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METHOD FOR REPLICATING OR AMPLIFYING CIRCULAR DNA NEW

外国特許コード F180009530
整理番号 (IP14P003)
掲載日 2018年11月2日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2018JP007485
国際公開番号 WO 2018159669
国際出願日 平成30年2月28日(2018.2.28)
国際公開日 平成30年9月7日(2018.9.7)
優先権データ
  • 特願2017-037489 (2017.2.28) JP
発明の名称 (英語) METHOD FOR REPLICATING OR AMPLIFYING CIRCULAR DNA NEW
発明の概要(英語) Provided is a method that enables replication or amplification of circular DNA, particularly long-chain circular DNA, in a cell-free system. Specifically, provided is a method for inhibiting generation of a DNA multimer which is a by-product, during replication or amplification of circular DNA having a replication initiation sequence (origin of chromosome (oriC)) by using the following enzymes: (1) first enzymes which catalyze replication of the circular DNA; (2) second enzymes which catalyze an Okazaki fragment linking reaction, to synthesize two sister circular DNA molecules forming a catenane; and (3) third enzymes which catalyze a separation reaction of the two sister circular DNA molecules. Further provided is a method that comprises introduction of oriC into circular DNA by using a transposon.
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 Escherichia coli plasmid DNA to be 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, E. coli chromosomal replication system is used in the mini-ring to produce amplified product of 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)
  • Masayuki Suji
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 JO 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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