Skip to main content

Advertisement

SpringerLink
Log in

Comparison of DNA Walking Methods for Isolation of Transgene-Flanking Regions in GM Potato

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

An important aspect in the safety assessment of transgenic plants is the exact location of transgene insertion sites within the host genome. However, robust standard operating procedures are not currently available. Using potato as a test species, different methodologies for the determination of insertion sites using a range of published protocols and commercially available kits were assessed in transgenic lines of varying degrees of complexity, from low copy number to complex re-transformed and co-transformed lines. Three commercial kits, APAgene™ GOLD Genome Walking Kit (BIO S&T), DNA Walking SpeedUp™ Kit II (Seegene), and Universal Vectorette™ System (Sigma) were compared with an adaptor-mediated PCR technique. Overall, the APAgene™ kit was used with a high success rate with low copy number potato lines, and also more complex co- and re-transformed lines, and adhering to key confirmation steps it was possible to obtain flanking sequence ranging in size from 300 to 2,500 bp and eliminate PCR artefacts from the analysis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Halpin, C. (2005). Gene stacking in transgenic plants—the challenge for 21st century plant biotechnology. Plant Biotechnology Journal, 3, 141–155.

    Article  CAS  Google Scholar 

  2. Rommens, C. M., Haring, M. A., Swords, K., Davies, H. V., & Belknap, W. R. (2007). The intragenic approach as a new extension to traditional plant breeding. Trends in Plant Science, 12, 397–403.

    Article  CAS  Google Scholar 

  3. Travella, S., Ross, S. M., Harden, J., Everett, C., Snape, J. W., & Harwood, W. A. A. (2005). Comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques. Plant Cell Reporter, 23, 780–789.

    Article  CAS  Google Scholar 

  4. Grevelding, C., Fantes, V., Kemper, E., Schell, J., & Masterson, R. (1993). Single-copy T-DNA insertions in Arabidopsis are the pre-dominant form of integration in root-derived transgenics, whereas multiple insertions are found in leaf discs. Plant Molecular Biology, 23, 847–860.

    Article  CAS  Google Scholar 

  5. Hui, E. K., Wang, P. C., & Lo, S. J. (1998). Strategies for cloning unknown cellular flanking DNA sequences from foreign integrants. Cellular and Molecular Life Sciences, 54, 1403–1411.

    Article  CAS  Google Scholar 

  6. Sallaud, C., Gay, C., Larmande, P., Bes, M., Piffanelli, P., Piégu, B., et al. (2004). High throughput T-DNA insertion mutagenesis in rice: A first step towards in silico reverse genetics. Plant Journal, 39, 450–464.

    Article  CAS  Google Scholar 

  7. An, S., Park, S., Jeong, D. H., Lee, D. Y., Kang, H. G., Yu, J. H., et al. (2003). Generation and analysis of end sequence database for T-DNA tagging lines in rice. Plant Physiology, 133, 2040–2047.

    Article  CAS  Google Scholar 

  8. Sha, Y., Li, S., Pei, Z., Luo, L., Tian, Y., & He, C. (2004). Generation and flanking sequence analysis of a rice T-DNA tagged population. Theoretical and Applied Genetics, 108, 306–314.

    Article  CAS  Google Scholar 

  9. Ducreux, L. J., Morris, W. L., Hedley, P. E., Shepherd, T., Davies, H. V., Millam, S., et al. (2005). Metabolic engineering of high carotenoid potato tubers containing enhanced levels of beta-carotene and lutein. Journal of Experimental Botany, 56, 81–89.

    CAS  Google Scholar 

  10. Morris, W. L., Ducreux, L. J. M., Hedden, P., Millam, S., & Taylor, M. A. (2006). Over-expression of a bacterial 1-deoxy-d-xylulose 5-phosphate synthase gene in potato tubers perturbs the isoprenoid metabolic network: Implications for the control of the tuber life-cycle. Journal of Experimental Botany, 57, 3007–3018.

    Article  CAS  Google Scholar 

  11. Spertini, D., Béliveau, C., & Bellemare, G. (1999). Screening of transgenic plants by amplification of unknown genomic DNA flanking T-DNA. BioTechniques, 27, 308–314.

    CAS  Google Scholar 

  12. Salvo-Garrido, H., Travella, S., Bilham, L. J., Harwood, W. A., & Snape, J. W. (2004). The distribution of transgene insertion sites in barley determined by physical and genetic mapping. Genetics, 167, 1371–1379.

    Article  CAS  Google Scholar 

  13. Smith, D. S., Maxwell, P. W., & De Boer, S. H. (2005). Comparison of several methods for the extraction of DNA from potatoes and potato-derived products. Journal of Agriculture and Food Chemistry, 53, 9848–9859.

