Nova Patents
CA2284552C

Detection of nucleic acids by multiple sequential invasive cleavages

Abstract

The present invention relates to means for the detection and characterizatio n of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure - specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for th e separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specifi c methods for the detection of nucleic acid from various viruses in a sample.

CA2284552C, drawing sheet 1
Sheet 1 of 139

Term

Term ended

Expired 24 March 2018, 8.5 years ago.

  1. Priority
  2. Filed
  3. Granted
  4. Expired
  5. Today

113 claims: 15 independent, 98 dependent

  1. 1
    CA 02284552 2007-08-14 74667-130 CLAIMS :1. A method of detecting the presence of a target nucleic acid molecule comprising: a) providing: 5 i) a cleavage means, ii) a source of a first target nucleic acid, said first target nucleic acid having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region 10 and wherein said second region is located adjacent to and downstream from said third region;iii) a first oligonucleotide having a 5' and a 3' portion wherein said 5' portion of said first oligonucleotide contains a sequence complementary to said 15 second region of said first target nucleic acid and wherein said 3' portion of said first oligonucleotide contains a sequence complementary to said third region of said first target nucleic acid;iv) a second oligonucleotide having a 5' and a 3' 20 portion wherein said 5' portion of said second oligonucleotide contains a sequence complementary to said first region of said first target nucleic acid and wherein said 3' portion of said second oligonucleotide contains a sequence complementary to said second region of said first 25 target nucleic acid;v) a source of a second target nucleic acid, said second target nucleic acid having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region 369 CA 02284552 2007-08-14 74667-130 and wherein said second region is located adjacent to and downstream from said third region;vi) a third oligonucleotide having a 5' and a 3' portion wherein said 5' portion of said third 5 oligonucleotide contains a sequence complementary to said second region of said second target nucleic acid and wherein said 3' portion of said third oligonucleotide contains a sequence complementary to said third region of said second target nucleic acid;10 b) generating a first cleavage structure wherein at least said 3' portion of said first oligonucleotide is annealed to said first target nucleic acid and wherein at least said 5' portion of said second oligonucleotide is annealed to said first target nucleic acid and wherein cleavage of said first 15 cleavage structure occurs via said cleavage means thereby cleaving said first oligonucleotide to generate a fourth oligonucleotide, said fourth oligonucleotide having a 5' and a 3' portion wherein said 5' portion of said fourth oligonucleotide contains a sequence complementary to said 20 first region of said second target nucleic acid and wherein said 31 portion of said fourth oligonucleotide contains a sequence complementary to said second region of said second target nucleic acid;c) generating a second cleavage structure under conditions 25 wherein at least said 3' portion of said third oligonucleotide is annealed to said second target nucleic acid and wherein at least said 5' portion of said fourth oligonucleotide is annealed to said second target nucleic acid oligonucleotide and wherein cleavage of said second 30 cleavage structure occurs to generate a fifth 370 CA 02284552 2007-08-14 74667-130 oligonucleotide, said fifth oligonucleotide having a 3'hydroxyl group;and d) detecting said fifth oligonucleotide thereby detecting the presence of said target nucleic acid molecule. 5 2. The method of Claim 1, wherein said first oligonucleotide has a length between eleven and fifteen nucleotides . 3. The method of Claim 1, wherein said cleavage means is a structure-specific nuclease. 10 4. The method of Claim 3, wherein said structurespecific nuclease is a thermostable structure-specific nuclease. 5. The method of Claim 4, wherein said thermostable structure-specific nuclease is a Pyrococcus woesii FEN-1 15 endonuclease. 6. The method of Claim 1, wherein one or more of said first, second, and said third oligonucleotides contain a dideoxynucleotide at the 3' terminus. 