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    • 2. 发明公开
    • PRIMER TECHNOLOGY
    • PRIMERTECHNOLOGIE
    • EP2951316A2
    • 2015-12-09
    • EP14703318.7
    • 2014-02-03
    • Selvi, OzanOrcan, Serkan
    • SELVI, OzanORCAN, SerkanTOKSÖZ, Sila
    • C12Q1/68
    • C12Q1/6853C12N15/1048C12P19/34C12Q1/6806C12Q1/6844C12Q2521/319C12Q2525/143C12Q2525/173C12Q2525/191
    • The present invention provides a method of nucleic acid manipulation comprising: (i) hybridizing a double stranded primer to the 3' end of a single stranded nucleic acid template, wherein said primer comprises a double stranded region and a single stranded region, wherein the single stranded region is a 3' overhang region and wherein the 3' overhang region enables the double stranded primer to target and hybridize to the 3' end of the nucleic acid template, wherein said single stranded 3' overhang region comprises a degenerate sequence or a sequence comprising universal bases, and wherein the double stranded primer is made up of two separate strands; (ii) at least one round of polymerization using a polypeptide with 5' to 3' DNA polymerization activity, wherein at least the first round of polymerization comprises using a polypeptide with 5' to 3' DNA polymerization activity to carry out a primer extension reaction to synthesise nucleotides in a template dependent manner from the 3' end of the single stranded region of said hybridized double stranded primer; wherein the hybridizing step (i) and at least the first primer extension reaction from the 3' end of the single stranded region of said hybridized double stranded primer in step (ii) takes place without the formation of a phosphodiester bond between the 3' end of the nucleic acid template and the double stranded primer of step (i). Products, kits and compositions suitable for use in such methods are also provided.
    • 本发明提供了一种核酸操作方法,包括:(i)将双链引物与单链核酸模板的3'末端杂交,其中所述引物包含双链区域和单链区域,其中单链引物 双链区是3'突出区,其中3'突出区使得双链引物靶向并与核酸模板的3'末端杂交,其中所述单链3'突出区包含简并序列或序列 包括通用碱基,并且其中双链引物由两条单独的链组成; (ii)使用具有5'至3'DNA聚合活性的多肽的至少一轮聚合,其中至少第一轮聚合包括使用具有5'至3'DNA聚合活性的多肽以进行引物延伸反应 以模板依赖的方式从所述杂交双链引物的单链区域的3'末端合成核苷酸; 其中所述杂交步骤(i)至少在步骤(ii)中所述杂交双链引物的单链区域的3'末端的第一引物延伸反应进行而不在3'末端之间形成磷酸二酯键 的核酸模板和步骤(i)的双链引物。 还提供适用于这些方法的产品,试剂盒和组合物。
    • 9. 发明公开
    • METHOD FOR MEASURING CYTOKERATIN-19 MRNA
    • 测量细胞角蛋白-19MRNA的方法
    • EP2351851A1
    • 2011-08-03
    • EP09829212.1
    • 2009-11-30
    • Tosoh Corporation
    • OMOTO, DaisukeSAITO, JuichiOONAKA, SatoruHAYASHI, Toshinori
    • C12Q1/68C12N15/09
    • C12Q1/6853C12Q1/6865C12Q2525/143C12Q2545/114C12Q2561/113C12Q2531/143C12Q2521/107
    • Disclosed is a method of amplifying and detecting cytokeratin 19 mRNA in RNA amplification process, comprising: a step for forming a double-stranded DNA containing a promoter sequence with a reverse transcriptase by use of a combination of oligonucleotides consisting of a first primer having a sequence homologous to a portion of cytokeratin 19 mRNA and a second primer having a complementary sequence, wherein the promoter sequence is added to the 5'-end of either the first primer or the second primer, forming an RNA transcription product by use of an RNA polymerase with using the double-stranded DNA as template, and forming the double-stranded DNA by use of a reverse transcriptase by continuing to use the RNA transcription product as a template of DNA synthesis, by measuring an amount of amplified RNA produced over time with an oligonucleotide probe designed so that signal properties change with the formation of a complementary double strand with the amplified RNA.
    • 本发明公开了在RNA扩增过程中扩增和检测细胞角蛋白19mRNA的方法,其包括:用逆转录酶形成含有启动子序列的双链DNA的步骤,其中使用由具有序列的第一引物 与细胞角蛋白19 mRNA的一部分同源,以及具有互补序列的第二引物,其中启动子序列被添加到第一引物或第二引物的5'末端,通过使用RNA聚合酶形成RNA转录产物 以双链DNA为模板,通过继续使用RNA转录产物作为DNA合成的模板,通过使用逆转录酶形成双链DNA,通过测量随时间产生的扩增RNA的量 寡核苷酸探针,其设计使得信号特性随着与扩增的RNA形成互补双链而改变。