Contribution of Taq polymerase-induced errors to the estimation of RNA virus diversity

被引:119
作者
Bracho, MA [1 ]
Moya, A [1 ]
Barrio, E [1 ]
机构
[1] Univ Valencia, Dept Genet, E-46100 Burjassot, Valencia, Spain
关键词
D O I
10.1099/0022-1317-79-12-2921
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
The genetic diversity of a vesicular stomatitis virus population was analysed by RT-PCR, cloning and sequencing of two similar to 500 nucleotide regions of the virus genome. PCR amplifications were performed in parallel experiments with both Tag and Pfu DNA polymerases, and important differences were observed. Between 10 and 22 mutations were detected when virus populations were analysed by Tag 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 Tag 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.
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页码:2921 / 2928
页数:8
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