公开(公告)号：US20130011840A1

公开(公告)日：2013-01-10

申请号：US13612485

申请日：2012-09-12

申请人： Caifu Chen ,  Dana Ridzon ,  Zhaohui Zhou ,  Kai Qin Lao ,  Neil A. Straus

发明人： Caifu Chen ,  Dana Ridzon ,  Zhaohui Zhou ,  Kai Qin Lao ,  Neil A. Straus

IPC分类号： C12Q1/68

CPC分类号： C12Q1/6876 ,  C07H21/04 ,  C12N9/00 ,  C12P19/34 ,  C12Q1/686 ,  C12Q2525/301 ,  C12Q2600/16 ,  C12Q2600/178

摘要： The present invention is directed to methods, reagents, kits, and compositions for identifying and quantifying target polynucleotide sequences. A linker probe comprising a 3′ target specific portion, a loop, and a stem is hybridized to a target polynucleotide and extended to form a reaction product that includes a reverse primer portion and the stem nucleotides. A detector probe, a specific forward primer, and a reverse primer can be employed in an amplification reaction wherein the detector probe can detect the amplified target polynucleotide based on the stem nucleotides introduced by the linker probe. In some embodiments a plurality of short miRNAs are queried with a plurality of linker probes, wherein the linker probes all comprise a universal reverse primer portion a different 3′ target specific portion and different stems. The plurality of queried miRNAs can then be decoded in a plurality of amplification reactions.

摘要翻译： 本发明涉及用于鉴定和定量靶多核苷酸序列的方法，试剂，试剂盒和组合物。 包含3'靶特异性部分，环和茎的接头探针与目标多核苷酸杂交并延伸以形成包含反向引物部分和茎核苷酸的反应产物。 可以在扩增反应中使用检测器探针，特异性正向引物和反向引物，其中检测器探针可以基于由接头探针引入的干核苷酸检测扩增的靶多核苷酸。 在一些实施方案中，使用多个接头探针查询多个短miRNA，其中所述连接物探针都包含不同3'靶特异性部分和不同茎的通用反向引物部分。 然后可以在多个扩增反应中解码多个查询的miRNA。

公开(公告)号：US20100112573A1

公开(公告)日：2010-05-06

申请号：US12543466

申请日：2009-08-18

申请人： Caifu Chen ,  Dana Ridzon ,  Zhaohui Zhou ,  Kai Qin Lao ,  Neil A. Straus

发明人： Caifu Chen ,  Dana Ridzon ,  Zhaohui Zhou ,  Kai Qin Lao ,  Neil A. Straus

IPC分类号： C12Q1/68

CPC分类号： C12Q1/6876 ,  C07H21/04 ,  C12N9/00 ,  C12P19/34 ,  C12Q1/686 ,  C12Q2525/301 ,  C12Q2600/16 ,  C12Q2600/178

摘要： The present invention is directed to methods, reagents, kits, and compositions for identifying and quantifying target polynucleotide sequences. A linker probe comprising a 3′ target specific portion, a loop, and a stem is hybridized to a target polynucleotide and extended to form a reaction product that includes a reverse primer portion and the stem nucleotides. A detector probe, a specific forward primer, and a reverse primer can be employed in an amplification reaction wherein the detector probe can detect the amplified target polynucleotide based on the stem nucleotides introduced by the linker probe. In some embodiments a plurality of short miRNAs are queried with a plurality of linker probes, wherein the linker probes all comprise a universal reverse primer portion a different 3′ target specific portion and different stems. The plurality of queried miRNAs can then be decoded in a plurality of amplification reactions.

摘要翻译： 本发明涉及用于鉴定和定量靶多核苷酸序列的方法，试剂，试剂盒和组合物。 包含3'靶特异性部分，环和茎的接头探针与目标多核苷酸杂交并延伸以形成包含反向引物部分和茎核苷酸的反应产物。 可以在扩增反应中使用检测器探针，特异性正向引物和反向引物，其中检测器探针可以基于由接头探针引入的干核苷酸检测扩增的靶多核苷酸。 在一些实施方案中，使用多个接头探针查询多个短miRNA，其中所述连接物探针都包含不同3'靶特异性部分和不同茎的通用反向引物部分。 然后可以在多个扩增反应中解码多个查询的miRNA。

