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1. 发明申请

US20020192677A1 Nucleic acid amplification with DNA-dependent RNA polymerase activity of RNA replicases 失效
标题翻译：核酸扩增与RNA复制酶的DNA依赖性RNA聚合酶活性
公开(公告)号：US20020192677A1
公开(公告)日：2002-12-19
申请号：US10071057
申请日：2002-02-07
申请人： Promega Corporation
发明人： Randall L. Dimond , Steven J. Ekenberg , James R. Hartnett , Geoffrey R. Hudson , Leopoldo G. Mendoza , Katharine M. Miller , John E. Monahan , Christopher L. Jones , Mark A. Maffitt , Richard A. Martinelli , Edward E. Pahuski , James W. Schumm
IPC分类号： C12Q001/68 , C12P019/34
CPC分类号： C12Q1/6867 , C12Q1/6806 , C12Q1/6853 , C12Q2521/501 , C12Q2521/325 , C12Q2521/119 , C12Q2521/107 , C12Q2563/137 , C12Q2531/149 , C12Q2525/121
摘要： The present invention entails methods, and kits for carrying them out, based on the discovery that an RNA replicase, such as Qnull replicase, has DNA-dependent RNA polymerase (nullDDRPnull) activity with nucleic acid segments, including DNA segments and DNA:RNA chimeric segments, which comprise a 2null-deoxyribonucleotide or an analog thereof and which have sequences of RNAs that are autocatalytically replicatable by the replicase. The discovery of this DDRP activity provides methods of the invention for nucleic acid amplification wherein a nucleic acid, with a DNA segment with the sequence of an RNA that is autocatalytically replicatable by an RNA replicase, is provided as a substrate for the replicase. The replicase catalyzes synthesis, from the DNA segment, of the RNA, which the replicase then autocatalytically replicates. The invention entails use of the amplification methods in detecting nucleic acid analytes, as in nucleic acid probe hybridization assays. Such assays of the invention include those wherein a nucleic acid analyte is hybridized with one or more nucleic acid probes, which include or are processed to generate a DNA segment which is amplifiable through production from the segment, catalyzed by the DDRP activity of an RNA replicase, of an autocatalytically replicatable RNA, which is autocatalytically replicated to provide an abundance of readily detectable reporter molecules. The invention permits replacement of an RNA, that is autocatalytically replicatable with an RNA replicase and employed as a reporter or label in prior art assays, such as nucleic acid probe hybridization assays or immunoassays, with a nucleic acid comprising a DNA segment with the same base sequence as the RNA. The invention also includes the methods of the invention with Mnnull2, Conull2, or Znnull2 in the solutions in which the DDRP activity occurs.
摘要翻译：基于以下发现，本发明涉及用于携带它们的方法和试剂盒：RNA复制酶（例如Qbeta复制酶）具有核酸片段（包括DNA片段和DNA）的DNA依赖性RNA聚合酶（“DDRP”）活性的发现： RNA嵌合片段，其包含2'-脱氧核糖核苷酸或其类似物，并且具有由复制酶自动催化复制的RNA序列。该DDRP活性的发现提供了用于核酸扩增的本发明的方法，其中提供具有可由RNA复制酶自动催化复制的RNA序列的DNA区段的核酸作为复制酶的底物。复制酶催化从DNA片段合成RNA，然后复制酶自动催化复制。本发明需要使用扩增方法来检测核酸分析物，如在核酸探针杂交测定中。本发明的这种测定包括其中核酸分析物与一个或多个核酸探针杂交的那些，其包括或被加工以产生可通过片段产生扩增的DNA片段，由RNA复制酶的DDRP活性催化的自催化可复制的RNA，其被自动催化复制以提供丰富的容易检测的报道分子。本发明允许用RNA复制酶自动催化复制并在现有技术测定中用作报告物或标记的RNA，例如核酸探针杂交测定或免疫测定，其中核酸包含具有相同碱基的DNA区段序列作为RNA。本发明还包括在发生DDRP活性的溶液中具有Mn +2，Co + 2或Zn + 2的本发明的方法。

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