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    • 1. 发明申请
    • MULTIPLEX NUCLEIC ACID REACTIONS
    • WO2004007755A3
    • 2004-01-22
    • PCT/US2003/022171
    • 2003-07-15
    • ILLUMINA, INC.CHEE, MarkFAN, Jian-BingGUNDERSON, Kevin
    • CHEE, MarkFAN, Jian-BingGUNDERSON, Kevin
    • C12Q1/68
    • The invention is directed to a variety of multiplexing methods used to amplify and/or genotype a variety of samples simultaneously. The invention provides a method of detecting a target sequence. The method consists of: (a) contacting a first and second probe with a target sequence under conditions where complementary probes form a hybridization complex with the target sequence, the first probe comprising an upstream universal priming site and a target­ specific sequence, the second probe comprising a downstream universal priming site and a target-specific sequence, wherein one of the first or second probes comprise an adapter sequence; (b) extending the first or second probe of the hybridization complex to form a modified probe; (c) amplifying the modified probe to form an amplicon, and (d) detecting the amplicon. A method of detecting the relative amounts of two or more target sequences is also provided. The method consists of (a) contacting a first and a second probe with first and second target sequences in an initial population under conditions where complementary probes form a hybridization complex with the target sequences, the first and second probes comprising a universal priming site, an adapter sequence and a target-specific sequence; (b) linearly amplifying the first and second probes forming the hybridization complex to produce first and second amplicons having distinctive adapter sequences, and (c) determining a relative amount of the first and second amplicons distinguishable by the adapter sequence, wherein the relative amount of the amplicons is indicative of the relative amounts of the first and second target sequences in the initial population. Further provided is a method of amplifying a target sequence to produce a signal within a dynamic range of a detection assay. The method consists of: (a) hybridizing a target-specific probe having an upstream universal priming site (UUP), a downstream universal priming site (DUP) and an adapter sequence with a set of differential primers, the set of differential primers comprising an upstream primer and first and second downstream primers, the second downstream primer having a lower Tm compared to the upstream primer and the first downstream primer; (b) amplifying the probe with the set of differential primers for two or more cycles of enzymatic polymerization; (c) increasing hybridization stringency to suppress hybridization of the second downstream primer, and (d) amplifying the probe from the upstream and the first downstream primers of the set for at least one cycle of enzymatic polymerization, wherein differential signals of amplicons produced from amplification of the first or the second downstream primers fall within a dynamic range of a detection assay.
    • 6. 发明申请
    • METHODS AND COMPOSITIONS FOR DIAGNOSING LUNG CANCER WITH SPECIFIC DNA METHYLATION PATTERNS
    • 用特异性DNA甲基化模式诊断肺癌的方法和组合物
    • WO2007050777A2
    • 2007-05-03
    • PCT/US2006/041821
    • 2006-10-25
    • ILLUMINA, INC.FAN, Jian-BingBIBIKOVA, Marina
    • FAN, Jian-BingBIBIKOVA, Marina
    • C12Q1/68G06F19/00
    • C12Q1/6827C12Q1/6886C12Q2600/112C12Q2600/118C12Q2600/154C12Q2600/16C12Q2523/125
    • The present invention provides a method for identification of differentially methylated genomic CpG dinucleotide sequences within genomic target sequences that are associated with cancer in an individual by obtaining a biological sample comprising genomic DNA from the individual measuring the level or pattern of methylated genomic CpG dinucleotide sequences for two or more of the genomic targets in the sample, and comparing the level of methylated genomic CpG dinucleotide sequences in the sample to a reference level of methylated genomic CpG dinucleotide sequences, wherein a difference in the level or pattern of methylation of the genomic CpG dinucleotide sequences in the sample compared to the reference level identifies differentially methylated genomic CpG dinucleotide sequences associated with cancer. As disclosed herein, the methods of the invention have numerous diagnostic and prognostic applications. The methods of the invention can be combined with a miniaturized array platform that allows for a high level of assay multiplexing and scalable automation for sample handling and data processing. Also provided by the invention are genomic targets and corresponding nucleic acid probes that are useful in the methods of the invention as they enable detection of differentially methylated genomic CpG dinucleotide sequences associated with adenocarcenomas of the lung.
