Production of closed linear dna

Abstract

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.