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Widespread nuclease contamination in commonly used oxygen-scavenging systems

Nature Methods volume 12, pages 901–902 (2015)Cite this article

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These observations suggested that DNA binding and/or aggregation, nuclease and other contaminants should be removed from the OSS before use in DNA-SM analysis. We developed a straightforward Superose 6 size-exclusion chromatography purification method for PCDa that takes advantage of the enzyme's large reported size, with 12 α-dimer (23.3 kDa) and β-dimer (26.6 kDa) subunits4 (Supplementary Notes 5 and 6). The majority of nuclease activity eluted as an ∼40-kDa protein, with a slightly higher molecular weight shoulder at ∼100 kDa (Fig. 1b and Supplementary Fig. 6), whereas peak PCD enzyme activity corresponded with the αβ-subunits, eluting at 417 kDa (Fig. 1b and Supplementary Figs. 6 and 7). The pooled PCD fractions (43–51) were free of nuclease activity and did not inhibit several DNA metabolic reactions even at high concentrations (100 nM; Supplementary Figs. 5 and 8–10 and Supplementary Note 7).

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Figure 1: Nuclease contamination of oxygen-scavenging enzymes; purification and analysis of PCD.

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Acknowledgements

We thank our laboratory members for their insights and helpful discussions. This work was supported by the National Research Foundation of Korea (grant 2011-0013901 to J.-B.L.) and the US National Institutes of Health (grant AI099854 to K.E.Y.; grants GM080176 and CA67007 to R.F.).

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Author notes

  1. Gayan Senavirathne and Jiaquan Liu: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University Medical Center, Columbus, Ohio, USA

    Gayan Senavirathne, Jiaquan Liu, Miguel A Lopez Jr, Jeungphill Hanne, Juana Martin-Lopez, Kristine E Yoder & Richard Fishel

  2. Department of Physics, Pohang University of Science and Technology, Pohang, Korea

    Jong-Bong Lee

  3. School of Interdisciplinary Bioscience and Bioengineering, Pohang, Korea

    Jong-Bong Lee

  4. Physics Department, The Ohio State University, Columbus, Ohio, USA

    Richard Fishel

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  1. Gayan Senavirathne
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Correspondence to Richard Fishel.

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Supplementary Figures 1–11, Supplementary Tables 1 and 2 and Supplementary Notes 1–7 and Supplementary Methods (PDF 3436 kb)

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Senavirathne, G., Liu, J., Lopez, M. et al. Widespread nuclease contamination in commonly used oxygen-scavenging systems. Nat Methods 12, 901–902 (2015). https://doi.org/10.1038/nmeth.3588

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