Dried composition for hot-start PCR with long-term stability
The present invention relates to a dried composition for hot-start PCR, more precisely a dried composition for hot-start PCR with improved stability and long-term storagability which is characteristically prepared by the steps of preparing a reaction mixture by mixing an aqueous solution containing reaction buffer, MgCl 2 , 4 types of dNTPs, DNA polymerase with pyrophosphate and pyrophosphatase in a reaction tube; and drying the reaction mixture prepared above, a preparation method of the same and a method for amplifying nucleic acid using the same. The dried composition for hot-start PCR is added with pyrophosphate and pyrophosphatase together before drying, so that it can have improved stability and long-term storagability as well as convenience in use, compared with the conventional compositions for hot-start PCR. Therefore, this composition can be effectively used for hot-start PCR, multiplex PCR or real-time quantitative PCR.
1. A dried composition in a reaction tube for hot-start PCR comprising reaction buffer, MgCl 2 , 4 types of dNTPs, DNA polymerase, pyrophosphate, pyrophosphatase which converts inorganic pyrophosphate to two phosphate ions, and one or more stabilizer selected from the group consisting of polyol, gelatin, bovine serum albumin (BSA), Thesit, and PET-8000.
2. The dried composition in a reaction tube for hot-start PCR according to claim 1 , wherein the composition further comprises a primer set or a probe.
3. The dried composition in a reaction tube for hot-start PCR according to claim 1 , wherein the composition further comprises a fluorescent dye binding to DNA.
4. The dried composition in a reaction tube for hot-start PCR according to claim 3 , wherein the fluorescent dye is selected from the group consisting of SyBr Green, EtBr and HRdye.
5. The dried composition in a reaction tube for hot-start PCR according to claim 1 , wherein the composition further comprises template nucleic acid.
6. The dried composition in a reaction tube for hot-start PCR according to claim 1 , wherein the pyrophosphate is included at the concentration of 0.3-5 mM in the final reaction solution and the pyrophosphatase is included at the concentration more than 30 to 200 mU in 20 μl d of the final reaction solution.
7. The dried composition in a reaction tube for hot-start PCR according to claim 6 , wherein the pyrophosphate is included at the concentration of 0.95-3.0 mM in the final reaction solution.
8. The dried composition in a reaction tube for hot-start PCR according to claim 6 , wherein the pyrophosphatase is included at the concentration of 50-100 mU in 20 μl of the final reaction solution.
9. The dried composition in a reaction tube for hot-start PCR according to claim 1 , wherein the DNA polymerase is one or more polymerases selected from the group consisting of the polymerase having the activity of 5′→3′ exonuclease, the polymerase having the activity of 3′→5′ exonuclease, and the polymerase having none of the activities of 5′→3′ exonuclease and 3′→5′ exonuclease.
10. The dried composition in a reaction tube for hot-start PCR according to claim 1 , wherein the composition further comprises a dye which is not reactive to nucleic acid.
11. The dried composition in a reaction tube for hot-start PCR according to claim 10 , wherein the dye is one or more materials selected from the group consisting of rhodamine, tamra, lax, bromophenol blue, xylene cyanole, bromocresol red, and cresol red.
12. The dried composition in a reaction tube for hot-start PCR according to claim 1 , wherein the composition is used for multiplex PCR, real time PCR or real-time quantitative PCR.
13. A kit for hot-start PCR comprising one of the dried compositions for hot-start PCR of claim 1 .
14. A method for amplifying nucleic acid using one of the dried compositions for hot-start PCR of claim 1 .
15. The method for amplifying nucleic acid according to claim 14 , wherein the method comprises the following steps: mixing a sample containing template nucleic acid with the dried composition for hot-start PCR; performing a reaction to amplify the reaction mixture; and analyzing the amplified product of the template nucleic acid.
16. The method for amplifying nucleic acid according to claim 14 , wherein the PCR is selected from the group consisting of multiplex PCR, real-time PCR and real-time quantitative PCR.