Plant Molecular Biology Reporter

, Volume 24, Issue 2, pp 237–243

A primer-based approach to genome walking

Protocols

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

Abbreviations

AP

adaptor primer

GSP

gene specific primer

NP

nested primer

OHP

overhanging primer

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Anonymous (2006) New England Biolabs catalogue and technical reference. http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/frequency_of_restriction_sites.aspGoogle Scholar
  2. Arnold C and Hodgson IJ (1991) Vectorette PCR: a novel approach to genomic walking. PCR Methods and Applications 1:39–42.PubMedGoogle Scholar
  3. Ashoub A (2003) Full-length Sequence of Egyptian Potato Leafroll Virus (PLRV) Isolate. Arab J Biotech 6: 173–182.Google Scholar
  4. Lundberg KS, Shoemaker DD, Short JM, Sorge JA, Adams MWW, and Mathur E (1991) High fidelity amplification with a thermostable DNA polymerase isolated fromPyrococcus furiosus. Gene 108: 1–6.PubMedCrossRefGoogle Scholar
  5. Ochman H, Gerber AS, and Hartl DL (1988) Genetic applications of an inverse polymerase chain reaction. Genetics 120: 621–623.PubMedGoogle Scholar
  6. Riley J, Butler R, Ogilvie D, Finniear R, Jenner D, Powell S, Anand R, Smith JC, and Markham AF (1990) A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones. Nucleic Acids Res 18: 2887–2890.PubMedCrossRefGoogle Scholar
  7. Rishi AS, Nelson ND, and Goyal A (2004) Genome walking of large fragments: an improved method. J Biotech 111: 9–15.CrossRefGoogle Scholar
  8. Töpfer R, Matzeit V, Gronenborn B, Schell J, and Steinbiss HH (1987) A set of plant expression vectors for transcriptional and translational fusions. Nucleic Acids Res 15: 5890.PubMedCrossRefGoogle Scholar
  9. Triglia T, Peterson MG, and Kemp D J (1988) A procedure forin vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucleic Acids Res 16: 8186.PubMedCrossRefGoogle Scholar
  10. Upcroft P and Healey A (1993) PCR priming from restriction endonuclease site 3′ extension. Nucleic Acids Res 21: 4854.PubMedCrossRefGoogle Scholar

Copyright information

© Springer 2006

Authors and Affiliations

  1. 1.Agricultural Genetic Engineering Research InstituteAgricultural Research CentreGizaEgypt

Personalised recommendations