Primer Walking


Primer walking is a step‐by‐step approach to sequencing long DNA templates from end to end that overcomes the inability of the Sanger chain termination method to read more than a few hundred bases in a single reaction. After an initial round of sequencing from a known sequence at one end of the template, each subsequent round is initiated from a new primer, which is based on the end of the sequence obtained from the previous reaction.

Keywords: primer walking; DNA sequencing; genome; gap closure; hexamer library

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.



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Further Reading

Sterky F and Lundeberg J (2000) Sequence analysis of genes and genomes. Journal of Biotechnology 76: 1–31.

Voss H, Schwager C, Wiemann S, et al. (1995) Efficient low redundancy large‐scale DNA sequencing at EMBL. Journal of Biotechnology 41: 121–129.

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How to Cite close
Sverdlov, Eugene, and Azhikina, Tatyana(Sep 2005) Primer Walking. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1038/npg.els.0005382]