Optimization of annealing temperature for amplification of the exon one region of the HPPD gene in HA1 accession sunflowers

Abstract

The HPPD gene is one of the genes that is actively involved in the biosynthesis of tocopherol, which is the main component of Vitamin E. Sunflower is raw materials for natural medicine since their oil contains vitamin E. To obtain complete information regarding the whole sequences of the 4-Hydroxyphenylpyruvate Dioxygenase (HPPD) gene in sunflower accession HA1, the exon one region needs to be identified using a PCR-based method. One factor affecting the success rate of PCR is the annealing temperature. The optimum annealing temperature will produce specific target gene products. This study aimed to determine the optimal temperature for amplifying the exon 1 region of the HPPD gene using the PCR method. This research began with primer design activities, followed by in vitro amplification (PCR), then optimization of annealing temperature using gradient PCR. The results showed that the optimum temperature for amplifying fragment one was an annealing temperature of 53 °C, fragment two was at an annealing temperature of 45 °C, and fragment three had an annealing temperature of 57 °C with the expected size of PCR products are 306 bp, 506 bp, and 534 bp respectively.