Figure 1. Basic principle of primer walking. (a) A primer complementary to a known sequence (shaded box; typically the cloning vector or part of an incomplete shotgun sequence) is used to initiate a first round of Sanger sequencing on a long template. (b) A new primer is designed, complementary to a region near to the end of the newly obtained sequence (solid box), to allow a second cycle of sequencing on the same template. (c) The process can be repeated as many times as necessary; each iteration yields a few hundred more bases of sequence, with minimal overlaps between consecutive reads.

Figure 2. Use of tandem hexamers in primer walking. For any given target sequence (top) obtained during a walk, a series of hexamers can be chosen from a presynthesized library, represented schematically beneath, that will anneal in a head-to-tail manner to the target. Cooperative interactions between the adjacent hexamers stabilize their binding to the target sequence and can be reinforced by ligating the annealed hexamers to one another. This forms a ‘segmented primer’ that is capable of initiating sequencing for the next step of the walk.