Normalization of polymerase activity
Provided herein is technology relating to the amplification-based detection of nucleic acids and particularly, but not exclusively, to methods and compositions for minimizing variability in the activity between different samples or manufacturing lots of DNA polymerases, such as Taq DNA polymerase.
1. A method for quantitating target nucleic acid, comprising:
a) providing a reaction mixture comprising:
i) an amount of target nucleic acid;
ii) purified exogenous non-target DNA isolated from fish at a concentration of approximately 2 to 20 nanograms per μl of reaction mixture; and
iii) PCR amplification assay reagents comprising:
thermostable DNA polymerase;
dNTPs;
a first primer and a second primer configured for amplifying a DNA product from the target nucleic acid;
a flap endonuclease; and
an oligonucleotide probe;
wherein the reaction mixture is characterized in that it can amplify the DNA product from the target nucleic acid and produce an amount of amplified DNA product proportional to the amount of the target nucleic acid in the reaction mixture;
b) conducting an amplification reaction with the reaction mixture and measuring DNA product amplified from the target nucleic acid during the amplification reaction; and
c) calculating the amount of the target nucleic acid in the reaction mixture.
2. The method of claim 1 , where the purified exogenous non-target DNA isolated from fish comprises DNA isolated from herring and/or cod and/or salmon.
3. The method of claim 1 , wherein the purified exogenous non-target DNA isolated from fish is added at a concentration of approximately 6 to 7 nanograms per μl of reaction mixture.
4. The method of claim 1 , wherein the oligonucleotide probe is a flap oligonucleotide.
5. The method of claim 1 , wherein the reaction mixture comprises an oligonucleotide with a fluorescent label.
6. The method of claim 1 , wherein the thermostable DNA polymerase is a thermostable eubacterial DNA polymerase.
7. The method of claim 6 , wherein the eubacterial DNA polymerase is from Thermus aquaticus.
8. The method of claim 1 , wherein the DNA polymerase is modified for hot start PCR.
9. The method of claim 1 , wherein the flap endonuclease is a FEN-1 endonuclease.
10. The method of claim 9 , wherein the FEN-1 endonuclease is from an archaeal organism.
11. The method of claim 1 , wherein the target nucleic acid is human nucleic acid.
12. The method of claim 11 , wherein the human nucleic acid comprises human genomic DNA.