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 reaction mixture that is at a temperature below 37° C., said mixture comprising a template nucleic acid, a primer having a nucleotide sequence that is complementary to a specific target portion of said template nucleic acid, an enzyme effective to catalyze primer extension, nucleotides necessary for enzyme-directed nucleic acid synthesis, and a single-stranded nucleic acid binding protein having no known enzymatic activity and that is effective to inhibit said primer from participating in a primer extension reaction up to at least a first temperature at or below 30° C., wherein the inhibitive capability of said single-stranded nucleic acid binding protein is lost at an inactivation temperature in the range of 50° C. to about 72° C. as a result of being subjected to said inactivation temperature, said template nucleic acid being unamplified and not denatured.
2. A reaction mixture according to claim 1 , said single-stranded nucleic acid binding protein comprising at least one of wild-type T7 gp2.5 and its mutant variants, or a combination thereof.
3. A reaction mixture according to claim 1 , said single-stranded nucleic acid binding protein comprising at least one of T7gp2.5-F232L and T7 gp2.5-Δ21C.
4. A reaction mixture according to claim 1 , said single-stranded nucleic acid binding protein comprising a mixture of proteins including wild-type T7 gp2.5 and T7 gp2.5-Δ26C.
5. A reaction mixture according to claim 1 , further comprising a divalent cation.
6. A method of duplicating a template nucleic acid or a portion thereof, comprising the steps of:
(a) providing the reaction mixture of claim 1 at said first temperature,
(b) at said second temperature, carrying out a hybridization reaction in said reaction mixture to produce a hybridized product of said primer and said template nucleic acid, 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 being inhibited from participating in at least one of the hybridization and extension reactions at said first temperature due to the presence of said single-stranded nucleic acid binding protein but becoming uninhibited from participating in at least one of said reactions as a result of said single-stranded nucleic acid binding protein losing its inhibitive capability as a result of being subjected to said inactivation temperature.
7. A method according to claim 6 , said reaction mixture further comprising a divalent cation at said first temperature.
8. A method according to claim 6 , 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.
9. A method according to claim 6 , 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).
10. A method according to claim 9 , 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.
11. A method according to claim 10 , said third temperature being in the range of 50° C. to about 72° C., and said fourth temperature being at or above 90° C.
12. A method according to claim 10 , said second and third temperatures being the same.
13. A method according to claim 6 , said single-stranded nucleic acid binding protein comprising at least one of wild-type T7 gp2.5 and its mutant variants, or a combination thereof.
14. A method according to claim 6 , said single-stranded nucleic acid binding protein comprising at least one of T7gp2.5-F232L and T7 gp2.5-Δ21C.
15. A method according to claim 6 , said single-stranded nucleic acid binding protein comprising a mixture of proteins including wild-type T7 gp2.5 and T7 gp2.5-Δ26C.
16. A method of duplicating a template nucleic acid or a portion thereof, comprising the steps of:
(a) at a first temperature below 37° C., providing the reaction mixture of claim 1 ,
(b) at a second temperature higher than said first temperature, carrying out a hybridization reaction in said reaction mixture to produce a hybridized product of said primer and said template nucleic acid, 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.