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    Quantitative amplification and detection of small numbers of target polynucleotides
    1.
    发明申请
    Quantitative amplification and detection of small numbers of target polynucleotides 审中-公开
    定量扩增和检测少量目标多核苷酸

    公开(公告)号：US20050208539A1

    公开(公告)日：2005-09-22

    申请号：US11026924

    申请日：2004-12-30

    申请人： Charles Vann ,  Kai Lao ,  Mark Reed ,  Seth Stern ,  Dennis Lehto

    发明人： Charles Vann ,  Kai Lao ,  Mark Reed ,  Seth Stern ,  Dennis Lehto

    IPC分类号： B01L3/00 ,  B01L7/00 ,  C12M1/34 ,  C12Q1/68

    CPC分类号： B01L7/52 ,  B01L3/502707 ,  B01L3/502715 ,  B01L2300/0861 ,  B01L2300/18 ,  B01L2400/06 ,  C12Q1/686 ,  C12Q2527/125

    摘要： Devices, assemblies, systems and methods described herein enable detection of as few as a single copy of a target nucleic acid molecule. Polynucleotides copied from a single or a small number of target nucleotide(s) within regions near to an initial copying site may be detected by optical or other methods as disclosed herein. Devices, assemblies and systems may comprise probes and/or primer molecules. Systems comprising optical assemblies, thermal assemblies and reaction assemblies (having reaction chambers for amplification of target nucleic acid molecules) are provided in which used reaction assemblies may be replaced to provide reusable devices. Systems comprising analytical assemblies and detection assemblies are provided in which an assay cartridge having assay chambers may engage a thermal assembly for amplification of target nucleic acid molecules. These devices, systems and methods offer the advantages of detection of as few as a single copy of a target nucleic acid molecule.

    摘要翻译： 本文描述的装置，组件，系统和方法能够检测目标核酸分子的单拷贝数。 从初始复制部位附近的区域内单个或少数目标核苷酸复制的多核苷酸可以通过本文公开的光学或其它方法检测。 装置，组件和系统可以包括探针和/或引物分子。 提供了包括光学组件，热组件和反应组件（具有用于扩增靶核酸分子的反应室）的系统，其中可以更换所使用的反应组件以提供可重复使用的装置。 提供了包括分析组件和检测组件的系统，其中具有测定室的测定盒可以接合热组件以扩增靶核酸分子。 这些装置，系统和方法提供了检测目标核酸分子的单拷贝的优点。

    Detection of gene expression
    3.
    发明申请
    Detection of gene expression 审中-公开
    检测基因表达

    公开(公告)号：US20060141518A1

    公开(公告)日：2006-06-29

    申请号：US11320440

    申请日：2005-12-28

    申请人： Kai Lao ,  Mark Reed

    发明人： Kai Lao ,  Mark Reed

    IPC分类号： C12Q1/68 ,  C12P19/34

    CPC分类号： C12Q1/686 ,  C12Q2537/143 ,  C12Q2549/119 ,  C12Q2561/113

    摘要： Methods for detection of nucleic acids such as a cDNA copy of an mRNA are disclosed. The methods comprise using a PCR to form a preamplification product which comprises cDNA sequence as well as primer target sequences and a detection probe sequence, which are introduced by the forward and reverse primers. In a second PCR, preamplification product is amplified using universal primers which hybridize to the primer target sequences or their complements. Amplification can be detected using a detection probe that hybridizes to the detection probe sequence or its complement.

