sequence that indicates the presence of the target nucleic acid in a sample.

    摘要翻译： 公开了用于体外核酸扩增的组合物，其包括目标特异性通用（TSU）启动子引物或启动子提供者寡核苷酸，其包括与扩增的靶序列特异性杂交的靶特异性（TS）序列和通用 （U）序列，通过使用通用序列的引物引入扩增的序列。 公开了体外核酸扩增方法，其中使用一种或多种TSU寡核苷酸将U序列连接到目标捕获步骤中的靶核酸，然后在基本上等温条件下进行的随后扩增步骤中使用U序列引物 制备含有指示样品中靶核酸存在的U序列的扩增产物。

    METHODS AND COMPOSITIONS FOR NUCLEIC ACID AMPLIFICATION
    7.
    发明申请
    METHODS AND COMPOSITIONS FOR NUCLEIC ACID AMPLIFICATION 有权
    用于核酸扩增的方法和组合物

    公开(公告)号：US20120178636A1

    公开(公告)日：2012-07-12

    申请号：US13380018

    申请日：2010-07-01

    申请人： Steven T. Brentano ,  Dmitry Lyakhov ,  James D. Carlson ,  Michael M. Becker ,  Norman C. Nelson ,  Lyle J. Arnold, JR.

    发明人： Steven T. Brentano ,  Dmitry Lyakhov ,  James D. Carlson ,  Michael M. Becker ,  Norman C. Nelson ,  Lyle J. Arnold, JR.

    IPC分类号： C40B30/00

    CPC分类号： C12Q1/6865 ,  C12Q1/6844 ,  C12Q1/6848 ,  C12Q1/6853 ,  C12Q2549/119 ,  C12Q2537/143 ,  C12Q2531/143

    摘要： Compositions, reaction mixtures, and methods for performing an amplification reaction, including multiplex amplification reaction, wherein the method comprises using one or more amplification oligomer complexes comprising linked first and second amplification oligomer members. In one aspect, the amplification oligomer complex is hybridized to a target nucleic acid, the target nucleic acid with hybridized amplification oligomer complex is then captured, and other components are washed away. Target sequences of the target nucleic acids are pre-amplified to generate a first amplification product. The first amplification product is amplified in one or more secondary amplification reactions to generate second amplification products.

    摘要翻译： 用于进行扩增反应的组合物，反应混合物和方法，包括多重扩增反应，其中所述方法包括使用包含连接的第一和第二扩增寡聚体成员的一种或多种扩增寡聚体复合物。 一方面，将扩增寡聚物复合物与靶核酸杂交，然后捕获具有杂交扩增寡聚物复合物的靶核酸，并洗涤其它组分。 靶核酸的靶序列被预扩增以产生第一扩增产物。 第一扩增产物在一个或多个二级扩增反应中扩增以产生第二扩增产物。

    Methods and compositions for nucleic acid amplification
    8.
    发明授权
    Methods and compositions for nucleic acid amplification 有权
    用于核酸扩增的方法和组合物

    公开(公告)号：US09169512B2

    公开(公告)日：2015-10-27

    申请号：US13380018

    申请日：2010-07-01

    申请人： Steven T. Brentano ,  Dmitry Lyakhov ,  James D. Carlson ,  Michael M. Becker ,  Norman C. Nelson ,  Lyle J. Arnold, Jr.

    发明人： Steven T. Brentano ,  Dmitry Lyakhov ,  James D. Carlson ,  Michael M. Becker ,  Norman C. Nelson ,  Lyle J. Arnold, Jr.

    IPC分类号： C12P19/34 ,  C12Q1/68

    CPC分类号： C12Q1/6865 ,  C12Q1/6844 ,  C12Q1/6848 ,  C12Q1/6853 ,  C12Q2549/119 ,  C12Q2537/143 ,  C12Q2531/143

    摘要： Compositions, reaction mixtures, and methods for performing an amplification reaction, including multiplex amplification reaction, wherein the method comprises using one or more amplification oligomer complexes comprising linked first and second amplification oligomer members. In one aspect, the amplification oligomer complex is hybridized to a target nucleic acid, the target nucleic acid with hybridized amplification oligomer complex is then captured, and other components are washed away. Target sequences of the target nucleic acids are pre-amplified to generate a first amplification product. The first amplification product is amplified in one or more secondary amplification reactions to generate second amplification products.

