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PCR Quantitative Analysis[ PCR Procedures | Qualitative PCR Analysis | Detection Limit & Quantification Limit ] Quantitative Real-Time PCR is used to determine the exact percentage of genetically modified DNA in a sample. All GeneScan labs utilize the ABI PRISMTM 7700 Sequence Detection System (TaqManTM) from Applied Biosystems. Currently, the TaqMan models 7700 and 7900 offer the most advanced and accurate DNA quantification by Real-Time PCR available on the market (Applied Biosystems states that the simplified model 5700 should not be used in a quantification format). In contrast to standard PCR technique with subsequent gel electrophoresis, TaqMan PCR offers the possibility to follow the progress of the PCR reaction real-time, directly in the reaction vessel, as measured by an increase in fluorescence. The time required for the generation of PCR products varies in relation to the number of target molecules present in the beginning of the reaction. The TaqMan apparatus is a PCR thermocycler that has two optical systems linked to each of its 96 reaction wells. Laser light is directed into the reaction mixtures, and a sensory pathway measures the fluorescence emitted from the wells. Besides the components of a conventional PCR reaction, the TaqMan reaction mixture contains a DNA probe that anneals within the amplified DNA region. It carries two fluorescent dyes, the 'reporter' and the 'quencher', in close proximity. When activated by the TaqMan's laser system, the reporter does not emit fluorescence but transfers the absorbed energy to the nearby quencher. During the PCR reaction, the probe molecules are cleaved and the quencher and the reporter are separated from each other. The laser's energy, absorbed by the 'reporter', no longer transferred to the 'quencher', is emitted via fluorescence. The number of emissions from the 'reporter' increases relative to the number of copies made during PCR. The simultaneous analysis of the sample DNA and standard DNA with defined numbers of target molecules allows for the calculation of the number of target molecules in the sample prior to the PCR reaction. In order to achieve a relative quantification, this is done in two independent PCR systems, one of which is designed to measure the amount of DNA molecules specific for the plant species (e.g. soy), regardless if GM or non GM. The second primer system determines the amount of GM molecules only (e.g. the Roundup Ready soy construct). From these two values, the relative amount of GM DNA in the sample is calculated and reported as a percentage. » Read about Detection Limit & Quantification Limit
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