Massive Mutagenesis: high-throughput combinatorial site-directed mutagenesis
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Updated: Jun 10, 2026

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing
Published on: July 3, 2016
1PhotoFuel SAS, Paris, France. julien.sylvestre@photofuel.fr
Abstract:
Massive Mutagenesis is a proprietary library creation method that enables the fast generation of high-quality genetic libraries. Starting from a single gene on a plasmid and hundreds to thousands of oligonucleotides, a one-step single-strand circular amplification method creates random combinations of several site-directed substitutions, insertions, or deletions. Libraries of up to a billion such variants have been routinely generated. Sequencing those variants demonstrated lower biases than alternative approaches such as error-prone PCR. Screening and selecting them has yielded improved biocatalysts, therapeutic proteins, and antibodies.
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