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Fidelity and mutational spectrum of Pfu DNA polymerase on a human mitochondrial DNA sequence
1Division of Toxicology, Massachusetts Institute of Technology, Cambridge 02139, USA.
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 from Pyrococcus 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) of Pfu was 6.5 x 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.
Insights
Researchers studied rare genetic mutations using Pfu DNA polymerase and constant denaturant capillary electrophoresis (CDCE). Pfu polymerase has a low error rate of 6.5 x 10(-7), with identified mutation "hot spots".
Area of Science:
- Genetics
- Molecular Biology
- Biochemistry
Background:
- Studying rare genetic changes in human tissues requires highly sensitive techniques.
- Point mutations at fractions as low as 10(-6) are crucial for understanding mutational spectra.
- Standard PCR amplification can introduce artificial mutations ('PCR noise'), necessitating careful characterization of DNA polymerase and conditions.
Purpose of the Study:
- To characterize the fidelity and mutation spectrum of Pfu DNA polymerase.
- To develop a sensitive method for detecting low-frequency mutations in human mitochondrial DNA.
- To identify specific mutation 'hot spots' generated by Pfu polymerase.
Main Methods:
- Utilized Pfu DNA polymerase, known for its high fidelity, for PCR amplification.
- Employed constant denaturant capillary electrophoresis (CDCE) to separate and isolate PCR products.
- Separated mutant/wild-type heteroduplexes using CDCE for enrichment and analysis.
- Incorporated an artificial mutant as an internal standard for accurate mutant fraction calculation.
Main Results:
- Identified point mutations generated by Pfu DNA polymerase in a 96-bp mitochondrial sequence.
- Determined the average error rate of Pfu DNA polymerase to be 6.5 x 10(-7) mutations per base pair duplication.
- Characterized five frequent mutation types (hot spots): three transversions, one transition, and one 1-bp deletion.
Conclusions:
- Pfu DNA polymerase exhibits high fidelity, making it suitable for studying tissue mutational spectra.
- CDCE is an effective strategy for enumerating and identifying low-frequency point mutations from PCR.
- Further reduction of Pfu-induced mutants is necessary to achieve even higher sensitivity in mutation detection.