Massively parallel single-nucleotide mutagenesis using reversibly terminated inosine

Nat Methods. 2016 Nov;13(11):923-924. doi: 10.1038/nmeth.4015. Epub 2016 Oct 3.


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.

MeSH terms

  • Ampicillin Resistance / genetics
  • DNA Mutational Analysis / methods*
  • Gene Library
  • High-Throughput Nucleotide Sequencing / methods*
  • Inosine Triphosphate / genetics*
  • Models, Molecular
  • Mutagenesis, Site-Directed*
  • Polymorphism, Single Nucleotide / genetics*
  • beta-Lactamases / genetics*


  • Inosine Triphosphate
  • beta-Lactamases
  • beta-lactamase TEM-1