Analysis of Transgenic Plants
Requirements
• Sequence of gene of interest• Gene construct
Gene construct
BamHI
gus-intron nptII T 35S P 35S T 35S LB RB
P SAG12 ipt P 35S T nos
GMO detection system
• Morphology• Physiology
• Yield characters• Confirmation selectable marker or
gene of interest• Gene expression• Gene sequence
1.Selection based on Selectable Marker Gene (Morphology)
Protein Based Testing Protein Based Testing MethodsMethods
• 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
DNA-Based MethodsDNA-Based Methods• Rely on 2 complementary
DNA strands that hybridize in a sequence-specific manner
• rDNA in the crop consists of several unique elements
35S promoter EPSPS CP4EPSPS CP4 NOS terminatorCaMV Agrobacterium tumefaciens
•Typically included in rDNA: promoter sequence, structural gene and a stop sequence
Southern Analysis
Principles of PCRPrinciples 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…
Cycles DNA molecules formed
1 - 2 1 3 2 4 4 5 8 6 16 7 32 8 64 9 128 10 256 11 512 12 1,024 13 2,048 14 4,096 15 8,192 16 16,384 17 32,768 18 65,536
Progeny Testing