Gene Cloning Using Degenerate Primers and Genome Walking

Abstract

Gene cloning is the first step of targeted gene replacement for functional studies, discovery of gene alleles, and gene expression among other applications. In this chapter, we will describe a cloning technique suitable for fungal species where the genomic information and sequences available are limited. This strategy involves obtaining protein sequences of the gene of interest from various organisms to identify at least two conserved regions. Degenerate primers are designed from these two conserved regions and the resulting PCR products are sequenced. The sequence of the PCR products can be analyzed using suitable databases to determine their similarity to the gene/protein of interest. In cases where the entire gene cannot be cloned directly using these primers, this initial nucleotide sequence can be used as a template for further primer design and genome walking in both directions for either the cloning of a longer fragment or even the cloning of the complete gene. Here, we describe the partial cloning of a reducing polyketide synthase gene from the fungal plant pathogen Ascochyta rabiei using this strategy.