TOP > 外国特許検索 > PRIMER, APPARATUS FOR PRODUCING DOUBLE-STRANDED DNA USING SAME, AND METHOD FOR PRODUCING DOUBLE-STRANDED DNA

PRIMER, APPARATUS FOR PRODUCING DOUBLE-STRANDED DNA USING SAME, AND METHOD FOR PRODUCING DOUBLE-STRANDED DNA

外国特許コード F210010395
整理番号 (SHW001-01)
掲載日 2021年5月6日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2020JP029441
国際公開番号 WO 2021020561
国際出願日 令和2年7月31日(2020.7.31)
国際公開日 令和3年2月4日(2021.2.4)
優先権データ
  • 特願2019-140851 (2019.7.31) JP
発明の名称 (英語) PRIMER, APPARATUS FOR PRODUCING DOUBLE-STRANDED DNA USING SAME, AND METHOD FOR PRODUCING DOUBLE-STRANDED DNA
発明の概要(英語) The present invention provides a primer which is used in amplifying a nucleic acid and has a structure represented by Formula (1). (wherein A1 represents -S-, -S-S-, or -Se-, B represents a base, and R1 represents a degradable protecting group. * means a bond to the sugar of an adjacent nucleotide.) The present invention is also an apparatus for producing a double-stranded DNA, the apparatus being provided with: a forward primer and reverse primer having the structure represented by Formula (1); a PCR apparatus for generating a double-stranded DNA having a 3’ recessed end by performing multiple PCR cycles using a template DNA as a template; a klenow fragment for making the 3’ end blunt; and a light irradiation means for forming an adhesion end in which R1 is eliminated and the 3’end protrudes.
従来技術、競合技術の概要(英語) BACKGROUND ART
In fields such as molecular biology, vectors in which a target DNA is incorporated into a host have been used to perform genetic recombination, transformation, and the like. Generally, the target DNA is amplified and used by polymerase chain reaction (PCR) when the amount is small. PCR uses a template DNA comprising a target DNA sequence and amplifies the template DNA by repeating thermal denaturation and annealing multiple times with primers that bind complementarily thereto.
The template DNA amplification product amplified in PCR is blunt ended and needs to be processed to bind (ligate) it to host DNA, such as plasmid DNA. Generally, a restriction enzyme that cleaves a particular sequence is used as such a treatment, but this method suffers from poor versatility because the DNA that can be attached depends on the sequence of the cleavage site of the restriction enzyme.
An example of a method that does not use a restriction enzyme is a technique in which the 3 ' end and the 5 ' end of the amplification product are used as cohesive ends (also referred to as cohesive ends, protruding ends, and the like), and the host side similarly forms cohesive ends to ligate the two ends to construct a vector. For example, in recent years, the Gibson assembly method, the In-Fusion method, the SLiCE method, and the like have been known. In these methods, the ends of the double-stranded DNA fragment are each provided with a homologous sequence of approximately 15 bp, and the strands on one side in the duplex are digested with exonuclease activity to generate adherent ends and then ligated. Note that in the Gibson assembly method, linking is performed using Taq DNA ligase at in vitro, and in the In-Fusion method, linking is performed using a repair system within e. coli.
In these methods, in addition to costly, site specificity may be inferior depending on reaction conditions and the like because exonuclease, which is an enzyme, is used, and quantitative adhesive end formation is difficult, leading to a problem in that efficiency of the linking reaction is low. Therefore, there is a need for a seamless rinsing method that does not use enzymes.
Therefore, a method for preparing a DNA having an adhesive terminal by a chemical method has been developed, and the primer described in Patent Document 1 is known as a primer for PCR for this method. In the primer of this document, the base corresponding to the 3 ' end in the base sequence of the non-complementary DNA portion is modified with a protecting group. This protecting group has a function of stopping the progression of DNA replication by an DNA polymerase, and can be desorbed from the base to be modified by photoirradiation, alkali treatment, or the like. In addition, in this document, a protecting group is introduced to the base of the primer using a substituent introduction agent for introducing the protecting group (substituent) into the biomolecule.
  • 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)
  • ABE Hiroshi
  • ABE Naoko
  • NAKAMOTO Kosuke
  • MURASE Hiroki
  • KIMURA Yasuaki
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 IT 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 ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN WS 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
ライセンスをご希望の方、特許の内容に興味を持たれた方は、問合せボタンを押してください。

PAGE TOP

close
close
close
close
close
close