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Massively parallel single-nucleotide mutagenesis using reversibly terminated inosine.

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 ?-lactamase gene. When combined with high-throughput functional assays, SAS mutational libraries can expedite the functional assessment of genetic variation.

SUBMITTER: Haller G

PROVIDER: S-EPMC5327618 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Massively parallel single-nucleotide mutagenesis using reversibly terminated inosine.

Haller Gabe G Alvarado David D McCall Kevin K Mitra Robi D RD Dobbs Matthew B MB Gurnett Christina A CA

Nature methods 20161003 11

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-throu ...[more]

PMID: 27694911

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