Low incorporation of dUMP by some thermostable DNA polymerases may limit their use in PCR amplifications.
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.
Slupphaug
G
G
UNIGEN Center for Molecular Biology, University of Trondheim, Norway.
Alseth
I
I
Eftedal
I
I
Volden
G
G
Krokan
H E
HE
D001419:Bacteria / Q000201:enzymology; D001483:Base Sequence; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D003856:Deoxyuracil Nucleotides / Q000737:chemistry*; D004795:Enzyme Stability; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000737:chemistry*
D003856:Deoxyuracil Nucleotides; D013942:Thymine Nucleotides; C027078:deoxyuridine triphosphate; C007267:2'-deoxyuridylic acid; D004259:DNA-Directed DNA Polymerase; C024157:thymidine 5'-triphosphate