Basic principles of the PCR

The quantity, quality and purity of the template DNA are important factors in successful PGR amplification. The PGR is an extremely sensitive method capable of detecting trace amounts of DNA in a crop or food sample, so PGR amplification is possible even if a very small quantity of DNA is isolated from the sample. DNA quality can be compromised in highly processed foods such as pastries, breakfast cereals, ready-to-eat meals or food additives owing to the DNA-degrading action of some manufacturing processes. DNA purity is a concern when substances that inhibit the PGR are present in the sample. For example, cocoa-containing foodstuffs contain high levels of plant secondary metabolites, which can lead to irreversible inhibition of the PGR. It is important that these substances are removed prior to PGR amplification. Extraction and purification protocols must be optimized for each type of sample. 

Several standard DNA isolation kits are commercially available, including the QIAamp DNA Stool Mini Kit and the DNeasy Plant Mini Kit made by Qiagen. Both of these products are based on silica gel membrane technology and allow for the extraction of total DNA from processed foods and raw foodstuffs, respectively. In 

Classical approaches to plant DNA isolation aim to produce large quantities of highly purified DNA. However, smaller quantities of crudely extracted plant DNA are often acceptable for PCR analysis. Another efficient method for preparation of plant DNA for PCR is a single-step protocol that involves heating a small amount of plant tissue in a simple solution. Several factors influence nucleic acid release from tissue salt, EDTA, pH, incubation time and temperature. These factors must be optimized for different sample substrates. EDTA in the sample solution binds the Mg + cofactor required by the Taq polymerase in the PCR, so the EDTA concentration in the solution, or the Mg + concentration in the PCR, must be carefully optimized. 

An optimized single-step protocol for the extraction of leaf tissue or seed embryos is given here. The template preparation solution (TPS) contains  

Add a l- uL portion of the supernatant (or dilution thereof, if inhibitors are present) to the 50- uL PCR reaction. 

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