Phylogenetic Footprinting to Find Functional DNA Elements

Abstract

Phylogenetic footprinting is powerful technique for finding functional elements from sequence data. Functional elements are thought to have greater sequence constraint than nonfunctional elements, and, thus, undergo a slower rate of sequence change through time. Phylogenetic footprinting uses comparisons of homologous sequences from closely related organisms to identify "phylogenetic footprints," regions with slower rates of sequence change than background. This does not require prior characterization of the sequence in question, therefore, it can be used in a wide range of applications. In particular, it is useful for the identification of functional elements in noncoding DNA, which are traditionally difficult to detect. Here, we describe in detail how to perform a simple yet powerful phylogenetic footprinting analysis. As an example, we use ribosomal DNA repeat sequences from various Saccharomyces yeasts to find functional noncoding DNA elements in the intergenic spacer, and explain critical considerations in performing phylogenetic footprinting analyses, including the number of species and species range, and some of the available software. Our methods are broadly applicable and should appeal to molecular biologists with little experience in bioinformatics.