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Low incorporation of dUMP by some thermostable DNA polymerases may limit their use in PCR amplifications
G Slupphaug1, I Alseth, I Eftedal
1UNIGEN Center for Molecular Biology, University of Trondheim, Norway.
Abstract:
Incorporation of dUMP instead of dTMP is frequently used to control carryover contamination during PCR amplifications. We have tested four thermostable DNA polymerases for their ability to utilize dUTP as a substrate in PCR. Amplification of products in the presence of dUTP instead of dTTP was good with Thermus aquaticus DNA polymerase but highly inefficient with three other thermostable DNA polymerases. The latter was due to: (a) lower incorporation of dUMP relative to dTMP, (b) increased proofreading toward dUMP in DNA, (c) relative termination at dUMP residues as verified by sequencing reactions in the presence of dUTP, (d) thermostable dUTPase activity in the commercial enzyme preparation. The last point only applies to Pyrococcus furiosus DNA polymerase. This study demonstrates that various thermostable DNA polymerases utilize dTTP and dUTP with highly different efficiencies and thus the choice of DNA polymerase may be critical for amplification of DNA.
Insights
Using deoxyuridine triphosphate (dUTP) instead of deoxythymidine triphosphate (dTTP) in PCR helps prevent contamination. However, DNA polymerase efficiency varies greatly when using dUTP, impacting DNA amplification success.
Area of Science:
- Molecular Biology
- Biochemistry
Background:
- Deoxyuridine triphosphate (dUTP) incorporation is a common strategy to control PCR contamination.
- Thermostable DNA polymerases are crucial for various molecular biology applications.
Purpose of the Study:
- To evaluate the efficiency of different thermostable DNA polymerases in utilizing dUTP for PCR.
- To identify factors influencing dUTP incorporation and DNA amplification.
Main Methods:
- Testing four thermostable DNA polymerases with dUTP instead of dTTP in PCR reactions.
- Analyzing dUTP incorporation, proofreading activity, and termination events via sequencing.
- Assessing dUTPase activity in commercial enzyme preparations.
Main Results:
- Thermus aquaticus DNA polymerase showed good amplification with dUTP.
- Three other polymerases exhibited inefficient amplification due to lower dUTP incorporation, increased proofreading, and termination at dUTP residues.
- Thermostable dUTPase activity was observed in one enzyme preparation (Pyrococcus furiosus DNA polymerase).
Conclusions:
- Thermostable DNA polymerases display significant variability in their ability to utilize dUTP.
- The choice of DNA polymerase is critical for successful PCR amplification when using dUTP for contamination control.