Primer Walking

Abstract

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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References

Fraser CM and Fleischmann RD (1997) Strategies for whole microbial genome sequencing and analysis. Electrophoresis 18: 1207–1216.

Kaczorowski T and Szybalski W (1998) Genomic DNA sequencing by SPEL‐6 primer walking using hexamer ligation. Gene 26: 83–91.

Kieleszawa J, Dunn JJ and Studier FW (1992) DNA sequencing by primer walking with strings of contiguous hexamers. Science 258: 1787–1791.

Strauss EC, Kobori JA, Siu G and Hood LH (1986) Specific‐primer‐directed DNA sequencing. Analytical Biochemistry 154: 353–360.

Studier FW (1989) A strategy for high‐volume sequencing of cosmid DNAs: random and directed priming with a library of oligonucleotides. Proceedings of the National Academy of Sciences of the United States of America 86: 6917–6921.

Szybalski W (1990) Proposal for sequencing DNA using ligation of hexamers to generate sequential elongation primers (SPEL‐6). Gene 90: 177–178.

Venter JC, Adams MD, Myers EW, et al. (2001) The sequence of the human genome. Science 291: 1304–1351.

Voss H, Wiemann S, Grothues D, et al. (1993) Automated low‐redundancy large‐scale DNA sequencing by primer walking. Biotechniques 15: 714–721.

Wiemann S, Rupp T, Zimmermann J, et al. (1995) Primer design for automated DNA sequencing utilising T7 DNA polymerase and internal labeling with fluorescein‐15‐dATP. Biotechniques 18: 688–696.

Zevin‐Sonkin D, Liberzon A, Ghochikyan A, et al. (1999) DENS (Differential extension with nucleotide subsets): application to the sequencing of human genomic DNA and cDNA. DNA Sequence 10: 245–254.

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. http://www.els.net [doi: 10.1038/npg.els.0005382]