Fluorescence in situ hybridization on extended DNA fibres as a tool to analyse complex T-DNA loci in potato
1998
Wolters, A.M.A. | Trinidade, L.M. | Jacobsen, E. | Visser, R.G.F.
In this study the T-DNA composition of four antisense potato transformants showing complete or very strong inhibition of granule-bound starch synthase (GBSS) activity was analysed in detail. By Southern blot hybridizations, it was determined that all four transformants contained T-DNAs on multiple linkage groups and that most linkage groups contained multiple T-DNA copies, often in combination with non-T-DNA vector sequences. Subsequently, fluorescence in situ hybridization was performed on extended DNA fibres ('fibre-FISH') of three progeny plants each containing a single linkage group with a complex T-DNA organization. By using two differently labelled probes, one consisting of T-DNA sequences and the other of vector DNA sequences, it was possible to visualize the composition of complex loci. DNA sequences of 5-6 kb were well distinguishable. With this technique it is possible to determine T-DNA copy number, and arrangement of T-DNA and vector DNA sequences in a locus, more accurately than by Southern blot analysis alone. Therefore, fibre-FISH is a valuable supplementary tool to study T-DNA integrations in detail.
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