Tagged oligonucleotides and their use in nucleic acid amplification methods
View Patent ↗The present invention provides kits containing tagged oligonucleotides for use in certain nucleic acid amplification methods to desirably reduce or eliminate false positive amplification signals resulting from contaminating biological material, e.g., nucleic acid, that may be present in one or more reagents used in an amplification reaction and/or that may be present in the environment in which an amplification reaction is performed. The kits containing tagged oligonucleotides can be used in purification and/or sterility efforts under less stringent conditions than conventionally needed to reduce or eliminate false positive results in a nucleic acid amplification method.
1. A kit for use in the selective amplification of at least one target nucleic acid sequence from a nucleic acid sample, said kit comprising:
(a) a tagged oligonucleotide comprising:
(i) a first region comprising a target hybridizing sequence which hybridizes to a 3′-end of a target nucleic acid sequence under a first set of conditions so that said first region can be extended in a template-dependent manner in the presence of a DNA polymerase; and
(ii) a second region comprising a tag sequence situated 5′ to said first region, wherein said second region does not stably hybridize to a target nucleic acid containing said target nucleic acid sequence under said first set of conditions;
(b) a tag closing sequence which hybridizes to said target hybridizing sequence under a second set of conditions, thereby blocking hybridization of said tagged oligonucleotide to said target nucleic acid sequence, wherein said tag closing sequence does not stably hybridize to said target hybridizing sequence under said first set of conditions; and
(c) a first priming oligonucleotide which hybridizes to the complement of said tag sequence under said second set of conditions so that said first priming oligonucleotide can be extended in a template-dependent manner in the presence of a DNA polymerase.
2. The kit of claim 1 , wherein said tagged oligonucleotide and said tag closing sequence constitute distinct molecules, said tag closing sequence being a tag closing oligonucleotide.
3. The kit of claim 1 , wherein said tagged oligonucleotide and said tag closing sequence are contained in the same molecule.
4. The kit of claim 3 , wherein said tag closing sequence is joined to said tagged oligonucleotide by a non-nucleotide linker.
5. The kit of claim 4 , wherein said non-nucleotide linker comprises at least one of abasic nucleotides and polyethylene glycol.
6. The kit of claim 1 , wherein said first priming oligonucleotide does stably hybridize to said target nucleic acid and, thereby, participate in detectable amplification of said target nucleic acid sequence under said second set of conditions.
7. The kit of claim 1 further comprising a second priming oligonucleotide which hybridizes to the complement of a 5′-end of said target nucleic acid sequence under said second set of conditions so that said second priming oligonucleotide can be extended in a template-dependent manner in the presence of a DNA polymerase.
8. The kit of claim 1 further comprising a promoter oligonucleotide comprising first and second regions, said first region comprising a hybridizing sequence which hybridizes to the complement of a 5′-end of said target nucleic acid sequence under said second set of conditions, and said second region comprising a promoter for an RNA polymerase which is situated 5′ to said first region.
9. The kit of claim 1 further comprising one or more of the following: a DNA polymerase, a reverse transcriptase, an RNA polymerase, a nucleotide triphosphate, and a solid support for binding a complex comprising said target nucleic acid and said tagged oligonucleotide.
10. The kit of claim 1 , wherein said tagged oligonucleotide is free in solution.
11. A kit for preparing a pre-amplification reaction mixture for selective amplification of one or more target nucleic acid sequences, said kit comprising:
(a) a tagged oligonucleotide comprising first and second regions, said first region comprising a target hybridizing sequence hybridized to a target region contained at a 3′-end of one or more target nucleic acid sequences present in said reaction mixture and said second region comprising a tag sequence situated 5′ to said target hybridizing sequence;
(b) a first oligonucleotide comprising a hybridizing sequence which hybridizes to a 3′-end of the complement of one or more of said target nucleic acid sequences; and
(c) a second oligonucleotide comprising a hybridizing sequence which hybridizes to the complement of said tag sequence,
wherein said tagged oligonucleotide is configured to have an active form and an inactive form, said active form of said tagged oligonucleotide has an available target hybridizing sequence for hybridization to said target region, and said inactive form of said tagged oligonucleotide has a target hybridizing sequence that is blocked from hybridization to said target region.
12. The kit of claim 11 , wherein said tagged oligonucleotide comprises a promoter for an RNA polymerase situated 5′ to said tag sequence.
13. The kit of claim 11 , wherein said first oligonucleotide comprises a blocking moiety situated at its 3′-terminus.
14. The kit of claim 11 , wherein said tagged oligonucleotide comprises a tag closing sequence joined to a 5′-end thereof.
15. The kit of claim 14 , wherein said tagged oligonucleotide comprises a promoter for an RNA polymerase situated 5′ to said tag sequence.
16. The kit of claim 14 , wherein said tagged oligonucleotide does not comprise a promoter for an RNA polymerase.
17. The kit of claim 11 further comprising a probe for detecting one or more amplification products comprising said one or more target nucleic acid sequences or complements thereof.
18. A kit for preparing a target capture reaction mixture for capturing a target nucleic acid from a sample, the kit comprising:
(a) a capture oligonucleotide comprising first and second regions, said first region comprising a target hybridizing sequence configured to hybridize to a target nucleic acid sequence and said second region comprising a moiety attached to a solid support;
(b) a tagged oligonucleotide comprising first and second regions, said first region comprising a target hybridizing sequence configured to hybridize to a 3′-end of said target nucleic acid sequence and said second region comprising a tag sequence situated 5′ to said target hybridizing sequence; and
wherein said tagged oligonucleotide is configured to have an inactive form when unhybridized to target nucleic acid and wherein the inactive form blocks or prevents said unhybridized tagged oligonucleotide from hybridizing to said target nucleic acid sequence.
19. The kit of claim 18 , wherein said tagged oligonucleotide comprises a promoter for an RNA polymerase situated 5′ to said tag sequence, and wherein said target capture reaction mixture further comprises a first oligonucleotide, said first oligonucleotide comprises a sequence that is complementary to said promoter sequence situated 5′ to a hybridizing sequence.
20. The kit of claim 18 , wherein said kit further comprises a first oligonucleotide, said first oligonucleotide comprises a promoter for an RNA polymerase situated 5′ to a hybridizing sequence and said tagged oligonucleotide comprises a sequence that is complementary to said promoter sequence situated 5′ to said tag sequence.
21. The kit of claim 18 , further comprising a chemical component for chemically lysing any cells or any virion particles present in said sample.
22. The kit of claim 18 , wherein said solid support is a magnetic bead.
23. The kit of claim 22 , wherein said magnetic bead comprises an immobilized nucleic acid probe sequence.
24. The kit of claim 23 , wherein said immobilized probe is hybridized to said capture oligonucleotide second region moiety to attach said capture probe to said solid support.