A new method for random mutagenesis by error-prone polymerase chain reaction using heavy water

► We developed a new method of epPCR using heavy water as a solvent instead of normal water (H2O). ► D2O or H218O was used as a solvent of PCR with/without Mn2+. ► A maximum error rate of 1.8 × 10−3 errors/bp was detected, without any particular hot-spot. ► All kinds of mutations could be introduced with the condition of 99% D2O/0.6 mM Mn2+. ► This new method may be a powerful random mutagenesis technique.