Genome walking by next generation sequencing approaches

The identification of unknown nucleotide sequences starting from a previously identified DNA region can be directly obtained by a number of Genome Walking (GW) methods all having in common a final PCR amplification in which an oligonucleotide specific for the known sequence is coupled with an oligonucleotide derived from the adopted GW strategy. The numerous GW methods can be classified into three main categories, according to the first step of the whole strategy: (1) Restriction-based GW methods, requiring a restriction digestion of genomic DNA and ligation of restriction fragments to DNA-cassettes; (2) Primer-based GW methods, in which PCR amplifications are directly carried out using a variously designed combinatorial (random or degenerate primer) coupled to a sequence specific primer; (3) Extension-based GW methods, in which the extension of a sequence specific primer and subsequent 3'-tailing of the resulting single strand DNA (ssDNA) provide the substrate for the final PCR amplification. Critical overviews of the conceived GW strategies, and their possible applications to both eukaryotic and prokaryotic genomes, have been recently reported [1-3].