Random Mutagenesis Library Construction to characterize structure-function relationships (CAT#: STEM-MB-0024-WXH)

Introduction

Random mutagenesis of DNA is a powerful tool to study gene function and protein structure and is a prerequisite for preparation of randomized DNA libraries, required for many genetic selections. Compared with the rational design, random mutagenesis is not targeted amino acid substitutions, and therefore do not require a large body of the knowledge about the biocatalyst being improved, including the three-dimensional structure and the chemical mechanism of the reaction. Random mutagenesis, introducing random mutations into the protein-coding gene and then screening the expressed mutant proteins, provides access to proteins having altered properties without human bias. Several methods have been developed to create random mutagenesis. Among these, error-prone PCR is one of the most widely used random mutagenesis. During DNA amplification, misincorporation of nucleotides can be promoted by altering the nucleotide ratio and the concentration of divalent cations in the reaction. It has been reported that, by adjusting template concentration, frequency of mutation could be controlled and a library with desired mutation rate could be obtained.