Patent ID: 8278051
Filing Date: 2012-10-02
Classification: C12Q

Abstract:
1. A method of amplifying a selected nucleic acid molecule, the method comprising subjecting a reaction mixture to rolling circle amplification to perform simultaneously a formation of a single stranded circular DNA molecule which is a copy of the selected nucleic acid molecule and an amplification of the single stranded circular DNA molecule, said reaction mixture comprising (a) a first hairpin oligonucleotide which comprises a first loop region, a first double-stranded linear region, and a first single-stranded end sequence (“first overhang sequence”), (b) a second hairpin oligonucleotide which comprises a second loop region, a second double stranded linear region, and a second single-stranded end sequence (“second overhang sequence”), wherein the second overhang sequence of the second hairpin oligonucleotide is complementary to the first overhang sequence of the first hairpin oligonucleotide, (c) the selected nucleic acid molecule having a single-stranded end sequence complementary to the first or second overhang sequence; (d) a DNA ligase capable of ligating the first hairpin oligonucleotide to the second hairpin oligonucleotide and capable of ligating the selected nucleic acid molecule to at least the first or second hairpin oligonucleotide; (e) a double-stranded DNA-specific endonuclease capable of cleaving hairpin oligonucleotide dimers at the DNA sequence formed by the ligation of the first hairpin oligonucleotide and the second hairpin nucleotide; and the DNA sequence formed at the site of the ligation between the second hairpin oligonucleotide and the selected nucleic acid molecule does not generate a recognition site for the endonuclease of step (e) (f) a primer oligonucleotide which is complementary to one or more elements selected from the group consisting of the first hairpin oligonucleotide, the second hairpin oligonucleotide and the selected nucleic acid molecule; and (g) a DNA polymerase capable of polymerizing a nucleotide chain extending from the primer oligonucleotide during rolling circle amplification, wherein the single stranded circular DNA molecule is formed by ligating the first hairpin oligonucleotide and the second hairpin oligonucleotide to the selected nucleic acid molecule, and wherein the DNA sequence formed at the site of the ligation between the first hairpin oligonucleotide and the selected nucleic acid molecule does not generate a recognition site for the endonuclease of step (e).