Fidelity and Mutational Spectrum of Pfu DNA Polymerase on a Human Mitochondrial DNA Sequence
Open Access
- 1 August 1997
- journal article
- Published by Cold Spring Harbor Laboratory in Genome Research
- Vol. 7 (8) , 843-852
- https://doi.org/10.1101/gr.7.8.843
Abstract
The study of rare genetic changes in human tissues requires specialized techniques. Point mutations at fractions at or below 10−6 must be observed to discover even the most prominent features of the point mutational spectrum. PCR permits the increase in number of mutant copies but does so at the expense of creating many additional mutations or “PCR noise”. Thus, each DNA sequence studied must be characterized with regard to the DNA polymerase and conditions used to avoid interpreting a PCR-generated mutation as one arising in human tissue. The thermostable DNA polymerase derived fromPyrococcus furiosus designated Pfu has the highest fidelity of any DNA thermostable polymerase studied to date, and this property recommends it for analyses of tissue mutational spectra. Here, we apply constant denaturant capillary electrophoresis (CDCE) to separate and isolate the products of DNA amplification. This new strategy permitted direct enumeration and identification of point mutations created by Pfu DNA polymerase in a 96-bp low melting domain of a human mitochondrial sequence despite the very low mutant fractions generated in the PCR process. This sequence, containing part of the tRNA glycine and NADH dehydrogenase subunit 3 genes, is the target of our studies of mitochondrial mutagenesis in human cells and tissues. Incorrectly synthesized sequences were separated from the wild type as mutant/wild-type heteroduplexes by sequential enrichment on CDCE. An artificially constructed mutant was used as an internal standard to permit calculation of the mutant fraction. Our study found that the average error rate (mutations per base pair duplication) ofPfu was 6.5 × 10−7, and five of its more frequent mutations (hot spots) consisted of three transversions (GC → TA, AT → TA, and AT → CG), one transition (AT → GC), and one 1-bp deletion (in an AAAAAA sequence). To achieve an even higher sensitivity, the amount of Pfu-induced mutants must be reduced.Keywords
This publication has 45 references indexed in Scilit:
- Mutational spectrum of chromium(VI) in human cellsMutation Research Letters, 1994
- Improved polymerase fidelity in PCR-SSCPAMutation Research Letters, 1993
- Mutational analysis using denaturing gradient gel electrophoresis and PCRMutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, 1993
- High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosusGene, 1991
- [6] Efficient site-directed mutagenesis using uracil-containing DNAPublished by Elsevier ,1991
- Estimation of in-vivo miscoding ratesJournal of Molecular Biology, 1988
- Depurination-induced infidelity of DNA synthesis with purified DNA replication proteins in vitroBiochemistry, 1983
- Isolation of a human lymphoblastoid line heterozygous at the thymidine kinase locus: Possibility for a rapid human cell mutation assayBiochemical and Biophysical Research Communications, 1978
- Studies on the biochemical basis of spontaneous mutationJournal of Molecular Biology, 1974
- Studies on polynucleotidesJournal of Molecular Biology, 1971