Assay, reagent and kit employing nucleic acid strand displacement and restriction endonuclease cleavage

摘要

A target nucleotide sequence having a half-restriction site is determined in the nucleic acid of a biological sample. It is contacted with a reagent complex of: (i) a labeled probe polynucleotide containing a target binding region which is substantially complementary to the target nucleotide sequence and which contains a unique half-restriction site completely complementary to the half-restriction site of the target nucleotide sequence; and (ii) a second polynucleotide hybridized to the labeled probe polynucleotide in at least a portion of the target binding region, the portion including the unique half-restriction site of the target binding region; The second polynucleotide contains at least one mismatched or unpaired nucleotide opposite to the unique half-restriction site of the labeled probe polynucleotide, whereby a restriction enzyme specific for the unique restriction site will not cleave the reagent complex. The target nucleotide sequence, if present, displaces the second polynucleotide from the target nucleotide region and forms the unique restriction site in double-stranded form. The labeled probe polynucleotide is then cleaved at the unique restriction site in double-stranded form to form labeled cleavage fragment, which is detected.