COMPOSITIONS AND METHODS FOR PRODUCING SINGLE-STRANDED CIRCULAR DNA

• US 20120164691A1
• Filed: 12/27/2011
• Published: 06/28/2012
• Est. Priority Date: 12/27/2010
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Eshoo, Mark W., Picuri, John
• Granted Patent: US 9,487,807 B2
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]

Abstract

The present invention provides methods, kits, and compositions for producing single-stranded circular DNA by PCR. In particular, hairpin primers are provided, and methods of use thereof to produce single-stranded circular DNA molecules.

Claims

1. A method of producing single-stranded circular DNA molecules comprising the steps of:

(a) annealing a pair of hairpin primers to a target nucleic acid, wherein said hairpin primers comprise a target-hybridization region complementary to a sequence of said target nucleic acid, wherein said hairpin primers are configured to hybridize to opposing strands at different ends of said target nucleic acid in such a manner as to prime polymerization along each strand, and wherein said hairpin primers comprise a hairpin-forming region capable of folding back on itself to form an intramolecular hairpin;

(b) amplifying said target nucleic acid with said hairpin primers to produce a duplex of amplicon sequences, wherein said amplicon sequences comprise target sequence flanked on both ends by hairpin primer sequences;

(c) altering the solution conditions to promote denaturation of said duplex and formation of said intramolecular hairpins;

(d) treating said amplicons with non-strand displacing polymerase to extend amplicon sequence and ligase to unite the 5′ and

3′

ends of the extended sequence.

2. The method of claim 1, wherein said target nucleic acid comprises DNA.

3. The method of claim 1, wherein said amplifying is by polymerase chain reaction.

4. The method of claim 1, wherein altering the solution conditions comprises the steps of:

(i) applying denaturing conditions;

(ii) altering said solution conditions to favor intramolecular interactions over intermolecular interactions; and

(iii) applying reannealing condition.

5. The method of claim 4, wherein denaturing conditions comprise heating above the T_m of said duplex.

6. The method of claim 4, wherein solution conditions that favor intramolecular interactions over intermolecular interactions comprise more dilute conditions.

7. The method of claim 4, wherein reannealing conditions comprise controlled cooling below the T_m of said intramolecular hairpins.

8. The method of claim 1, wherein said non-strand displacing polymerase comprises a DNA polymerase.

9. The method of claim 8, wherein said non-strand displacing polymerase lacks 5′

to 3′

exonuclease activity.

10. The method of claim 9, wherein said non-strand displacing polymerase comprises T4 or T7 DNA polymerase.

11. The method of claim 1, wherein said non-strand displacing polymerase extends the amplicon sequence from the 3′

end of the hairpin primer until the extended 3′

end reaches the 5′

end of the other hairpin primer, using the target-hybridization regions of the hairpin primers and the target sequence as a template.

12. The method of claim 1, wherein said ligase comprises a DNA ligase.

13. The method of claim 12, wherein said ligase comprises T4, T. aquaticus, or E. coli DNA ligase.

14. The method of claim 1, wherein said ligase repairs the nick between the 3′

end and the 5′

end of said extended sequence.

15. A composition comprising:

a) a target nucleic acid, and

b) a pair of hairpin primers, wherein said hairpin primers comprise a target-hybridization region complementary to a sequence of said target nucleic acid, wherein said hairpin primers are configured to hybridize to opposing strands at different ends of said target nucleic acid in such a manner as to prime polymerization along each strand, and wherein said hairpin primers comprise a hairpin-forming region capable of folding back on itself to form an intramolecular hairpin.

16. The composition of claim 15, further comprising a polymerase.

17. The composition of claim 15, further comprising a non-strand displacing polymerase.

18. The composition of claim 15, further comprising a ligase enzyme.

19. A system comprising:

a) a target nucleic acid, and

b) a pair of hairpin primers, wherein said hairpin primers comprise a target-hybridization region complementary to a sequence of said target nucleic acid, wherein said hairpin primers are configured to hybridize to opposing strands at different ends of said target nucleic acid in such a manner as to prime polymerization along each strand, and wherein said hairpin primers comprise a hairpin-forming region capable of folding back on itself to form an intramolecular hairpin.

20. The system of claim 19, further comprising at least one reagent selected from the group consisting of:

a polymerase, a non-strand displacing polymerase, and ligase.