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Identification of DNA sequences flanking T-DNA insertions by PCR-walking

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In recent years, concerns over genetic modification issues have resulted in regulatory authorities requiring comprehensive analysis of transgene insertion events in the plants that are to be commercialized. Determining that plants are devoid of vector backbone sequences is a trivial task that is best achieved by Southern blot analysis; however, identifying the DNA sequences flanking the T-DNA insertions can be arduous. In this paper, we present a robust method of characterizing this insertion event. We have applied and modified a genomic walking method that combines vectorette and suppression PCR walking.

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Correspondence to Nicola A. Ramsay.

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Cottage, A., Yang, A., Maunders, H. et al. Identification of DNA sequences flanking T-DNA insertions by PCR-walking. Plant Mol Biol Rep 19, 321–327 (2001).

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