Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1997 Aug;7(8):843-52.
doi: 10.1101/gr.7.8.843.

Fidelity and Mutational Spectrum of Pfu DNA Polymerase on a Human Mitochondrial DNA Sequence

Affiliations
Free PMC article

Fidelity and Mutational Spectrum of Pfu DNA Polymerase on a Human Mitochondrial DNA Sequence

P André et al. Genome Res. .
Free PMC article

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.

Figures

Figure 1
Figure 1
Analysis by CDCE of the purified homoduplex mutant fractions after 14 and 34 doublings using Pfu DNA polymerase. This CDCE was run using a 5% polyacrylamide gel in a 35-cm capillary with a 15-cm heated zone at a temperature of 70.6°C with a 5-μA current. Many small peaks after 14 doublings may be seen to increase significantly after 34 doublings. Of the major peaks for which the mutant sequences were determined, peak A is an AT → CG transversion at bp 10071 of human mitochondrial DNA; peak B is an AT → GC transition at bp 10108; peak C is an AT deletion occurring in a run of six consecutive AT base pairs at positions 10048–10053; peak D is an AT → TA transversion at bp 10071; and peak E is a GC → TA transversion at bp 10070.
Figure 2
Figure 2
The relationship between the number of doublings and the mutant fraction of all mutants created by Pfu. The mutant fractions from three independent experiments were estimated by comparing the total area of all mutant peaks with the area of an internal standard. A linear regression analysis was applied to all data points. By dividing the estimated slope by the length of the low-temperature melting domain of our DNA fragment (96 bp), we calculated an error rate of 6.5 ± 3.1 × 10−7 mutants per base pair per doubling (95% confidence interval).
Figure 3
Figure 3
Summary of the kinds and positions of the mutations induced by Pfu DNA polymerase. The low melting domain of 96 bp in which we can detect mutations extends from the mitochondrial genome position at bp 10,031 to 10,126. The symbol Δ is used to indicate a base deletion.
Figure 4
Figure 4
Plots of the mutant hot spot fractions vs. the amount of DNA doublings using data from three independent experiments. A linear regression analysis was applied to all data points. The mutation rate per doubling for each hot spot was estimated from the slope. The mutation rate for hot spot A was 2.8 ± 1.1 × 10−6; for hot spot B, 1.6 ± 1.3 × 10−6; for hot spot C, 5.6 ± 3.3 × 10−6; for hot spot D, 1.9 ± 0.9 × 10−6; and for hot spot E, 3.6 ± 1.7 × 10−6 (95% confidence intervals for the best estimates of the slopes).

Similar articles

See all similar articles

Cited by 22 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback