Lakhasly

Polymerase chain reaction (PCR) is a biochemical and molecular biology technique for isolating a segment of DNA and amplifying it exponentially, by enzymatic replication, without using a living organism.Although this technique requires advanced machinery and data processing tools, the non-chemical approach could have some advantages such as lower cost and enhanced speed and portability.Despite the variety of methods used for DNA analysis, only PCR in its various formats has been widely applied in the detection and analysis of GMOs and is generally accepted by the regulator.Annealing temperatures must be optimized for each primer set to work properly within a single reaction, and the sizes of the amplicons , i.e. their base pair lengths , must be different enough to form distinct bands when visualized by gel electrophoresis .Shortcomings of current detection methods Currently, it is highly unlikely that unexpected or even unknown GMOs will be detected, as the DNA sequence of the modified gene or resulting protein must be known in order to detect them.Protein-based methods detect the product of the transgene, for example the Bt toxin.

Polymerase chain reaction (PCR) is a biochemical and molecular biology technique for isolating a segment of DNA and amplifying it exponentially, by enzymatic replication, without using a living organism. It enables the detection of specific DNA sequences by making millions of copies of the target genetic sequence. The target sequence is essentially copied at an exponential rate, and simple 3D imaging techniques can make it easy to visualize millions of copies.

This method works by pairing the target gene with specially designed complementary pieces of DNA called primers . In the presence of the target gene, the primers match with it and trigger a chain reaction. DNA replication enzymes use the primers as binding points and begin to replicate the target sequence. The process is repeated over and over again by sequential heating and cooling until the target sequence has been replicated and re-replicated several million times. The millions of identical fragments are then purified in a stained gel, visible under ultraviolet light. They are free from contamination. Despite the variety of methods used for DNA analysis, only PCR in its various formats has been widely applied in the detection and analysis of GMOs and is generally accepted by the regulator. DNA-based detection methods rely on the complementarity of two strands of the DNA double helix that are hybridized in a sequence-specific manner. The DNA of GMOs consists of several elements that control its operation. The elements are the promoter codon, the structural gene, and the stop codon of the gene.

Quantitative detection
Quantitative PCR (Q-PCR) is used to measure the amount of PCR product (best known as real-time, QRT-PCR). It is the preferred method for quantitatively measuring the amounts of modified DNA in a food or feed sample. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and how many copies of it are present in the sample. Currently, the method with the highest level of accuracy is quantitative real-time PCR. QRT-PCR uses fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time. If the target gene is unique to a genetically modified organism, a positive PCR test proves that the genetically modified organism is present in the sample.

Qualitative detection
Whether or not GMOs are present in a sample can be tested by Q-PCR, but also by multiplex PCR. Multiplex PCR uses multiple, unique primers within a single PCR reaction to produce amplicons of different sizes specific to different DNA codons, i.e. different transgenes. By targeting different genes simultaneously, additional information can be obtained from a single test run that would otherwise require several times more reagents and more time to perform. Annealing temperatures must be optimized for each primer set to work properly within a single reaction, and the sizes of the amplicons , i.e. their base pair lengths , must be different enough to form distinct bands when visualized by gel electrophoresis .

Event-specific vs. construction-specific disclosure
When manufacturers, importers, or authorities test a sample for the unintended presence of GMOs, they often do not know what GMOs to expect. While EU authorities prefer an event-specific approach to dealing with this issue, US authorities rely on construction-specific testing schemes.

Event Disclosure
Event detection looks for the presence of a DNA sequence unique to a particular GMO, usually the junction between the transgene and the original DNA of the organism. This approach is ideal for accurately identifying GMOs, however very similar GMOs will go completely unnoticed. Event detection is based on PCR.

Building specific disclosure
Construct detection methods can be either DNA-based or protein- based . DNA-based detection looks for a foreign DNA fragment inserted into a GMO. For technical reasons, certain DNA codes are shared by many GMOs. Protein-based methods detect the product of the transgene, for example the Bt toxin. Since different GMOs may produce the same protein, construct detection can test a sample for many GMOs in one step, but it is unable to identify the specific GMOs present. Protein-based detection of the construct approach is used particularly in the USA.

Shortcomings of current detection methods
Currently, it is highly unlikely that unexpected or even unknown GMOs will be detected, as the DNA sequence of the modified gene or resulting protein must be known in order to detect them. In addition, even testing for known GMOs is time-consuming and expensive, as current reliable detection methods can only test for one GMO at a time. Research programs such as Co-Extra are therefore developing improved and alternative testing methods, such as DNA microarrays .

Alternative detection methods
Improved PCR-based detection
Improving PCR-based detection of GMOs is a further goal of the European Co-Extra research programme. Research is underway to develop multiplex PCR methods that can simultaneously detect many different GMO lines. Another major challenge is the increasing prevalence of stacked GM crops. This refers to GM varieties derived from crosses between genetically modified parent lines, combining the genetically modified traits of both parents. One GM maize variety currently awaiting a decision from the European Commission, MON863 x MON810 x NK603, has three stacked traits. It is resistant to herbicides and two different insect pests. Some combination test methods can give results that triple the actual GMO content of a sample containing that GMO.

Detection of unknown GMOs
Almost all genetically modified plants have a few common building blocks that make it easy to find unknown GMOs. Although discovering a new gene in a GMO can be like finding a needle in a haystack, the fact that needles are usually similar makes it much easier. To trigger gene expression, scientists pair the gene they want to add with what is known as a transcription promoter . The high-performance 35S promoter is a common feature of many GMOs. In addition, the stop signal for gene transcription in most GMOs is often the same: a NOS terminator . Researchers are now assembling a set of genetic sequences that are characteristic of GMOs. After selecting the genetic characteristics of the GMOs, methods and tools are developed to detect them in test samples. Methods under consideration include microarrays and anchorage PCR profiling.

Near Infrared (NIR) Fluorescence
Near-infrared ( NIR ) detection is a method that can detect many types of chemicals in a sample based on their unique physical properties. This is done by shining near- infrared light on the sample, which causes chemical bonds in the sample to vibrate and re-emit light energy at the wavelength characteristic of a particular molecule or chemical bond. It is not yet known whether the differences between GMOs and conventional plants are large enough to be detected using NIR imaging. Although this technique requires advanced machinery and data processing tools, the non-chemical approach could have some advantages such as lower cost and enhanced speed and portability.