| 摘要 |
Described is a method of preparing recombinant nucleic acids, which comprises the steps of: (a) amplification of a 1st nucleic acid fragment (x) in the presence of a polymerization agent, the various deoxynucleoside triphosphates, and two primers (x1/x2) hybridizing to form opposite strands of the nucleic acid, wherein after being separated from its template strand the extension product synthesized by one primer (xl) serves as a template for the synthesis of an extension product of the other primer (x2), the primer (x2) determining the terminus, to be linked, of the 1st nucleic acid and the 3' end of primer (x2) being substantially complementary to a strand of the desired terminus of the 1st nucleic acid and the 5' end of primer (x1) being not complementary to this strand and containing a recognition sequence for a type IIS restriction endonuclease; (b) amplification of a 2nd nucleic acid fragment (y) in the presence of a polymerization agent, the various deoxynucleoside triphosphates and two primers (y1/y2) hybridizing to form opposite strands of the nucleic acid, wherein after being separated from its template strand the extension product synthesized by one primer (y1) serves as a template for the synthesis of an extension product of the other primer (y2), the primer (y1) determining the terminus, to be linked, of the 2nd nucleic acid and the 3' end of primer (y1) being substantially complementary to a strand of the desired terminus of the 2nd nucleic acid and the 5' end of primer (y1) being not complementary to this strand and containing a recognition sequence for a type IIS restriction endonuclease; wherein (c) a sequence identical in both primers is additionally present between this 3' end and the 5' end of primers (x2) and (y1), which corresponds to the sequence cleaved by the restriction enzyme; (d) restriction of the amplified 1st and 2nd nucleic acids with (a) restriction endonuclease(s) recognizing the recognition sequences defined in (a) and (b); and (e) ligation of the nucleic acid fragments obtained in (d). |