    摘要翻译： 公开了检测核酸例如mRNA的cDNA拷贝的方法。 所述方法包括使用PCR形成包含cDNA序列以及通过正向和反向引物引入的引物靶序列和检测探针序列的预扩增产物。 在第二次PCR中，使用与引物靶序列或其互补序列杂交的通用引物扩增前扩增产物。 可以使用与检测探针序列或其补体杂交的检测探针来检测扩增。

    Encoding and decoding reactions for determining target polynucleotides
    4.
    发明申请
    Encoding and decoding reactions for determining target polynucleotides 失效
    用于确定靶多核苷酸的编码和解码反应

    公开(公告)号：US20050272071A1

    公开(公告)日：2005-12-08

    申请号：US11090468

    申请日：2005-03-24

    申请人： Kai Lao ,  Mark Reed

    发明人： Kai Lao ,  Mark Reed

    IPC分类号： C12Q1/68

    CPC分类号： C12Q1/6869 ,  C12Q1/6851 ,  C12Q2525/191 ,  C12Q2525/185 ,  C12Q2521/501 ,  C12Q2525/155 ,  C12Q2521/313 ,  C12Q2537/143 ,  C12Q2525/161

    摘要： The present invention is directed to methods, reagents, and kits for detecting the presence or absence of (or quantifying) target polynucleotide sequences in at least one sample using encoding and decoding reactions. When a particular target polynucleotide is present in a sample, a reaction product is formed in the encoding reaction that includes addressable primer portions. At least one label and at least one address primer is employed in the decoding amplification reaction thereby providing a detectable signal value depending upon whether a sequence is present or absent. In some embodiments universal bases and reduced libraries of probes can be employed for highly multiplexed analysis of target polynucleotides.

    摘要翻译： 本发明涉及用于使用编码和解码反应检测至少一个样品中靶多核苷酸序列的存在或不存在（或定量）的方法，试剂和试剂盒。 当特定靶多核苷酸存在于样品中时，在编码反应中形成包含可寻址引物部分的反应产物。 在解码扩增反应中使用至少一个标签和至少一个地址引物，从而根据序列是否存在提供可检测的信号值。 在一些实施方案中，探针的通用碱基和还原型文库可用于靶多核苷酸的高度多重分析。

    Methods for normalizing and for identifying small nucleic acids
    6.
    发明申请
    Methods for normalizing and for identifying small nucleic acids 审中-公开
    归一化和鉴定小核酸的方法

    公开(公告)号：US20060223066A1

    公开(公告)日：2006-10-05

    申请号：US11093587

    申请日：2005-03-29

    申请人： Kai Lao ,  Neil Straus ,  John Burns

    发明人： Kai Lao ,  Neil Straus ,  John 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。

    Ligation assay
    8.
    发明申请
    Ligation assay 有权
    连接试验

    公开(公告)号：US20050064459A1

    公开(公告)日：2005-03-24

    申请号：US10866012

    申请日：2004-06-10

    申请人： Kai Lao

    发明人： Kai Lao

    IPC分类号： C12Q1/68

    CPC分类号： C12Q1/682 ,  C12Q1/6827 ,  C12Q2561/125 ,  C12Q2531/113 ,  C12Q2525/155 ,  C12Q2525/125

    摘要： Disclosed are methods and kits for detecting or quantifying one or more target polynucleotide sequences in a sample. Embodiments of the invention employ a first ligation reaction, a subsequent optional amplification reaction, and a second ligation reaction. Embodiments of the invention combine the specificity of both hybridization and ligation reactions along with universal probe polynucleotide sequences to achieve specific and multiplexed detection of a plurality of target polynucleotide sequences.

    摘要翻译： 公开了用于检测或定量样品中一种或多种靶多核苷酸序列的方法和试剂盒。 本发明的实施方案采用第一连接反应，随后任选的扩增反应和第二连接反应。 本发明的实施方案将杂交和连接反应的特异性与通用探针多核苷酸序列结合，以实现多个靶多核苷酸序列的特异性和多重检测。

    MULTIPLEXED AMPLIFICATION OF SHORT NUCLEIC ACIDS
    9.
    发明申请
    MULTIPLEXED AMPLIFICATION OF SHORT NUCLEIC ACIDS 审中-公开
    短期核酸的多重放大

