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.
Department of Orthopaedic Surgery, Washington University, St. Louis, Missouri, USA.
Gabe
http://orcid.org/0000-0002-3884-5776
G
Haller
Department of Orthopaedic Surgery, Washington University, St. Louis, Missouri, USA.
David
D
Alvarado
Department of Orthopaedic Surgery, Washington University, St. Louis, Missouri, USA.
Kevin
K
McCall
Center for Genome Sciences and Systems Biology, Department of Genetics, Washington University School of Medicine, St. Louis, Missouri, USA.
Robi D
RD
Mitra
Department of Orthopaedic Surgery, Washington University, St. Louis, Missouri, USA.|Shriners Hospital for Children, St. Louis, Missouri, USA.
Matthew B
MB
Dobbs
Department of Orthopaedic Surgery, Washington University, St. Louis, Missouri, USA.|Department of Neurology, Washington University, St. Louis, Missouri, USA.|Department of Pediatrics, Washington University, St. Louis, Missouri, USA.
Christina A
CA
Gurnett
D000668:Ampicillin Resistance / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D007293:Inosine Triphosphate / Q000235:genetics*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D001618:beta-Lactamases / Q000235:genetics*
D007293:Inosine Triphosphate; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1
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