Method for making available a priming oligonucleotide
View Patent ↗The present invention is directed to novel methods of synthesizing multiple copies of a target nucleic acid sequence which are autocatalytic (i.e., able to cycle automatically without the need to modify reaction conditions such as temperature, pH, or ionic strength and using the product of one cycle in the next one). In particular, the present invention discloses a method of nucleic acid amplification which is robust and efficient, while reducing the appearance of side-products. The method uses only one primer, the “priming oligonucleotide,” a promoter oligonucleotide modified to prevent polymerase extension from its 3′-terminus and, optionally, a means for terminating a primer extension reaction, to amplify RNA or DNA molecules in vitro, while reducing or substantially eliminating the formation of side-products. The method of the present invention minimizes or substantially eliminates the emergence of side-products, thus providing a high level of specificity. Furthermore, the appearance of side-products can complicate the analysis of the amplification reaction by various molecular detection techniques. The present invention minimizes or substantially eliminates this problem, thus providing an enhanced level of sensitivity.
1. A method for making available a priming oligonucleotide for extension in the presence of a target nucleic acid, the method comprising the steps of:
(a) forming a reaction mixture comprising the priming oligonucleotide and the target nucleic acid, the priming oligonucleotide having a cap hybridized to a region at the 3′-end thereof, wherein the 5′-terminal base of the cap is complementary to the 3′-terminal base of the priming oligonucleotide, and wherein the cap is modified to prevent the initiation of DNA synthesis therefrom;
(b) hybridizing the priming oligonucleotide to a target sequence contained within the target nucleic acid, thereby displacing the cap from the priming oligonucleotide; and
(c) extending the priming oligonucleotide in a primer extension reaction with a DNA polymerase to give a DNA primer extension product complementary to the target sequence.
2. The method of claim 1 , wherein the cap is complementary to at least 3 nucleotides at the 3′-end of the priming oligonucleotide.
3. The method of claim 1 , wherein the cap is complementary to no more than 8 nucleotides at the 3′-end of the priming oligonucleotide.
4. The method of claim 1 , wherein the cap is a capping oligonucleotide modified to include a blocking moiety situated at its 3′-terminus.
5. The method of claim 1 , wherein the 3′-end of the cap is covalently attached to the 5′-end of the priming oligonucleotide, and wherein the cap hybridizes to the 3′-end of the priming oligonucleotide by forming a loop.
6. The method of claim 5 , wherein the cap is joined to the priming oligonucleotide via a linker region.
7. The method of claim 6 , wherein the linker region comprises at least 5 nucleotides.
8. The method of claim 6 , wherein the linker region comprises at least 5 abasic nucleotides.
9. The method of claim 1 further comprising providing a detectably labeled probe to the reaction mixture, wherein the probe is complementary to a region of the target sequence or its complement.
10. The method of claim 9 , wherein the probe is provided to the reaction mixture prior to step (c).
11. The method of claim 10 , wherein the probe comprises interacting labels.
12. The method of claim 11 , wherein the probe is a self-hybridizing probe.
13. The method of claim 11 , wherein the interacting labels comprise a fluorophore and a quencher.
14. A method for making available a priming oligonucleotide for extension in the presence of a target nucleic acid, the method comprising the steps of:
(a) forming a reaction mixture comprising the priming oligonucleotide and the target nucleic acid, the priming oligonucleotide having a cap hybridized to a region at the 3′-end thereof, wherein the 5-terminal base of the cap is complementary to the 3′-terminal base of the priming oligonucleotide, wherein the cap is modified to prevent the initiation of DNA synthesis therefrom, and wherein the cap is complementary to at least 3 nucleotides but no more than 8 nucleotides at the 3′-end of the priming oligonucleotide;
(b) hybridizing the priming oligonucleotide to a target sequence contained within the target nucleic acid, thereby displacing the cap from the priming oligonucleotide;
(c) extending the priming oligonucleotide in a primer extension reaction with a DNA polymerase to give a DNA primer extension product complementary to the target sequence; and
(d) providing a detectably labeled probe to the reaction mixture, wherein the probe is complementary to a region of the target sequence or its complement.
15. The method of claim 14 , wherein the cap is a capping oligonucleotide modified to include a blocking moiety situated at its 3′-terminus.
16. The method of claim 14 , wherein the 3′-end of the cap is covalently attached to the 5′-end of the priming oligonucleotide, and wherein the cap hybridizes to the 3′-end of the priming oligonucleotide by forming a loop.
17. The method of claim 16 , wherein the cap is joined to the priming oligonucleotide via a linker region.
18. The method of claim 17 , wherein the linker region comprises at least 5 nucleotides.
19. The method of claim 17 , wherein the linker region comprises at least 5 abasic nucleotides.
20. The method of claim 14 , wherein the probe is provided to the reaction mixture prior to step (c).
21. The method of claim 20 , wherein the probe comprises interacting labels.
22. The method of claim 21 , wherein the probe is a self-hybridizing probe.
23. The method of claim 21 , wherein the interacting labels comprise a fluorophore and a quencher.