METHOD FOR PREPARING DNA FRAGMENT CONTAINING INVERTED REPEAT SEQUENCE DERIVED FROM FOREIGN DNA FRAGMENT AND METHOD FOR PREPARING DNA-TYPE RNAi LIBRARY

摘要

PPROBLEM TO BE SOLVED: To provide a method for simultaneously forming inverted repeat sequences in a high forming rate in one test tube for long-chain cDNAs having various kinds of lengths and sequences, to provide a method for preparing the DNA-type RNAi library utilizing the method, and to provide a utilization method. PSOLUTION: There is provided the method for preparing the plasmid having a target gene DR (direct repeat) structure. The method includes (1) a step of preparing the plasmid having one target gene (plasmid S: having nicking endonuclease-recognizing sites at both sides of the target gene, and when the DR structure is formed at the target gene side of the nicking endonuclease-recognizing site, having a site for forming a restriction enzyme-recognizing site, for forming a restriction enzyme-recognizing site in the spacer region therebetween), (2) a step of causing the nicking in the plasmid S, (3) a step of preparing the plasmid (two restriction enzyme-recognizing sites are newly formed by the sites for forming the restriction enzyme-recognizing sites) having the target gene converted to the DR structure by the chain-substitution reaction of the nicked plasmid S and the successively carried out self ligation reaction, (4) a step of introducing the plasmid group containing the plasmid having the target gene DR structure to a host, and cloning the plasmid group by using the obtained transformant, (5) a step of digesting the cloned plasmid group with two restriction enzymes corresponding to the newly formed two restriction enzyme-recognizing sites, and (6) a step of collecting the plasmids formed into linear shapes by being digested by the restriction enzymes from the digested plasmid group. PCOPYRIGHT: (C)2011,JPO&INPIT