7. The method of Claim 1, wherein detecting said 20 fifth oligonucleotide comprises: a) incubating said fifth oligonucleotide with a templateindependent polymerase and at least one labelled nucleoside triphosphate under conditions such that at least one labelled nucleotide is added to the 3'-hydroxyl group of 25 said fifth oligonucleotide to generate a labelled fifth oligonucleotide;and b) detecting the presence of said labelled fifth oligonucleotide. 371 CA 02284552 2007-08-14 74667-130 8. The method of Claim 7, wherein said templateindependent polymerase is selected from the group consisting of terminal deoxynucleotidyl transferase and poly A polymerase. 5 9. The method of Claim 8, wherein said third oligonucleotide contains a 5' end label, said 5' end label being a different label than the label present upon said labelled nucleoside triphosphate. 10. The method of Claim 1, wherein detecting said 10 fifth oligonucleotide comprises: a) incubating said fifth oligonucleotide with a templateindependent polymerase and at least one nucleoside triphosphate under conditions such that at least one nucleotide is added to the 31-hydroxyl group of said fifth 15 oligonucleotide to generate a tailed oligonucleotide;and b) detecting the presence of said tailed fifth oligonucleotide . 11. The method of Claim 10, wherein said templateindependent polymerase is selected from the group consisting 20 of terminal deoxynucleotidyl transferase and poly A polymerase. 12. The method of Claim 11, wherein said third oligonucleotide contains a 5' end label. 13. The method of Claim 1, wherein detecting said 25 fifth oligonucleotide comprises: a) providing: i) said fifth oligonucleotide;372 CA 02284552 2007-08-14 74667-130 ii) a composition comprising two single-stranded nucleic acids annealed so as to define a single-stranded portion of a protein binding region;iii) a DNA or RNA polymerase;5 b) exposing said fifth oligonucleotide to said singlestranded portion of said protein binding region under conditions such that at least a portion of said fifth oligonucleotide anneals to at least a portion of said single-stranded portion of said protein binding region to 10 form a duplex protein binding region and said DNA or RNA polymerase binds to said duplex protein binding region and produces nucleic acid that is detected, thereby detecting the presence of said fifth oligonucleotide. 14. The method of Claim 13, wherein said single15 stranded portion of said protein binding region comprises: a) a first single continuous strand of nucleic acid comprising a sequence defining the template strand of an RNA polymerase binding region;and b) a second single continuous strand of nucleic acid having 20 a 5' and a 3' end, said second nucleic acid comprising a region complementary to a portion of said first nucleic acid, wherein said second nucleic acid is annealed to said first nucleic acid so as to define said single-stranded portion of said protein binding region. 25 15. The method of Claim 14, wherein said protein binding region is a template-dependent RNA polymerase binding region. 373 CA 02284552 2007-08-14 74667-130 16. The method of Claim 15, wherein said templatedependent RNA polymerase binding region is a T7 RNA polymerase binding region. 17. The method of Claim 1, wherein detecting said 5 fifth oligonucleotide comprises: a) providing: i) said fifth oligonucleotide;ii) a single continuous strand of nucleic acid comprising a sequence defining a single strand of an RNA 10 polymerase binding region;iii) a tempiate-dependent DNA polymerase;iv) a template-dependent RNA polymerase;b) exposing said fifth oligonucleotide to said RNA polymerase binding region under conditions such that said 15 fifth oligonucleotide binds to a portion of said single strand of said RNA polymerase binding region;c) exposing said bound fifth oligonucleotide to said template-dependent DNA polymerase under conditions such that a double-stranded RNA polymerase binding region is produced;
  2. 2
    2 0 and d) exposing said double-stranded RNA polymerase binding region to said template-dependent RNA polymerase under conditions such that RNA transcripts are produced and detected, thereby detecting the presence of said fifth 25 oligonucleotide. 18. The method of Claim 17, wherein said templatedependent RNA polymerase is a T7 RNA polymerase. 374 CA 02284552 2007-08-14 74667-130 19. A method of detecting the presence of a target nucleic acid molecule comprising:a) providing: i) a cleavage means,
