Screening of transgenic plants by amplification of unknown genomic DNA flanking T-DNA.
For the screening of transfer DNA (T-DNA) integration in transgenic plant material, we developed a method based on specific amplification of genomic plant DNA flanking T-DNA borders. This approach is possible because the length of the region flanking T-DNA extremity on a restriction fragment is specific to the integration locus. We have modified an adaptor ligation PCR technique developed for amplification of unknown DNA flanking known sequence. The PCR patterns obtained were specific and reproducible for different plants from a given transgenic line. Furthermore, the number of PCR products obtained could be considered a good estimation of the T-DNA copy number. When compared to Southern blot analysis, the PCR results give valuable complementary information about the complexity of the T-DNA integration pattern and also about the integrity of the T-DNA borders. We describe the applications of this approach to populations of transgenic Arabidopsis thaliana plants.
Spertini
D
D
Université Laval Québec, Canada.
Béliveau
C
C
Bellemare
G
G
D017360:Arabidopsis / Q000235:genetics; D015139:Blotting, Southern / Q000379:methods; D004269:DNA, Bacterial / Q000032:analysis*; D018744:DNA, Plant / Q000032:analysis*; D005784:Gene Amplification; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction / Q000379:methods*
D004269:DNA, Bacterial; D018744:DNA, Plant; C036483:T-DNA