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METHOD FOR AMPLIFYING CYCLIC DNA NEW

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 NEW
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.
特許請求の範囲(英語) [claim1]
1. Being expansion method of annulation DNA, process below:
(1) annulation DNA and below it becomes the mold:
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;
The third enzyme group which the separation reaction of two sisters annulations DNA the catalyst is done;
Buffer;
ATP;
GTP, CTP and UTP;
dNTP;
Magnesium ion source;
And
Alkaline metal ion source;
The process which forms the reaction blend of the reaction liquid which is included,
Here as for particular annulation DNA the enzyme and the connection possible duplication start arrangement which possess DnaA activity (origin of chromosome (oriC))It includes;
And
The process which insulates the reaction blend which was formed (2) in process (1) under isothermal condition;
It includes, the aforementioned method.
[claim2]
2. Being expansion method of annulation DNA, process below:
(1) annulation DNA and below it becomes the mold:
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;
The third enzyme group which the separation reaction of two sisters annulations DNA the catalyst is done;
Buffer;
ATP;
GTP, CTP and UTP;
dNTP;
Magnesium ion source;
And
Alkaline metal ion source;
The process which forms the reaction blend of the reaction liquid which is included,
Here as for particular annulation DNA the enzyme and the connection possible duplication start arrangement which possess DnaA activity (origin of chromosome (oriC))It includes;
And
The reaction blend which was formed (2) in process (1), under the temperature cycle which repeats inkiyubeshiyon above the 30.deg.C and inkiyubeshiyon below the 27.deg.C, in queue/cue bait the process which is done;
It includes, the aforementioned method.
[claim3]
3. The reaction liquid, furthermore the non unique adsorption inhibiter of the protein, and/or includes the non unique adsorption inhibiter of nuclear acid, in claim 1 or 2 method of statement.
[claim4]
4. The reaction liquid, furthermore direct condition DNA unique ekisonukureaze and/or includes the RecG type helicase, in claim 1 or 2 method of statement.
[claim5]
5. The reaction liquid, furthermore includes the ammonium salt, in claim 1 or 2 method of statement.
[claim6]
6. The first enzyme group, 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 the enzyme or enzyme which possess DnaB type helicase activity group which possesses DNA polymerase III* activity, implication combination,
The second enzyme group, implication the combination of the enzyme which possesses DNA polymerase I activity and the enzyme which possesses DNA ligase activity,
The third enzyme group, the enzyme which possesses topoisomerase III activity and/or includes the enzyme which possesses topoisomerase IV activity,
In claim 1 or 2 method of statement.
[claim7]
7. The second enzyme group furthermore, includes the enzyme which possesses RNaseH activity, in claim 6 method of statement.
[claim8]
8. The third enzyme group furthermore, includes the enzyme which possesses RecQ type helicase activity, in claim 6 method of statement.
[claim9]
9. In the first enzyme group,
Nuclear like 1 kinds or more the body protein is IHF or HU,
The enzyme or enzyme group which possesses DNA jiyairesu activity, is the mixture which consists of GyrA and GyrB,
The enzyme which possesses DnaB type helicase activity is the DnaB helicase,
The enzyme which possesses DNA helicase loader activity is the DnaC helicase loader,
The enzyme which possesses DNA primase activity is the DnaG primase,
The enzyme which possesses DNA clamp activity is the DnaN clamp,
The enzyme or enzyme group which possesses DNA polymerase III* activity, is the enzyme or enzyme group which includes either DnaX, HolA, HolB, HolC, HolD, DnaE, DnaQ, and HolE,
In claim 6 method of statement.
[claim10]
10. 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 claim 1 method of statement.
[claim11]
11. The reaction liquid, furthermore the RecG type helicase and/or includes one chain DNA unique ekisonukureaze, in claim 1 or 2 method of statement.
[claim12]
12. The reaction liquid, furthermore direct condition DNA unique ekisonukureaze and/or includes one chain DNA unique ekisonukureaze, in claim 1 or 2 method of statement.
[claim13]
13. The reaction liquid, furthermore includes the stabilization factor of DNA, in claim 1 or 2 method of statement.
[claim14]
14. Process (1)
(1-1) Below:
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;
The third enzyme group which the separation reaction of two sisters annulations DNA the catalyst is done;
Buffer;
ATP;
GTP, CTP and UTP;
dNTP;
Magnesium ion source;
And
Alkaline metal ion source;
The process which the reaction liquid which is included pureinkiyubeshiyon is done;
(1-2) The process which forms the reaction blend of the annulation DNA which becomes the particular reaction liquid and the mold;
And
It includes, in claim 1 or 2 method of statement.
[claim15]
15. Process (2), is done inside the waterdrop type emulsion in the oil, in claim 1 or 2 method of statement.
[claim16]
16. Continuing in process (2), furthermore,
(3) the process which processes after the reacting;
To include, here, as for after-treatment the particular reacting,
(i) From the first third enzyme group after in the reaction liquid which is not included diluting five times or more, the processing which re-insulates;
(ii) Direct condition DNA unique ekisonukureaze and/or the processing with one chain DNA unique ekisonukureaze;
And/or
(iii) The processing by the gap repair enzyme;
So it is,
In claim 1 or 2 method of statement.
[claim17]
17. Being the composition for expansion of annulation DNA,
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;
The third enzyme group which the separation reaction of two sisters annulations DNA the catalyst is done;
Buffer;
ATP;
GTP, CTP and UTP;
dNTP;
Magnesium ion source;
And
Alkaline metal ion source;
It includes, the aforementioned composition.
[claim18]
18. Furthermore the non unique adsorption inhibiter of the protein, and/or the non unique adsorption inhibiter of nuclear acid is included, in claim 17 the composition of statement.
[claim19]
19. Furthermore direct condition DNA unique ekisonukureaze and/or the RecG type helicase is included, in claim 17 the composition of statement.
[claim20]
20. Furthermore the RecG type helicase and/or one chain DNA unique ekisonukureaze is included, in claim 17 the composition of statement.
[claim21]
21. Furthermore direct condition DNA unique ekisonukureaze and/or one chain DNA unique ekisonukureaze is included, in claim 17 the composition of statement.
[claim22]
22. Furthermore the stabilization factor of DNA is included, in claim 17 the composition of statement.
[claim23]
23. Being the kit for expansion of annulation DNA,
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;
The third enzyme group which the separation reaction of two sisters annulations DNA the catalyst is done;
Buffer;
ATP;
GTP, CTP and UTP;
dNTP;
Magnesium ion source;
And
Alkaline metal ion source;
Combination is included, the aforementioned kit.
[claim24]
24. Furthermore the non unique adsorption inhibiter of the protein, and/or combination with the non unique adsorption inhibiter of nuclear acid is included, in claim 23 the kit of statement.
[claim25]
25. Furthermore direct condition DNA unique ekisonukureaze and/or combination with the RecG type helicase is included, in claim 23 the kit of statement.
[claim26]
26. Furthermore the RecG type helicase and/or one chain DNA unique ekisonukureaze is included, in claim 23 the kit of statement.
[claim27]
27. Furthermore direct condition DNA unique ekisonukureaze and/or one chain DNA unique ekisonukureaze is included, in claim 23 the kit of statement.
[claim28]
28. Furthermore the stabilization factor of DNA is included, in claim 23 the kit of statement.
[claim29]
29. Furthermore the gap repair enzyme is included, in claim 23 the kit of statement.
  • 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 (WO2017199991)
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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