A Rapid and Versatile PCR-Based Site-Directed Mutagenesis Protocol for Generation of Mutations Along the Entire Length of a Cloned cDNA

Abstract

Deciphering protein function is a major challenge in modern biology and continues to remain at the frontier of investigations into the molecular basis of cell behavior. With the explosion in our bioinformatics knowledge base and the now widespread use of associated software tools and database resources, we have an enormous logistic capability to identify protein domains of interest and the compelling desire to introduce mutations within these sequences in order to ultimately understand the functional aspects of a given protein and/or test its therapeutic applications. Faced with this ultimate task, a quick and efficient means to introduce desired mutations anywhere along the protein length is necessary as a first step. Here, HIF1α and HIF2α are used as examples to demonstrate the simplicity, speed, and versatility of the PCR-based mutagenesis method.