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 PCR 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 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 via denaturation thereof at a second temperature in the range of 50° C. to about 72° C. as a result of being subjected to said second temperature, said template nucleic acid being unamplified and not denatured, said single-stranded nucleic acid binding protein being present in said reaction mixture in a stoichiometric excess relative to said primer.
2. A PCR 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 PCR 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 PCR 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 T7gp2.5-Δ26C.
5. A PCR reaction mixture according to claim 1 , said stoichiometric excess being at least 2-fold.
6. A PCR reaction mixture according to claim 1 , said stoichiometric excess being at least 3-fold.
7. A PCR reaction mixture according to claim 1 , said stoichiometric excess being at least 4-fold.
8. A PCR reaction mixture according to claim 1 , said stoichiometric excess being at least 50%.
9. A PCR reaction mixture according to claim 1 , said stoichiometric excess being at least 1-fold.