    摘要翻译： 用于进行扩增反应的组合物，反应混合物和方法，包括多重扩增反应，其中所述方法包括使用包含连接的第一和第二扩增寡聚体成员的一种或多种扩增寡聚体复合物。 一方面，将扩增寡聚物复合物与靶核酸杂交，然后捕获具有杂交扩增寡聚物复合物的靶核酸，并洗涤其它组分。 靶核酸的靶序列被预扩增以产生第一扩增产物。 第一扩增产物在一个或多个二级扩增反应中扩增以产生第二扩增产物。

    Methods and compositions for nucleic acid amplification
    9.
    发明授权
    Methods and compositions for nucleic acid amplification 有权
    用于核酸扩增的方法和组合物

    公开(公告)号：US08642268B2

    公开(公告)日：2014-02-04

    申请号：US13460341

    申请日：2012-04-30

    申请人： Steven T. Brentano ,  Dmitry Lyakhov ,  James D. Carlson ,  Norman C. Nelson ,  Lyle J. Arnold, Jr. ,  Michael M. Becker

    发明人： Steven T. Brentano ,  Dmitry Lyakhov ,  James D. Carlson ,  Norman C. Nelson ,  Lyle J. Arnold, Jr. ,  Michael M. Becker

    IPC分类号： C12Q1/68 ,  C12P19/34 ,  C07H19/00

    CPC分类号： C12Q1/6881 ,  C12Q1/6834 ,  C12Q1/6844 ,  C12Q1/6865 ,  C12Q2525/155 ,  C12Q2525/186 ,  C12Q2525/197 ,  C12Q2531/143 ,  C12Q2565/543

    摘要： Compositions that are used in nucleic acid amplification in vitro are disclosed, which include a target specific universal (TSU) promoter primer or promoter provider oligonucleotide that includes a target specific (TS) sequence that hybridizes specifically to a target sequence that is amplified and a universal (U) sequence that is introduced into the sequence that is amplified, by using a primer for the universal sequence. Methods of nucleic acid amplification in vitro are disclosed that use one or more TSU oligonucleotides to attached a U sequence to a target nucleic acid in a target capture step and then use a primer for a U sequence in subsequent amplification steps performed in substantially isothermal conditions to make amplification products that contain a U sequence that indicates the presence of the target nucleic acid in a sample.

    摘要翻译： 公开了用于体外核酸扩增的组合物，其包括目标特异性通用（TSU）启动子引物或启动子提供者寡核苷酸，其包括与扩增的靶序列特异性杂交的靶特异性（TS）序列和通用 （U）序列，通过使用通用序列的引物引入扩增的序列。 公开了体外核酸扩增方法，其中使用一种或多种TSU寡核苷酸将U序列连接到目标捕获步骤中的靶核酸，然后在基本上等温条件下进行的随后扩增步骤中使用U序列引物 制备含有指示样品中靶核酸存在的U序列的扩增产物。

    Composition kits and methods for performing amplification reactions
    10.
    发明授权
    Composition kits and methods for performing amplification reactions 有权
    用于进行扩增反应的组合物试剂盒和方法

    公开(公告)号：US07696337B2

    公开(公告)日：2010-04-13

    申请号：US11574307

    申请日：2005-08-26

    申请人： Michael M. Becker ,  Steven T. Brentano ,  Daniel P. Kolk ,  Wai-Chung Lam ,  Kristin W. Livezey ,  Norman C. Nelson ,  Astrid R. W. Schroder ,  Gary P. Schroth

    发明人： Michael M. Becker ,  Steven T. Brentano ,  Daniel P. Kolk ,  Wai-Chung Lam ,  Kristin W. Livezey ,  Norman C. Nelson ,  Astrid R. W. Schroder ,  Gary P. Schroth

    IPC分类号： C07H21/04 ,  C12Q1/68

    CPC分类号： C12P19/34 ,  C12Q1/6865 ,  C12Q2537/163 ,  C12Q2521/325 ,  C12Q2521/107 ,  C12Q2525/186 ,  C12Q2533/101 ,  C12Q2525/143

    摘要： The present invention is directed to novel methods of synthesizing multiple copies of a target nucleic acid sequence which are autocatalytic (i.e., able to cycle automatically without the need to modify reaction conditions such as temperature, pH, or ionic strength and using the product of one cycle in the next one). In particular, the present invention discloses a method of nucleic acid amplification which is robust and efficient, while reducing the appearance of side products. The method uses only one primer, the “priming oligonucleotide,” a 3′blocked promoter oligonucleotide and optionally, a means for terminating a primer extension reaction, to amplify RNA or DNA molecules in vitro, while reducing or eliminating the formation of side products. The method of the present invention minimizes or eliminates the emergence of side products, thus providing a high level of specificity. Furthermore, the appearance of side products can complicate the analysis of the amplification reaction by various molecular detection techniques. The present invention minimizes or eliminates this problem, thus providing an enhanced level of sensitivity.

    摘要翻译： 本发明涉及合成目标核酸序列的多个拷贝的新型方法，所述目标核酸序列是自动催化的（即，能够自动循环而不需要修饰诸如温度，pH或离子强度的反应条件，并且使用一种 循环在下一个）。 特别地，本发明公开了一种鲁棒有效的核酸扩增方法，同时减少了副产物的外观。 该方法仅使用一种引物，即引物寡核苷酸，3'封闭的启动子寡核苷酸和任选的终止引物延伸反应的手段，以体外扩增RNA或DNA分子，同时减少或消除副产物的形成。 本发明的方法使副产物的出现最小化或消除，从而提供高水平的特异性。 此外，副产物的出现可能通过各种分子检测技术使扩增反应的分析复杂化。 本发明使这个问题最小化或消除了这一点，从而提供了增强的灵敏度。