Production of closed linear DNA

1. A process for production of linear double stranded deoxyribonucleic (DNA) covalently closed at both ends by hairpin loops, comprising:

(a) amplifying by rolling circle amplification a DNA template comprising at least one protelomerase target sequence to produce a product comprising multiple protelomerase target sequences; and

(b) producing linear double stranded DNA covalently closed at both ends by hairpin loops by contacting the product comprising multiple protelomerase target sequences produced in (a) with at least one protelomerase under suitable conditions,

wherein steps (a) and (b) occur in an in vitro cell-free environment.

2. The process of claim 1 , wherein primers for the amplification are random primers.

3. The process of claim 1 , wherein a DNA polymerase for the amplification is phi29 of SEQ ID NO: 2 or a variant thereof which comprises a sequence having at least 95% identity to SEQ ID NO: 2 and/or said protelomerase is bacteriophage N15 TelN of SEQ ID NO: 15 or a variant thereof which comprises a sequence having at least 95% identity to SEQ ID NO: 15.

4. The process of claim 1 , wherein said at least one protelomerase target sequence comprises a perfect inverted repeat DNA sequence.

5. The process of claim 1 , wherein said DNA template is a closed circular DNA.

6. The process of claim 1 , wherein said DNA template is a linear double stranded DNA covalently closed at both ends by hairpin loops.

7. The process according to claim 1 , wherein said DNA template comprises an expression cassette comprising a eukaryotic promoter operably linked to a coding sequence of interest.

8. The process according to claim 7 , wherein said expression cassette is flanked on either side by a protelomerase target sequence.

9. A process according to claim 1 , which produces a linear double stranded expression cassette DNA that is covalently closed at both ends by hairpin loops.

10. The process of claim 1 , which further comprises purifying the linear double stranded DNA covalently closed at both ends by hairpin loops produced in (b).

11. A process according to claim 1 comprising formulating the resulting linear double stranded DNA covalently closed at both ends by hairpin loops with a pharmaceutically acceptable carrier or excipient to make a pharmaceutical composition comprising a linear double stranded DNA covalently closed at both ends by hairpin loops.

12. The process according to claim 6 , wherein said DNA template is incubated under denaturing conditions to form a closed circular DNA.

13. The process according to claim 6 , wherein said expression cassette comprises a eukaryotic transcription termination sequence.

14. The process according to claim 7 , wherein said coding sequence of interest is a human coding sequence or a coding sequence from a pathogen that infects humans.

15. A process for production of linear double stranded deoxyribonucleic (DNA) covalently closed at both ends by hairpin loops, comprising:

(a) amplifying a DNA template comprising more than one protelomerase target sequence to produce a product comprising multiple protelomerase target sequences; and

(b) producing linear double stranded DNA covalently closed at both ends by hairpin loops by contacting the product comprising multiple protelomerase target sequences produced in (a) with at least one protelomerase under suitable conditions,

wherein steps (a) and (b) occur in an in vitro cell-free environment.

16. The process of claim 15 , wherein said DNA template is a closed circular DNA.

17. The process of claim 15 , wherein said DNA template is a linear double stranded DNA covalently closed at both ends by hairpin loops.

18. The process according to claim 15 , wherein said DNA template comprises an expression cassette comprising a eukaryotic promoter operably linked to a coding sequence of interest.

19. The process according to claim 18 , wherein said expression cassette is flanked on either side by a protelomerase target sequence.

20. A process according to claim 15 , which produces a linear double stranded expression cassette DNA that is covalently closed at both ends by hairpin loops.

21. The process of claim 15 , which further comprises purifying the linear double stranded DNA covalently closed at both ends by hairpin loops produced in (b).

22. A process according to claim 15 comprising formulating the resulting linear double stranded DNA covalently closed at both ends by hairpin loops with a pharmaceutically acceptable carrier or excipient to make a pharmaceutical composition comprising a linear double stranded DNA covalently closed at both ends by hairpin loops.

23. The process according to claim 17 , wherein said DNA template is incubated under denaturing conditions to form a closed circular DNA.

24. The process according to claim 15 , wherein said DNA template is amplified by polymerase chain reaction.

25. The process of claim 1 , wherein a DNA polymerase for the amplification is phi 29 of SEQ ID NO: 2 or a variant thereof which comprises a sequence having at least 95% identity to SEQ ID NO: 2 and said protelomerase is bacteriophage N15 TelN of SEQ ID NO: 15 or a variant thereof which comprises a sequence having at least 95% identity to SEQ ID NO: 15.

26. A process according to claim 25 , wherein the amplifying step occurs at a temperature of about 25 to about 35 degrees centigrade and wherein the producing step occurs at a temperature of about 25 to about 35 degrees centigrade.

27. A process according to claim 26 , wherein said protelomerase target sequence comprises the sequence of SEQ ID NO 25.

28. The process of claim 1 , wherein steps (a) and (b) occur simultaneously.

29. The process of claim 1 , wherein steps (a) and (b) occur consecutively.