  3. 3
    5 ii) a source of a first target nucleic acid, said first target nucleic acid having a first region, a second region, a third region and a fourth region, wherein said first region is located adjacent to and downstream from said second region, said second region is located adjacent to and
  4. 4
    10 downstream from said third region and said third region is located adjacent to and downstream from said fourth region; iii) a first oligonucleotide complementary to said fourth region of said first target nucleic acid; iv) a second oligonucleotide having a 5' portion 15 and a 3' portion wherein said 5' portion of said second oligonucleotide contains a sequence complementary to said second region of said first target nucleic acid and wherein said 31 portion of said second oligonucleotide contains a sequence complementary to said third region of said first 20 target nucleic acid; v) a third oligonucleotide having a 5' and a 3' portion wherein said 5' portion of said third oligonucleotide contains a sequence complementary to said first region of said first target nucleic acid and wherein 25 said 3' portion of said third oligonucleotide contains a sequence complementary to said second region of said first target nucleic acid; vi) a source of a second target nucleic acid, said second target nucleic acid having a first region, a second 375 CA 02284552 2007-08-14 74667-130 region and a third region, wherein said first region is located adjacent to and downstream from said second region and wherein said second region is located adjacent to and downstream from said third region; vii) a fourth oligonucleotide having a 5' and a 3' portion wherein said 5' portion of said fourth oligonucleotide contains a sequence complementary to said second region of said second target nucleic acid and wherein said 31 portion of said fourth oligonucleotide contains a sequence complementary to said third region of said second target nucleic acid; b) generating a first cleavage structure wherein said first oligonucleotide is annealed to said fourth region of said first target nucleic acid and wherein at least said 3' portion of said second oligonucleotide is annealed to said first target nucleic acid and wherein at least said 5' portion of said third oligonucleotide is annealed to said first target nucleic acid and wherein cleavage of said first cleavage structure occurs thereby cleaving said second oligonucleotide to generate a fifth oligonucleotide, said fifth oligonucleotide having a 5' and a 3' portion wherein said 5' portion of said fifth oligonucleotide contains a sequence complementary to said first region of said second target nucleic acid and wherein said 3' portion of said fifth oligonucleotide contains a sequence complementary to said second region of said second target nucleic acid; c) generating a second cleavage structure under conditions wherein at least said 3' portion of said fourth oligonucleotide is annealed to said second target nucleic acid and wherein at least said 5' portion of said fifth oligonucleotide is annealed to said second target nucleic 376 CA 02284552 2007-08-14 74667-130 acid and wherein cleavage of said second cleavage structure occurs to generate a sixth oligonucleotide, said sixth oligonucleotide having a 3'-hydroxyl group; and d) detecting said sixth oligonucleotide thereby detecting 5 the presence of said target nucleic acid molecule. 20. A method of detecting the presence of a target nucleic acid molecule comprising:a) providing: i) a cleavage means, 10 ii) a source of a target nucleic acid, said target nucleic acid having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region and wherein said second region is located adjacent to and downstream from
  5. 5
    15 said third region;iii) a first oligonucleotide having a 5' and a 3' portion wherein said 5' portion of said first oligonucleotide contains a sequence complementary to said second region of said target nucleic acid and wherein said
  6. 6
