A versatile, simple, and rapid (VSR) method for generating gene clones, diverse mutants, and short gene fragments for...

Gene cloning, site-directed mutagenesis (SDM), and short gene synthesis are essential tools in molecular biology, yet existing approaches remain constrained by efficiency, flexibility, and cost. Here, we developed a versatile, simple, and rapid (VSR) 3-in-1 method that integrates seamless cloning, mutagenesis, and short gene assembly in a single platform. This approach works on the principle of overlap extension polymerase chain reaction (O_PCR), uses high-fidelity DNA polymerase and short overlapping primers. Target genes can be directly amplified from genomic DNA or complementary DNA (cDNA) templates and cloned into vectors without intermediate purification, restriction enzyme digestion, or ligation. VSR supports the insertion of DNA fragments ranging from 80 bp to 33 kb at any plasmid locus while enabling the introduction of single or multiple nucleotide substitutions at one or more sites in a single reaction. Using this method, we cloned 35 genes of diverse lengths and two complete viral genomes with efficiencies ranging from 60 to 100% and introduced up to 17 substitutions in a single mutagenesis reaction. We further demonstrate efficient de novo synthesis of short genes from overlapping oligonucleotides. The VSR method is a reliable, rapid, and cost-effective approach with a wide range of applications for both routine and high-throughput workflows.