Compositions and methods for producing single-stranded circular dna

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.

We claim: 
     
         1 . 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, wherein said hairpin primers comprise a hairpin-forming region capable of folding back on itself to form an intramolecular hairpin, and wherein at least one primer of said pair of hairpin primers comprises a 5′ terminal phosphate in the hairpin-forming region of said primer.   
     
     
         2 . The composition of  claim 1 , further comprising a polymerase. 
     
     
         3 . The composition of  claim 1 , further comprising a non-strand displacing polymerase. 
     
     
         4 . The composition of  claim 1 , further comprising a ligase enzyme. 
     
     
         5 . 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, wherein said hairpin primers comprise a hairpin-forming region capable of folding back on itself to form an intramolecular hairpin and wherein at least one primer of said pair of hairpin primers comprises a 5′ terminal phosphate in the hairpin-forming region of said primer.   
     
     
         6 . The system of  claim 5 , further comprising at least one reagent selected from the group consisting of: a polymerase, a non-strand displacing polymerase, and ligase. 
     
     
         7 . The composition of  claim 1 , wherein said target nucleic acid comprises DNA. 
     
     
         8 . The composition of  claim 3 , wherein said non-strand displacing polymerase lacks 5′-3′ exonuclease activity 
     
     
         9 . The composition of  claim 8 , wherein said non-strand displacing polymerase comprises T4 or T7 DNA polymerase. 
     
     
         10 . The composition of  claim 4 , wherein said ligase comprises T4,  T. aquaticus , or  E. coli  DNA ligase 
     
     
         11 . The system of  claim 6 , wherein said non-strand displacing polymerase lacks 5′-3′ exonuclease activity. 
     
     
         12 . The system of  claim 11 , wherein said non-strand displacing polymerase comprises T4 or T7 DNA polymerase. 
     
     
         13 . The system of  claim 6 , wherein said ligase comprises a DNA ligase. 
     
     
         14 . The system of  claim 13 , wherein said ligase comprises T4,  T. aquaticus , or  E. coli  DNA ligase. 
     
     
         15 . The system of  claim 6 , further comprising an exonuclease. 
     
     
         16 . The system of  claim 15 , wherein said exonuclease comprises exo III and/or exo VII. 
     
     
         17 . The system of  claim 6 , further comprising a thermal cycler.