    20 5' portion of said first oligonucleotide contains a region of self-complementarity and wherein said 3' portion of said first oligonucleotide contains a sequence complementary to said third region of said target nucleic acid;iv) a second oligonucleotide having a 5' and a 3' 25 portion wherein said 5' portion of said second oligonucleotide contains a sequence complementary to said first region of said target nucleic acid and wherein said 3' portion of said second oligonucleotide contains a sequence 377 CA 02284552 2007-08-14 74667-130 complementary to said second region of said target nucleic acid;v) a third oligonucleotide having a 5' and a 3' portion wherein said 3' portion of said third oligonucleotide contains a sequence complementary to said 5' portion of said first oligonucleotide;b) generating a first cleavage structure wherein at least said 3' portion of said first oligonucleotide is annealed to said target nucleic acid and wherein at least said 5' portion of said second oligonucleotide is annealed to said target nucleic acid and wherein cleavage of said first cleavage structure occurs thereby cleaving said first oligonucleotide to generate a fourth oligonucleotide, said fourth oligonucleotide having a first region, a second region and a third region, wherein said first region is located adjacent to and upstream of said second region and wherein said second region is located adjacent to and upstream of said third region and wherein said third region of said fourth oligonucleotide contains a region of selfcomplementarity;c) generating a second cleavage structure under conditions wherein at least said 3' portion of said third oligonucleotide is annealed to said first region of said fourth oligonucleotide and wherein said third region of said fourth oligonucleotide forms a hairpin structure and wherein cleavage of said second cleavage structure occurs to generate a fifth oligonucleotide, said fifth oligonucleotide having a 3'-hydroxyl group;and d) detecting said fifth oligonucleotide thereby detecting the presence of said target nucleic acid molecule. 378 CA 02284552 2007-08-14 74667-130
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    21. A method of detecting the presence of human cytomegalovirus nucleic acid in a sample comprising:a) providing: i) a cleavage means, 5 ii) a sample suspected of containing human cytomegalovirus target nucleic acid, said target nucleic acid having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region and wherein said second 10 region is located adjacent to and downstream from said third region;iii) a first oligonucleotide having a 5' and a 3' portion wherein said 5' portion of said first oligonucleotide contains a sequence complementary to said 15 second region of said target nucleic acid and wherein said 3' portion of said first oligonucleotide contains a sequence complementary to said third region of said target nucleic acid;iv) a second oligonucleotide having a 5' and a 3' 20 portion wherein said 5' portion of said second oligonucleotide contains a sequence complementary to said first region of said target nucleic acid and wherein said 3' portion of said second oligonucleotide contains a sequence complementary to said second region of said target nucleic 25 acid;b) generating a cleavage structure wherein at least said 3' portion of said first oligonucleotide is annealed to said target nucleic acid and wherein at least said 5 ' portion of said second oligonucleotide is annealed to said target 30 nucleic acid and wherein cleavage of said cleavage structure 379 CA 02284552 2007-08-14 74667-130 occurs via said cleavage means to generate non-target cleavage products, each non-target cleavage product having a 3' hydroxyl group;and c) detecting said non-target cleavage products and thereby 5 detecting the presence of human cytomegalovirus nucleic acid in said sample.
  8. 19
    33. A method of detecting the presence of human cytomegalovirus nucleic acid in a sample comprising:15 a) providing: i) a cleavage means, ii) a sample suspected of containing human cytomegalovirus target nucleic acid, said target nucleic acid having a first region, a second region, a third region 20 and a fourth region, wherein said first region is located adjacent to and downstream from said second region, said second region is located adjacent to and downstream from said third region and said third region is located adjacent to and downstream from said fourth region;25 iii) a first oligonucleotide complementary to said fourth region of said target nucleic acid;iv) a second oligonucleotide having a 5' portion and