One-step random mutagenesis by error-prone rolling circle amplification

Abstract

In vitro random mutagenesis is a powerful tool for altering properties of enzymes. We describe here a novel random mutagenesis method using rolling circle amplification, named error-prone RCA. This method consists of only one DNA amplification step followed by transformation of the host strain, without treatment with any restriction enzymes or DNA ligases, and results in a randomly mutated plasmid library with 3-4 mutations per kilobase. Specific primers or special equipment, such as a thermal-cycler, are not required. This method permits rapid preparation of randomly mutated plasmid libraries, enabling random mutagenesis to become a more commonly used technique.

Figure 1

Distribution of mutations. The seven clones produced by RCA in the presence of 1.5 mM MnCl₂ from 25 pg pUC19 were sequenced. Lines indicate mutations in the pUC19 sequence. Types of mutations are described in Table 4.

Figure 2

Schematic diagram of error-prone RCA in comparison with the conventional random mutagenesis methods.