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DNA PRODUCTION METHOD AND DNA FRAGMENT JOINING KIT NEW

外国特許コード F190009779
整理番号 (IP14P004)
掲載日 2019年5月7日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2018JP025528
国際公開番号 WO 2019009361
国際出願日 平成30年7月5日(2018.7.5)
国際公開日 平成31年1月10日(2019.1.10)
優先権データ
  • 特願2017-132084 (2017.7.5) JP
  • 特願2017-231732 (2017.12.1) JP
発明の名称 (英語) DNA PRODUCTION METHOD AND DNA FRAGMENT JOINING KIT NEW
発明の概要(英語) The present invention provides: a method that makes it possible to produce straight-chain or annular DNA by the joining of two or more types of DNA fragments at regions that have homologous base sequences; and a DNA fragment joining kit that uses said method. The present invention is a DNA production method that involves: preparing a reaction solution that includes two or more types of DNA fragments and a protein that acts as a RecA-family recombination enzyme; and producing straight-chain or annular DNA in the reaction solution by causing the two or more types of DNA fragments to be joined at regions that have homologous base sequences or regions that have complementary base sequences.
従来技術、競合技術の概要(英語) BACKGROUND ART
A plurality of linear DNA fragments were ligated to the 2 single-stranded, linear or cyclic double-stranded DNA 2 is a method of producing. According to the method, chemical synthesis is a more difficult to synthesize a long-chain 2-stranded DNA can be obtained. 2 Stranded DNA fragments were ligated linear method is, primarily, In fusion method (see Patent Document 1.) And Gibson Assembly method (see Patent Document 2 and Patent Document 3.) Is present.
In fusion method, each end of the stranded DNA fragments 2 15 base sequence homologous to the recognition function that fuzes the coupling reaction using an enzyme In fusion is a method. Specifically, first, by using PCR, DNA fragments of 2 for the purpose of the present chain is connected to the end of the same nucleotide sequence of the homologous region is added. At both ends of the homologous region of the base 15 is added to each of the stranded DNA fragments 2 2 can be mixed and incubated In fusion enzymes can be linked by.
On the other hand, Gibson Assembly method, first, the first distal region 1 of the first DNA molecule DNA molecule 2 of the proximal region of the enzymes with exonuclease activity, the same area of each of the (mutually-specific hybridization of a sufficient length of the region of sequence identity) 1 and the single-stranded state. Then, both the anneal after the couple, to repair a nick and a gap, a complete DNA strand 2 to obtain the connecting body. For example, to Patent Document 2, 1.6kbp and 2 of the double-stranded DNA fragments 1.4kbp, T4 DNA was digested with exonuclease activity of the polymerase comprises a stranded state 1 and then, after coupled in the presence of RecA, dNTP and T4 DNA DNA ligase was added and this fills the gap in the polymerase activity to repair the nick, double-stranded DNA of 3kbp 2 as described was obtained.
Other, using 2 linear RecA DNA strand as a method of concatenating a plurality of fragments, in Patent Document 4, or recombinant polypeptide of the recombinant enzyme family RecA protein activity in the presence of, a plurality of linear DNA single-stranded DNA fragment 2 can be incubated with a ligase, a plurality of linear DNA fragments 2 are coupled in series is double-stranded linear DNA strand 2 have been disclosed methods for making the same. In this method, the recombinant enzyme family RecA the like is used, the stranded DNA fragment 2 of a linear combination of both ends is suppressed, and, single-stranded DNA fragments between the linear 2 to facilitate the coupling of the terminal.
  • 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)
  • SUETSUGU Masayuki
  • KURATA Tatsuaki
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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