Isothermal DNA amplification
View Patent ↗Provided herein are nucleic acid synthesis methods and agents that employ an endonuclease for example, endonuclease V, to introduce a nick into a target DNA including one or more inosine, and uses a DNA polymerase to generate amplicons of the target DNA.
1. A method of producing at least one amplicon based on a target DNA comprising:
(a) providing the target DNA;
(b) annealing at least one inosine-containing primer to the target DNA to create a target DNA:primer hybrid;
(c) nicking the inosine-containing primer in the target DNA:primer hybrid at a residue 3′ to the inosine residue using a mutant endonuclease V; and
(d) extending the nicked inosine-containing primer via a nucleic acid amplification reaction to produce at least one amplicon complementary to at least one portion of the target DNA;
wherein extending the nicked inosine-containing primer uses a dNTP mixture which is devoid of deoxyinosine triphosphate.
2. The method of claim 1 , wherein steps (a)-(d) occur substantially simultaneously.
3. The method of claim 1 , wherein multiple forward and reverse inosine-containing primers are annealed to the target DNA.
4. The method of claim 3 , wherein the multiple inosine-containing primers include at least one extender template.
5. The method of claim 1 , wherein at least step (d) is performed under isothermal conditions wherein variation in temperature is within a range of 1° C., 5° C., or 10° C.
6. The method of claim 1 , wherein the inosine is positioned at least 4 nucleotides from the 5′ end of the at least one inosine-containing primer.
7. The method of claim 1 , wherein the nucleic acid amplification reaction is performed using a DNA polymerase selected from exonuclease-deficient T7 DNA polymerase, Bst DNA polymerase, exo (−) Klenow, delta Tts DNA polymerase, or combinations thereof.
8. The method of claim 1 , further comprising a step of denaturing the target DNA prior to the annealing step.
9. The method of claim 8 , wherein the denaturing step comprises a chemical or a thermal denaturing of the target DNA.
10. The method of claim 1 , wherein reaction mixture for the nucleic acid amplification includes a buffer selected from Tris, HEPES, or MOPS.
11. The method of claim 1 , wherein reaction mixture for the nucleic acid amplification includes a surfactant selected from Tween-20, NP-40, Triton-X-100, or combinations thereof.
12. The method of claim 1 , wherein reaction mixture for the nucleic acid amplification includes a reducing agent.
13. The method of claim 1 , wherein reaction mixture for the nucleic acid amplification includes at least one single stranded DNA binding protein.
14. The method of claim 1 , wherein reaction mixture for the nucleic acid amplification includes at least one blocking agent including albumin.
15. The method of claim 1 , wherein reaction mixture for the nucleic acid amplification includes at least one topoisomerase.
16. The method of claim 15 , wherein the at least one topoisomerase comprises type 1 topoisomerase.
17. The method of claim 1 , wherein the target DNA is of eukaryotic origin, prokaryotic origin, viral origin, bacteriophage origin, or synthetic origin.
18. The method of claim 1 , wherein the at least one inosine-containing primer is 5 to 100 nucleotides in length, 5 to 30 nucleotides in length, or 5 to 20 nucleotides in length.
19. The method of claim 1 , wherein the at least one inosine-containing primer demonstrates a melting temperature of 25° C. to 70° C., 30° C. to 65° C., or 40° C. to 55° C. in reaction mixture for the nucleic acid amplification.
20. The method of claim 1 , wherein at least one inosine-containing primer demonstrates a melting temperature of 45° C. in reaction mixture for the nucleic acid amplification.
21. A method of producing at least one amplicon based on a target DNA comprising:
(a) providing a target DNA;
(b) annealing at least one inosine-containing primer to the target DNA to create a target DNA:primer hybrid;
(c) extending the primer strand via a nucleic acid amplification reaction to produce a complementary strand to at least one portion of the target DNA and to generate a nicking site in the extended primer strand at a residue 3′ to the inosine residue;
wherein extending the nicked inosine-containing primer uses a dNTP mixture which is devoid of deoxyinosine triphosphate;
(d) nicking the extended primer strand at the nicking site using a mutant endonuclease V to generate an initiation site in the primer strand for a subsequent nucleic acid amplification reaction; and
(e) repeating steps (c) and (d) employing a strand displacement nucleic acid polymerase for the nucleic acid amplification reaction to produce the at least one amplicon based on the target DNA.
22. The method of claim 21 , wherein steps (a)-(e) occur substantially simultaneously.
23. The method of claim 21 , wherein the amplicon is produced under isothermal conditions.
24. The method of claim 21 , wherein the strand displacement nucleic acid polymerase is an exonuclease-deficient, strand displacement nucleic acid polymerase.
25. A method of producing at least one amplicon based on a target DNA comprising:
(a) providing the target DNA;
(b) providing a DNA amplification reaction mixture comprising at least one inosine-containing primer, at least one 5′→3′ exonuclease-deficient DNA polymerase having strand displacement activity, at least one mutant endonuclease V that is capable of nicking a DNA at a residue 3′ to an inosine residue, and dNTP mixture;
wherein the dNTP mixture is devoid of deoxyinosine triphosphate; and
(c) amplifying at least one portion of the target DNA using the DNA amplification reaction mixture of step (b) to produce the at least one amplicon.
26. The method of claim 25 , wherein amplification reaction is performed under isothermal conditions.
27. The method of claim 25 , wherein the inosine-containing primer comprises at least 2 inosine residues.
28. The method of claim 27 , wherein the inosine-containing primer comprises inosine residues at both the penultimate 3′ residue and ultimate 3′ residue.
29. The method of claim 25 , wherein the inosine-containing primer comprises at least one inosine residue positioned at least 4 nucleotides, at least 5 nucleotides or at least 10 nucleotides from the 5′ end.
30. The method of claim 25 , wherein the at least one 5′→3′ exonuclease-deficient DNA polymerase is selected from 5′→3′ exonuclease-deficient T7 DNA polymerase, 5′→3′ exonuclease-deficient Bst DNA polymerase, 5′→3′ exonuclease-deficient Klenow, 5′→3′ exonuclease-deficient delta Tts DNA polymerase, or combinations thereof.
31. The method of claim 1 , wherein the mutant endonuclease V comprises amino acid sequence of SEQ ID NO: 2, or conservative variants thereof.
32. The method of claim 1 , wherein the mutant endonuclease V comprises amino acid sequence of SEQ ID NO: 3, or conservative variants thereof.
33. The method of claim 21 , wherein the mutant endonuclease V comprises amino acid sequence of SEQ ID NO: 2, or conservative variants thereof.
34. The method of claim 21 , wherein the mutant endonuclease V comprises amino acid sequence of SEQ ID NO: 3, or conservative variants thereof.
35. The method of claim 25 , wherein the mutant endonuclease V comprises amino acid sequence of SEQ ID NO: 2, or conservative variants thereof.
36. The method of claim 25 , wherein the mutant endonuclease V comprises amino acid sequence of SEQ ID NO: 3, or conservative variants thereof.