Plant Molecular Biology Reporter

, Volume 24, Issue 2, pp 237–243

A primer-based approach to genome walking



A genome walking strategy based on annealing and ligation of single-stranded DNA primers to 3′ overhangs following restriction endonuclease digestion was developed. A set of primers contains 4 nucleotides at the 3′ end that are complementary to overhangs formed by restriction endonucleasesApaI;PstI;SacI andSphI. Following ligation, 5′ end overhangs are formed on the DNA, which serves as sites for the adaptor primers and nested primers for PCR amplification in combination with the gene-specific primers. This strategy was verified by the amplification of up to 4 kb of a potato leafroll virus full-length infectious clone. The procedure could be adopted to target any upstream and downstream regions flanking known sequences within the plant genome.

Key words

genome walking 3′ overhangs primer ligation restriction endonucleases single stranded DNA primers 



adaptor primer


gene specific primer


nested primer


overhanging primer


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Copyright information

© Springer 2006

Authors and Affiliations

  1. 1.Agricultural Genetic Engineering Research InstituteAgricultural Research CentreGizaEgypt

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