Nucleic acid detection employing charged adducts
First Claim
1. A method of detecting the presence of a target RNA molecule by detecting non-target cleavage products comprising:
- a) providing;
i) a cleavage means, ii) a source of target RNA, said target RNA 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 RNA and wherein said 3′
portion of said first oligonucleotide contains a sequence complementary to said third region of said target RNA;
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 RNA and wherein said 3′
portion of said second oligonucleotide contains a sequence complementary to said second region of said target nucleic acid;
b) mixing said cleavage means, said target RNA, said first oligonucleotide and said second oligonucleotide to create a reaction mixture under reaction conditions such that at least said 3′
portion of said first oligonucleotide is annealed to said target RNA and wherein at least said 5′
portion of said second oligonucleotide is annealed to said target RNA so as to create a cleavage structure and wherein cleavage of said cleavage structure occurs to generate non-target cleavage products; and
c) detecting said non-target cleavage products.
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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 5′ 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 by charge.
62 Citations
52 Claims
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1. A method of detecting the presence of a target RNA molecule by detecting non-target cleavage products comprising:
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a) providing;
i) a cleavage means, ii) a source of target RNA, said target RNA 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 RNA and wherein said 3′
portion of said first oligonucleotide contains a sequence complementary to said third region of said target RNA;
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 RNA and wherein said 3′
portion of said second oligonucleotide contains a sequence complementary to said second region of said target nucleic acid;
b) mixing said cleavage means, said target RNA, said first oligonucleotide and said second oligonucleotide to create a reaction mixture under reaction conditions such that at least said 3′
portion of said first oligonucleotide is annealed to said target RNA and wherein at least said 5′
portion of said second oligonucleotide is annealed to said target RNA so as to create a cleavage structure and wherein cleavage of said cleavage structure occurs to generate non-target cleavage products; and
c) detecting said non-target cleavage products. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41)
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16. A method of separating nucleic acid molecules, comprising:
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a) providing;
i) a charge-balanced oligonucleotide and ii) a reactant;
b) mixing said charge-balanced oligonucleotide with said reactant to create a reaction mixture under conditions such that a charge-unbalanced oligonucleotide is produced; and
c) separating said charge-unbalanced oligonucleotide from said reaction mixture.
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42. A method of detecting cleaved nucleic molecules, comprising:
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a) providing;
i) a homogeneous plurality of charge-balanced oligonucleotides;
ii) a sample suspected of containing a target nucleic acid having a sequence comprising a first region complementary to said charge-balanced oligonucleotide;
iii) a cleavage means; and
iv) a reaction vessel;
b) adding to said vessel, in any order, said sample, said charge-balanced oligonucleotides and said cleavage means to create a reaction mixture under conditions such that a portion of said charge-balanced oligonucleotides binds to said complementary target nucleic acid to create a bound population, and such that said cleavage means cleaves at least a portion of said bound population of charge-balanced oligonucleotides to produce a population of unbound, charge-unbalanced oligonucleotides; and
c) separating said unbound, charge-unbalanced oligonucleotides from sid reaction mixture. - View Dependent Claims (43, 44, 45, 46, 47, 48, 49, 50, 51, 52)
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Specification