    公开(公告)号：US20070077570A1

    公开(公告)日：2007-04-05

    申请号：US11421449

    申请日：2006-05-31

    申请人： Kai Lao ,  Kenneth Livak ,  Neil Straus

    发明人： Kai Lao ,  Kenneth Livak ,  Neil Straus

    IPC分类号： C12Q1/68 ,  C12P19/34

    CPC分类号： C12N15/1065 ,  C12Q1/6851 ,  C12Q1/686 ,  C12Q2525/301 ,  C12Q2525/161 ,  C12Q2563/179 ,  C12Q2537/143 ,  C12Q2525/207

    摘要： The present teachings provide methods, compositions, and kits for reverse transcribing and amplifying small nucleic acids such as micro RNAs. High levels of multiplexing are provided by the use of a zip-coded stem-loop reverse transcription primer, along with a PCR-based pre-amplification reaction that comprises a zip-coded forward primer. Detector probes in downstream decoding PCRs can take advantage of the zip-code introduced by the stem-loop reverse transcription primer. In some embodiments, further amplification is achieved by cycling the reverse transcription reaction. The present teachings also provide compositions and kits useful for performing the reverse transcription and amplification reactions described herein.

    摘要翻译： 本教导提供用于逆转录和扩增小核酸如微RNA的方法，组合物和试剂盒。 通过使用zip编码的茎 - 环逆转录引物以及包含经翻译的正向引物的基于PCR的预扩增反应来提供高水平的复用。 下游解码PCR中的检测器探针可以利用茎环逆转录引物引入的zip码。 在一些实施方案中，通过循环逆转录反应来实现进一步的扩增。 本教导还提供可用于进行本文所述的逆转录和扩增反应的组合物和试剂盒。

    Pure miRNA Sample Preparation Method
    10.
    发明申请
    Pure miRNA Sample Preparation Method 审中-公开
    纯miRNA样品制备方法

    公开(公告)号：US20070059739A1

    公开(公告)日：2007-03-15

    申请号：US11458077

    申请日：2006-07-17

    申请人： Kai Lao

    发明人： Kai Lao

    IPC分类号： C12Q1/68 ,  C07H21/02 ,  C12P19/34 ,  C12N1/08

    CPC分类号： C12N15/1003 ,  C12N1/06 ,  C12N1/08 ,  C12N15/111 ,  C12N2310/14 ,  C12N2310/315 ,  C12N2310/321 ,  C12N2310/341 ,  C12N2310/346 ,  C12N2330/10 ,  C12N2330/30 ,  C12P19/34 ,  C12Q1/68 ,  C12N2310/3521

    摘要： The present teachings provide novel methods, compositions, and kits for analyzing mature micro RNAs (miRNAs). By taking advantage of the observation that most mature miRNAs in cells are tightly associated with RISCs, the present teachings provide approaches for studying mature miRNAs without the complications of additional nucleic acids. For example, in some embodiments the present teachings provide a method of purifying mature miRNAs comprising heating a sample to form a lysate, and, degrading the additional nucleic acids. The resulting mixture lacks the additional nucleic acids, and contains mature miRNAs associated with RISCs. Liberating the mature miRNAs from RISCs, for example by a protease, a detergent, and/or heat, can result in a pure collection of mature miRNAs.

    摘要翻译： 本教导提供用于分析成熟微RNA（miRNA）的新型方法，组合物和试剂盒。 通过利用细胞中大多数成熟miRNA与RISC紧密相关的观察，本教导提供了研究成熟miRNA而没有附加核酸并发症的方法。 例如，在一些实施方案中，本发明提供纯化成熟miRNA的方法，包括加热样品以形成裂解物，并降解其它核酸。 所得混合物缺乏额外的核酸，并且含有与RISC相关的成熟miRNA。 从RISC解离成熟的miRNA，例如通过蛋白酶，洗涤剂和/或加热，可导致成熟miRNA的纯收集。