Within three billion years of evolution, nature has produced a plethora of proteins simply by repeated cycles of random mutagenesis followed by in vivo selection. This example of natural evolution has guided researchers to develop strategies for in vitro permutation of genes and proteins. Among a variety of strategies, three powerful techniques have emerged:

Mutagenesis by dNTP Analogs

The method achieves mutagenicity of up to 20% and is based on incorporation of mutagenic dNTP analogs into an amplified DNA fragment. The analogs are eliminated by a second PCR in the presence of the four natural dNTPs, leaving highly mutated DNA ready for further investigation.

Mutagenesis by Error-Prone PCR

Mutagenesis is performed by a PCR reaction under modified conditions that induce an increased error-rate of the DNA-polymerase. Simply run the PCR protocol provided in the manual and achieve mutagenicity in the range of 0.6-2.0% in a single PCR reaction!

Mutagenesis by DNA Shuffling

DNA shuffling generates libraries by random fragmentation of one gene or a pool of related genes, followed by reassembly of the fragments in a self-priming PCR reaction. The mutagenicity is similar to error-prone PCR but DNA shuffling allows recombination of sequences from different, related genes.

For each of these techniques we offer a ready-to-go kit, accompanied by detailed manuals and background information.

Random Mutagenesis is a common approach for directed evolution of proteins and for analysis of protein-structure/function relationships. In contrast to site-directed approaches, random mutagenesis is a promising tool for identification of beneficial mutations without prior structural and functional information about the protein of interest. Randomly introduced DNA mutations such as nucleotide exchange (substitution), insertion and deletion of one or multiple nucleotides result in amino acid sequence changes thus, producing proteins with altered characteristics such as enhanced or novel activities.

A powerful approach for random mutagenesis is PCR-based incorporation of mutagenic nucleotide analogs into a DNA fragment^[1-7]. The mutagenic potential of these nucleotide analogs relies on their alternate base pairing properties that lead to the introduction of several mutations during multiple rounds of a PCR reaction (Tab. 1). Elimination of the modified analogs is achieved by a second PCR in the presence of the four natural dNTPs, leaving highly mutated DNA ready for further investigation

Table 1: Nucleotide selection guide for random mutagenesis approaches.

Taq: Thermus aquaticus; transition: exchange of purine for purine (A ↔ G) or pyrimidine for pyrimidine (C ↔ T); transversion: exchange of purine for pyrimidine or vice versa (C/T ↔ A/G).