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

The determination of nucleotide sequences adjacent to a known region is a recurring need in many genome scale studies. To fulfil this aim, and to overcome the time consuming approach of the screening of genomic libraries, many methods have been developed based on PCR techniques. Usually these protocols resort to specific requirements and strategies, as the presence of suitable nucleotide restriction sites and the ligation of specific single- or double-strand linkers, which make their application somehow limited. In this paper we present an alternative PCR based protocol, consisting of four principal steps: extension of a sequence specific primer; 3’-tailing of extended single-strand DNA; PCR and nested PCR amplifications. The method, which appears as a valid alternative to the other PCR based protocols, has been used for the identification of sequences flanking in the spinach genome the cDNA encoding region of the Lhcb1.1 gene (one member of the multigene family coding for the light harvesting protein Lhcb1).