FIDELITY AND MUTATIONAL SPECTRUM OF PFU DNA-POLYMERASE ON A HUMAN MITOCHONDRIAL-DNA SEQUENCE

The study of rare generic changes in human tissues requires specialize d techniques. Point mutations at fractions at or below 10(-6) must be observed to discover even the most prominent features of the point mut ational 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 wit h regard to the DNA polymerase and conditions used to avoid interpreti ng a PCR-generated mutation as one arising in human tissue. The thermo stable 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 mutation al spectra. Here, we apply constant denaturant capillary electrophores is [CDCE] to separate and isolate the products of DNA amplification. T his new strategy permitted direct enumeration and identification of po int mutations created by Pfu DNA polymerase in a 96-bp low melting dom ain of a human mitochondrial sequence despite the very low mutant frac tions 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 tissue s. 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 a verage error rate (mutations per base pair duplication) of Pfu was 6.5 x 10(-7), and five of its more Frequent mutations (hot spots) consist ed of three transversions (CC --> TA, AT --> TA, and AT --> CG), one t ransition (AT --> CC), and one I-bp deletion (in an AAAAAA sequence). To achieve an even higher sensitivity, the amount of Pfu-induced mutan ts must be reduced.