Analysis of Transgenic Plants

1. Analysis of Transgenic Plants

2. Requirements • Sequence of gene of interest • Gene construct

4. Gene construct

5. GMO detection system • Morphology • Physiology • Yield characters • Confirmation selectable marker or gene of interest • Gene expression • Gene sequence

6. Selection based on • Selectable Marker Gene (Morphology)

7. Protein Based Testing Methods • Transgenes encode for novel proteins • Immunoassay techniques using antibodies • Ideal for qualitative and quantitative needs • Detects specific proteins in complex matrices • Analyte must be known • Interference from non-specific interactions of proteins, surfactants (saponins), phenolics, fatty acids and phosphatases • Polyclonal antibodies • Sensitive but less specific recognition • Monoclonal antibodies • Highly specific, slightly less sensitive recognition

8. 35S promoter EPSPS CP4 NOS terminator CaMV Agrobacterium tumefaciens • Typically included in rDNA: promoter sequence, structural gene and a stop sequence DNA-Based Methods • Rely on 2 complementary DNA strands that hybridize in a sequence-specific manner • rDNA in the crop consists of several unique elements

9. Southern Analysis

10. Principles of PCR • Allows millifold amplification of target DNA • DNA polymerase makes exact copy of template • Synthetic oligonucleotide primers • Frame desired target sequence • Complimentary to DNA • Allows Taq-polymerase enzyme to generate complimentary sequence between primer sets • Sequence # grows exponentially • In theory…

11. Progeny Testing