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A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing
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A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Article DOI: 10.3791/54119-v • 11:36 min • July 3rd, 2016
July 3rd, 2016 •
1, 1, 1, 1
1Green Center for Systems Biology, University of Texas Southwestern Medical Center

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Summary
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We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 β-lactamase selected at a clinically relevant dosage of ampicillin are provided.

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Functional Assessment Whole-protein Saturation Mutagenesis Libraries High-throughput Sequencing Biochemistry Molecular Evolution Protein Engineering PCR Master Mix Sense Mutagenesis Primers PCR Plates Thermocycler Diluting Reactions Mutagenic PCR Products
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