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 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 in a reaction mixture comprising PCR-invasive cleavage assay reagents, wherein said PCR-invasive cleavage assay reagents comprise
i) DNA polymerase;
ii) a flap endonuclease; and
iii) a hairpin oligonucleotide;
iv) a primer
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 hairpin oligonucleotide is a FRET cassette oligonucleotide.
3 . The method of claim 1 , wherein said non-target DNA is genomic DNA.
4 . The method of claim 1 , wherein said non-target DNA is isolated from fish.
5 . The method of claim 4 , wherein said non-target DNA isolated from fish comprises DNA isolated from herring and/or cod and/or salmon.
6 . The method of claim 1 , wherein said non-target DNA is added at a concentration of approximately 2 to approximately 20 nanograms per μl of reaction mixture.
7 . The method of claim 1 , wherein said non-target nucleic acid is added at a concentration of approximately 6 to 7 nanograms per μl of reaction mixture
8 . The method of claim 1 , wherein said DNA polymerase is a thermostable DNA polymerase.
9 . The method of claim 8 , wherein said thermostable DNA polymerase is a eubacterial DNA polymerase.
10 . The method of claim 9 , wherein said eubacterial DNA polymerase is from Thermus aquaticus.
11 . The method of claim 1 , wherein the DNA polymerase is modified for hot start PCR.
12 . The method of claim 1 , wherein said flap endonuclease is thermostable.
13 . The method of claim 1 , wherein said flap endonuclease is a FEN-1 endonuclease.
14 . The method of claim 13 , wherein said FEN-1 endonuclease is from an archaeal organism.
15 . The method of claim 1 , wherein the target nucleic acid is human.
16 . A reaction mixture comprising:
a) target nucleic acid;
b) purified exogenous non-target DNA;
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.
17 . A set of reaction mixtures, each of said reaction mixtures comprising 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 each 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;
wherein said set of reaction mixtures comprises a dilution series, wherein each member of said dilution series comprises a different known amount of said target nucleic acid, and essentially the same concentration of reaction components i-vi.
18 . The composition of claim 16 or claim 17 . wherein said thermostable DNA polymerase is from Thermus aquaticus.
19 . The composition of claim 16 or claim 17 , wherein said purified exogenous, non-target DNA is isolated from fish.
20 . The composition of claim 19 , wherein said purified exogenous non-target DNA isolated from fish comprises DNA isolated from herring and/or cod and/or salmon.