Patent CA-3094983-A1 (CA)
Title: Methods using enzymatic amplification cascades for detecting target nucleic acid in a sample
Inventor: SMITH KENNETH D
IPC: C12Q1/6876
Filed: 2009-08-04
Abstract: ABSTRACT  A method for assessing a sample for target nucleic acid, said method comprising: (a) contacting said sample with a probe nucleic acid comprising an initial amplifying restriction endonuclease and a nucleotide sequence complementary to a sequence of said target nucleic acid under conditions wherein, if said target nucleic acid is present in said sample, at least a portion of said target nucleic acid 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 double-stranded portion of nucleic acid with a recognition restriction endonuclease having the ability to cut said double-stranded portion of nucleic acid at said restriction endonuclease cut site under conditions wherein said recognition restriction endonuclease cleaves said double-stranded portion of nucleic acid at said restriction endonuclease cut site, thereby separating a portion of said probe nucleic acid comprising said initial amplifying restriction endonuclease from at least another portion of said probe nucleic acid, (c) contacting said portion of said probe nucleic acid cornprising said initial amplifying restriction endonuclease with a first reporter nucleic acid comprising a secondary amplifying restriction endonuclease and a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site of said initial amplifying restriction endonuclease under conditions wherein said initial amplifying restriction endonuclease cleaves said first reporter nucleic acid at said restriction endonuclease cut site of said initial amplifying restriction endonuclease, thereby separating a portion of said first nucleic acid cornprising said secondary amplifying restriction endonuclease from at least another portion of said first nucleic acid, (d) contacting said portion of said first reporter nucleic acid comprising said secondary arnplifying restriction endonuclease with a second reporter nucleic acid comprising said initial amplifying restriction endonuclease and a double-stranded portion of nucleic acid comprising a restriction endonuclease cut site of said secondary amplifying restriction endonuclease under conditions wherein said initial arnplifying restriction endonuclease cleaves said second nucleic acid at said restriction endonuclease cut site of said secondary amplifying restriction endonuclease, thereby separating a portion of said second nucleic acid comprising said initial amplifying restriction endonuclease from at least another portion of said second nucleic acid, and (e) determining the presence or absence of said portion of said first reporter nucleic acid, said second reporter nucleic acid, or both said first reporter nucleic acid and said second reporter nucleic acid, wherein said presence indicates that said sample contains said target nucleic acid, and wherein said absence indicates that said sample does not contain said target nucleic acid. Date Recue/Date Received 2020-10-01