Patent ID: 8883420

Claim:
A process of inserting a nucleic acid sequence of interest into an acceptor nucleic acid, comprising the following steps: (i) amplifying by PCR a DNA, said DNA comprising in the following order a sequence segment U, a nucleic acid sequence segment of known nucleotide sequence K2 and a nucleic acid sequence segment of known sequence K3, said PCR using a forward primer defining a first end of the amplified DNA and a reverse primer defining a second end of the amplified DNA, said reverse primer terminating at its 3′-end in a nucleotide sequence of nucleic acid sequence segment K3; (ii) treating said DNA amplified in the previous step, said DNA being linear double-stranded and comprising in the following order said sequence segment U, said nucleic acid sequence segment of known nucleotide sequence K2 and said nucleic acid sequence segment of known sequence K3, with an exonuclease to obtain a single-stranded overhang at the first end of the DNA and a single-stranded overhang comprising nucleic acid segments K2 and K3 at the second end of the DNA; (iii) annealing the DNA obtained in the previous step and having the single-stranded overhangs at the first and the second end thereof as defined in step (ii) to a linearized double-stranded acceptor nucleic acid having at a first end thereof a single-stranded overhang complementary to the single-stranded overhang of the first end of the DNA and at a second end thereof a single-stranded overhang complementary to the single-stranded sequence segment K2 of the second end of the DNA, wherein segment K3 is not inserted into the acceptor nucleic acid; and (iv) transforming the reaction product obtained in the previous step into a host cell.