Abstract
Background:
Tumour protein 53 gene (TP53) is a critical factor that controls different cell activities such as cell cycle, DNA repair mechanism, autophagy, apoptosis and metabolism. The TP53 gene is the most commonly mutated gene, especially in the 4-8 exons region. This mutation enhances the development of many abnormalities, such as the initiation of different types of cancer.

Aim:
The main objective of this study was to design and evaluate the efficacy of three different primer sets that targeted TP53 gene at the hotspot regions (4-8 exons).

Methods:
To do that, twelve blood samples were collected from dogs belonging to the German shepherd breed/K9 aged between 8-12 years. Then the DNA extraction and polymerase chain reaction (PCR) took place by using the three primer sets, which were designed using SnapGene. The first primer set targeted exons 4-8, while the second and the third targeted exons 5-8 located in the canine TP53 gene. The following step, all the PCR products were sent for Sanger sequencing and then phylogenetic analysis.

Results:
Our findings indicated that the first primer set was consistently showed higher amplification signal efficiency and reduced dimer formation compared with the second and third primer sets respectively, with a 60 ºC annealing temperature. In addition, all the sequenced samples aligned with the reference canine TP53 gene in the phylogenetic tree.

Conclusion:
This study was offering the best TP53 primer design that targeted the hotspot regions of canine TP53 gene for researchers who are interested in targeting such regions in this gene.

Key words: Canine, Exons 5-9, PCR, TP53 gene, TP53 hot spot regions