Method of DNA synthesis
The present invention relates to an in vitro cell-free process for production of deoxyribonucleotides (DNAs) comprising at least one hairpin, corresponding DNA products and uses thereof, and oligonucleotides and kits useful in the process of the invention.
1. A single-stranded circular DNA comprising at least one hairpin, wherein the hairpin comprises a portion of a protelomerase target sequence, and wherein the portion is at least 14 bases in length.
2. The single-stranded circular DNA of claim 1 , wherein the single-stranded circular DNA molecule comprises a first hairpin comprising a portion of a first protelomerase target sequence and a second hairpin comprising:
(i) a portion of a second protelomerase target sequence at least 14 bases in length, wherein the first and second protelomerase target sequences are for the same or different protelomerases, or
(ii) neighbouring complementary sequences.
3. The single-stranded circular DNA of claim 1 , wherein said single-stranded circular DNA comprises a loop of single stranded DNA.
4. The single stranded circular DNA of claim 1 , wherein said single-stranded circular DNA comprises an aptamer.
5. The single stranded circular DNA of claim 1 wherein said single-stranded circular DNA encodes an antigen.
6. The single stranded circular DNA of claim 1 , wherein said single-stranded circular DNA includes a DNA sequence which encodes any one of a DNA aptamer, a protein, a peptide, or an RNA.
7. The single stranded circular DNA of claim 1 wherein said single-stranded circular DNA comprises an expression cassette comprising one or more promoter or enhancer elements and a gene or coding sequence.
8. The single stranded circular DNA of claim 2 , wherein said single-stranded sections of the circular DNA are separated by the hairpin structures do not anneal to form a double stranded structure.
9. The single stranded circular DNA of claim 1 , wherein said protelomerase target sequence is selected from bacteriophage N15 TelN of SEQ ID NO: 15 or a variant thereof, Agrobacterium tumefaciens TelA of SEQ ID NO: 20 or a variant thereof, or Vibrio parahaemolyticus plasmid Vp58.5 of SEQ ID NO: 47 or a variant thereof.
10. The single-stranded circular DNA of claim 1 , wherein the portion of the protelomerase target sequence is a palindrome.
11. The single-stranded circular DNA of claim 10 , wherein the palindrome is a perfect inverted repeat and is flanked by inverted repeat sequences on both sides.
12. A single-stranded circular DNA comprising at least one hairpin, wherein the hairpin comprises at least 14 consecutive bases of a protelomerase target sequence, the protelomerase target sequence being selected from bacteriophage N15 TelN of SEQ ID NO: 15, Agrobacterium tumefaciens TelA of SEQ ID NO: 20, or Vibrio parahaemolyticus plasmid Vp58.5 of SEQ ID NO: 47.
13. A single-stranded circular DNA comprising at least one hairpin, wherein the hairpin comprises a portion of a protelomerase target sequence, and wherein the portion is at least 14 bases in length and is a perfect inverted repeat.