5.3.5. DNA based methods – PCR application

DNA-based methods primary rely on multiplication of a specific DNA piece by PCR technique [14]. For visualization of the amplification products gel electrophoresis is routinely used. It may be solely performed or coupled with restriction endonuclease digestion (RFLP-PCR). A more sophisticated variant of the basic PCR protocol involves determination of the Tm profile by means of a dye intercalating double stranded DNA and emitting fluorescent light. With increasing the temperature the two strands of DNA begin to separate and correspondingly – the light emission, which can be measured, decreases. Tm is a specific characteristic of a DNA sequence rather than DNA length. At last, but not leased an alternative is to use probes and perform hybridization with DNA or RNA. If appropriately designed, a probe can discriminate between the native and any foreign sequence. Labeling the probe with radioactive or non-radioactive compounds facilitates the detection of the present molecule. For GMO analysis gel electrophoresis and hybridization techniques are currently the most commonly exploited techniques.

Screening of food samples for the presence of GMOs by use of basic PCR protocol comprises the following procedure;

• extract of DNA from the sample and standards of unknown GMO content;
• assembly of several PCR with specific primers (usually for well known regulatory sequences, as the viral 35SCaMV or Tnos promoters);
• visualization of the DNA fragments on an agarose gel electrophoresis;
• analysis and semi-quantitative assay using image analysis software.

With multiplex PCR-based methods several DNA sequences can be screened for and detected in a single reaction. However, the development of a multiplex assay requires careful testing and approving. The pool of amplification fragments needs to be further analyzed to distinguish between the various amplicons. This can be done with the aid of specific hybridization probes by gel electrophoresis and comparison of the fragments size or using specifically labeled primers.

A great advantage of this technique is the fact that fewer reactions are needed to test a sample for presence of GMO-derived DNA. Additionally if it is necessary to further perform quantification assays it will be good if you know which GMO to quantify since the procedure is relatively expensive. The identification of a certain GMO is important also in the context of our knowledge about the approved and aunapproved GMOs.

Another approach is to apply PCR-based quantification methods. PCR-based quantification can be performed both during the amplification process (the real-time PCR) and at its end (end-point PCR).

The end product analyses are commonly based on comparison of the mount of amplified DNA of two DNA targets: the one to be quantified and a competitor (in known small quantity) added to the amplification mixture before the PCR and co-amplified with the target to be quantified. This process is also called competitive quantitative PCR. It is based on the presumption that if both target DNA and competitive DNA yield the same amount of amplification product, the starting amount of DNA is also assumed to be the same.

In real-time PCR analysis the amount of a product synthesized during PCR is estimated directly by measurement of the fluorescence in a PCR. There are commercially available hybridization probes emitting fluorescence corresponding to the amount of the synthesized DNA. The amount of the synthesized product can be also estimated by the emitting of intercalated fluorescent dye but here it is not possible to distinguish between specific and not-specific products. The advantage of this method is that not only the quantity of the formed product can be followed in dynamics but also the defined number of cycles, which are needed to produce a certain amount of PCR product, can be determined.

The real-time PCR requires more sophisticated and expensive equipment; it is faster than competitive PCR and more specific.