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

Area of Science:

• Molecular Biology
• Biotechnology
• Protein Engineering

Background:

• Error-prone PCR (epPCR) is a key technique for random mutagenesis in protein engineering.
• Traditional epPCR methods often face limitations such as positional bias and template dependency.
• Developing new epPCR strategies is crucial for advancing protein engineering capabilities.

Purpose of the Study:

• To develop a novel error-prone PCR (epPCR) method utilizing heavy water (D2O) as a solvent.
• To evaluate the efficiency and characteristics of D2O-based epPCR for random mutagenesis.
• To assess the potential of D2O and H2(18)O in epPCR for protein engineering applications.

Main Methods:

• Developed a new epPCR protocol using heavy water (D2O) instead of normal water (H2O).
• Amplified Ayu fish rhodopsin cDNA under five different conditions, including varying D2O concentrations and Mn2+ presence.
• Sequenced a significant number of base pairs (13,960–33,504) to analyze mutation rates and types.

Main Results:

• The D2O-based epPCR method introduced random mutations with a maximum error rate of 1.8×10(-3) errors/bp.
• Observed specific mutation preferences: AT→GC transitions in D2O/Mn2+ conditions and transitions over transversions in D2O alone.
• H2(18)O solvent exclusively induced transversions, while D2O-based methods showed no positional bias or template dependency.

Conclusions:

• Heavy water (D2O) as a solvent in epPCR enables random mutagenesis without positional bias, template dependency, or decreased yield.
• The novel D2O-based epPCR method is a powerful tool for random mutagenesis in protein engineering.
• Combining D2O and H2(18)O may offer further advantages for advanced mutagenesis strategies.