Summary auto-generated
This study investigated how DNA polymerase errors during PCR amplification affect estimates of genetic diversity in RNA viruses. Researchers analyzed vesicular stomatitis virus (VSV) populations using RT-PCR with two different polymerases: Taq (error-prone) and Pfu (high-fidelity with proofreading activity). When sequencing two ~500 nucleotide regions of the VSV genome, Taq amplification detected 10-22 mutations per region, while Pfu detected only 0-5 mutations in the same samples. Control experiments showed Taq's error rate was approximately 0.27×10⁻⁴ misincorporations per base pair per cycle, about 9-10 times higher than Pfu's rate. Most mutations detected with Taq in early samples (0h, 28h, 48h) appeared to be PCR errors rather than genuine viral mutations, since they were unique to individual clones and absent in Pfu-amplified products. In contrast, sample 40d (from 40 days of viral passage) showed higher genetic diversity, with some mutations detected by both polymerases, indicating genuine viral variation. The study demonstrates that polymerase choice significantly impacts diversity estimates, particularly at within-population levels where natural mutation rates may be similar to PCR error rates.
Key findings
- Taq polymerase introduces approximately 9-10 times more errors than Pfu during PCR amplification of viral templates
- Most mutations detected with Taq in early VSV samples were PCR artifacts rather than authentic viral mutations, identifiable by their absence in Pfu-amplified products
- Early-passage VSV samples showed minimal genuine genetic diversity, suggesting previously published VSV mutation rates may be overestimated due to PCR errors
- High-fidelity Pfu polymerase with proofreading activity is substantially more reliable for accurately assessing virus genetic diversity within populations
- PCR reaction conditions significantly affect Taq error rates, and many published studies may report inflated viral diversity due to suboptimal amplification conditions
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Abstract
The genetic diversity of a vesicular stomatitis virus population was analysed by RT-PCR, cloning and sequencing of two approximately 500 nucleotide regions of the virus genome. PCR amplifications were performed in parallel experiments with both Taq and Pfu DNA polymerases, and important differences were observed. Between 10 and 22 mutations were detected when virus populations were analysed by Taq amplification (20 clones from each region), whereas amplification of the same samples with Pfu revealed between 0 and 5 mutations. PCR fidelity assays, performed under the same PCR conditions as those used in the population analysis, showed that the Taq error-rate estimate of 0.27 x 10(-4) misincorporations per bp per cycle was within the range estimated elsewhere from PCR amplification of recombinant plasmids (0.27-0.85 x 10(-4) errors per bp per cycle) or from functional assays (0.2-2 x 10(-4) errors per bp per cycle). The error rate of Taq was found to be 9.3 times higher than the error rate of Pfu with DNA as a template, and about 10 times higher with cDNAs obtained by reverse transcription of viral RNA templates from natural populations. In the present study, we discuss (i) the implications of Taq errors on the analysis of genetic variability, based on both the frequency and nature (replacement vs synonymous) of the observed substitutions and (ii) the sample size required to assess the genetic variability in a virus population generated by a single infection.