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

Massively parallel single-nucleotide mutagenesis using reversibly terminated inosine.

Author information

1
Department of Orthopaedic Surgery, Washington University, St. Louis, Missouri, USA.
2
Center for Genome Sciences and Systems Biology, Department of Genetics, Washington University School of Medicine, St. Louis, Missouri, USA.
3
Shriners Hospital for Children, St. Louis, Missouri, USA.
4
Department of Neurology, Washington University, St. Louis, Missouri, USA.
5
Department of Pediatrics, Washington University, St. Louis, Missouri, USA.

Abstract

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.

PMID:
27694911
PMCID:
PMC5327618
DOI:
10.1038/nmeth.4015
[Indexed for MEDLINE]
Free PMC Article

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