Synthetic gene technology: Applications to ancestral gene reconstruction and structure-function studies of receptors

Abstract

Publisher Summary A novel and important application of synthetic gene technology in studies of G protein-coupled receptors (GPCRs) is the recreation of ancestral receptors. The superfamily of GPCRs consists of a large number of related seven transmembrane proteins of highly varied function. Recreating fossil receptors for functional studies in the laboratory can provide important insights into the constraints that shaped molecular structure and function in this diverse superfamily that are difficult to attain by using more traditional mutagenesis methods. In addition, in studying receptor function via heterologous expression of mutant receptors, it is often useful to create chimeric receptors in which putative functional domains are exchanged. This chapter presents a summary of methods and applications of synthetic gene technology, with an emphasis on synthesizing ancestral genes. Algorithms for inferring ancestral sequences and general considerations for designing synthetic genes are discussed in detail in the chapter. Detailed laboratory procedures for the various steps in gene synthesis, from oligonucleotide preparation to cassette mutagenesis, are also given.