Patent ID: 8143006

Claim:
A method for amplification and detection of a nucleic acid sequence, comprising: a) providing a reaction mixture comprising at least one target nucleic acid sequence having an amplifiable target sequence, a forward external nick-directing oligonucleotide primer (ND-primer) and a reverse external ND-primer, the external primers suitable to hybridize to the target nucleic acid sequence at nucleotide positions external to the amplifiable target sequence, at least one internal ND-primer suitable to hybridize to an external ND-primer extension product comprising the amplifiable target sequence or a portion thereof, but lacking a respective ND primer sequence or a portion thereof, a strand-displacing DNA polymerase suitable for primer extension of the hybridized primers, to provide respective primer-extension products, a nick-directing endonuclease suitable for strand-specific cleavage of the ND-primer-extension products to provide for primer-extension products lacking the respective ND-primers or portions thereof, a cleavable FRET probe, and a mixture of deoxynucleoside 5′-triphosphates; b) incubating the reaction mixture in the presence of reagents, and under reaction conditions suitable to support primer hybridization, DNA polymerase-mediated primer extension and strand displacement, and nick-directing endonuclease-mediated strand-specific cleavage of the extension products, to provide for amplification of the amplifiable target sequence, wherein the amplification comprises primer extension, by least one internal ND-primer, of an external ND-primer extension product comprising the amplifiable target sequence or a portion thereof but lacking the respective external ND-primer sequence or a portion thereof; and c) detecting, based on the cleavable FRET probe, the amplified target sequence or portion thereof.