Sequence-specific detection of nucleotide sequences

摘要

A method for detecting the presence of a target nucleotide sequence in a sample of DNA is described herein in which a test sample comprising single stranded DNA is exposed to a DNA probe and a nicking endonuclease under conditions that would permit sequence-specific hybridization of the probe to a complementary target sequence. The probe comprises a sequence complementary to the target sequence to be detected and this sequence also includes a recognition sequence for the nicking endonuclease. If the sample contains the target sequence, the probe hybridizes to the target and is cleaved by the nicking endonuclease, which leaves the target intact. Observing the presence of probe cleaved by the nicking endonuclease indicates the presence of the target nucleotide sequence in the sample of DNA.

主权项

1. A method for detecting the presence of a first target nucleotide sequence in a sample of single stranded DNA, comprising;
(a) exposing the sample to a first full-length DNA probe molecule and a first nicking endonuclease under conditions that would permit sequence-specific hybridization of the first full-length probe molecule to the first target nucleotide sequence, wherein the first full-length probe molecule comprises a sequence complementary to the first target nucleotide sequence that also includes a first recognition sequence for the first nicking endonuclease with complete complementarity between the recognition sequence and the first target nucleotide sequence, whereby a first recognition site is created for the first nicking endonuclease, whereby the first full-length DNA probe molecule is cleaved into a pair of first cleaved DNA probe molecules and the first target nucleotide sequence remains intact; (b) disassociating the pair of the first cleaved DNA probe molecules from the first target nucleotide sequence, whereby the first target nucleotide sequence is free for hybridization; (c) repeating steps (a) and (b) once or more, whereby two or more pairs of the first cleaved DNA molecules are dissociated from the first target nucleotide sequence; and (d) detecting the dissociated two or more pairs of the first cleaved DNA probe molecules, wherein the presence of the dissociated two or more pairs of the first cleaved DNA probe molecules indicates the presence of the first target nucleotide sequence in the sample.