Methods for detecting and identifying DNA using PCR technique in Vietnam

Methods for detecting and identifying DNA using PCR technique in Vietnam (Illustration)

According to the Appendix on qualitative and quantitative analysis procedures of Deoxyribonucleic Acid issued together with Circular 13/2013/TT-BTNMT, it stipulates the methods for detecting and identifying DNA of genetically modified organisms (GMOs) using PCR technique, including:

Taxon-specific method: Taxon is a classification unit. Target taxon is the classification unit of the genetically modified organism. This is a common method to detect DNA sequences in a target taxon, usually a species but can be at a lower or higher taxonomic rank. Methods based on target taxonomy are used to assess the presence, quality, and quantity of DNA originating from the taxon and are sometimes used as a standard unit for relative quantification of genetically modified material.

PCR screening method for detecting GMO DNA: Detect common genetic elements among some genetically modified organisms (such as promoter genes, terminator genes, or some other genetic elements of interest). This is a quick and reliable screening method for a large number of test samples.

Detecting GMOs using PCR technique primarily relies on specific primer pairs for promoter and terminator regions. Most DNA sequences transferred into plant genomes are under the control of the CaMV 35S promoter, with only a few cases using tissue-specific promoters expressed in areas such as roots, stems, seeds, or inducible promoters. Currently, many genetically modified plants have been licensed for cultivation bearing at least one copy of the 35S promoter and T-Nos terminator (isolated from the nopaline synthase gene in Agrobacterium tumefaciens). Another commonly used gene is the kanamycin resistance nptII gene isolated from Escherichia coli transposon 5. Both Agrobacterium tumefaciens and Cauliflower mosaic virus are listed as harmful plant pathogens and, specifically, the Cauliflower mosaic virus not only causes diseases in cauliflower but also other members of the Brassicaceae (Cruciferae) as well as the Resedaceae and Solanaceae families. Therefore, positive results from Brassicaceae, Resedaceae, and Solanaceae samples need to be handled with care. To distinguish between viral infection and genetically modified material, a virus detection method is needed.

For genetically modified animals, promoters used often originate from animals such as metallothionein (MT), thymidine kinase, b-actin, amylase, insulin, b-lactoglobulin, adipocyte P2, or from animal viruses such as Simian virus (SV40), Rous sarcoma virus (RSV), etc. However, due to the potential for animal-derived promoters to be confused with endogenous promoters of the host, animal virus-derived promoters can be used to screen for the presence of animal-derived DNA.

Event-specific method to detect target gene sequences in genetically modified products: Used to detect the combination of DNA sequences introduced into the host genome, this structure is found only in the lines originating from genetically modified organisms. For instance, to detect T25/"LibertyLink" corn resistant to herbicide due to genetic modification in raw materials by amplifying the junction between DNA sequences originating from the 35S-CaMV promoter and the pat gene (herbicide resistance gene). This method does not distinguish corn varieties but is used to detect the presence of the transferred gene segment in kernels and corn plants.

The size of the target gene segment after PCR amplification is determined when the size of the PCR product corresponds to the expected target DNA segment size. PCR products are analyzed and processed using agarose gel electrophoresis and stained by ethidium bromide. A 1.5% agarose gel is suitable for analyzing PCR products from 150-1000 bp. A DNA ladder with sizes ranging from 0.1

More details can be found in Circular 13/2013/TT-BTNMT, which comes into effect in Vietnam from August 5, 2013.

Le Vy

>> CLICK HERE TO READ THIS ARTICLE IN VIETNAMESE