Patent ID: 8470533
Filing Date: 2013-06-25
Classification: C12Q

Abstract:
1. A method for determining whether a target RNA or a cDNA of said target RNA is present in a sample, said method comprising: (a) contacting a sample which may contain a target RNA or a cDNA of said target RNA with a probe nucleic acid and forming a first solution mixture, wherein the probe nucleic acid is attached to an amplifying restriction endonuclease and comprises a nucleotide sequence complementary to a sequence of said target RNA or a cDNA of said target RNA, if said target RNA or said cDNA is present in said sample, at least a portion of said target RNA or said cDNA hybridizes to at least a portion of said probe nucleic acid to form a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site, (b) contacting said first solution mixture with a recognition restriction endonuclease having the ability to cut said restriction endonuclease cut site of said double-stranded portion of nucleic acid and forming a second solution mixture, wherein said recognition restriction endonuclease cleaves said double-stranded portion of nucleic acid, thereby separating a portion of said probe nucleic acid with attached said amplifying restriction endonuclease from at least another portion of said probe nucleic acid and forming a reaction product comprising said portion of said probe nucleic acid with attached said amplifying restriction endonuclease if said target RNA or said cDNA is present in said sample, (c) contacting said second solution mixture with a first nucleic acid, wherein the first nucleic acid is attached to an amplifying restriction endonuclease and comprises a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site of the amplifying restriction endonuclease of said portion of said probe nucleic acid and forming a third solution mixture, wherein the amplifying restriction endonuclease of said portion of said probe nucleic acid cleaves said first nucleic acid, thereby separating a portion of said first nucleic acid with attached said amplifying restriction endonuclease from at least another portion of said first nucleic acid and forming a reaction product comprising said portion of said first nucleic acid with attached said amplifying restriction endonuclease if said target RNA or said cDNA is present in said sample, (d) contacting said third solution mixture with a second nucleic acid, wherein the second nucleic acid is attached to an amplifying restriction endonuclease and comprises a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site of the amplifying restriction endonuclease of said portion of said first nucleic acid and forming a fourth solution mixture, wherein the amplifying restriction endonuclease of said portion of said first nucleic acid cleaves said second nucleic acid, thereby separating a portion of said second nucleic acid with attached said amplifying restriction endonuclease from at least another portion of said second nucleic acid and forming reaction products comprising said portion of said second nucleic acid with attached said amplifying restriction endonuclease and said portion of said first nucleic acid with attached said amplifying restriction endonuclease if said target RNA or said cDNA is present in said sample, (e) contacting said fourth solution mixture with a reporter nucleic acid comprising a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site of the amplifying restriction endonuclease of said portion of said second nucleic acid or a restriction endonuclease cut site of the amplifying restriction endonuclease of said portion of said first nucleic acid and forming a fifth solution mixture, wherein the amplifying restriction endonuclease of said portion of said second nucleic acid or the amplifying restriction endonuclease of said portion of said first nucleic acid cleaves said reporter nucleic acid, thereby forming a cleaved reporter nucleic acid if said target RNA or said cDNA is present in said sample, and (f) determining the presence or absence of said cleaved reporter nucleic acid, wherein the presence of said cleaved reporter nucleic acid in the fifth solution mixture indicates that said target RNA or said cDNA is present in said sample, and wherein the absence of said cleaved reporter nucleic acid indicates that said target RNA or said cDNA is not present in said sample.