Generation of single-strand circular DNA from linear self-annealing segments

Abstract

The present invention provides a method for the rapid simultaneous production of a plurality of single-stranded DNA circles having a predetermined size and nucleotide sequence using pre-designed hairpin oligonucleotides containing complementary sequences for directing ligation to form dumbbell-shaped monomers followed by heat denaturation to yield single-stranded DNA circles.

1 - 20 . (canceled)  
     
     
         21 . A method for simultaneous synthesis of multiple copies of different single-stranded circular DNA molecules, comprising: 
 (a) contacting a plurality of universal hairpin oligonucleotides with at least two different target hairpin oligonucleotides, wherein each said hairpin oligonucleotide contains a segment that does not hybridize with any segment of the same or another of said hairpin oligonucleotides and wherein each of said hairpin oligonucleotides comprises a single stranded terminal non-palindromic sequence (segment C) such that said segment C of each universal hairpin oligonucleotide is complementary to the segment C of each target hairpin oligonucleotide but said segment C of any target oligonucleotide is not complementary to segment C of the same or another target hairpin oligonucleotide, and wherein said contacting occurs under conditions promoting said contacting and promoting hybridization of said segments C,    (b) ligating the resulting hybridized hairpin oligonucleotides to form a population of monomers each comprising a duplex linear segment with a single-stranded loop at each end.    
     
     
         22 . The method of  claim 21  further comprising amplifying the monomers via rolling circle amplification.  
     
     
         23 . The method of  claim 22  wherein said duplex linear segment of step (b) comprises at least one endonuclease restriction site, the method further comprising contacting the product of rolling circle amplification with an endonuclease specific for said endonuclease restriction site to form a new set of hairpin oligonucleotides and then repeating steps (a) through (c) using said new set of hairpin oligonucleotides.  
     
     
         24 . The method of  claim 21  wherein the target hairpin oligonucleotides of step (a) are of different sizes.  
     
     
         25 . The method of  claim 21  wherein at least one of the hairpin oligonucleotides contains a pre-selected nucleotide sequence.  
     
     
         26 . The method of  claim 21  wherein said duplex linear segment of step (b) comprises at least one endonuclease restriction site.  
     
     
         27 . The method of  claim 26  wherein the endonuclease restriction site contains a phosphorothioate derivative.  
     
     
         28 . The process of  claim 21  wherein said restriction site is a BamHI restriction site and said endonuclease is Bam HI.  
     
     
         29 . The method of  claim 21  wherein the ligation step employs an enzyme.  
     
     
         30 . The method of  claim 29  wherein the enzyme is selected from the group consisting of T4 ligase, Ampligase and  E. coli  ligase.  
     
     
         31 . The method of  claim 21  further comprising ligation at a temperature of between 4° C. to 65° C.  
     
     
         32 . The method of  claim 21  wherein the ligation step is non-enzymatic.  
     
     
         33 . The process of  claim 32  wherein the ligation step requires the formation of a phosphorothioate derivative of a hairpin oligonucleotide.  
     
     
         34 . The method of  claim 21  wherein said double-stranded linear segment formed in step (b) comprises at least two endonuclease restriction sites.  
     
     
         35 . The method of  claim 21  wherein said single-stranded circular DNA molecules are produced in a ratio equal to that of the respective starting target hairpin oligonucleotides used to form said circular DNA molecules.  
     
     
         36 . The method of  claim 21  wherein at least 3 different target hairpin oligonucleotides are contacted with said universal hairpin oligonucleotides.  
     
     
         37 . The method of  claim 21  wherein at least 4 different target hairpin oligonucleotides are contacted with said universal hairpin oligonucleotides.  
     
     
         38 . A method for simultaneous synthesis of multiple copies of different single-stranded circular DNA molecules, comprising: 
 (a) contacting a plurality of universal hairpin oligonucleotides with at least two different target hairpin oligonucleotides, wherein each said hairpin oligonucleotide contains a segment that does not hybridize with any segment of the same or another of said hairpin oligonucleotides and wherein each of said hairpin oligonucleotides comprises a single stranded terminal non-palindromic sequence (segment C) such that said segment C of each universal hairpin oligonucleotide is complementary to the segment C of each target hairpin oligonucleotide but said segment C of any target oligonucleotide is not complementary to segment C of the same or another target hairpin oligonucleotide, and wherein said contacting occurs under conditions promoting said contacting and promoting hybridization of said segments C,    (b) ligating the resulting hybridized hairpin oligonucleotides to form a population of monomers each comprising a duplex linear segment with a single-stranded loop at each end,    (c) contacting said monomers with a plurality of deoxynucleoside triphosphates (dNTPs), multiple primer oligonucleotides comprising at least one oligonucleotide complementary to at least one segment of each of the monomers and a DNA polymerase capable of supporting rolling circle amplification (RCA), said contacting occurring under conditions promoting said contacting and promoting said rolling circle amplification to form an RCA product, thereby simultaneously generating multiple copies of different single-stranded circular DNA molecules.