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METHOD FOR AMPLIFYING CYCLIC DNA 新技術説明会

外国特許コード F170009298
整理番号 IP14P002
掲載日 2017年12月7日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2017JP018472
国際公開番号 WO 2017199991
国際出願日 平成29年5月17日(2017.5.17)
国際公開日 平成29年11月23日(2017.11.23)
優先権データ
  • 特願2016-099157 (2016.5.17) JP
発明の名称 (英語) METHOD FOR AMPLIFYING CYCLIC DNA 新技術説明会
発明の概要(英語) Provided is a method for amplifying cyclic DNA, particularly long-chain cyclic DNA, simply and exponentially in a cell-free system. More specifically, a cyclic DNA amplification method is provided, which comprises allowing to react a reaction mixture produced by mixing cyclic DNA having an origin sequence of chromosomal replication (an origin of chromosome (oriC)) with a reaction solution, wherein the reaction solution contains enzymes (1) to (3) as mentioned below, a buffer solution, NTP, dNTP, a magnesium ion source and an alkali metal ion source: (1) a first group enzyme which can catalyze the replication of cyclic DNA; (2) a second group enzyme which can catalyze an Okazaki fragment connection reaction to synthesize two sister cyclic DNA molecules capable of forming a catenane; and (3) a third group enzyme which can catalyze a reaction of separating two sister cyclic DNA molecules from each other.
従来技術、競合技術の概要(英語) BACKGROUND ART
Development of biotechnology is DNA cloning techniques are based, DNA fragments were prepared by the paste within the cells such as E. coli DNA plasmid annular as a method of amplifying. DNA cloning techniques were used to amplify the DNA in the case of annular, cell culture and the extraction of the amplification product, such as purified and a complicated procedure is required. In addition, cells were used for cloning the gene DNA is necessary to produce a recombinant organism, to limitations in the experimental environment.
In vitro DNA amplification methods include, polymerase chain reaction (PCR) is generally used. However, the test tube by PCR DNA amplification method, the DNA is amplified as it is not annular. In vitro DNA amplification methods include an annular, rolling circle amplification (RCA) method and the like (Non-Patent Document 1, Patent Document 1, Patent Document 2, Patent Document 3). However, the rolling-circle amplification method for amplifying DNA is annular, every target DNA with primers specific for the design. In addition, direct rolling circle DNA amplification products is straight-chain, the amplification products obtained in order to make an annular shape, such as recombinant enzyme incubated with an annular step is further required. E. coli chromosome replication (oriC cyclic DNA) mini and then, this was separated, the single amount of the annular body to obtain the replication products have been reported (Non-Patent Document 2-5). However, these documents used in the reaction conditions, as the DNA molecule is a cyclic copy of the efficiency, of the template DNA were added to about 15-40% and remains, as does not reach twice the amplification amount can be shown experimentally (Non-Patent Document 3-6). Further, in these documents is used as the template 10 kbp remains less than the size of the circular DNA.
In this way, the in vitro DNA amplification method in the prior art for amplifying DNA is annular, the binding of the primer DNA template is necessary, and DNA amplification products is straight-chain, in addition, the number of amplifiable DNA was kbp size remains. Further, the mini chromosome replication system is used in the E. coli cyclic attempts to generate an amplification product in the case, an annular template DNA is not amplified even times a problem.
  • 出願人(英語)
  • ※2012年7月以前掲載分については米国以外のすべての指定国
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • 発明者(英語)
  • SU'ETSUGU, Masayuki
  • TSUJIMOTO, Hiroko
  • SHINOHARA, Takeshi
国際特許分類(IPC)
指定国 National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KH KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
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