METHOD FOR IDENTIFICATION AND ENUMERATION OF NUCLEIC ACID SEQUENCE, EXPRESSION, COPY, OR DNA METHYLATION CHANGES, USING COMBINED NUCLEASE, LIGASE, POLYMERASE, AND SEQUENCING REACTIONS
    1.
    发明申请
    METHOD FOR IDENTIFICATION AND ENUMERATION OF NUCLEIC ACID SEQUENCE, EXPRESSION, COPY, OR DNA METHYLATION CHANGES, USING COMBINED NUCLEASE, LIGASE, POLYMERASE, AND SEQUENCING REACTIONS 审中-公开
    用核酸核酸序列,表达,拷贝或DNA甲基化改变的鉴定和产生的方法,使用组合核酸酶,配体,聚合酶和序列反应

    公开(公告)号:WO2015188192A2

    公开(公告)日:2015-12-10

    申请号:PCT/US2015/034724

    申请日:2015-06-08

    Abstract: The present invention relates to a method for the highly specific, targeted capture of regions of human genomes and transcriptomes from the blood, i.e. from cell free circulating DNA, exosomes, microRNA, circulating tumor cells, or total blood cells, to allow for the highly sensitive detection of mutation, expression, copy number, translocation, alternative splicing, and methylation changes using combined nuclease, ligation, polymerase, and massively parallel sequencing reactions. The method generates a collection of different circular chimeric single- stranded nucleic acid constructs, suitable for sequencing on multiple platforms. In some embodiments, each construct of the collection comprised a first single stranded segment of original genomic DNA from a host organism and a second single stranded synthetic nucleic acid segment that is linked to the first single stranded segment and comprises a nucleotide sequence that is exogenous to the host organism. These chimeric constructs are suitable for identifying and enumerating mutations, copy changes, translocations, and methylation changes. In other embodiments, input mRNA, IncRNA, or miRNA is used to generate circular DNA products that reflect the presence and copy number of specific mRNA's, IncRNA's splice-site variants, translocations, and miRNA.

    Abstract translation: 本发明涉及一种用于高度特异性靶向捕获来自血液的人类基因组和转录组的区域的方法,即来自无细胞循环的DNA,外来体,微小RNA,循环肿瘤细胞或全血细胞,以允许高度的 使用组合核酸酶,连接,聚合酶和大规模平行测序反应敏感检测突变,表达,拷贝数,易位,选择性剪接和甲基化变化。 该方法产生适合于在多个平台上进行测序的不同的环状嵌合单链核酸构建体的集合。 在一些实施方案中,集合的每个构建体包含来自宿主生物体的原始基因组DNA的第一单链区段和连接到第一单链区段的第二单链合成核酸区段,并且包含外源至 宿主生物体。 这些嵌合构建体适用于鉴定和列举突变,拷贝变化,易位和甲基化变化。 在其他实施方案中,使用输入mRNA,IncRNA或miRNA来产生反映特异性mRNA's,IncRNA的剪接位点变体,易位和miRNA的存在和拷贝数的环状DNA产物。

    DETECTION OF TARGET NUCLEIC ACID SEQUENCES USING FLUORESCENCE RESONANCE ENERGY TRANSFER
    3.
    发明申请
    DETECTION OF TARGET NUCLEIC ACID SEQUENCES USING FLUORESCENCE RESONANCE ENERGY TRANSFER 审中-公开
    使用荧光共振能量转移检测目标核酸序列

    公开(公告)号:WO2009126678A2

    公开(公告)日:2009-10-15

    申请号:PCT/US2009/039855

    申请日:2009-04-08

    Abstract: The present invention is directed to a method for identifying one or more of a plurality of target nucleic acid molecules in a sample. This method includes providing a sample potentially containing one or more target nucleic acid molecules and a plurality of oligonucleotide probe sets. Each probe set is characterized by (a) a first oligonucleotide probe, having a target-specific portion and a tunable portion with an endcapped hairpin and (b) a second oligonucleotide probe having a target specific portion and a tunable portion. One of the first and second oligonucleotide probe has an acceptor group and the other of the first and second probe has a donor group. A ligase is provided and blended with the sample and the plurality of oligonucleotide probe sets to form a ligase detection reaction mixture. The mixture is subjected to one or more ligase detection reaction cycles with each cycle comprising a denaturation and hybridization treatment. During the denaturation treatment any hybridized oligonucleotides are separated from the target nucleic acid sequences, and, during the hybridization treatment, the set of oligonucleotide probes hybridize in a base-specific manner to their respective target nucleotide sequences, if present in the sample, and ligate to one another to form a ligation product. The ligation product contains the tunable portions, the endcapped hairpin, the target-specific portions, the acceptor group, and the donor group. The ligation products are subjected to conditions effective to permit hybridization of the tunable portions of the ligation product to one another to form an internally hybridized ligation product. The fluorescence resonance energy transfer (FRET) between the donor and acceptor groups of the internally hybridized ligation product is detected, thereby indicating the presence of a target nucleic acid molecule in the sample. An alternative embodiment of the present invention involves the use of an initial PCR amplification procedure with primers provided with universal tails. Finally, FRET detection can be carried out through the use of the above-described oligonucleotide probes without any ligation.

