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, preferable 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 cell-free method of producing closed linear deoxyribonucleic acid (DNA) molecules, the method comprising:
(a) contacting a DNA template comprising a linear, double stranded DNA molecule covalently closed at each of its ends by a portion of a protelomerase recognition sequence and comprising at least one stem loop motif with a strand-displacing polymerase under conditions promoting amplification of said DNA template in the presence of at least one primer which is capable of hybridizing specifically to a primer binding site within said stem loop motif; and
(b) producing the closed linear DNA molecules by contacting DNA produced in step (a) with at least one protelomerase under conditions promoting production of the closed linear DNA molecules;
wherein a sequence of the stem loop motif comprises a central section and two flanking sequences, and wherein the central section comprises the primer binding site, wherein the sequence of the stem loop motif is capable of forming a stem loop secondary structure under conditions promoting formation of a secondary structure,
wherein the central section of the sequence of the stem loop motif is a single stranded DNA region in the stem loop secondary structure, and
wherein the sequence of the stem loop motif is different from the portion of the protelomerase recognition sequence of the DNA template, and the stem loop motif and the portion of the protelomerase recognition sequence are located in different parts of the DNA template.
2 . The method of claim 1 , wherein the sequence of the stem loop motif is adjacent or near to the portion of the protelomerase recognition sequence covalently closed at one of ends of the linear, double stranded DNA molecule.
3 . The method of claim 1 , wherein the sequence of the stem loop motif includes self-complementary sequences within the two flanking sequences which allow the formation of the stem loop secondary structure.
4 . The method of claim 1 , wherein, the sequences within the two flanking sequences are being complementary to a bridging oligonucleotide.
5 . The method of claim 1 , wherein each of the ends of the DNA template comprises the portion of protelomerase recognition sequence and the portion of protelomerase recognition sequence on each of the ends of the DNA template has a different sequence.
6 . The method of claim 1 , wherein the DNA template comprises one or more additional protelomerase recognition sequences.
7 . The method of claim 6 , wherein the one or more additional protelomerase recognition sequences comprises two additional protelomerase recognition sequences.
8 . The method of claim 7 , wherein each of the two additional protelomerase recognition sequences is separated from one of the ends of the linear, double stranded DNA molecule by the at least one stem loop motif.