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All Rights Reserved AEIC 2004

All Rights Reserved AEIC 2004. All Rights Reserved AEIC 2004. All Rights Reserved AEIC 2004. All Rights Reserved AEIC 2004. All Rights Reserved AEIC 2004. All Rights Reserved AEIC 2004. All Rights Reserved AEIC 2004. All Rights Reserved AEIC 2004.

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All Rights Reserved AEIC 2004

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  1. All Rights Reserved AEIC 2004

  2. All Rights Reserved AEIC 2004

  3. All Rights Reserved AEIC 2004

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  6. All Rights Reserved AEIC 2004 All Rights Reserved AEIC 2004

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  9. Transgene Flanking Primer Flanking Primer All Rights Reserved AEIC 2004

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  12. CaMV 35S Promoter RR Soya: Petunia CTP CP4 EPSPS NOS Terminator 118 bp 123 bp 128 bp BT11 Corn: ivs 2 - Intron pat-Gene 118 bp 123 bp CaMV 35S Promoter 211 bp NOS Terminator 189 bp CDPK Promoter CaMV 35S Terminator BT176 Corn: PEPC Intron #9 crylA(b)-Gene All Rights Reserved AEIC 2004

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  16. 10000 10000 2000 2000 400 Rn 400 80 80 Copy-number Cycle All Rights Reserved AEIC 2004

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  19. Problem Control Measures During validation: • Search data base for potential unwanted (homologous) annealing sites • Test new primer system on a wide range of DNA types that appear in food samples • Verify the identity of the PCR product by Southern blot or restriction enzyme digestion Artifact PCR products from templates other than the target sequence in question (worst scenario: artifacts of the same size as the expected PCR product) During routine analysis: • Follow strict rules how to work cleanly in a molecular biology lab • Use one way sample flow lab design to prevent contamination with positive DNA from down- stream steps (especially PCR products), strict separation of certain lab areas from each other • Use duplicate analysis of each sample • Use negative controls on homogenization, DNA extraction and PCR setup Corruption of sample, extracted sample DNA or PCR setup contains positive DNA All Rights Reserved AEIC 2004

  20. Problem Control Measures During design and validation: • Use computer aided primer design • Test the sensitivity of new primer systems for the intended concentration range Insufficient sensitivity of primer system During validation: • Test primer system on sample DNA solutions known to contain inhibitory compounds During routine analysis: • Test each PCR reaction for inhibition by an individual positive control (a spiked counterpart) • Use duplicate analysis of each sample Inhibition of PCR reaction During design and validation: • Choose appropriate extraction protocol for sample type • Run controls to monitor efficiency of each extraction series DNA extraction failed All Rights Reserved AEIC 2004

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