Improving directed evolution strategies: error-prone PCR optimization for SARS-CoV-2 spike receptor binding domain.

Area of Science:

• Virology
• Molecular Biology
• Biotechnology

Background:

• Human coronaviruses, including SARS-CoV-2, pose significant health risks.
• The SARS-CoV-2 receptor binding domain (RBD) is crucial for viral entry via ACE-2 interaction.
• Understanding RBD mutations is key to combating viral transmission and developing treatments.

Purpose of the Study:

• To develop and optimize an error-prone PCR (ep-PCR) protocol for the SARS-CoV-2 RBD.
• To analyze mutation patterns and identify optimal conditions for generating diversity in the RBD sequence.

Main Methods:

• Application and optimization of ep-PCR for SARS-CoV-2 RBD.
• Systematic variation of dNTP ratios, MgCl₂, MnCl₂, and cycle numbers.
• Sequencing to analyze mutation distribution and patterns.

Main Results:

• Identified MnCl₂ concentration as the most influential parameter for mutation generation.
• Determined optimal conditions yielding 12 specific protocols for mutations in the 13-17 bp range.
• Characterized mutation distribution within the RBD sequence.

Conclusions:

• The optimized ep-PCR method provides combinatorial mutation diversity in a single round.
• This accessible technique, using standard PCR and Sanger sequencing, aids in evaluating vaccine candidates.
• Findings enhance understanding of SARS-CoV-2 virulence and transmission mechanisms.