Low Incorporation of dUMP by Some Thermostable DNA Polymerases May Limit Their Use in PCR Amplifications
| Autor: | Ingrun Alseth, Gunnar Volden, Geir Slupphaug, Ingrid Eftedal, H.E. Krokan |
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| Rok vydání: | 1993 |
| Předmět: |
Hot Temperature
DNA polymerase DNA polymerase II Molecular Sequence Data Biophysics DNA-Directed DNA Polymerase Polymerase Chain Reaction Biochemistry Substrate Specificity law.invention chemistry.chemical_compound law Enzyme Stability Thymine Nucleotides Molecular Biology Polymerase chain reaction Bacteria Base Sequence Thermus aquaticus biology Cell Biology biology.organism_classification Molecular biology chemistry biology.protein Pyrococcus furiosus Proofreading Deoxyuracil Nucleotides Uracil nucleotide DNA |
| Zdroj: | Analytical Biochemistry. 211:164-169 |
| ISSN: | 0003-2697 |
| DOI: | 10.1006/abio.1993.1248 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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