    • 本发明提供了通过获得包含来自个体的基因组DNA的生物样品来鉴定与个体癌症相关的基因组靶序列内的差异甲基化基因组CpG二核苷酸序列的方法,所述生物样品包含测量甲基化基因组CpG二核苷酸序列的水平或模式, 样品中的两个或更多个基因组靶标,并将样品中甲基化的基因组CpG二核苷酸序列的水平与甲基化的基因组CpG二核苷酸序列的参考水平进行比较,其中基因组CpG二核苷酸的甲基化水平或模式的差异 与参考水平相比,样品中的序列鉴定与癌症相关的差异甲基化的基因组CpG二核苷酸序列。 如本文所公开的,本发明的方法具有许多诊断和预后应用。 本发明的方法可以与微型阵列平台组合,其允许用于样品处理和数据处理的高水平的测定复用和可扩展的自动化。 本发明还提供了可用于本发明方法的基因组靶和相应的核酸探针,因为它们能够检测与肺腺癌有关的差异甲基化的基因组CpG二核苷酸序列。
    • 7. 发明申请
    • METHODS AND COMPOSITIONS FOR DIAGNOSING CONDITIONS ASSOCIATED WITH SPECIFIC DNA METHYLATION PATTERNS
    • 用于特异性DNA甲基化模式诊断条件的方法和组合
    • WO2004110246A2
    • 2004-12-23
    • PCT/US2004/015382
    • 2004-05-14
    • ILLUMINA, INC.FAN, Jian-BingBIBIKOVA, Marina
    • FAN, Jian-BingBIBIKOVA, Marina
    • A61B
    • C12Q1/6886C12Q2600/112C12Q2600/154
    • The present invention provides a method for identification of differentially methylated genomic CpG dinucleotide sequences associated with cancer in an individual by obtaining a biological sample comprising genomic DNA from the individual measuring the level or pattern of methylated genomic CpG dinucleotide sequences for two or more of the genomic targets in the sample, and comparing the level of methylated genomic CpG dinucleotide sequences in the sample to a reference level of methylated genomic CpG dinucleotide sequences, wherein a difference in the level or pattern of methylation of the genomic CpG dinucleotide sequences in the sample compared to the reference level identifies differentially methylated genomic CpG dinucleotide sequences associated with cancer. As disclosed herein, the methods of the invention have numerous diagnostic and prognostic applications. The methods of the invention can be combined with a miniaturized array platform that allows for a high level of assay multiplexing and scalable automation for sample handling and data processing. Also provided by the invention are genomic targets and corresponding nucleic acid probes that are useful in the methods of the invention as they enable detection of differentially methylated genomic CpG dinucleotide sequences associated with cancer, for example, adenocarcenomas and sqamous cell carcinomas of the lung.
    • 本发明提供了通过获得包含来自个体的基因组DNA的生物学样品来鉴定与个体相关的癌症的差异甲基化的基因组CpG二核苷酸序列的方法,所述基因组DNA测量两个或多个基因组的甲基化基因组CpG二核苷酸序列的水平或模式 样本中的靶标,并将样品中甲基化基因组CpG二核苷酸序列的水平与甲基化基因组CpG二核苷酸序列的参考水平进行比较,其中样品中基因组CpG二核苷酸序列的甲基化水平或模式差异与 参考水平鉴定与癌症相关的差异甲基化的基因组CpG二核苷酸序列。 如本文所公开的,本发明的方法具有许多诊断和预后应用。 本发明的方法可以与微型阵列平台组合,其允许用于样品处理和数据处理的高水平的测定复用和可扩展的自动化。 本发明还提供了可用于本发明方法的基因组靶标和相应的核酸探针,因为它们能够检测与癌症相关的差异甲基化的基因组CpG二核苷酸序列,例如肺腺癌细胞癌和鳞状细胞癌。