A genome walking strategy for the identification of eukaryotic nucleotide sequences adjacent to known regions

Abstract

Determination of nucleotide sequences adjacent to a known region is a recurring need in many genome scale studies. Various methods have been developed based on PCR techniques in order to fulfill this aim and overcome the time-consuming approach of screening genomic libraries. Usually these protocols rely on specific requirements and strategies, such as the presence of suitable nucleotide restriction sites and ligation of specific single- or double- strand linkers, thus limiting their application to a certain extent. In this paper we present an alternative PCR-based protocol, consisting of four main steps: (i) extension of a sequence-specific primer; (ii) 3′-tailing of extended single-strand DNA; (iii) PCR; and (iv) nested PCR amplifications. This method, which appears to be a valid alternative to the other PCR-based protocols, was used for the identification of sequences flanking the cDNA encoding region of the Lhcb1.1 gene (one member of the multigene family coding for the light harvesting protein Lhcb1) in the spinach genome.