analysis of transgenic plants

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