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Third and final step involves making two identical copies of original DNA strand by adding exact nucleotides with the help of DNA polymerase at an appropriate temperature. Amplification of target gene occur in-vitro through a reaction catalyzed by a DNA polymerase in the presence of oligonucleotide primers and deoxyribonucleoside triphosphates in a defined reaction buffer [ 23 , 24 ].
Public perception of genetically-modified (GM) food: A Nationwide Chinese Consumer Study
This amplified DNA can be visualized by using gel electrophoresis techniques. The results of this method will be either positive or negative for specific GM elements. There are four testing methods which includes i Target-taxon specific ii Screening iii Construct-specific and iv Event-specific, these methods are generally used for the detection and identification of GM crops using PCR.
Selection of specific and suitable primers is the most critical step in GMO detection which depends upon the testing method used. Brief detail of qualitative PCR based testing methods is given below:. PCR with various barcoding methods normally used for plant identification from mixed food samples particularly prepared from different plants. DNA barcode is broadly used technique for the detection and identification plants, animals or fungi texa by sequencing an optimized short DNA fragment. PCR and barcoding approaches identify specific texa very intelligently within samples of different origins [ 25 , 26 , 27 ].
This approach also plays very important role in the detection of mislabeled species and accidental or intentional species exchanges in food samples [ 28 , 29 ]. The success of this method for identification and detection of species depends on the selected loci, because DNA barcode constitute a small portion of genome coupled with other PCR limiting factors, no single locus has been selected as universal DNA barcode region for all plant identification. For example lectin gene Le1 for soybean [ 30 ], chloroplast trnL intron for the identification of multicopy DNA sequences in plants [ 31 ], polygalacturonase gene PG gene codes for a PG-enzyme that is linked with ripening in GM Zeneca tomato etc.
This is a most generalized method and widely used for the screening of GM crops from non-GM materials. This is not crop specific and can detect the GM elements even in raw and processed matrices like food and feed products developed from GM crops. In this method promoter, terminator and selection marker genes are the target elements in PCR. These are the bacterial gene sequences used to regulate the transgenes and selection of transgenic cells on artificial plant media [ 33 , 34 ].
Hence, one can easily detect and identify the presence of GM crop by using specific primers of these genetic elements in PCR [ 35 , 36 ]. In this method specific primer pairs normally got designed from the transformed gene construct. These construct could be transformed more than one crop for genetic improvement. The construct-specific detection method involves targeting the junction between two elements, and it is not able to distinguish two different events transformed with the same plasmid [ 37 ]. These methods either DNA or protein based. Since different GM crops may produce the identical protein, this test method can detect a sample for several GMOs in one step.
The junction sequences in the transgene integration points in the plant genome can be used to identify and detect the specific transformation event. The transgene integration site usually unique and specific for each transformation event due to lack of homologous recombination. Hence, different GM crops could be produced with similar gene construct and this event-specific detection method will be the only approach to differentiate between GM crops having similar transgenic cassette.
Another DNA based GM crops identification techniques is southern blotting which was described by Southern in [ 38 ]. This test method is frequently used for the identification of specific DNA fragments transformed into the genome of transgenic plants or its products. This method could also be used in gene discovery and mapping, evolution and developmental studies, diagnostics and forensics etc.
This test method involved five steps i DNA isolation and enzyme restriction ii electrophoresis for DNA separation iii shifting and fixing of separated DNA on suitable membrane iv hybridization with labeled probe and v detection by chemiluminescence or radioactive methods. This is very reliable method that provides the molecular evidence of the transgene integration and also estimates the copy number of introduced gene into the GMO genome.
In comparison with PCR, this method associated with some limitations like it requires large amount of DNA, expensive, requires more time, proper infrastructure and trained manpower etc. A microarray is a laboratory method used to identify the expression of more than one gene in a single test. It is DNA based and new in comparison to previous protocols.
This method consists of pre-amplification step of the desired targets, followed by hybridization on a chip having specific probes, and then detection step [ 42 , 43 ]. So far, it is used for qualitative information of GMO, sometime semi-quantitative. Use of microarray technology for the GMO detection is restrained as it require very special and costly equipment for scanning microarrays, chances of cross contamination and laborious in comparison with other techniques.
Transgenic DNA must be translated into protein to be an effective and have effects in an organism.
The presence of mRNA is directly associated with gene expression. Different molecular biology techniques used to monitor and study the gene expression in GMOs include real-time PCR, northern etc. These methods could be used to identify the transgene expression in various plant tissues and at different developmental phases in GMOs.
The amplified fragment electrophoresed and visualized using agarose gel under UV. Intensity of amplified band in agarose gel give some indications of target mRNA in tested sample [ 44 ]. It is more robust, specific and sensitive, provides good quantitative results. The process of amplification is presented in real-time by capturing a fluorescent signal in more sophisticated way. In real-time assay of transgene in GMOs, the amplification and detection occur simultaneously [ 45 ]. It gives comparative amount of gene expression at the RNA level. This is comparatively simple to perform, cheap and not overwhelmed by artifacts [ 46 ].
