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METHOD OF AMPLIFYING CIRCULAR DNA NEW

外国特許コード F170009138
整理番号 IP14B001
掲載日 2017年8月8日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2015JP082356
国際公開番号 WO 2016080424
国際出願日 平成27年11月18日(2015.11.18)
国際公開日 平成28年5月26日(2016.5.26)
優先権データ
  • 特願2014-233563 (2014.11.18) JP
発明の名称 (英語) METHOD OF AMPLIFYING CIRCULAR DNA NEW
発明の概要(英語) Provided is a method capable of simply and exponentially amplifying a circular DNA, especially a long-chain circular DNA in a cell-free system. Specifically, provided is a method of amplifying a circular DNA, the method including isothermally reacting a reaction mixture which is produced by mixing a circular DNA having a replication initiation sequence (origin of chromosome (oriC)) with a reaction solution including the following enzyme groups: (1) a first enzyme group catalyzing the replication of the circular DNA; (2) a second enzyme group catalyzing a linking reaction of Okazaki fragments and synthesizing two sister circular DNAs which form catenane; and (3) a third enzyme group catalyzing a separation reaction of two sister circular DNAs.
特許請求の範囲(英語) [claim1]
1. Expansion method of the annulation DNA which includes the process below:
(1) the first enzyme group which duplication of annulation DNA the catalyst is done;
The catalyst doing the Okazaki fragment connection reaction, the second enzyme group which synthesizes two sisters annulations DNA which form [katenan];
And
The third enzyme group which the separation reaction of two sisters annulations DNA the catalyst is done
The process which forms the reaction blend of the annulation DNA which becomes the reaction liquid and the mold which are included, the enzyme and the connection possible duplication start arrangement where annulation DNA has DnaA activity here (origin of chromosome (oriC))It includes;
And
The process which insulates the reaction blend which was formed (2) in process (1) under isothermal condition.
[claim2]
2. Being method of statement in claim 1,
The first enzyme group which duplication of annulation DNA the catalyst is done, nuclear like above the enzyme and 1 kinds which possess DnaA activity the body protein, the enzyme or enzyme group which possesses DNA [jiyairesu] activity, the one chain DNA connection protein (single-strand binding protein (SSB))The enzyme, possess DNA helicase loader activity the enzyme, possess DNA primase activity the enzyme, possess DNA clamp activity the enzyme, and it is combination of the enzyme or enzyme which possess DNA helicase activity group which possesses DNA polymerase III activity,
The catalyst doing the Okazaki fragment connection reaction, the second enzyme group which synthesizes two sisters annulations DNA which form [katenan], is combination of the enzyme, the enzyme which possesses DNA polymerase I activity, and possesses DNA ligase activity the enzyme which possess RNaseH activity,
The third enzyme group which the separation reaction of two sisters annulations DNA the catalyst is done, is combination of the enzyme, the enzyme which possesses topoisomerase III activity, and possesses RecQ activity the enzyme which possess topoisomerase IV activity, method.
[claim3]
3. The enzyme and the connection possible duplication start arrangement which possess DnaA activity, are duplication start arrangement of the colon bacillus, in claim 2 method of statement.
[claim4]
4. Size of annulation DNA is above 50kb, claim in either 2 or 3 method of statement.
[claim5]
5. Nuclear like 1 kinds or more the body protein is the combination of IhfA and IhfB, in either of the claim 2-4 method of statement.
[claim6]
6. The enzyme or enzyme group which possesses DNA [jiyairesu] activity, is the combination of GyrA and GyrB, in either of the claim 2-5 method of statement.
[claim7]
7. The enzyme which possesses DNA helicase activity is the DnaB helicase, in either of the claim 2-6 method of statement.
[claim8]
8. The enzyme which possesses DNA helicase loader activity is the DnaC helicase loader, in either of the claim 2-7 method of statement.
[claim9]
9. The enzyme which possesses DNA primase activity is the DnaG primase, in either of the claim 2-8 method of statement.
[claim10]
10. The enzyme which possesses DNA clamp activity is the DnaN clamp, in either of the claim 2-9 method of statement.
[claim11]
11. The enzyme or enzyme group which possesses DNA polymerase III activity, is the combination of DnaX, HolA, HolB, HolC, HolD, DnaE, DnaQ, and HolE, in either of the claim 2-10 method of statement.
