Invasion assays
First Claim
1. 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 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;
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 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′
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 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 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 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;
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 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 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 target nucleic acid;
c) generating a second cleavage structure under conditions wherein said 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 cleavage structure occurs to generate a fifth oligonucleotide, said fifth oligonucleotide having a 3′
-hydroxyl group; and
d) detecting said fifth oligonucleotide.
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Accused Products
Abstract
The present invention relates to means for the detection and characterization 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 the 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 specific methods for the detection of human cytomegalovirus nucleic acid in a sample.
58 Citations
34 Claims
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1. A method of detecting the presence of a target nucleic acid molecule comprising:
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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 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;
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 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′
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 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 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 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;
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 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 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 target nucleic acid;
c) generating a second cleavage structure under conditions wherein said 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 cleavage structure occurs to generate a fifth oligonucleotide, said fifth oligonucleotide having a 3′
-hydroxyl group; and
d) detecting said fifth oligonucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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20. A method of detecting the presence of a target nucleic acid molecule comprising:
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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) a second oligonucleotide having a 5′
portion 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 3′
portion of said second oligonucleotide contains a sequence complementary to said third region of said first target nucleic acid;
iv) 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 said 3′
portion of said third oligonucleotide contains a sequence complementary to said second region of said first 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 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 said 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 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.
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21. A method of detecting the presence of a target nucleic acid molecule comprising:
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a) providing;
i) a cleavage means, 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 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 5′
portion of said first oligonuceotide 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′
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 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 a region of self-complementarity;
c) generating a second cleavage structure under conditions wherein said at least said 3′
portion of said third oligonucleotide is annealed to said first region of said fourth oligonucleotide and wherein at least said 5′
portion of said fourth oligonucleotide is annealed to said second region of said third olignucleotide 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 oligonuceotide, said fifth oligonucleotide having a 3′
-hydroxyl group; and
d) detecting said fifth oligonucleotide.
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22. A method of detecting the presence of human cytomegalovirus nucleic acid in a sample comprising:
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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 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;
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 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′
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 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 nucleic acid and wherein cleavage of said cleavage structure 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 detecting the presence of human cytomegalovirus nucleic acid in said sample. - View Dependent Claims (23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33)
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34. A method of detecting the presence of human cytomegalovirus nucleic acid in a sample comprising:
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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 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 target nucleic acid;
iv) a second oligonucleotide having a 5′
portion and a 3′
portion wherein said 5′
portion of said second oligonucleotide contains a sequence complementary to said 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;
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′
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 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 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 detecting the presence of human cytomegalovirus nucleic acid in said sample.
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Specification