analysis of transgenic plants
DESCRIPTION
Analysis of Transgenic Plants. Requirements. Sequence of gene of interest Gene construct. Gene construct. GMO detection system. Morphology Physiology Yield characters Confirmation selectable marker or gene of interest Gene expression Gene sequence. Selection based on - PowerPoint PPT PresentationTRANSCRIPT
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