Recent advancements of hybridization membranes and buffers have resulted in increased sensitivity, closing the gap to the more laborious nuclease protection experiments.ficolviporal.cf
How to Make a GMO - Science in the News
This can be very useful to monitor the up- or down regulation of transgene for specific problem, but is not useful in monitoring the up- or down regulated genes are unknown. Immunoassay protocols for the detection of GMOs by antibodies are the impressive for the detection of various types of proteins either qualitatively or quantitatively [ 47 ]. Two types of antibodies, i. Most common antibody based test for GMO screening is strip test method also known as lateral flow or dipstick test. It is qualitative in nature and gives the information about the presence or absence of specific proteins in tested samples.
In this method, thin strip made-up of nitrocellulose membrane used which protected by a sample pad on one end and a wicking pad on other end. Test samples normally homogenized in suitable buffer solutions and membrane on strip wicks up the solution and it will move upward via capillary movement and protein will bind to its specific antibody.
The results shown in the form of visible lines on the strip depicting that the specific protein is present in test sample. There are normally two lines appears on the strip, one for tested protein and second of control line showing the authenticity of all test procedure and strip used. The appearance of only control line on the strip, shows that sample is negative for transgenic protein, but the test was performed accurately [ 48 ]. In addition, it is cheap, easy to perform and not require specific equipment and special trained manpower.
It can be performed in open field as well. Currently, strips are available to detect multiple proteins in single assay [ 50 ].
It gives information about the quantity of protein in tested samples. In this assay protein specific antibody coated multi-well plate is used to identify and quantify the specific protein. Specific protein present will bind to antibody, following washing, another antibody specific for protein of interest and tagged with an enzyme is added to well [ 51 ].
The enzyme linked identification antibody will bind with specific protein and unbound antibody removed by washing. The color of the solution will change from blue to yellow by the addition of substrate for enzyme. Intensity of yellow color is directly proportional to amount of protein present in well.
This GMO test method is more sensitive in comparison with strip test and can detect target protein even in very low concentrations. However, it requires more time, trained manpower and good laboratory facilities in contrast to strip test. This is very specific method and provides the qualitative results of the target protein in GM crop sample.
This method is very useful to analyze the insoluble proteins [ 47 , 50 ]. Like other blotting techniques samples are solubilized with detergents and reducing agents and separated by electrophoresis and shifted to membrane. Binding immunoglobulin sites on membrane are blocked by dried nonfat milk and specific sites are probed with antibodies.
Detection carried out using different staining agents silver nitrate of Coomassie, alkaline phosphatase etc. Its detection limit varies with test ample like 0.
Genetically Modified Governance Issues
In comparison with other protein based assays, it is difficult method, and is capable of studying only a few samples at a time. Therefore, it is not frequently used in GMO testing activities but it is more used in research purpose to verify initial results generated by other testing method.
Validity and authenticity of GMO testing results is doubtful until the use of positive and negative controls at each testing step. Use of certified reference material CRM or standard reference material SRM during testing produce not only validate the testing results but at the same time, assess the performance of test method, equipment, personnel and other environmental conditions in which testing being performed [ 52 ].
CRM must contain the certificate of analysis, should be prepared by following ISO-Guide 34, have information about which GM events or elements present and what is its concentration, storage requirements, preparation and expiry date etc.
Journal of Genetic Engineering and Biotechnology
While SRM have all the similar information but lacks the certificate of analysis and was not prepared by a certified company. Normally seeds of GM and Non-GM crops are mixed at specific percentage and homogenized to make powder before analysis [ 51 ]. The most common improvement by the introduction of GM crops is the increase in yield and quality. Conventional approaches like irrigations, sprays and use of fertilizers etc.
By the introduction of recombinant DNA technology in agricultural sector, scientists successfully develop the new face of existing cultivars with improved and desirable traits. The GM technologies increase the opportunities for plant breeders to develop crops that are protected from climatic stresses and attacks of insects and diseases [ 54 , 55 ].
Furthermore, this technology helping us to improve the nutritional quality, longer shelf life, foods that are more appealing to eat and easier to transport. Development of various biopharmaceuticals and expression of human therapeutic proteins in plants also a great contribution of GM technology to improve the human life [ 56 ].
On the other hand there are also some biosafety issues linked with the use of GM crops. Biosafety means the need to protect human and animal health from possible adverse effects of GM technology. There are some reports about the potential threats linked with the use of GMOs like risks of allergineicity, development of herbicide tolerant weeds and resistant insects, harms to non-target organisms, selection marker gene could induce antibiotic resistant and reduce the effectiveness of antibiotics to cure disease etc. Biosafety is an essential to modern biotechnology and the adoption of biotech products requires to be balanced with acceptable biosafety safeguards.
Participation of different stakeholders and dissemination of information and knowledge in public about GM products is much important to safe use of this technology. Agriculture sector of Pakistan plays a dominant role in the economy with It is also a chief source of foreign exchange earnings and provide raw material for progress of other sectors [ 59 ].