Detection of nucleic acid sequences by invader-directed cleavage
First Claim
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1. A method of detecting cleaved nucleic molecules, comprising:
- 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 reaction vessel, in any order, said sample, said charge-balanced oligonucleotides and said cleavage means to create a reaction mixture under conditions such that at least a portion of said charge-balanced oligonucleotides binds to the 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;
c) separating said unbound, charge-unbalanced oligonucleotides from said reaction mixture in an electric field, wherein said separating comprises migration of said unbound charge-unbalanced oligonucleotide in a different direction in said electric field than said charge-balanced oligonucleotide; and
d) detecting the separated charge-unbalanced oligonucleotides.
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Abstract
The present invention relates to methods 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.
141 Citations
26 Claims
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1. 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 reaction vessel, in any order, said sample, said charge-balanced oligonucleotides and said cleavage means to create a reaction mixture under conditions such that at least a portion of said charge-balanced oligonucleotides binds to the 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;
c) separating said unbound, charge-unbalanced oligonucleotides from said reaction mixture in an electric field, wherein said separating comprises migration of said unbound charge-unbalanced oligonucleotide in a different direction in said electric field than said charge-balanced oligonucleotide; and
d) detecting the separated charge-unbalanced oligonucleotides. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A method of separating nucleic acid molecules, comprising:
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a) providing;
i) a cleavage means, ii) 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 downstream from said second region and wherein said second region is contiguous to and downstream from said third region;
iii) a charge-balanced oligonucleotide and a second oligonucleotide having 3′ and
5′
portions, wherein the 3′
portion of said charge-balanced oligonucleotide contains a sequence complementary to said third region of said target nucleic acid and wherein the 5′
portion of said charge-balanced oligonucleotide and the 3′
portion of said second oligonucleotide each contain sequence fully complementary to said second region of said target nucleic acid, and wherein the 5′
portion of said second oligonucleotide contains a sequence complementary to said first region of said target nucleic acid;
b) mixing said cleavage means, said target nucleic acid, said charge-balanced oligonucleotide and said second oligonucleotide to create a reaction mixture under reaction conditions such that at least said 3′
portion of said charge-balanced 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 so as to create a cleavage structure and wherein cleavage of said cleavage structure occurs to generate a charge-unbalanced oligonucleotide; and
c) separating said charge-unbalanced oligonucleotide from said reaction mixture. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
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Specification