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 and detection 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;
(b) 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;
(c) after step (b), initiating a nucleic acid polymerase dependent primer extension reaction from the 3′ end of the tagged oligonucleotide hybridized to the target nucleic acid, thereby producing an extension product;
(d) separating the primer extension product from the target nucleic acid; and
(e) producing amplification products in an isothermal 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; and
(f) detecting the amplification products generated in step (e), wherein detecting the amplification products comprises exposing the amplification products to a probe having a hybridizing sequence which hybridizes to the amplification product.
2. A method for the selective amplification and detection 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;
(b) 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;
(c) after step (b), initiating a nucleic acid polymerase dependent primer extension reaction from the 3′ end of the tagged oligonucleotide hybridized to the target nucleic acid, thereby producing an extension product;
(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; and
(f) detecting in a real-time detection reaction the amplification products generated in step (e), wherein the real-time detection reaction is a continuous reaction that occurs during the amplification reaction.
3. The method of claim 1 , where the target nucleic acid sequence is immobilized on a solid support during step (b).
4. The method of claim 1 , wherein step (b) comprises removing unhybridized tagged oligonucleotide from the nucleic acid sample.
5. The method of claim 1 , wherein the nucleic acid amplification reaction is a transcription-based amplification reaction.
6. The method of claim 5 , wherein the transcription-based amplification reaction is TMA.
7. The method of claim 5 , wherein the first oligonucleotide comprises a promoter for an RNA polymerase which is situated 5′ to the hybridizing sequence.
8. The method of claim 7 , wherein the first oligonucleotide is modified to prevent initiation of DNA synthesis therefrom.
9. The method of claim 5 , wherein the second oligonucleotide comprises a promoter for an RNA polymerase which is situated 5′ to the hybridizing sequence, and wherein the tagged oligonucleotide further comprises a promoter for an RNA polymerase which is situated 5′ to the second region.
10. The method of claim 1 , wherein the probe comprises a label.
11. The method of claim 10 , wherein the label is a fluorescent label or a chemiluminescent label.
12. The method of claim 10 , wherein the probe comprises interacting labels which emit different signals depending on whether the probe is hybridized to its target sequence.
13. The method of claim 2 , wherein the conditions of the nucleic acid amplification reaction are isothermal.
14. The method of claim 13 , wherein the nucleic acid amplification reaction is a transcription-based amplification reaction.
15. The method of claim 14 , wherein the transcription-based amplification reaction is TMA.
16. The method of claim 2 , wherein the amplification reaction is a PCR reaction.
17. The method of claim 2 , wherein the amplification products are detected through the use of a self-hybridizing probe.
18. The method of claim 17 , wherein the self-hybridizing probe is a molecular torch or a molecular beacon.
19. The method of claim 14 , wherein the amplification products are detected through the use of a self-hybridizing probe.
20. The method of claim 19 , wherein the self-hybridizing probe is a molecular torch or a molecular beacon.
21. The method of claim 10, wherein the label is acridinium.