Patent ID: 7041480

Claim:
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) denaturing said monomers to form single-stranded circles, (d) contacting said single stranded circles of step (c) 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 single stranded circles formed in step (c) 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.