公开(公告)号：US09068222B2

公开(公告)日：2015-06-30

申请号：US12543466

申请日：2009-08-18

申请人： Caifu Chen ,  Dana Ridzon ,  Zhaohui Zhou ,  Kai Qin Lao ,  Neil A. Straus

发明人： Caifu Chen ,  Dana Ridzon ,  Zhaohui Zhou ,  Kai Qin Lao ,  Neil A. Straus

IPC分类号： C07H21/04 ,  C12N9/00 ,  C12Q1/68

CPC分类号： C12Q1/6876 ,  C07H21/04 ,  C12N9/00 ,  C12P19/34 ,  C12Q1/686 ,  C12Q2525/301 ,  C12Q2600/16 ,  C12Q2600/178

摘要： The present invention is directed to methods, reagents, kits, and compositions for identifying and quantifying target polynucleotide sequences. A linker probe comprising a 3′ target specific portion, a loop, and a stem is hybridized to a target polynucleotide and extended to form a reaction product that includes a reverse primer portion and the stem nucleotides. A detector probe, a specific forward primer, and a reverse primer can be employed in an amplification reaction wherein the detector probe can detect the amplified target polynucleotide based on the stem nucleotides introduced by the linker probe. In some embodiments a plurality of short miRNAs are queried with a plurality of linker probes, wherein the linker probes all comprise a universal reverse primer portion a different 3′ target specific portion and different stems. The plurality of queried miRNAs can then be decoded in a plurality of amplification reactions.

摘要翻译： 本发明涉及用于鉴定和定量靶多核苷酸序列的方法，试剂，试剂盒和组合物。 包含3'靶特异性部分，环和茎的接头探针与目标多核苷酸杂交并延伸以形成包含反向引物部分和茎核苷酸的反应产物。 可以在扩增反应中使用检测器探针，特异性正向引物和反向引物，其中检测器探针可以基于由接头探针引入的干核苷酸检测扩增的靶多核苷酸。 在一些实施方案中，使用多个接头探针查询多个短miRNA，其中所述连接物探针都包含不同3'靶特异性部分和不同茎的通用反向引物部分。 然后可以在多个扩增反应中解码多个查询的miRNA。

公开(公告)号：US07575863B2

公开(公告)日：2009-08-18

申请号：US10947460

申请日：2004-09-21

申请人： Caifu Chen ,  Dana Ridzon ,  Zhaohui Zhou ,  Kai Qin Lao ,  Neil A. Straus

发明人： Caifu Chen ,  Dana Ridzon ,  Zhaohui Zhou ,  Kai Qin Lao ,  Neil A. Straus

IPC分类号： C12Q1/68 ,  C12P19/34

CPC分类号： C12Q1/6876 ,  C07H21/04 ,  C12N9/00 ,  C12P19/34 ,  C12Q1/686 ,  C12Q2525/301 ,  C12Q2600/16 ,  C12Q2600/178

摘要： The present invention is directed to methods, reagents, kits, and compositions for identifying and quantifying target polynucleotide sequences. A linker probe comprising a 3′ target specific portion, a loop, and a stem is hybridized to a target polynucleotide and extended to form a reaction product that includes a reverse primer portion and the stem nucleotides. A detector probe, a specific forward primer, and a reverse primer can be employed in an amplification reaction wherein the detector probe can detect the amplified target polynucleotide based on the stem nucleotides introduced by the linker probe. In some embodiments a plurality of short miRNAs are queried with a plurality of linker probes, wherein the linker probes all comprise a universal reverse primer portion a different 3′ target specific portion and different stems. The plurality of queried miRNAs can then be decoded in a plurality of amplification reactions.

公开(公告)号：US08993240B2

公开(公告)日：2015-03-31

申请号：US12504633

申请日：2009-07-16

申请人： Kai Qin Lao ,  Neil A. Straus ,  Kenneth J. Livak

发明人： Kai Qin Lao ,  Neil A. Straus ,  Kenneth J. Livak

IPC分类号： C12Q1/68 ,  C12P19/34

CPC分类号： C12Q1/6876 ,  C07H21/02 ,  C07H21/04 ,  C12N9/00 ,  C12Q1/6844 ,  C12Q1/6848 ,  C12Q1/686 ,  C12Q2549/101 ,  C12Q2533/101 ,  C12Q2525/301

摘要： The present teachings provide methods, compositions, and kits for performing primer extension reactions. In some embodiments, a reverse transcription reaction is performed on a target polynucleotide with a hot start primer comprising a blunt-ended self-complementary stem, and a loop, and extension products form at high temperatures but reduce extension product formation at low temperatures.

