PRODUCTION OF CLOSED LINEAR DNA
An in vitro process for the production of closed linear deoxyribonucleic acid (DNA) comprises (a) contacting a DNA template comprising at least one protelomerase target sequence with at least one DNA polymerase in the presence of one or more primers under conditions promoting amplification of the template; and (b) contacting amplified DNA produced in (a) with at least one protelomerase under conditions promoting production of closed linear DNA. A kit provides components necessary in the process.
1 . A kit suitable for use in an in vitro cell-free process for production of a closed linear deoxyribonucleic acid (DNA), comprising at least one DNA polymerase and at least one protelomerase.
2 . The kit according to claim 1 , wherein said DNA polymerase is a strand displacement-type DNA polymerase.
3 . The kit according to claim 2 , wherein said DNA polymerase is a rolling circle amplification (RCA) DNA polymerase.
4 . The kit according to claim 3 , wherein said DNA polymerase is phi29 of SEQ ID NO: 2 or a variant thereof which comprises at least 80% identity to full-length SEQ ID NO: 2.
5 . The kit according to claim 1 , wherein said protelomerase is bacteriophage N15 TelN of SEQ ID NO: 15 or a variant thereof which comprises at least 80% identity to full-length SEQ ID NO:15.
6 . The kit according to claim 1 , further comprising dNTPs, suitable buffers and one or more primers.
7 . The kit according to claim 6 , wherein said primers are random primers.
8 . The kit according to claim 6 , wherein said primers are specific primers.
9 . The kit according to claim 6 , wherein said primers comprise chemically modified nucleotides.
10 . The kit according to claim 1 , further comprising a pyrophosphatase.
11 . Concatameric DNA comprising multiple repeats of a DNA template, said DNA template comprising at least one protelomerase target sequence.
12 . The concatameric DNA of claim 11 , wherein said DNA template comprises one or more expression cassettes flanked on either side by protelomerase target sequence.
13 . The concatameric DNA of claim 12 , wherein said expression cassette comprises a eukaryotic promoter operably linked to a sequence encoding an mRNA or protein.
14 . The concatameric DNA of claim 12 , wherein said expression cassette further comprises a eukaryotic transcription termination sequence.
15 . The concatameric DNA of claim 12 , wherein the expression cassette lacks one or more bacterial or vector sequences selected from the group consisting of:
(i) bacterial origins of replication;
(ii) bacterial selection markers; and
(iii) unmethylated CpG motifs.
16 . The concatameric DNA of claim 11 , wherein the concatamer comprises 10 or more units of amplified DNA template.
17 . The concatameric DNA of claim 11 , wherein the concatamer is at least 5 kb in size.
18 . The concatameric DNA of claim 11 , wherein said DNA is linear single stranded DNA with multiple repeats of template DNA.
19 . The concatameric DNA of claim 11 , wherein said DNA is in vitro.
20 . The concatameric DNA of claim 11 , wherein said DNA template comprises more than one protelomerase target sequence.