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Massively parallel single-nucleotide mutagenesis using reversibly terminated inosine


Large-scale mutagenesis of target DNA sequences allows researchers to comprehensively assess the effects of single-nucleotide changes. Here we demonstrate the construction of a systematic allelic series (SAS) using massively parallel single-nucleotide mutagenesis with reversibly terminated deoxyinosine triphosphates (rtITP). We created a mutational library containing every possible single-nucleotide mutation surrounding the active site of the TEM-1 β-lactamase gene. When combined with high-throughput functional assays, SAS mutational libraries can expedite the functional assessment of genetic variation.

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Figure 1: Schematic and library characteristics of SAS mutagenesis.

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We thank C. Cruchaga and J. Budde and members of the Gurnett/Dobbs Lab for helpful discussion. This work was supported by a postdoctoral research fellowship (84291-STL) from the Shriners Hospital for Children (G.H.); US National Institutes of Health (NIH) grants (R01AR067715-01) (C.A.G. and M.B.D.) and (R01NS076993) (R.D.M.); and Shriners Hospital for Children research grant (85200-STL) (C.A.G. and M.B.D.)

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G.H., D.A., R.D.M., M.B.D. and C.A.G. designed the study and wrote the manuscript. G.H. and K.M. performed experiments. All authors contributed to and approved the final manuscript.

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Correspondence to Christina A Gurnett.

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Washington University in St. Louis has filed a provisional patent application on this method, with G.H., D.A., C.A.G. and M.B.D. as inventors.

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Supplementary Figures 1–7, Supplementary Tables 1 and 2, and Supplementary Note. (PDF 915 kb)

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Haller, G., Alvarado, D., McCall, K. et al. Massively parallel single-nucleotide mutagenesis using reversibly terminated inosine. Nat Methods 13, 923–924 (2016).

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