Specific Primer Design and Annealing Temperature for Glutathione Peroxidase (GPX) Gene Amplification on Oryza sativa
Abstract
Glutathione peroxidase (GPX) is one of the antioxidant enzymes for the protection of proteins and cell membranes against oxidative stress in plants. Plants will activate the antioxidant defense system in plant cells, namely enzymatic and nonenzymatic components when experiencing oxidative stress, and rice is a plant whose growth has a serious impact due to drought which has an impact on oxidative stress. One method that can be used for glutathione peroxidase (GPX) gene amplification is qRT-PCR and this method requires a specific primer for the target gene. But currently there is no known primary design of the gene. The purpose of this study was to design specific primers to be used in PCR amplification so as to amplify the GPX gene and determine the optimal conditions of the annealing temperature. The search for optimal conditions of the annealing temperature is very important, as it relates to the specificity and sensitivity of the PCR product. Primary design is done on the PrimerQuest program. The results of the analysis were viewed using GeneiousPrime, then checked primary specificity with primerBLAST. The results of the primary design with the best criteria are Forward GPX 5'- GGCTTTGAGATATTGGCTTTCC-3' and Reverse GPX 5'- AGCCCACCTTTGTTAGACTTC-3' primers. The primary pair can amplify the GPX gene from Oryza sativa with a product size of 185 bp. PCR performance is optimum at an annealing temperature of 60ºC and results will be specific using the Touchdown PCR program in vitro tests.
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DOI: https://doi.org/10.24036/bsc.v8i1.122697
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