a 3' portion wherein said 5' portion of said second 383 CA 02284552 2007-08-14 74667-130 oligonucleotide contains a sequence complementary to said second region of said target nucleic acid and wherein said 3' portion of said second oligonucleotide contains a sequence complementary to said third region of said target 5 nucleic acid;v) a third oligonucleotide having a 5' portion and a 3' portion wherein said 5' portion of said third oligonucleotide contains a sequence complementary to said first region of said target nucleic acid and wherein said 3' 10 portion of said third oligonucleotide contains a sequence complementary to said second region of said target nucleic acid;b) generating a cleavage structure wherein said first oligonucleotide is annealed to said fourth region of said 15 target nucleic acid and wherein at least said 3' portion of said second oligonucleotide is annealed to said target nucleic acid and wherein at least said 5' portion of said third oligonucleotide is annealed to said target nucleic acid and wherein cleavage of said cleavage structure occurs 20 via said cleavage means to generate non-target cleavage products, each non-target cleavage product having a 3' hydroxyl group;and detecting said non-target cleavage products and thereby detecting the presence of human cytomegalovirus nucleic acid in said sample. 25
  9. 20
    34. A method of detecting the presence of a target nucleic acid molecule comprising:a) providing: i) a cleavage means, ii) a source of a first target nucleic acid, said 30 first target nucleic acid having a first region, a second 384 CA 02284552 2007-08-14 74667-130 region and a third region, wherein said first region is located adjacent to and downstream from said second region and wherein said second region is located adjacent to and downstream from said third region;5 iii) first and second oligonucleotides having 3' and 5' portions, wherein said 3' portion of said first oligonucleotide contains a sequence complementary to said third region of said target nucleic acid and wherein said 51 portion of said first oligonucleotide and said 3' portion of 10 said second oligonucleotide each contain sequence full complementary to said second region of said target nucleic acid, and wherein said 5' portion of said second oligonucleotide contains sequence complementary to said first region of said target nucleic acid;15 iv) a source of a second target nucleic acid, said second target nucleic acid having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region and wherein said second region is located adjacent to and 20 downstream from said third region;v) a third oligonucleotide having a 5' and a 3' portion wherein said 5' portion of said third oligonucleotide contains a sequence complementary to said second region of said second target nucleic acid and wherein 25 said 3' portion of said third oligonucleotide contains a sequence complementary to said third region of said second target nucleic acid;b) generating a first cleavage structure wherein at least said 3' portion of said first oligonucleotide is annealed to 30 said first target nucleic acid and wherein at least said 5' portion of said second oligonucleotide is annealed to said 385 CA 02284552 2007-08-14 74667-130 first target nucleic acid and wherein cleavage of said first cleavage structure occurs via said cleavage means thereby cleaving said first oligonucleotide to generate a fourth oligonucleotide, said fourth oligonucleotide having a 5' and 5 a 3' portion wherein said 51 portion of said fourth oligonucleotide contains a sequence complementary to said first region of said second target nucleic acid and wherein said 3' portion of said fourth oligonucleotide contains a sequence complementary to said second region of said second 10 target nucleic acid;c) generating a second cleavage structure under conditions wherein at least said 3' portion of said third oligonucleotide is annealed to said second target nucleic acid and wherein at least said 5' portion of said fourth 15 oligonucleotide is annealed to said second target nucleic acid oligonucleotide and wherein cleavage of said second cleavage structure occurs to generate a fifth oligonucleotide, said fifth oligonucleotide having a 3'hydroxyl group;and 20 d) detecting said fifth oligonucleotide thereby detecting the presence of said target nucleic acid molecule.
