All Rights Reserved AEIC 2004 Flanking Primer Transgene.

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All Rights Reserved AEIC 2004

Flanking Primer Transgene

All Rights Reserved AEIC 2004

CaMV 35S Promoter Petunia CTPCP4 EPSPSNOS Terminator RR Soya: 118 bp 128 bp 123 bp 211 bp CDPK Promoter crylA(b)-Gene PEPC Intron #9 CaMV 35S Terminator BT176 Corn: 189 bp ivs 2 - Intronpat-Gene BT11 Corn: 118 bp 123 bp CaMV 35S Promoter NOS Terminator All Rights Reserved AEIC 2004

Cycle Copy-number Rn

All Rights Reserved AEIC 2004

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 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) Control MeasuresProblem All Rights Reserved AEIC 2004

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 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 Control MeasuresProblem All Rights Reserved AEIC 2004