Fast and precise multi-site mutagenesis on linear DNA fragments

The effectiveness and scalability of site-directed mutagenesis are constrained by the limited number of mutations and the intricate cloning process required for isolation of the target sequence. Here, we present a method for precise introduction of multiple non-contiguous mutations. The mutant strands are collected through one specially designed magnetic bead separation in alkaline conditions, efficiently removing their complementary partner strands with the original sequences. In a proof-of-concept test, a green fluorescent protein (GFP) was simultaneously mutated in 1–3 specific amino acids, successfully shifting its fluorescence spectrum. The precise mutation rates for single-, double- and triple-site mutations reached 100%, 76% and 70%, respectively. This multiple non-contiguous mutagenesis method may offer a fast and cost-effective approach for customizable construction of gene library.