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

Foreign code F210010395
File No. SHW001-01WO
Posted date 2021年5月6日
Country 世界知的所有権機関(WIPO)
International application number 2020JP029441
International publication number WO 2021020561
Date of international filing 令和2年7月31日(2020.7.31)
Date of international publication 令和3年2月4日(2021.2.4)
Priority data
  • 特願2019-140851 (2019.7.31) JP
Title PRIMER, APPARATUS FOR PRODUCING DOUBLE-STRANDED DNA USING SAME, AND METHOD FOR PRODUCING DOUBLE-STRANDED DNA
Abstract 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.
Outline of related art and contending technology 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.
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • Inventor
  • ABE Hiroshi
  • ABE Naoko
  • NAKAMOTO Kosuke
  • MURASE Hiroki
  • KIMURA Yasuaki
IPC(International Patent Classification)
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