Rapid gene splicing and multi-sited mutagenesis by one-step overlap extension polymerase chain reaction.

Gene splicing and site-directed mutagenesis (SDM) are important to introduce desired sequences in target DNA. However, introducing mutations at multiple sites requires multiple steps of DNA manipulation, which is time-consuming and labor-intensive. Here, we present a rapid efficient gene splicing and multi-sited mutagenesis method that introduces mutations at two distant sites via sequential connection of DNA fragments by one-step overlap extension polymerase chain reaction (OE-PCR). This bottom-up approach for DNA engineering can be broadly used to study protein structure-function, to optimize codon use for protein expression, and to assemble genes of interest.