Tagged oligonucleotides and their use in nucleic acid amplification methods
View Patent ↗The present invention provides nucleic acid amplification systems and methods that 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 invention offers the further advantage of requiring less stringent purification and/or sterility efforts than conventionally needed in order to ensure that enzymes and other reagents used in amplification reactions, and the environment in which an amplification reaction is performed, are free of bacterial or other nucleic acid contamination that may yield false positive results.
1. A method for the selective amplification of at least one target nucleic acid sequence from a nucleic acid sample, said method comprising the steps of:
(a) treating a nucleic acid sample comprising a target nucleic acid sequence with a tagged oligonucleotide comprising first and second regions, said first region comprising a target hybridizing sequence which hybridizes 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, wherein said second region does not stably hybridize to a target nucleic acid containing said target nucleic acid sequence, and wherein said target nucleic acid sequence is contained in each of a plurality of ribosomal nucleic acids from multiple species of microorganisms;
(b) prior to initiating a primer extension reaction, reducing in said nucleic acid sample the effective concentration of unhybridized tagged oligonucleotide having an active form in which a target hybridizing sequence of said unhybridized tagged oligonucleotide is available for hybridization to said target nucleic acid sequence; and
(c) after step (b), initiating an extension reaction from the 3′-end of the tagged oligonucleotide with a DNA polymerase to produce a primer extension product comprising a region complementary to the target nucleic acid sequence;
(d) separating the primer extension product from the target nucleic acid; and
(e) producing amplification products in a nucleic acid amplification reaction using first and second oligonucleotides, wherein said first oligonucleotide comprises a hybridizing sequence which hybridizes to a 3′-end of the complement of said target nucleic acid sequence and said second oligonucleotide comprises a hybridizing sequence which hybridizes to the complement of said tag sequence, wherein said second oligonucleotide does stably hybridize to said target nucleic acid, and wherein each of said amplification products comprises a base sequence which is substantially identical or complementary to the base sequence of said target nucleic acid sequence and further comprises a base sequence which is substantially identical or complementary to all or a portion of said tag sequence.
2. The method of claim 1 , wherein step (b) comprises removing unhybridized tagged oligonucleotide from said nucleic acid sample.
3. The method of claim 2 , wherein said target nucleic acid sequence is immobilized on a solid support during step (b).
4. The method of claim 1 , wherein step (b) comprises inactivating unhybridized tagged oligonucleotide so that said unhybridized tagged oligonucleotide does not stably hybridize to said target nucleic acid sequence during step (c).
5. The method of claim 4 further comprising removing unhybridized tagged oligonucleotide from said nucleic acid sample during step (b).
6. The method of claim 4 , wherein said tagged oligonucleotide has an active form during step (a) which permits said target hybridizing sequence to hybridize to said target nucleic acid sequence, and wherein unhybridized tagged oligonucleotide is converted to an inactive form in step (b) which blocks or prevents said tagged oligonucleotide from hybridizing to said target nucleic acid sequence during step (c).
7. The method of claim 6 , wherein the conditions of steps (b)-(e) are less stringent than the conditions of step (a).
8. The method of claim 7 , wherein the temperature of said nucleic acid sample is lowered between steps (a) and (b).
9. The method of claim 6 , wherein unhybridized tagged oligonucleotide from step (a) is converted from a single-stranded form to a duplexed form in step (b).
10. The method of claim 9 , wherein the duplexed form is hairpin tag molecule comprising a tag closing sequence joined to a 5′-end of said tagged oligonucleotide, wherein said tag closing sequence hybridizes to said target hybridizing sequence under the conditions of step (b), thereby blocking hybridization of unhybridized tagged oligonucleotide from step (a) to said target nucleic acid sequence in steps (b)-(e).
11. The method of claim 10 , wherein said tag closing sequence is joined to said tagged oligonucleotide by a non-nucleotide linker.
12. The method of claim 10 , wherein a 3′-end of said tag closing sequence is joined to a 5′-end of said tagged oligonucleotide.
13. The method of claim 4 , wherein said target hybridizing sequence is hybridized to a tag closing oligonucleotide in step (b), said tagged oligonucleotide and said tag closing oligonucleotide being distinct molecules.
14. The method of claim 13 , wherein said tag closing oligonucleotide is modified to prevent the initiation of DNA synthesis therefrom.
15. The method of claim 14 , wherein a 3′-terminal base of said target hybridizing sequence is hybridized to a 5′-terminal base of said tag closing oligonucleotide.
16. The method of claim 14 , wherein said tagged oligonucleotide and said tag closing oligonucleotide are both present in said nucleic acid sample during step (a), and wherein said target hybridizing sequence favors hybridization to said target nucleic acid sequence over said tag closing oligonucleotide in step (a).
17. The method of claim 1 , wherein the conditions of said nucleic acid amplification reaction are isothermal.
18. The method of claim 17 , wherein said nucleic acid amplification reaction is a transcription-based amplification reaction.
19. 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;
wherein said target nucleic acid sequence is contained in each of a plurality of ribosomal nucleic acids from multiple species of microorganisms;
(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.
20. A pre-amplification reaction mixture for selective amplification of one or more target nucleic acid sequences, said reaction mixture comprising:
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 a target nucleic acid sequence present in said reaction mixture and said second region comprising a tag sequence situated 5′ to said target hybridizing sequence, wherein said target nucleic acid sequence is contained in each of a plurality of ribosomal nucleic acids from multiple species of microorganisms;
a first oligonucleotide comprising a hybridizing sequence which hybridizes to a 3′-end of the complement of said target nucleic acid sequence; and
a second oligonucleotide comprising a hybridizing sequence which hybridizes to the complement of said tag sequence,
wherein said reaction mixture is substantially free of an active form of said tagged oligonucleotide which is not hybridized to said target region contained in said target nucleic acid sequence present in said reaction mixture,
wherein said active form of said tagged oligonucleotide has an available target hybridizing sequence for hybridization to said target region present in a non-target nucleic acid added to said reaction mixture, and
wherein said reaction mixture does not comprise a nucleic acid polymerase capable of extending any of said oligonucleotides in a template-dependent manner.