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Identification of unknown nucleotide sequences flank-ing already characterized DNA regions can be pursued by a number of different PCR-based methods com-monly known as genome walking (GW). In times of high-throughput DNA sequencing tech-nologies, when more than 1000 genomes have been completely sequenced, the development of GW strategies can appear as an out-of-date laboratory activity. Nevertheless, papers describing applications of GW methods and improvements of several available strategies continue to be published with a steady positive trend. Reasons for such constant interest can be found both in the relatively low difficulty of the different strategies 更多：https://www.bmcx.com/ , which do not require expensive equipment or highly trained personnel, and in the increasing possibilities of applying GW methods to eukaryotic genomes. Furthermore, in some highly sophisticated applications, GW strategies have recently been com-bined with pyrosequencing technology allowing the production of hundreds of thousands of sequences per