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    • 21. 发明申请
    • METHOD FOR MEASURING TARGET POLYNUCLEOTIDES AND NOVEL ASTHMA BIOMOLECULES
    • 测量目标多核苷酸和新型哮喘生物分子的方法
    • WO0029621A3
    • 2000-11-09
    • PCT/US9926931
    • 1999-11-12
    • GENELABS TECH INC
    • DOLGANOV GREGORYNOVIKOV ALEXANDER
    • C07K14/52C12Q1/68C12N15/63C07K16/24
    • C12Q1/6883C07K14/521C12Q1/6809C12Q1/683C12Q2600/158C12Q2539/103
    • The present invention provides a method for simultaneously determining the levels of selected target polynucleotide sequences in a sample. In the method, selected target sequences are amplified using sequence-selective primer pairs to form double-stranded copies. The copies are cleaved with one or more endonucleases, and first and second aliquots of the cleaved fragment mixture are ligated to first and second adaptors, respectively, to form mixtures of first and second adaptor-fragment conjugates. The aliquots are combined and ligated to form conjugate dimers containing a conjugate from each of the first and second conjugate mixtures. After optional amplification, the conjugates are treated to release the adaptor segments, yielding target-identifier dimers. The dimers are polymerized to form target-identifier dimer multimers, and the relative abundances of the target-identifiers in one or more dimer multimers to provide an estimate of the levels of the selected target sequences in the sample. The present invention is particularly useful for determining expression levels of mRNA gene sequences in a sample, and for evaluating changes in mRNA expression levels among different samples or in response to changes in sample conditions. Also disclosed are certain polynucleotides and polypeptides that are useful for a variety of applications, particularly relating to asthma.
    • 本发明提供了用于同时测定样品中选定的靶多核苷酸序列水平的方法。 在该方法中,使用序列选择性引物对扩增选定的靶序列以形成双链拷贝。 用一种或多种内切核酸酶切割拷贝,并将切割的片段混合物的第一和第二等分试样分别连接到第一和第二衔接子上以形成第一和第二衔接子 - 片段缀合物的混合物。 将等分试样组合并连接以形成含有来自第一和第二缀合物混合物中的每一种的缀合物的缀合物二聚体。 在任选扩增后,处理缀合物以释放衔接子片段,产生目标 - 识别二聚体。 二聚体聚合形成靶标识物二聚体多聚体,并且靶标识别物在一个或多个二聚体多聚体中的相对丰度提供样品中所选靶序列水平的估计。 本发明对确定样品中mRNA基因序列的表达水平和评估不同样品中mRNA表达水平的变化或响应样品条件的变化特别有用。 还公开了可用于多种应用,特别是与哮喘有关的某些多核苷酸和多肽。
    • 22. 发明申请
    • MICROASSAY FOR SERIAL ANALYSIS OF GENE EXPRESSION AND APPLICATIONS THEREOF
    • 用于串联分析基因表达及其应用的微阵列
    • WO00044936A1
    • 2000-08-03
    • PCT/IB2000/000111
    • 2000-01-25
    • C12N15/09C12Q1/68
    • C12Q1/6809C12Q2539/103
    • Method of obtaining a library of tags able to define a specific state of a biological sample, comprising the following successive steps: (1) extracting in a single-step mRNA from a small amount of a biological sample using oligo(dT)25 covalently bound to paramagnetic beads, (2) generating a double strand cDNA library, from said mRNA, (3) cleaving the obtained cDNAs using Sau3A I, (4) separating the cleaved cDNAs in two aliquots, (5) ligating the cDNA contained in each of said two aliquots via said Sau3A I restriction site to a linker consisting of one double-strand cDNA molecule having one of the following formulas: GATCGTCCC-X1 or GATCGTCCC-X2, wherein X1 and X2, which comprise 30-37 nucleotides and are different, include a 20-25 bp PCR priming site with a Tm of 55 DEG C-65 DEG C, (6) digesting the products obtained in step (5) with the tagging enzyme BsmF I, (7) blunt-ending said BsmF I tags with a DNA polymerase and mixing the tags ligated with the different linkers, (8) ligating the tags obtained in step (7) to form ditags with a DNA ligase, (9) amplifying the ditags obtained in step (8) with primers comprising 20-25 bp and having a Tm of 55 DEG -65 DEG C, (10) isolating the ditags having between 20 and 28 bp from the amplification products obtained in step (9) by digesting said amplification products with Sau3A I and separating the digested products, (11) ligating the ditags obtained in step (10) to form concatemers, purifiying said concatemers and separating the concatemers having more than 300 bp, (12) cloning and sequencing said concatemers and (13) analysing the different obtained tags.