  10. 42
    56. A method of detecting the presence of a target nucleic acid molecule comprising:a) providing: i) a cleavage means, ii) a source of a first target nucleic acid, said first target nucleic acid having a first region, a second region, a third region and a fourth region, wherein said first region is located adjacent to and downstream from said second region, said second region is located adjacent to and downstream from said third region and said third region is located adjacent to and downstream from said fourth region;iii) a first oligonucleotide complementary to said fourth region of said first target nucleic acid;iv) second and third oligonucleotides having 3' and 5' portions, wherein said 3' portion of said second oligonucleotide contains a sequence complementary to said third region of said target nucleic acid and wherein said 5' portion of said second oligonucleotide and said 3' portion of said third oligonucleotide each contain sequence completely complementary to said second region of said target nucleic acid, and wherein said 5' portion of said third oligonucleotide contains sequence complementary to said first region of said target nucleic acid;391 CA 02284552 2007-08-14 74667-130 v) a source of a second target nucleic acid, said second target nucleic acid having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region and wherein said second region is located adjacent to and downstream from said third region;vi) a fourth oligonucleotide having a 5' and a 3' portion wherein said 5' portion of said fourth oligonucleotide contains a sequence complementary to said second region of said second target nucleic acid and wherein said 3' portion of said fourth oligonucleotide contains a sequence complementary to said third region of said second target nucleic acid;b) generating a first cleavage structure wherein said first oligonucleotide is annealed to said fourth region of said first target nucleic acid and wherein at least said 3' portion of said second oligonucleotide is annealed to said first target nucleic acid and wherein at least said 5' portion of said third oligonucleotide is annealed to said first target nucleic acid and wherein cleavage of said first cleavage structure occurs thereby cleaving said second oligonucleotide to generate a fifth oligonucleotide, said fifth oligonucleotide having a 5' and a 3' portion wherein said 5' portion of said fifth oligonucleotide contains a sequence complementary to said first region of said second target nucleic acid and wherein said 3' portion of said fifth oligonucleotide contains a sequence complementary to said second region of said second target nucleic acid;c) generating a second cleavage structure under conditions wherein at least said 31 portion of said fourth oligonucleotide is annealed to said second target nucleic 392 CA 02284552 2007-08-14 74667-130 acid and wherein at least said 5' portion of said fifth oligonucleotide is annealed to said second target nucleic acid and wherein cleavage of said second cleavage structure occurs to generate a sixth oligonucleotide, said sixth oligonucleotide having a 3'-hydroxyl group;and d) detecting said sixth oligonucleotide thereby detecting the presence of said target nucleic acid molecule.
  11. 53
    67. A method of detecting the cleavage of nucleic acid cleavage structures comprising:a) providing: i) a cleavage means having 5' nuclease activity;ii) a first target nucleic acid, said first target nucleic acid comprising a first region, a second region, and a third region, said first region downstream of and contiguous to said second region, and said second region downstream of and contiguous to said third region;iii) a first oligonucleotide comprising a 3' portion and a 5' portion, wherein at least a section of said 31 portion of said first oligonucleotide is completely complementary to said third region of said first target 394 CA 02284552 2007-08-14 74667-130 nucleic acid, and wherein a section of said 5' portion of said first oligonucleotide adjacent to said 3' portion of said first oligonucleotide is completely complementary to said second region of said first target nucleic acid;iv) a second oligonucleotide comprising a 3' portion and a 5' portion, wherein said 5' portion is completely complementary to said first region of said first target nucleic acid, and wherein said 3' portion is either complementary to said second region of said first target nucleic acid or is not 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, a second region, and a third region, said first region downstream of and contiguous to said second region, and said second region downstream of and contiguous to said third region, wherein said first region is complementary to said 5' portion of said first oligonucleotide;vi) a third oligonucleotide comprising a 5' portion and a 3' portion, wherein a section of said 3' portion of said third oligonucleotide is completely complementary to said third region of said second target nucleic acid, and wherein a section of said 5' portion of said third oligonucleotide adjacent to said 3' portion of said third oligonucleotide is completely complementary to said second region of said second target nucleic acid;and vii) an arrestor agent selected to inhibit interaction between said first oligonucleotide and said second target nucleic acid;395 CA 02284552 2007-08-14 74667-130 b) generating a first cleavage structure wherein at least a portion of said section of said 3' portion of said first oligonucleotide is