Nucleic acid detection assays employing blocker oligonucleotides
View Patent ↗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 composition, 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 within a target region, and wherein said first and second nucleic acid molecules are also able to form a second invasive cleavage structure with an interfering sequence within an interfering region, and
b) blocker oligonucleotides configured to at least partially hybridize with said interfering sequence such that formation of said second invasive cleavage structure is at least partially inhibited.
2. The composition of claim 1 , further comprising c) a cleavage agent, wherein said cleavage agent is capable of cleaving said first nucleic acid molecules when said first nucleic acid molecules form an invasive cleavage structure comprising said second nucleic acid molecules and said target sequence or said interfering sequence.
3. The composition of claim 1 , further comprising said target region.
4. The composition of claim 3 , further comprising said interfering region.
5. The composition of claim 1 , wherein said first and second nucleic acid molecules and said blocker oligonucleotides are configured such that at least 70 percent of invasive cleavage structures formed in the presence of said target sequence and said interfering sequence are said first invasive cleavage structures.
6. The composition 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.
7. The composition 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.
8. The composition 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.
9. The composition 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.
10. The composition of claim 1 , wherein said interfering sequence is identical to said target sequence.
11. The composition of claim 1 , wherein said interfering sequence is at least 97 percent homologous to said target sequence.
12. The composition of claim 1 , wherein said interfering sequence has five or fewer nucleotide base difference compared to said target sequence.
13. The composition of claim 1 , wherein said target sequence and said interfering sequence are each at least 15 bases in length.
14. The composition of claim 1 , wherein the Tm of said second oligonucleotide with regard to said interfering region less than 63° C.
15. The composition of claim 1 , wherein said target region comprises a first gene, and wherein said interfering region comprises a first pseudo-gene.
16. The composition of claim 1 , wherein said target sequence comprises a targeted polymorphic sequence.
17. The composition of claim 16 , wherein said targeted polymorphic sequence is a single nucleotide polymorphism.
18. A composition for analyzing a target sequence, comprising:
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.