A two-step PCR assembly for construction of gene variants across large mutational distances

Abstract

Abstract Construction of empirical fitness landscapes has transformed our understanding of genotype–phenotype relationships across genes. However, most empirical fitness landscapes have been constrained to the local genotype neighbourhood of a gene primarily due to our limited ability to systematically construct genotypes that differ by a large number of mutations. Although a few methods have been proposed in the literature, these techniques are complex owing to several steps of construction or contain a large number of amplification cycles that increase chances of non-specific mutations. A few other described methods require amplification of the whole vector, thereby increasing the chances of vector backbone mutations that can have unintended consequences for study of fitness landscapes. Thus, this has substantially constrained us from traversing large mutational distances in the genotype network, thereby limiting our understanding of the interactions between multiple mutations and the role these interactions play in evolution of novel phenotypes. In the current work, we present a simple but powerful approach that allows us to systematically and accurately construct gene variants at large mutational distances. Our approach relies on building-up small fragments containing targeted mutations in the first step followed by assembly of these fragments into the complete gene fragment by polymerase chain reaction (PCR). We demonstrate the utility of our approach by constructing variants that differ by up to 11 mutations in a model gene. Our work thus provides an accurate method for construction of multi-mutant variants of genes and therefore will transform the studies of empirical fitness landscapes by enabling exploration of genotypes that are far away from a starting genotype.