Abstract
Polymerase chain reaction (PCR) is used as an in vitro model system of DNA replication to assess the genotoxicity of nanoparticles (NPs). Prior results showed that several types of NPs inhibited PCR efficiency and increased amplicon error frequency. In this study, we examined the effects of various metal oxide NPs on inhibiting PCR, using high- vs. low-fidelity DNA polymerases; we also examined NP-induced DNA mutation bias at the single nucleotide level. The effects of seven major types of metal oxide NPs (Fe2O3, ZnO, CeO2, Fe3O4, Al2O3, CuO, and TiO2) on PCR replication via a low-fidelity DNA polymerase (Ex Taq) and a high-fidelity DNA polymerase (Phusion) were tested. The successfully amplified PCR products were subsequently sequenced using high-throughput amplicon sequencing. Using consistent proportions of NPs and DNA, we found that the effects of NPs on PCR yield differed depending on the DNA polymerase. Specifically, the efficiency of the high-fidelity DNA polymerase (Phusion) was significantly inhibited by NPs during PCR; such inhibition was not evident in reactions with Ex Taq. Amplicon sequencing showed that the overall error rate of NP-amended PCR was not significantly different from that of PCR without NPs (p > 0.05), and NPs did not introduce single nucleotide polymorphisms during PCR. Thus, overall, NPs inhibited PCR amplification in a DNA polymerase-specific manner, but mutations were not introduced in the process.
Highlights
Metal oxide nanoparticles (NPs) are the most widely used engineered NPs in consumer products (Hansen et al, 2016)
We determined whether the effects of metal oxide NPs in inhibiting two DNA polymerase Polymerase chain reaction (PCR) systems—one containing high-fidelity and another low fidelity polymerase, were different, and investigated NP-induced DNA mutant bias in different systems at the single nucleotide level
ZnO, CeO2, and TiO2 NPs completely inhibited the DNA replication of Ex Taq, while CeO2, Al2O3, and TiO2 NPs completely inhibited that of Phusion, suggesting that the tolerance of DNA polymerase to DNA-bound NPs varied between Ex Taq and Phusion
Summary
Metal oxide nanoparticles (NPs) are the most widely used engineered NPs in consumer products (Hansen et al, 2016). Metal oxide nanominerals or mineral NPs are common and widely distributed in diverse environments such as soil, atmosphere, and waters (Hochella et al, 2008). These NPs can exhibit remarkable antimicrobial activity, and cytotoxicity and genotoxicity to different types of organisms (Magdolenova et al, 2014; Golbamaki et al, 2015; Mahaye et al, 2017). The proposed toxicity mechanisms of metal oxide NPs include binding of NPs to genetic material (e.g., DNA and RNA), indirect damage from NP-generated reactive oxygen species (ROS), and toxic ions released from. The interaction between metal oxide NPs and DNA could play an important role in NP toxicity, and a complete elucidation or delineation of the underlying mechanisms is needed (Peng et al, 2017)
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