Nucleic acid detection assays employing blocker oligonucleotides
The present invention provides methods, compositions, and kits for detecting the presence or absence of target sequences in a sample, where the sample also contains interfering sequences that are similar or identical to the target sequences. In particular, the present invention provides blocker oligonucleotides that at least partially inhibit the formation of invasive cleavage structures with the interfering sequences but do not substantially inhibit the formation of invasive cleavage structures with the target sequences.
1. A method of detecting the presence of absence of a target sequence in a sample comprising;
a) providing;
i) first and second nucleic acid molecules, wherein said first and second nucleic acid molecules are configured to form a first invasive cleavage structure with a target sequence, and wherein said first and second nucleic acid molecules are also able to form a second invasive cleavage structure with an interfering sequence,
ii) a sample comprising: A) a target region suspected of containing said target sequence, and B) an interfering region comprising said interfering sequence,
iii) a cleavage agent, and
iv) blocker oligonucleotides configured to at least partially hybridize with said interfering region such that formation of said second invasive cleavage structure is at least partially inhibited;
b) contacting said sample with said first and second nucleic acid molecules, said cleavage agent, and said blocker oligonucleotides, and
c) detecting the presence or absence of said target sequence in said sample.
2. The method of claim 1 , wherein said detecting comprises observing a signal generated by cleavage of said first nucleic acid molecules, wherein at least 70 percent of said signal results from cleavage of said first nucleic acid molecules in said first invasive cleavage structure.
3. The method of claim 1 , wherein the melting temperature of said blocker oligonucleotides when hybridized to said interfering sequence is greater than the melting temperature of said second nucleic acid molecules when hybridized to said interfering sequence.
4. The method of claim 1 , wherein said blocker oligonucleotides are configured to hybridize to at least a portion of said interfering region.
5. The method of claim 1 , wherein said blocker oligonucleotides are configured to hybridize to at least a portion of said interfering sequence and to at least a portion of said interfering region that is not part of said interfering sequence.
6. The method of claim 1 , wherein said first nucleic acid molecules comprise a 5′ portion and a 3′ portion, wherein said 3′ portion is configured to hybridize to said target sequence, and wherein said 5′ portion is configured to not hybridize to said target sequence.
7. The method of claim 1 , wherein said second nucleic acid molecules comprise a 5′ portion and a 3′ portion, wherein said 5′ portion is configured to hybridize to said target sequence, and wherein said 3′ portion is configured to not hybridize to said target sequence.
8. The method of claim 1 , wherein said contacting is conducted under conditions such that said first nucleic acid molecules hybridize about equally to said target and said interfering sequences.
9. The method of claim 1 , wherein said blocker oligonucleotides do not substantially alter the ability of said first nucleic acid molecules to hybridize to said target and interfering sequences.
10. The method of claim 1 , wherein said first nucleic acid molecules when hybridized to said target sequence have a first Tm, and wherein said first nucleic acid molecules when hybridized to said interfering sequence have a second Tm that is substantially the same as said first Tm, and wherein during said contacting step, said blocker oligonucleotides do not cause any difference between said first and second Tm's to substantially change.
11. The method of claim 1 , wherein said interfering sequence is identical to said target sequence.
12. The method of claim 1 , wherein said interfering sequence is at least 97 percent homologous to said target sequence.
13. The method of claim 1 , wherein said interfering sequence has five or less nucleotide base difference compared to said target sequence.
14. The method of claim 1 , wherein said target sequence and said interfering sequence are each at least 15 bases in length.
15. The method of claim 1 , wherein the Tm of said second oligonucleotide with regard to said interfering region during said contacting step is less than 63° C.
16. The method of claim 1 , wherein said target region comprises a first gene, and wherein said interfering region comprises a first pseudo-gene.
17. The method of claim 1 , wherein said target sequence comprises a targeted polymorphic sequence.
18. The method of claim 17 , wherein said targeted polymorphic sequence is a single nucleotide polymorphism.
19. The method of claim 1 , wherein said cleavage agent is capable of cleaving said first nucleic acid molecules when said invasive cleavage structures are formed.
20. A kit for detecting the presence or absence of a target sequence in a sample comprising;
a) first and second nucleic acid molecules, wherein said first and second nucleic acid molecules are configured to form a first invasive cleavage structure with a target sequence, and wherein said first and second nucleic acid molecules are also able to form a second invasive cleavage structure with an interfering sequence, and
b) blocker oligonucleotides configured to at least partially hybridize with said interfering region such that formation of said second invasive cleavage structure is at least partially inhibited.
21. The kit of claim 20 , further comprising c) a cleavage agent, wherein said cleavage agent is capable of cleaving said first nucleic acid molecules when said invasive cleavage structures are formed.