    Article  CAS  Google Scholar 

  14. Wu, H., Sparks, C. A., & Jones, H. D. (2006). Characterisation of T-DNA loci and vector backbone sequences in transgenic wheat produced by Agrobacterium-mediated transformation. Molecular Breeding, 18, 195–208.

    Article  Google Scholar 

  15. Kovřík, A., Matzke, M. A., Matzke, A. J. M., & Koukalová, B. (2001). Transposition of IS10 from the host Escherichia coli genome to a plasmid may lead to cloning artefacts. Molecular Genetics and Genomics, 266, 216–222.

    Article  Google Scholar 

  16. Bogosian, G., Bilyeu, K., & O’Neil, J. P. (1993). Genome rearrangements by residual IS10 elements in strains of Escherichia coli K-12 which have undergone Tn10 mutagenesis and fusaric acid selection. Molecular Genetics and Genomics, 233, 17–22.

    Google Scholar 

  17. Petti, C., Wendt, T., Meade, C., & Mullins, E. (2009). Evidence of genotype dependency within Agrobacterium tumefaciens in relation to the integration of vector backbone sequence in transgenic Phytophthora infestans-tolerant potato. Journal of Bioscience and Bioengineering, 107, 301–306.

    Article  CAS  Google Scholar 

  18. Kononov, M. E., Bassuner, B., & Gelvin, S. B. (1997). Integration of T-DNA binary vector backbone sequences into the tobacco genome: Evidence for multiple complex pattern of integration. Plant Journal, 11, 945–957.

    Article  CAS  Google Scholar 

  19. Afolabi, A. S., Worland, B., Snape, J. W., & Vain, P. (2004). A large-scale study of rice plants transformed with different T-DNAs provides new insights into locus composition and T-DNA linkage configurations. Theoretical and Applied Genetics, 109, 815–826.

    Article  CAS  Google Scholar 

  20. Meza, T. J., Strageland, B., Mercy, I. S., Skarn, M., Nymoen, D. A., Berg, A., et al. (2002). Analyses of single-copy Arabidopsis T-DNA-transformed lines show that the presence of vector backbone sequences, short inverted repeats and DNA methylation is not sufficient or necessary for the induction of transgene silencing. Nucleic Acids Research, 30, 4556–4566.

    Article  CAS  Google Scholar 

  21. Abdal-Aziz, S. A., Pliego-Alfaro, F., Quesada, M. A., & Mercado, J. A. (2006). Evidence of frequent integration of non-T-DNA vector backbone sequences in transgenic strawberry plant. Journal of Bioscience and Bioengineering, 101, 508–510.

    Article  CAS  Google Scholar 

  22. Lange, M., Vincze, E., Moller, M. J., & Holm, P. B. (2006). Molecular analysis of transgene and vector backbone integration into the barley genome following Agrobacterium-mediated transformation. Plant Cell Reporter, 25, 815–820.

    Article  CAS  Google Scholar 

  23. Wolters, A.-M., Trindale, L. M., Jacobsen, E., & Visser, R. G. V. (1998). Fluorescence in situ hybridization on extended DNA fibres as a tool to analyse complex T-DNA loci in potato. Plant Journal, 13, 837–847.

    Article  CAS  Google Scholar 

  24. Matzke, A. J. M., & Matzke, M. A. (1998). Position effects and epigenetic silencing of plant transgenes. Current Opinion in Plant Biology, 1, 142–148.

    Article  CAS  Google Scholar 

  25. Matzke, M. A., Mette, M. F., & Matzke, A. J. M. (2000). Transgene silencing by the host genome defense: Implications for the evolution of epigenetic control mechanisms in plants and vertebrates. Plant Molecular Biology, 43, 401–415.

    Article  CAS  Google Scholar 

Download references

Acknowledgment

We gratefully acknowledge Wayne L. Morris and Laurence J. M. Ducreux for providing transgenic material and several DNA extracts. This work was supported by the Food Standards Agency of the United Kingdom.

Author information

Authors and Affiliations

  1. Plant Products and Food Quality, Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK

    Danny Cullen, Howard Davies & Mark Taylor

  2. John Innes Centre, Crop Genetics, Norwich Research Park, Colney, Norwich, NR4 7UH, UK

    Wendy Harwood & Mark Smedley

Authors
  1. Danny Cullen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Wendy Harwood
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mark Smedley
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Howard Davies
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mark Taylor
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Danny Cullen.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cullen, D., Harwood, W., Smedley, M. et al. Comparison of DNA Walking Methods for Isolation of Transgene-Flanking Regions in GM Potato. Mol Biotechnol 49, 19–31 (2011). https://doi.org/10.1007/s12033-010-9371-5

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-010-9371-5

Keywords

Search

Navigation