    Abstract translation: 本发明涉及用于鉴定样品中多个靶核酸分子中的一种或多种的方法。 该方法包括提供潜在地含有一个或多个靶核酸分子和多个寡核苷酸探针组的样品。 每个探针组的特征在于:(a)具有靶特异性部分的第一寡核苷酸探针和具有封端发夹的可调部分,和(b)具有目标特异部分和可调部分的第二寡核苷酸探针。 第一和第二寡核苷酸探针之一具有受体基团,第一和第二探针中的另一个具有供体基团。 提供连接酶并与样品和多个寡核苷酸探针组混合以形成连接酶检测反应混合物。 将混合物进行一个或多个连接酶检测反应循环,每个循环包含变性和杂交处理。 在变性处理期间,将任何杂交的寡核苷酸与靶核酸序列分离,并且在杂交处理期间,寡核苷酸探针组如果存在于样品中,则以碱基特异性方式与其各自的靶核苷酸序列杂交,并连接 彼此形成连接产物。 连接产物包含可调部分,封端的发夹,靶特异性部分,受体基团和供体基团。 连接产物经受有效的条件,允许连接产物的可调部分彼此杂交以形成内部杂交的连接产物。 检测内部杂交连接产物的供体和受体组之间的荧光共振能量转移(FRET),从而表明样品中存在靶核酸分子。 本发明的替代实施方案涉及使用具有通用尾部的引物的初始PCR扩增程序。 最后,可以通过使用上述寡核苷酸探针而不进行任何连接来进行FRET检测。

    METHOD FOR IDENTIFICATION AND QUANTIFICATION OF NUCLEIC ACID EXPRESSION, SPLICE VARIANT, TRANSLOCATION, COPY NUMBER, OR METHYLATION CHANGES
    4.
    发明申请
    METHOD FOR IDENTIFICATION AND QUANTIFICATION OF NUCLEIC ACID EXPRESSION, SPLICE VARIANT, TRANSLOCATION, COPY NUMBER, OR METHYLATION CHANGES 审中-公开
    鉴定和定量核酸表达,拼接变异,易位,拷贝数或甲基化变化的方法

    公开(公告)号:WO2016057832A3

    公开(公告)日:2016-04-14

    申请号:PCT/US2015/054759

    申请日:2015-10-08

    Abstract: The present invention relates to methods and devices for identifying and quantifying, including low abundance, nucleotide base mutations, insertions, deletions, translocations, splice variants, miRNA variants, alternative transcripts, alternative start sites, alternative coding sequences, alternative non-coding sequences, alternative splicings, exon insertions, exon deletions, intron insertions, or other rearrangement at the genome level and/or methylated nucleotide bases.

    Abstract translation: 本发明涉及用于鉴定和定量的方法和装置,包括低丰度核苷酸碱基突变,插入,缺失,易位,剪接变体,miRNA变体,替代转录物,替代起始位点,替代编码 序列,可选的非编码序列,可选剪接,外显子插入,外显子缺失,内含子插入或基因组水平的其他重排和/或甲基化的核苷酸碱基。

    METHOD FOR RELATIVE QUANTIFICATION OF CHANGES IN DNA METHYLATION, USING COMBINED NUCLEASE, LIGATION, AND POLYMERASE REACTIONS
    5.
    发明申请
    METHOD FOR RELATIVE QUANTIFICATION OF CHANGES IN DNA METHYLATION, USING COMBINED NUCLEASE, LIGATION, AND POLYMERASE REACTIONS 审中-公开
    使用组合核酸酶,引物和聚合酶反应相关量化DNA甲基化变化的方法

    公开(公告)号:WO2014160199A1

    公开(公告)日:2014-10-02

    申请号:PCT/US2014/026027

    申请日:2014-03-13

    Abstract: The present invention is directed to methods for identifying the presence of one or more methylated or unmethylated target nucleotide sequences in a sample that involve a nuclease-ligation reaction. In some embodiments, the ligation products formed in the nuclease-ligation process of the present invention are subsequently amplified using a polymerase chain reaction. The ligated product sequences or extension products thereof are detected, and the presence of one or more methylated or unmethylated target nucleotide sequences in the sample is identified based on the detection.

