A Rapid, High-Throughput Method for the Construction of Mutagenesis Libraries

As synthetic biology advances toward precise design, the construction of high-quality mutant libraries has become essential for large-scale functional screening. Traditional approaches, such as random and saturation mutagenesis, often suffer from low accuracy, high bias, and limited coverage. An ideal method should offer controlled mutagenesis, comprehensive coverage, high throughput, operational simplicity, and controllable outcomes, enabling effective large-scale screening. Here, we developed a high-throughput, precisely controlled method for constructing a mutagenesis library based on chip-based oligonucleotide synthesis. Using <i>PSMD10</i> as a model, we constructed a full-length amber codon scanning mutagenesis library with 93.75% mutation coverage. Among the five polymerases evaluated, KAPA HiFi HotStart, Platinum SuperFi II and Hot-Start Pfu DNA Polymerase demonstrated higher amplification efficiency and lower chimera formation rates, making them preferred enzymes for optimized library construction. Analysis of unmapped reads highlighted key technical factors, such as oligonucleotide synthesis errors and chimeric sequence formation caused by incomplete extension of DNA polymerase or synthesis across discontinuous templates during PCR. To improve efficiency and fidelity, we recommend refining PCR conditions and strengthening oligo synthesis quality control. We establish an efficient, scalable, precisely controlled mutagenesis library construction strategy tailored for high-throughput functional research and recommend using a high-fidelity, low-bias polymerase to ensure quality.