Patent ID: 6498023
Filing Date: 2002-12-24
Classification: C12N,C12Q

Abstract:
A process for preparing multiple copies of single-stranded circular DNA, comprising:(a) contacting at least two 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 at least two hairpin oligonucleotides comprises a single stranded terminal sequence complementary to that of the other hairpin oligonucleotide, and under conditions permitting said complementary terminal sequences to hybridize, (b) ligating the resulting hybridized hairpin oligonucleotides to form a monomer comprising a duplex linear segment containing at least one endonuclease restriction site with a single-stranded hairpin loop at each end, (c) denaturing said monomer to form a single-stranded circle, (d) contacting said single stranded circle of (c) with a plurality of deoxynucleoside triphosphates (dNTPs), at least one primer oligonucleotide complementary to at least one segment of the single stranded circle formed in step (c) and a DNA polymerase capable of supporting rolling circle amplification (RCA) under conditions promoting the formation of RCA product, (e) contacting said RCA product of (d) with an endonuclease specific for said endonuclease restriction site of (b) to form a new set of hairpin oligonucleotides, and (f) repeating steps (a) through (c) using the hairpin oligonucleotides formed in step (e), thereby generating multiple copies of single-stranded circular DNA.