[claim12]
12. Nuclear like above the enzyme and 1 kinds which possess DnaA activity the body protein, the enzyme or enzyme group which possesses DNA [jiyairesu] activity, the one chain DNA connection protein (single-strand binding protein (SSB))The enzyme, possess DNA helicase loader activity the enzyme, possess DNA primase activity the enzyme, possess DNA clamp activity the enzyme, the enzyme or enzyme which possess DNA helicase activity group which possesses DNA polymerase III activity, the enzyme, possess DNA polymerase I activity the enzyme, possess DNA ligase activity the enzyme, possess topoisomerase IV activity the enzyme, the enzyme which possesses topoisomerase III activity, and possesses RecQ activity the enzyme which possess RNaseH activity are colon bacillus origin, in either of the claim 2-11 method of statement.
[claim13]
13. In process (1) the reaction liquid, furthermore includes the component which controls the formation of the subsidiary product, in either of the claim 1-12 method of statement.
[claim14]
14. In process (2) the isothermal condition, is the fixed temperature which is included in the range of the 25.deg.C-50.deg.C, in either of the claim 1-13 method of statement.
[claim15]
15. The first enzyme group which duplication of annulation DNA the catalyst is done;
The catalyst doing the Okazaki fragment connection reaction, the second enzyme group which synthesizes two sisters annulations DNA which form [katenan];
And
The third enzyme group which the separation reaction of two sisters annulations DNA the catalyst is done
It includes, the composition for expansion of annulation DNA.
[claim16]
16. Being the composition of statement in claim 15,
The first enzyme group which duplication of annulation DNA the catalyst is done, nuclear like above the enzyme and 1 kinds which possess DnaA activity the body protein, the enzyme or enzyme group which possesses DNA [jiyairesu] activity, the one chain DNA connection protein (single-strand binding protein (SSB))The enzyme, possess DNA helicase loader activity the enzyme, possess DNA primase activity the enzyme, possess DNA clamp activity the enzyme, and it is combination of the enzyme or enzyme which possess DNA helicase activity group which possesses DNA polymerase III activity,
The catalyst doing the Okazaki fragment connection reaction, the second enzyme group which synthesizes two sisters annulations DNA which form [katenan], is combination of the enzyme, the enzyme which possesses DNA polymerase I activity, and possesses DNA ligase activity the enzyme which possess RNaseH activity,
The third enzyme group which the separation reaction of two sisters annulations DNA the catalyst is done, is combination of the enzyme, the enzyme which possesses topoisomerase III activity, and possesses RecQ activity the enzyme which possess topoisomerase IV activity, the composition.
[claim17]
17. The first enzyme group which duplication of annulation DNA the catalyst is done;
The catalyst doing the Okazaki fragment connection reaction, the second enzyme group which synthesizes two sisters annulations DNA which form [katenan];
And
The third enzyme group which the separation reaction of two sisters annulations DNA the catalyst is done
It includes, the kit for expansion of annulation DNA.
[claim18]
18. Being the kit of statement in claim 17,
The first enzyme group which duplication of annulation DNA the catalyst is done, nuclear like above the enzyme and 1 kinds which possess DnaA activity the body protein, the enzyme or enzyme group which possesses DNA [jiyairesu] activity, the one chain DNA connection protein (single-strand binding protein (SSB))The enzyme, possess DNA helicase loader activity the enzyme, possess DNA primase activity the enzyme, possess DNA clamp activity the enzyme, and it is combination of the enzyme or enzyme which possess DNA helicase activity group which possesses DNA polymerase III activity,
The catalyst doing the Okazaki fragment connection reaction, the second enzyme group which synthesizes two sisters annulations DNA which form [katenan], is combination of the enzyme, the enzyme which possesses DNA polymerase I activity, and possesses DNA ligase activity the enzyme which possess RNaseH activity,
The third enzyme group which the separation reaction of two sisters annulations DNA the catalyst is done, is combination of the enzyme, the enzyme which possesses topoisomerase III activity, and possesses RecQ activity the enzyme which possess topoisomerase IV activity, the kit.
  • 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
  • KOBAYASHI HIROKO
国際特許分類(IPC)
指定国 (WO201680424)
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 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 KN KP KR 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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