摘要翻译： 本教导提供了用于进行引物延伸反应的方法，组合物和试剂盒。 在一些实施方案中，对靶多核苷酸进行逆转录反应，所述靶多核苷酸具有包含平端自补偿干杆的热起始引物，并且循环和延伸产物在高温下形成，但是在低温下减少延伸产物形成。

公开(公告)号：US20100112581A1

公开(公告)日：2010-05-06

申请号：US12604792

申请日：2009-10-23

申请人： Kai Qin Lao ,  Neil A. Straus ,  John W. Burns

发明人： Kai Qin Lao ,  Neil A. Straus ,  John W. Burns

IPC分类号： C12Q1/68 ,  C12P19/34

CPC分类号： C12Q1/6855 ,  C12Q1/6851 ,  C12Q2535/138 ,  C12Q2525/121 ,  C12Q2525/301

摘要： The present teachings are generally directed to methods for normalizing at least one species of small nucleic acid that is present in a population of small nucleic acid species, wherein the relative concentration of at least one small nucleic acid species is substantially greater than the relative concentration of at least one other small nucleic acid species in the population. At least one small nucleic acid species is normalized using a multiplicity of primers comprising degenerate sequences. In some embodiments, a small nucleic acid species is identified by inserting at least part of an extension product from a normalized population into a vector and subsequently sequencing the insert. In some embodiments, a small nucleic acid species is identified by determining the sequence of at least part of an extension product.

摘要翻译： 本教导通常涉及用于归一化存在于小核酸物种群中的至少一种小核酸的方法，其中至少一种小核酸物质的相对浓度基本上大于 群体中至少一种其他小核酸物种。 使用包含简并序列的多个引物将至少一个小核酸物质归一化。 在一些实施方案中，通过将来自标准化群体的至少部分延伸产物插入载体并随后对插入物进行测序来鉴定小核酸物种。 在一些实施方案中，通过确定延伸产物的至少一部分的序列来鉴定小核酸物种。

公开(公告)号：US20090325183A1

公开(公告)日：2009-12-31

申请号：US12553928

申请日：2009-09-03

申请人： Kai Qin Lao ,  Neil A. Straus

发明人： Kai Qin Lao ,  Neil A. Straus

IPC分类号： C12Q1/68

CPC分类号： C12Q1/6872 ,  C12Q2537/143 ,  C12Q2535/101

摘要： The present teachings provide methods and compositions for sequencing one or more target nucleic acids. High levels of multiplexing are provided by the use of an emulsion PCR comprising primer-immobilized beads. The resulting reaction products can be sequenced by any of a variety of mobility-dependent analytical techniques, such as mass spectrometry. In some embodiments, a first collection of amplification products on a first collection of beads are transferred to a second collection of beads. In some embodiments, a first collection of amplification products on a first collection of beads is amplified in a rolling circle amplification reaction. The present teachings also provide compositions, kits, and devices for performing and sequencing the products of the emulsion amplification reactions as described herein.

摘要翻译： 本教导提供了用于测序一种或多种靶核酸的方法和组合物。 通过使用包含引物固定的珠粒的乳液PCR提供了高水平的复用。 所得到的反应产物可以通过各种迁移依赖性分析技术如质谱法中的任何一种进行测序。 在一些实施方案中，将第一集合珠上的扩增产物的第一集合转移到第二集合珠。 在一些实施方案中，在滚动圆扩增反应中扩增第一集合珠上的扩增产物的第一集合。 本教导还提供用于对本文所述的乳液扩增反应的产物进行和测序的组合物，试剂盒和装置。

