Plant Molecular Biology Reporter

, Volume 24, Issue 2, pp 237–243

A primer-based approach to genome walking

Protocols

DOI: 10.1007/BF02914062

Cite this article as:
Ashoub, A. & Abdalla, K.S. Plant Mol Biol Rep (2006) 24: 237. doi:10.1007/BF02914062
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Abstract

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 walking3′ overhangsprimer ligationrestriction endonucleasessingle stranded DNA primers

Abbreviations

AP

adaptor primer

GSP

gene specific primer

NP

nested primer

OHP

overhanging primer

Copyright information

© Springer 2006

Authors and Affiliations

  1. 1.Agricultural Genetic Engineering Research InstituteAgricultural Research CentreGizaEgypt