Amplification of orthologous genes using degenerate primers

Abstract

This chapter describes the use of degenerate primers for PCR amplification of orthologous DNA from related species. While several methods for designing degenerate primers have been described, an important consideration is to base the design on a short region of highly conserved amino acids. Here, we present the use of a degenerate primer design strategy called Consensus-degenerate hybrid oligonucleotide primer (CODEHOP). This strategy is very useful when there is low conservation in the multiple alignments of sequences across species. We demonstrate the use of CODEHOP to amplify the entire coding region of the flightin gene from multiple Drosophila species. © 2010 Springer Science+Business Media, LLC.