annealed to said third region of said first target nucleic acid, and wherein said section of said 5' portion of said first oligonucleotide is annealed to said second region of said first target nucleic acid, to form a double-stranded region, and wherein at least said 5' portion of said second oligonucleotide is annealed to said first 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 comprising a 5' nuclease activity, thereby cleaving said first oligonucleotide to generate a fourth oligonucleotide containing said 5' portion of said first oligonucleotide, said fourth oligonucleotide further comprising a 3' portion and a 5' portion wherein said 5' portion of said fourth oligonucleotide is complementary to said first region of said second target nucleic acid, and wherein said 3' portion is either complementary to said second region of said second target nucleic acid or is not complementary to said second region of said second target nucleic acid;c) generating a second cleavage structure under conditions wherein said arrestor agent inhibits interaction between said first oligonucleotide and said second target nucleic acid, and wherein at least said portion of said third oligonucleotide is annealed to said second region and said third region of said second target nucleic acid and wherein at least said 5' portion of said fourth oligonucleotide is annealed to said first 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. 396 CA 02284552 2007-08-14 74667-130
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    90. A set of reagents for detecting a first target nucleic acid comprising a first oligonucleotide, a second oligonucleotide, a third oligonucleotide, a second target 25 nucleic acid and an arrestor agent, said first and second oligonucleotides configured to form a first invasive cleavage structure in the presence of said first target nucleic acid, and said third oligonucleotide configured to form a second invasive cleavage structure in the presence of 399 CA 02284552 2007-08-14 74667-130 said second target nucleic acid and a cleavage product produced by the cleavage of said first invasive cleavage structure, and said arrestor agent configured to inhibit interaction of either of said first or said second 5 oligonucleotide with said second target nucleic acid, and a purified thermostable archaebacterial FEN-1 endonuclease, said first target nucleic acid comprising a first region, a second region, and a third region, said first region downstream of and contiguous to said second region, 10 and said second region downstream of and contiguous to said third region;said first oligonucleotide comprising a 3' portion and a 5' portion, wherein at least a section of said 3' portion of said first oligonucleotide is completely 15 complementary to said third region of said first target nucleic acid, and wherein a section of said 5' portion of said first oligonucleotide adjacent to said 3' portion of said first oligonucleotide is completely complementary to said second region of said first target nucleic acid;20 said second oligonucleotide comprising a 3' portion and a 5' portion, wherein said 5' portion is completely complementary to said first region of said first target nucleic acid, and wherein said 31 portion is either complementary to said second region of said first target 25 nucleic acid or is not complementary to said second region of said first target nucleic acid;said second target nucleic acid comprising a first region, second region, and a third region, said first region downstream of and contiguous to said second region, and said 30 second region downstream of and contiguous to said third region, wherein said first region is complementary to a 400 CA 02284552 2007-08-14 74667-130 sequence contained in said 5' portion of said first oligonucleotide ;said third oligonucleotide comprising a 5' portion and a 31 portion, wherein a section of said 3' portion of 5 said third oligonucleotide is completely complementary to said third region of said second target nucleic acid, and wherein a section of said 5' portion of said third oligonucleotide adjacent to said 3' portion of said third oligonucleotide is completely complementary to said second 10 region of said second target nucleic acid.
  13. 87
    101. A method of detecting the cleavage of nucleic acid cleavage structures comprising:a) providing: i) a cleavage agent;15 ii) a first target nucleic acid, said first target nucleic acid comprising a first region, a second region, and a third region, said first region downstream of and contiguous to said second region, and said second region downstream of and contiguous to said third region;20 iii) a first oligonucleotide, wherein at least a section of said first oligonucleotide is completely complementary to said second and said third regions of said first target nucleic acid;iv) a second oligonucleotide comprising a 3' 25 portion and a 5' portion, wherein said 5' portion is completely complementary to said first region of said first target nucleic acid, and wherein said 3' portion is either complementary to said second region of said first target 402 CA 02284552 2007-08-14 74667-130 nucleic acid or is not 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, a second 5 region, and a third region, said first region downstream of and contiguous to said second region, and said second region downstream of and contiguous to said third region, wherein said first region is complementary to a sequence contained in said 5' portion of said first