[A simple error-prone PCR method through dATP reduction].

Area of Science:

• Molecular Biology
• Biotechnology
• Genetic Engineering

Background:

• Error-prone PCR is a key technique for in vitro gene mutagenesis.
• Conventional methods often involve adding Mn2+, increasing Mg2+, and adjusting dCTP/dTTP concentrations.

Purpose of the Study:

• To investigate PCR mutagenesis by reducing dATP concentration.
• To analyze the impact of dATP reduction on the antifungal protein gene Ace-AMP1 and Bt toxin gene cry1A(c).

Main Methods:

• Performed PCR mutagenesis on Ace-AMP1 and cry1A(c) genes.
• Reduced dATP concentration without adding Mn2+ or altering other PCR components.
• Varied dTTP/dCTP/dGTP to dATP ratios from 20:1 to 40:1.

Main Results:

• Decreased dATP concentrations led to increased base mutation and sequence variation rates.
• At dTTP/dCTP/dGTP: dATP ratios of 20:1-40:1, base mutation rates ranged from 1.4% to 1.8%.
• Sequence variation rates reached 77.8% to 100% under these conditions.

Conclusions:

• The method is simple, practical, and allows optimization of error-prone PCR factors.
• The primary mutation type observed was A x T --> G x C transition, facilitating GC content increase.
• Reducing single dNTP concentration is an expansion of error-prone PCR for targeted gene modification.