公开(公告)号：US20080145898A1

公开(公告)日：2008-06-19

申请号：US11956100

申请日：2007-12-13

申请人： Kai Qin Lao ,  Neil A. Straus

发明人： Kai Qin Lao ,  Neil A. Straus

IPC分类号： C12P19/34

CPC分类号： C12Q1/6872 ,  C12Q2537/143 ,  C12Q2535/101

摘要： The present teachings provide methods and compositions for sequencing one or more target nucleic acids. High levels of multiplexing are provided by the use of an emulsion PCR comprising primer-immobilized beads. The resulting reaction products can be sequenced by any of a variety of mobility-dependent analytical techniques, such as mass spectrometry. In some embodiments, a first collection of amplification products on a first collection of beads are transferred to a second collection of beads. In some embodiments, a first collection of amplification products on a first collection of beads is amplified in a rolling circle amplification reaction. The present teachings also provide compositions, kits, and devices for performing and sequencing the products of the emulsion amplification reactions as described herein.

摘要翻译： 本教导提供了用于测序一种或多种靶核酸的方法和组合物。 通过使用包含引物固定的珠粒的乳液PCR提供了高水平的复用。 所得到的反应产物可以通过各种迁移依赖性分析技术如质谱法中的任何一种进行测序。 在一些实施方案中，将第一集合珠上的扩增产物的第一集合转移到第二集合珠。 在一些实施方案中，在滚动圆扩增反应中扩增第一集合珠上的扩增产物的第一集合。 本教导还提供用于对本文所述的乳液扩增反应的产物进行和测序的组合物，试剂盒和装置。

公开(公告)号：US20120064530A1

公开(公告)日：2012-03-15

申请号：US13175595

申请日：2011-07-01

申请人： Kai Qin Lao ,  Neil A. Straus

发明人： Kai Qin Lao ,  Neil A. Straus

IPC分类号： C12Q1/68 ,  C07H21/04

CPC分类号： C12Q1/6853 ,  C12Q1/6848 ,  C12Q1/6876 ,  C12Q2549/119 ,  C12Q2525/301 ,  C12Q2525/155

摘要： The present disclosure relates to the amplification of target nucleic acid sequences. This can be accomplished via the use of various primers. The use of these primers, as described herein, results in nucleic acid structures that can reduce the amplification of nonspecific hybridization events (such as primer dimerization) while allowing the amplification of the target nucleic acid sequences.

摘要翻译： 本公开涉及靶核酸序列的扩增。 这可以通过使用各种引物来实现。 如本文所述，使用这些引物导致可以减少非特异性杂交事件（例如引物二聚化）的扩增同时允许扩增靶核酸序列的核酸结构。

公开(公告)号：US07745122B2

公开(公告)日：2010-06-29

申请号：US11458089

申请日：2006-07-17

申请人： Kai Qin Lao ,  Neil A. Straus

发明人： Kai Qin Lao ,  Neil A. Straus

IPC分类号： C12Q1/68 ,  C12P19/34

CPC分类号： C12Q1/686 ,  C12Q1/6809 ,  C12Q1/6844 ,  C12Q1/6848 ,  C12Q1/6853 ,  C12Q2549/101 ,  C12Q2537/143 ,  C12Q2527/107 ,  C12Q2525/301 ,  C12Q2525/207 ,  C12Q2533/101

摘要： The present teachings provide methods, compositions, and kits for performing primer extension reactions on at least two target polynucleotides in the same reaction mixture. In some embodiments, a reverse transcription reaction is performed on a first target polynucleotide with a hot start primer comprising a self-complementary stem and a loop, and extension products form at high temperatures but extension products form less so at low temperatures since the self-complementary stem of the hot start primer prevents hybridization of the target specific region to the target. However, non-hot start primers with free target specific regions can hybridize to their corresponding targets at the low temperature and extension can happen at the low temperature.

摘要翻译： 本教导提供用于在相同反应混合物中的至少两个靶多核苷酸上进行引物延伸反应的方法，组合物和试剂盒。 在一些实施方案中，对具有包含自身互补茎和环的热起始底物的第一靶多核苷酸进行逆转录反应，并且在高温下形成延伸产物，但延伸产物在低温下形成较少， 热启动引物的互补干扰物可以防止目标特异性区域与靶标的杂交。 然而，具有游离目标特异性区域的非热启动引物可以在低温下与其相应的靶标杂交，并且延伸可以在低温下发生。