Patent ID: 6458535
Filing Date: 2002-10-01
Classification: C12N,C12Q

Abstract:
A method of detecting the cleavage of nucleic acid cleavage structures comprising:a) providing: i) a cleavage agent; ii) a first target nucleic acid, said first target nucleic acid comprising a first region and a second region, said second region downstream of and contiguous to said first region; iii) a first oligonucleotide, wherein at least a section of said first oligonucleotide is completely complementary to said first region of said first target nucleic acid; iv) a second oligonucleotide comprising a 3â€² portion and a 5â€² portion, wherein said 5â€² portion is completely complementary to said second region of said first target nucleic acid; v) a second target nucleic acid, said second target nucleic acid comprising a first region and a second region, said second region downstream of and contiguous to said first region; and vi) a third oligonucleotide, wherein at least a portion of said third oligonucleotide is completely complementary to said first region of said second target nucleic acid; b) generating a first cleavage structure wherein at least a portion of said section of said first oligonucleotide is annealed to said first region of said first target nucleic acid to form a double-stranded region and wherein at least said 5â€² portion of said second oligonucleotide is annealed to said second region of said first target nucleic acid and wherein cleavage of said first cleavage structure within said double-stranded region occurs via said cleavage agent thereby cleaving said first oligonucleotide to generate a fourth oligonucleotide, said fourth oligonucleotide comprising a 3â€² portion and a 5â€² portion, wherein said 5â€² portion is completely complementary to said second region of said second target nucleic acid; c) generating a second cleavage structure under conditions wherein at least said portion of said third oligonucleotide is annealed to said first region of said second target nucleic acid and wherein at least said 5â€² portion of said fourth oligonucleotide is annealed to said second region of said second target nucleic acid and wherein cleavage of said second cleavage structure occurs to generate a cleavage fragment; and d) detecting the cleavage of said second cleavage structure.