    • 获得能够定义生物样品的特定状态的标签库的方法,包括以下连续步骤:(1)使用共价结合的寡聚(dT)25从少量生物样品中提取单步mRNA (2)从所述mRNA产生双链cDNA文库,(3)使用Sau3AⅠ切割获得的cDNA,(4)以两个等分试样分离切割的cDNA,(5)连接包含在每个 将所述两个等分试样通过所述Sau3AⅠ限制性位点连接到由具有下式之一的一条双链cDNA分子组成的接头:GATCGTCCC-X1或GATCGTCCC-X2,其中X1和X2包含30-37个核苷酸并且不同, 包括Tm为55℃-65℃的20-25bp PCR引发位点,(6)用标记酶BsmF I消化步骤(5)中获得的产物,(7)将所述BsmF I标签平端化 用DNA聚合酶混合与不同接头连接的标签,(8)连接 (7)中获得的e标签以用DNA连接酶形成二价,(9)用包含20-25bp,Tm为55-65℃,(10)的引物扩增步骤(8)中获得的ditag, 通过用Sau3AI消化所述扩增产物并分离消化产物,分离步骤(9)中获得的扩增产物具有20至28bp的二价残基,(11)连接步骤(10)中获得的ditag以形成并发物 所述并列物质并且分离具有超过300bp的(2)克隆和测序所述并列物质的并列物质,(13)分析不同获得的标签。
    • 23. 发明申请
    • P53-INDUCED APOPTOSIS
    • P53诱导的细胞凋亡
    • WO99014356A2
    • 1999-03-25
    • PCT/US1998/019300
    • 1998-09-17
    • C12N15/09C07K16/32C12P21/08C12Q1/68C12Q
    • C12Q1/6809C12Q1/6886C12Q1/6897C12Q2539/103C12Q2600/136C12Q2600/142
    • The most well-documented biochemical property of p53 is its ability to transcriptionally activate genes. Many of the genes which are activated by p53 expression prior to the onset of apoptosis are predicted to encode proteins which could generate or respond to oxidative stress, including one that is implicated in apoptosis within plant meristems. p53 may result in apoptosis through a three-step process: (i) the transcriptional induction of specific redox-related genes; (ii) the formation of reactive oxygen species (ROS); and (iii) the oxidative degradation of mitochondrial components, rapidly leading to cell death. Transcription of other genes is decreased by p53. Examination of the level of transcription of p53-induced or -repressed genes can be used to determine p53 status, to diagnose cancer, and to evaluate cytotoxicity or carcinogenicity of a test agent.
    • p53的最佳记录性质是其转录激活基因的能力。 据预测,编码的细胞凋亡蛋白,可能产生氧化应激和反应,因此,包括在分生组织参与细胞凋亡的蛋白质的开始之前通过p53的表达被激活的许多基因 植物。 P53可以通过包括以下三个步骤的方法导致凋亡:(i)与氧化还原有关的特定基因的转录诱导; (ii)形成活性氧物质(ROS); 和(iii)氧化降解线粒体成分,迅速导致细胞死亡。 其他基因的转录被p53降低。 检测p53诱导的或p53抑制的基因转录水平可用于诊断癌症并评估测试药剂的细胞毒性和致癌性。
    • 28. 发明申请
    • MOLECULAR ANALYSIS
    • 分子分析
    • WO2005071112A3
    • 2005-12-08
    • PCT/GB2005000244
    • 2005-01-26
    • ISIS INNOVATIONVAUX DAVID
    • VAUX DAVID
    • C12Q1/68
    • C12Q1/6811C12Q2539/103C12Q2531/113C12Q2525/151
    • The present invention relates to a method of determining the sequence and/or occurrence frequency of a number of variable gene inserts from a gene library, which inserts exhibit a desired specific characteristic, wherein each variable gene insert is flanked (5') and (3') by known sequences, the method comprising; selecting the number of inserts by their ability to exhibit the desired specific characteristic, conducting polymerase chain reaction to amplify the selected number of variable gene inserts to produce components of a mixed PCR product, ligating the components of the mixed PCR product to produce a concatenated sequence and sequencing or determining the occurrence of the gene inserts in the concatenated sequence.
    • 本发明涉及确定来自基因文库的许多可变基因插入片段的序列和/或出现频率的方法,该插入片段表现出所需的特异性特征,其中每个可变基因插入片段(5')和(3' '),该方法包括: 通过它们显示所需特异性特征的能力来选择插入物的数目,进行聚合酶链式反应以扩增选定数目的可变基因插入物以产生混合PCR产物的组分,连接混合PCR产物的组分以产生连接序列 并测序或确定连锁序列中基因插入的出现。