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Quantification of random genomic mutations

Nature Methods volume 2, pages 285–290 (2005)Cite this article

Abstract

Cancer cells contain numerous clonal mutations. It has been theorized that malignant cells sustain an elevated mutation rate and, as a consequence, harbor yet larger numbers of random point mutations. Testing this hypothesis has been precluded by lack of an assay to measure random mutations—that is, mutations that occur in only one or a few cells of a population. We have established a method that has permitted us to detect and identify rare random mutations in human cells, at a frequency of 1 per 108 base pairs. The assay is based on gene capture, by hybridization with a uracil-containing probe, followed by magnetic separation. Mutations that render the mutational target sequence non-cleavable by a restriction enzyme are quantified by dilution to single molecules and real-time quantitative PCR amplification. The assay can be extended to quantify mutation in any DNA-based organism, at different sites in the genome, in introns and exons, in unselected and selected genes, and in proliferating and quiescent cells.

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Figure 1: Protocol for the random mutation capture assay.
Figure 2: Quantifying mutations in normal diploid human fibroblasts treated with ENU (500 ÎĽg/ml).
Figure 3: Enhancement of mutagenesis as a function of ENU exposure.
Figure 4: Mutation target neutrality.
Figure 5: Reconstruction experiment.

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Acknowledgements

The authors thank A. Blank for insightful comments and critical reading of the manuscript, E. Glick and C. Heindel for advice, and N. Griner for excellent technical assistance. This work was funded by grants CA78885 and CA102029 from the US National Institutes of Health. J.H.B. is supported by a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada.

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  1. Department of Pathology, The Joseph Gottstein Memorial Cancer Research Laboratory, University of Washington School of Medicine, 1959 N.E. Pacific Street, Seattle, 98195-7705, Washington, USA

    Jason H Bielas & Lawrence A Loeb

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  2. Lawrence A Loeb
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Correspondence to Lawrence A Loeb.

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Bielas, J., Loeb, L. Quantification of random genomic mutations. Nat Methods 2, 285–290 (2005). https://doi.org/10.1038/nmeth751

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