Hot start nucleic acid amplification
View Patent ↗Methods and compositions for performing nucleic acid duplication and amplification reactions are provided. A single-stranded nucleic acid binding protein is selected and provided in the reaction mixture which is assembled at a low, nonstringent temperature to include all of the necessary reagents for successful nucleic acid duplication or amplification reactions. By incorporating a single-stranded nucleic acid binding protein into the reaction mixture at low temperature, the generation of nonspecific products such as amplification products is improved despite the reaction mixture having been fully assembled at a nonstringent temperature.
1. A method of duplicating a template nucleic acid, or a portion thereof, wherein a primer having a nucleotide sequence that is complementary to a target portion of the template nucleic acid is hybridized to the template nucleic acid and then extended via an enzyme, the method comprising the steps of:
(a) at a first temperature, preparing a reaction mixture comprising a primer, a template nucleic acid, an enzyme effective to catalyze primer extension and single-stranded nucleic acid binding protein comprising at least one of wild-type T7 gp2.5 and its mutant variants, or a combination thereof, wherein said single-stranded nucleic acid binding protein inhibits the generation of nonspecific primer extension products at said first temperature,
(b) at a second temperature higher than said first temperature, carrying out a hybridization reaction in said reaction mixture to produce a hybridized product, wherein at said second temperature said primer is substantially uninhibited by said single-stranded nucleic acid binding protein from participating in the hybridization reaction, and
(c) at a third temperature higher than said first temperature, carrying out a primer extension reaction to produce from said hybridized product an extended product;
said primer becoming uninhibited from participating in at least one of the hybridization and extension reactions as a result of or in conjunction with said single-stranded nucleic acid binding protein being denatured at a temperature above said first temperature.
2. A method according to claim 1 , said enzyme being a polymerase, said reaction mixture farther comprising a divalent cation at said first temperature.
3. A method according to claim 1 , wherein at said third temperature said primer is uninhibited by said single-stranded nucleic acid binding protein from participating in the primer extension reaction.
4. A method according to claim 1 , said first temperature being at or below 37° C. and said second and third temperatures each being in the range of 50° C. to about 72° C.
5. A method according to claim 1 , further comprising the following step performed intermediate said steps (a) and (b):
(a.1) initially heating the reaction mixture to a fourth temperature, higher than said second and third temperatures, to denature double-stranded template nucleic acids present in the reaction mixture.
6. A method according to claim 1 , further comprising the following step performed subsequent to said step (c):
(d) heating the reaction mixture to a fourth temperature, higher than said second and third temperatures, to denature double-stranded extended products present in the reaction mixture which were produced during said step (c).
7. A method according to claim 6 , comprising carrying out an amplification reaction by repeating said steps (b) and (c) at least once to generate an amplified product, wherein the generation of specific amplified product is improved as a result of incorporating said single-stranded nucleic acid binding protein into said reaction mixture at said first temperature.
8. A method according to claim 7 , said first temperature being at or below 37° C., said second and third temperatures each being in the range of 50° C. to about 72° C., and said fourth temperature being at or above 90° C.
9. A method according to claim 7 , said second and third temperatures being the same.
10. A method according to claim 1 , wherein said single-stranded nucleic acid binding protein does not have any known enzymatic activity.
11. A method of duplicating a template nucleic acid, or a portion thereof, wherein a primer having a nucleotide sequence that is complementary to a target portion of the template nucleic acid is hybridized to the template nucleic acid and then extended via an enzyme, the method comprising the steps of:
(a) at a first temperature, preparing a reaction mixture comprising a primer, a template nucleic acid, an enzyme effective to catalyze primer extension and single-stranded nucleic acid binding protein comprising at least one of wild-type T7 gp2.5 and its mutant variants, or a combination thereof
(b) at a second temperature higher than said first temperature, carrying out a hybridization reaction in said reaction mixture to produce a hybridized product, and
(c) at a third temperature higher than said first temperature, carrying out a primer extension reaction to produce from said hybridized product an extended product;
wherein the generation of specific extended product is improved as a result of incorporating said single-stranded nucleic acid binding protein into said reaction mixture at said first temperature.
12. A method according to claim 11 , said single-stranded nucleic acid binding protein comprising T7 gp2.5-F232L.
13. A method according to claim 11 , said single-stranded nucleic acid binding protein comprising T7 gp2.5-Δ21C.
14. A method according to claim 11 , said single-stranded nucleic acid binding protein comprising a mixture of proteins including wild-type T7 gp2.5 and 17 gp2.5-Δ26C.
15. A method according to claim 1 , excluding the introduction of any additional reagent to the reaction mixture subsequent to the reaction mixture being prepared at said first temperature and prior to carrying out said primer extension reaction.
16. A method according to claim 1 , said single-stranded nucleic acid binding protein being present in said reaction mixture in a stoichiometric excess relative to said primer.
17. A method according to claim 16 , said stoichiometric excess being at least 1-fold.
18. A method according to claim 1 , wherein said single-stranded nucleic acid binding protein is supplied from a composition of it in a buffer solution, said buffer solution comprising 1-100 mM Tris-HCl pH 7.5, 1-100 mM EDIA, 0.005-200 mM DTT, 10-80 mass percent glycerol, balance water.
19. A method according to claim 1 , said reaction mixture further comprising, at said first temperature, nucleotides necessary for enzyme-directed nucleic acid synthesis.
20. A method according to claim 4 , said reaction mixture further comprising, at said first temperature, nucleotides necessary for enzyme-directed nucleic acid synthesis.
21. A method according to claim 7 , said reaction mixture further comprising, at said first temperature, nucleotides necessary for enzyme-directed nucleic acid synthesis.
22. A method according to claim 8 , said reaction mixture further comprising, at said first temperature, nucleotides necessary for enzyme-directed nucleic acid synthesis.
23. A method according to claim 11 , said reaction mixture further comprising, at said first temperature, nucleotides necessary for enzyme-directed nucleic acid synthesis.
24. A method according to claim 16 , said stoichiometric excess being at least 2-fold.
25. A method according to claim 16 , said stoichiometric excess being at least 3-fold.
26. A method according to claim 16 , said stoichiometric excess being at least 4-fold.
27. A method according to claim 11 , said reaction mixture at said first temperature comprising a stoichiometric excess of single-stranded nucleic acid binding protein relative to primer in said reaction mixture.
28. A method according to claim 27 , said stoichiometric excess being at least 50%.
29. A method according to claim 27 , said stoichiometric excess being at least 2-fold.
30. A method according to claim 27 , said stoichiometric excess being at least 3-fold.
31. A method according to claim 27 , said stoichiometric excess being at least 4-fold.
32. A method according to claim 1 , wherein the generation of specific extended product is improved as a result of incorporating said single-stranded nucleic acid binding protein into said reaction mixture at said first temperature.