    Abstract translation: 本发明涉及用于鉴定涉及核酸酶连接反应的样品中一种或多种甲基化或非甲基化靶核苷酸序列的存在的方法。 在一些实施方案中,随后使用聚合酶链式反应扩增在本发明的核酸酶连接方法中形成的连接产物。 检测连接的产物序列或其延伸产物,并且基于检测鉴定样品中一个或多个甲基化或未甲基化的靶核苷酸序列的存在。

    COFLUORONS AND METHODS OF MAKING AND USING THEM
    6.
    发明申请
    COFLUORONS AND METHODS OF MAKING AND USING THEM 审中-公开
    COFLORONS AND METHODS OF MAKING AND USING THEM

    公开(公告)号:WO2012154213A1

    公开(公告)日:2012-11-15

    申请号:PCT/US2012/000198

    申请日:2012-04-09

    Abstract: The present invention is directed to method of using a collection of monomers capable of forming multimers as a fluorescence reporter in different applications such as ligand detection/screening, disease diagnosis, drug discovery or screening, fluorescent labeling and imaging, or other fluorescent methodologies. Each monomer in the collection includes one or more ligand elements useful for binding to a target molecule with a dissociation constant of less than 300 μΜ and a linker element connected to the ligand elements directly or indirectly through a connector. Association of linker elements of different combinations of monomers, with their ligand elements bound to the target molecule to form a multimer, will generate a unique fluorescent signature different from that produced by those monomers either alone or in association with each other in the absence of the target molecule, when subjected to electromagnetic excitement.

    Abstract translation: 本发明涉及在不同应用如配体检测/筛选,疾病诊断,药物发现或筛选,荧光标记和成像或其它荧光方法中使用能够形成多聚体的单体的集合的方法。 收集物中的每个单体包括一个或多个配体元件,其用于结合靶分子,解离常数小于300μ? 以及通过连接器直接或间接连接到配体元件的连接体元件。 不同单体组合的连接体元件与其与靶分子结合以形成多聚体的连接体元件的关联将产生独特的荧光特征,其不同于单独或彼此相关联的单体,不存在 目标分子,当受到电磁兴奋时。

    SILYL MONOMERS CAPABLE OF MULTIMERIZING IN AN AQUEOUS SOLUTION, AND METHODS OF USING SAME
    10.
    发明申请
    SILYL MONOMERS CAPABLE OF MULTIMERIZING IN AN AQUEOUS SOLUTION, AND METHODS OF USING SAME 审中-公开
    可在水溶液中多元化的SILYL单体及其使用方法

    公开(公告)号:WO2013058825A1

    公开(公告)日:2013-04-25

    申请号:PCT/US2012/032813

    申请日:2012-04-09

    CPC classification number: A61K31/695 A61K47/481 A61K47/55 C07F7/10

    Abstract: Described herein are silyl monomers capable of forming a biologically useful multimer when in contact with one, two, three or more other monomers in an aqueous media. Such multimer forming associations of monomers may be promoted by the proximal binding of the monomers to their target biomolecule(s). In one aspect, such monomers may be capable of binding to another monomer in an aqueous media (e.g. in vivo) to form a multimer, (e.g. a dimer). Contemplated monomers may include a ligand moiety, a linker element, and a connector element that joins the ligand moiety and the linker element. In an aqueous media, such contemplated monomers may join together via each linker element and may thus be capable of modulating one or more biomolecules substantially simultaneously, e.g., modulate two or more binding domains on a protein or on different proteins.

    Abstract translation: 本文描述的是当在水性介质中与一种,两种,三种或更多种其它单体接触时能够形成生物学上有用的多聚体的甲硅烷基单体。 单体的这种多聚体形成关联可以通过单体与其目标生物分子的近端结合来促进。 在一个方面,这样的单体可以能够在水性介质中(例如在体内)结合另一单体以形成多聚体(例如二聚体)。 考虑的单体可以包括配体部分,连接体元件和连接配体部分和连接体元件的连接器元件。 在水性介质中,这样预期的单体可以经由每个连接体元件连接在一起,因此可以基本上同时调节一种或多种生物分子,例如调节蛋白质上或不同蛋白质上的两个或多个结合结构域。

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