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 reaction mixture for producing an amplified DNA product from a target nucleic acid, comprising:
a) an amount of target nucleic acid;
b) purified exogenous non-target DNA isolated from fish; and
c) PCR amplification assay reagents comprising:
i) thermostable DNA polymerase;
ii) dNTPs;
iii) a first primer and a second primer configured for amplifying a DNA product from the target nucleic acid; and
iv) a flap endonuclease;
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.
2. The reaction mixture of claim 1 , wherein the purified exogenous non-target DNA isolated from fish comprises DNA isolated from herring and/or cod and/or salmon.
3. The reaction mixture of claim 1 , wherein the PCR amplification assay reagents further comprise an oligonucleotide probe.
4. The reaction mixture of claim 3 , wherein the oligonucleotide probe is a flap oligonucleotide.
5. The reaction mixture of claim 1 , wherein the purified exogenous non-target DNA isolated from fish is at a concentration of 6 to 7 nanograms per μl of reaction mixture.
6. The reaction mixture of claim 1 , wherein the thermostable DNA polymerase is a eubacterial DNA polymerase.
7. The reaction mixture of claim 6 , wherein the eubacterial DNA polymerase is a Pol A type repair DNA polymerase from Thermus aquaticus.
8. The reaction mixture of claim 1 , wherein the thermostable DNA polymerase is modified for hot start PCR.
9. The reaction mixture of claim 1 , wherein the flap endonuclease is a FEN-1 endonuclease.
10. The reaction mixture of claim 1 , wherein the target nucleic acid is human nucleic acid.
11. The reaction mixture of claim 10 , wherein the human nucleic acid comprises human genomic DNA.
12. A set of reaction mixtures for producing amplified DNA product from a target nucleic acid, each of the reaction mixtures comprising:
a) an amount of target nucleic acid;
b) purified exogenous non-target DNA isolated from fish; and
c) PCR amplification assay reagents comprising:
i) thermostable DNA polymerase;
ii) dNTPs;
iii) a first primer and a second primer configured for amplifying a DNA product from the target nucleic acid; and
iv) a flap endonuclease;
wherein each 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; and
wherein the set of reaction mixtures comprises a dilution series, wherein each member of the dilution series comprises a different known amount of the same target nucleic acid, and essentially the same concentration of reaction components b) and c).
13. The set of reaction mixtures of claim 12 , wherein the purified exogenous non-target DNA isolated from fish comprises DNA isolated from herring and/or cod and/or salmon.
14. The set of reaction mixtures of claim 12 , wherein the PCR amplification assay reagents further comprise an oligonucleotide probe.
15. The set of reaction mixtures of claim 14 , wherein the oligonucleotide probe is a flap oligonucleotide.
16. The set of reaction mixtures of claim 12 , wherein the DNA polymerase is a eubacterial repair DNA polymerase from Thermus aquaticus.
17. The set of reaction mixtures of claim 12 , wherein the thermostable DNA polymerase is modified for hot start PCR.
18. The set of reaction mixtures of claim 12 , wherein the flap endonuclease is a FEN-1 endonuclease.
19. The set of reaction mixtures of claim 12 , wherein the target nucleic acid is human nucleic acid.
20. A method for amplifying a target nucleic acid in a PCR amplification assay, comprising:
a) preparing a reaction mixture comprising PCR amplification assay reagents, the reaction mixture comprising:
i) target nucleic acid;
ii) purified exogenous non-target DNA isolated from fish;
iii) thermostable DNA polymerase;
iv) dNTPs;
v) a first primer and a second primer configured for amplifying a DNA product from the target nucleic acid; and
vi) a flap endonuclease;
b) amplifying an amount of the DNA product from the target nucleic acid; and
c) detecting amplification of the DNA product from the target nucleic acid,
wherein the reaction mixture is characterized in that it can amplify an amount of DNA product from the target nucleic acid that is proportional to the amount of the target nucleic acid in the reaction mixture.