A frequency-controlled random mutagenesis method for GC-rich genes

A novel random mutagenesis strategy was developed by combining sodium bisulfite modification with polymerase chain reaction (PCR). This method introduced the predominant substitution of GC to AT, meaning that it was more suitable for mutagenesis of GC-rich genes and helped to decrease the GC content of target DNA. Mutation efficiency correlated with modification time and different mutation frequency could easily be obtained by controlling modification time. The results indicated that this method could yield a desired and adequate frequency of random mutation to the DNA of interest, especially GC-rich genes, and provided a powerful tool for directed molecular evolution.