Normalization of polymerase activity
View Patent ↗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 in a PCR-invasive cleavage assay having attenuated variability in DNA polymerase performance, comprising:
a) providing a sample comprising a target nucleic acid;
b) combining said sample with purified exogenous non-target DNA isolated from fish in a reaction mixture comprising PCR-invasive cleavage assay reagents, wherein said PCR-invasive cleavage assay reagents comprise
i) thermostable DNA polymerase;
ii) dNTPs;
iii) a first primer and a second primer configured for amplifying a product from said target nucleic acid;
iv) a flap endonuclease;
v) a FRET cassette;
vi) a flap oligonucleotide;
wherein said reaction mixture is characterized in that it can amplify said target nucleic acid and produce a detectable signal proportional to the amount of said target nucleic acid in said reaction mixture
c) detecting amplification of said target nucleic acid during an amplification reaction; and
d) calculating the amount of said target nucleic acid in said reaction mixture.
2. The method of claim 1 , wherein said 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 said purified exogenous non-target DNA isolated from fish is added at a concentration of approximately 2 to approximately 20 nanograms per μl of reaction mixture.
4. The method of claim 1 , wherein said purified exogenous non-target DNA isolated from fish is added at a concentration of approximately 6 to 7 nanograms per μl of reaction mixture.
5. The method of claim 1 , wherein said thermostable DNA polymerase is a thermostable eubacterial DNA polymerase.
6. The method of claim 5 , wherein said eubacterial DNA polymerase is from Thermus aquaticus.
7. The method of claim 1 , wherein the DNA polymerase is modified for hot start PCR.
8. The method of claim 1 , wherein said flap endonuclease is a FEN-1 endonuclease.
9. The method of claim 8 , wherein said FEN-1 endonuclease is from an archaeal organism.
10. The method of claim 1 , wherein the target nucleic acid is human nucleic acid.
11. A reaction mixture comprising:
a) target nucleic acid;
b) purified exogenous non-target DNA isolated from fish;
c) PCR-invasive cleavage assay reagents comprising:
i) thermostable DNA polymerase;
ii) dNTPs;
iii) a first primer and a second primer configured for amplifying a product from said target nucleic acid;
iv) a flap endonuclease;
v) a FRET cassette; and
vi) a flap oligonucleotide;
wherein said reaction mixture is characterized in that it can amplify said target nucleic acid and produce a detectable signal proportional to the amount of said target nucleic acid in said reaction mixture.
12. The reaction mixture of claim 11 , wherein said thermostable DNA polymerase is from Thermus aquaticus.
13. The reaction mixture of claim 11 , wherein said purified exogenous non-target DNA isolated from fish comprises DNA isolated from herring and/or cod and/or salmon.
14. The reaction mixture of claim 11 , wherein said purified exogenous non-target DNA isolated from fish is added at a concentration of approximately 2 to approximately 20 nanograms per μl of reaction mixture.
15. The reaction mixture of claim 11 , wherein said purified exogenous non-target DNA isolated from fish is added at a concentration of approximately 6 to 7 nanograms per μl of reaction mixture.
16. The reaction mixture of claim 11 , wherein the DNA polymerase is modified for hot start PCR.
17. The reaction mixture of claim 11 , wherein said flap endonuclease is a FEN-1 endonuclease.
18. The reaction mixture of claim 17 , wherein said FEN-1 endonuclease is from an archaeal organism.
19. The reaction mixture of claim 11 , wherein the target nucleic acid is human nucleic acid.