Closed linear DNA production
The present invention relates to improved processes for production of closed linear deoxyribonucleic acid (DNA), in particular cell-free enzymatic production of closed linear DNA molecules, preferably using a closed linear DNA as a template for DNA synthesis. The invention further relates to a novel closed linear DNA species, suitable for use as a template in the improved processes for production of closed linear DNA. Further, the invention pertains to the intermediate products of the processes, since this enables the production of larger quantities of closed linear DNA from the template than with methods known in the art.
1. A linear, double stranded DNA molecule covalently closed at each of its ends by a portion of a protelomerase recognition sequence, wherein the portion comprises at least 14 nucleotides of a double stranded palindromic sequence, the linear, double stranded DNA molecule comprises at least one eukaryotic promoter, and wherein the linear, double stranded DNA molecule includes at least one stem loop motif comprising a central non-complementary loop section flanked by two complementary sequences.
2. The linear double stranded DNA molecule as claimed in claim 1 , wherein said molecule comprises one or more additional protelomerase recognition sequences.
3. The linear double stranded DNA molecule as claimed in claim 2 , wherein said additional protelomerase recognition sequences are separated from the closed ends by the at least one stem loop motif.