oligonucleotide;and 10 vi) a third oligonucleotide, comprising a 5' portion and a 3' portion, wherein a section of said 3' portion of said third oligonucleotide is completely complementary to said third region of said second target nucleic acid, and wherein a section of said 5' portion of 15 said third oligonucleotide adjacent to said 3' portion of said third oligonucleotide is completely complementary to said second region of said second target nucleic acid;b) generating a first cleavage structure wherein at least a portion of said section of said 3' portion of said first 20 oligonucleotide is annealed to said third region of said first target nucleic acid and wherein said section of said 5' portion of said first oligonucleotide is annealed to said second region of said first target nucleic acid, to form a double-stranded region and wherein at least said 5' 25 portion of said second oligonucleotide is annealed to said first 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 30 fourth oligonucleotide, said fourth oligonucleotide comprising a 3' portion and a 5' portion, wherein said 5' 403 CA 02284552 2007-08-14 74667-130 portion is complementary to said first region of said second target nucleic acid, and wherein said 3' portion is either complementary to said second region of said second target nucleic acid or is not complementary to said second region 5 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 second region and said third region of said second target nucleic acid and wherein at least said 51 10 portion of said fourth oligonucleotide is annealed to said first 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. 15
  14. 112
    126. A method of detecting the cleavage of nucleic acid 20 cleavage structures comprising:a) providing: i) a cleavage agent;ii) a first target nucleic acid, said first target nucleic acid comprising a first region, a second region, and 25 a third region, said second region downstream of and contiguous to said first region and said third region upstream of said first region;408 CA 02284552 2007-08-14 74667-130 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;5 iv) a second oligonucleotide comprising a 31 portion and a 5' portion, wherein said 5' portion is completely complementary to said second region of said first target nucleic acid;v) a second target nucleic acid, said second 10 target nucleic acid comprising a first region and a second region, said second region downstream of and contiguous to said first region;vi) a third oligonucleotide, wherein at least a portion of said third oligonucleotide is completely 15 complementary to said first region of said second target nucleic acid;and vii) a fourth oligonucleotide complementary to said third portion of said first target nucleic acid;and b) generating a first cleavage structure wherein said fourth 20 oligonucleotide is annealed to said third region of said first target nucleic acid and 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 5' 25 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 occurs within said double-stranded region via said cleavage agent thereby cleaving said first oligonucleotide to generate a fifth 30 oligonucleotide, said fifth oligonucleotide comprising a 3' 409 CA 02284552 2007-08-14 - 74667-130 portion and a 5' portion, wherein said 5' portion is completely complementary to said second region of said second target nucleic acid;c) generating a second cleavage structure under conditions 5 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 5' portion of said fifth oligonucleotide is annealed to said second region of said second target nucleic acid and wherein cleavage of said 10 second cleavage structure occurs to generate a cleavage fragment;and d) detecting the cleavage of said second cleavage structure.
  15. 113
    127. A method of detecting the cleavage of nucleic acid cleavage structures comprising:15 a) providing: i) a cleavage agent;ii) a first target nucleic acid, said first target nucleic acid comprising a first region, a second region, and a third region, said second region downstream of and 20 contiguous to said first region and said third region upstream of said first region;iii) a first oligonucleotide comprising a 5' end, wherein at least a section of said first oligonucleotide is completely complementary to said first region of said first 25 target nucleic acid and wherein said 5' end contains a region of self-complementarity;iv) a second oligonucleotide comprising a 3' portion and a 5' portion, wherein said 5' portion is 410 CA 02284552 2007-08-14 74667-130 completely complementary to said second region of said first target nucleic acid;v) a third oligonucleotide comprising a 3' portion complementary to said 5' end of said first oligonucleotide;5 and 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 10 said 5' 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 occurs within said double-stranded region via said cleavage agent thereby cleaving said first oligonucleotide to generate a 15 fourth oligonucleotide, said fourth oligonucleotide comprising said 5' end of said first oligonucleotide;c) generating a second cleavage structure under conditions wherein at least said 3' portion of said third oligonucleotide is annealed to said fourth oligonucleotide 20 and wherein cleavage of said second cleavage structure occurs to generate a cleavage fragment;and d) detecting the cleavage of said second cleavage structure. SMART & BIGGAR OTTAWA, CANADA PATENT AGENT 411
Independent claims15