Massively parallel single-nucleotide mutagenesis using reversibly terminated inosine

Journal name:
Nature Methods
Volume:
13,
Pages:
923–924
Year published:
DOI:
doi:10.1038/nmeth.4015
Received
Accepted
Published online

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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References

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Affiliations

  1. Department of Orthopaedic Surgery, Washington University, St. Louis, Missouri, USA.

    • Gabe Haller,
    • David Alvarado,
    • Kevin McCall,
    • Matthew B Dobbs &
    • Christina A Gurnett
  2. Center for Genome Sciences and Systems Biology, Department of Genetics, Washington University School of Medicine, St. Louis, Missouri, USA.

    • Robi D Mitra
  3. Shriners Hospital for Children, St. Louis, Missouri, USA.

    • Matthew B Dobbs
  4. Department of Neurology, Washington University, St. Louis, Missouri, USA.

    • Christina A Gurnett
  5. Department of Pediatrics, Washington University, St. Louis, Missouri, USA.

    • Christina A Gurnett

Contributions

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.

Competing financial interests

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 information

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  1. Supplementary Text and Figures (937 KB)

    Supplementary Figures 1–7, Supplementary Tables 1 and 2, and Supplementary Note.

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