Compositions and methods for inhibiting terminal transferase activity
The present invention relates to systems and methods for amplifying nucleic acid. In particular, systems and methods are provided for inhibiting polymerase based terminal transferase activity within a polynucleotide amplification setting (e.g., polymerase chain reaction). In addition, systems and methods are provided for generating amplified products generated with polynucleotide amplification techniques having reduced 3′ non-templated nucleotide addition.
1. A method for generating amplification products, comprising: a) combining boric acid and a reaction solution comprising a nucleic acid template, primers complementary to the nucleic acid template, deoxynucleotide triphosphates, a polymerase having terminal transferase activity and a buffer solution comprising KCl and Tris-HCl; and b) performing a polymerase chain (PCR) with said combined boric acid and said reaction solution such that amplification products are generated.
2. The method of claim 1 , wherein said polymerase having terminal transferase activity is Taq polymerase or a fragment thereof.
3. The method of claim 1 , further comprising the step of detecting said amplification products via mass spectroscopy.
4. The method of claim 1 , wherein said reaction solution further comprises divalent cations.
5. The method of claim 4 , wherein said divalent cations are selected from the group consisting of Mg 2+ and Mn 2 + divalent cations.
6. The method of claim 1 , further comprising the step of analyzing said generated amplification product.
7. The method of claim 6 , wherein said analyzing comprises the use of mass spectroscopy.
8. A method for generating amplification products, comprising: a) combining boric acid and a reaction solution comprising a nucleic acid template, primers complementary to the nucleic acid template, deoxynucleotide triphosphates, a polymerase having terminal transferase activity and a buffer solution comprising (NH) 4 SO 4 and Tris-HCl; and b) performing a polymerase chain (PCR) with said combined boric acid and said reaction solution such that amplification products are generated.
9. The method of claim 8 , wherein said polymerase having terminal transferase activity is Taq polymerase or a fragment thereof.
10. The method of claim 8 , further comprising the step of detecting said amplification products via mass spectroscopy.
11. The method of claim 8 , wherein said reaction solution further comprises divalent cations.
12. The method of claim 11 , wherein said divalent cations are selected from the group consisting of Mg 2+ and Mn 2+ divalent cations.
13. The method of claim 8 , further comprising the step of analyzing said generated amplification product.
14. The method of claim 13 , wherein said analyzing comprises the use of mass spectroscopy.
15. A method for generating amplification products, comprising: a) combining boric acid and a reaction solution comprising a nucleic acid template, primers complementary to the nucleic acid template, deoxynucleotide triphosphates, a polymerase having terminal transferase activity wherein said boric acid is present at a final concentration of 100 mM to 200 mM in said reaction solution; and b) performing a polymerase chain (PCR) with said combined boric acid and said reaction solution such that amplification products are generated.
16. The method of claim 15 , wherein said polymerase having terminal transferase activity is Taq polymerase or a fragment thereof.
17. The method of claim 15 , further comprising the step of detecting said amplification products via mass spectroscopy.
18. The method of claim 15 , wherein said reaction solution further comprises divalent cations.
19. The method of claim 18 , wherein said divalent cations are selected from the group consisting of Mg 2+ and Mn 2+ divalent cations.
20. The method of claim 15 , further comprising the step of analyzing said generated amplification product.
21. The method of claim 20 , wherein said analyzing comprises the use of mass spectroscopy.
22. A method for generating amplification products, comprising: a) combining boric acid and a reaction solution comprising a nucleic acid template, primers complementary to the nucleic acid template, deoxynucleotide triphosphates, a polymerase having terminal transferase activity wherein said boric acid is present at a final concentration of 50 mM to 500 mM in said reaction solution; and b) performing a polymerase chain (PCR) with said combined boric acid and said reaction solution such that amplification products are generated.
23. The method of claim 22 , wherein said polymerase having terminal transferase activity is Taq polymerase or a fragment thereof.
24. The method of claim 22 , further comprising the step of detecting said amplification products via mass spectroscopy.
25. The method of claim 22 , wherein said reaction solution further comprises divalent cations.
26. The method of claim 25 , wherein said divalent cations are selected from the group consisting of Mg 2+ and Mn 2+ divalent cations.
27. The method of claim 22 , further comprising the step of analyzing said generated amplification product.
28. The method of claim 27 , wherein said analyzing comprises the use of mass spectroscopy.