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Objective: The aim of this study is to do a sequencing analysis of RUNX2 in non-syndromic patients with supernumerary tooth. Materials and methods: Fifty-three patients with supernumerary tooth were identified retrospectively from 1,275 radiographic reviews who attended the Hospital Universiti Sains Malaysia (USM) Dental Clinic. Informed consent was obtained from the patients prior to the study. Blood samples were collected from 41 patients and DNA extractions were performed out of which 10 samples were chosen randomly for PCR amplification using designated primers for RUNX2 followed by DNA sequencing analysis. Results: This study involved 28 male patients (68.3%) and 13 female patients (31.7%) with a gender ratio of 2.2:1 and mean age of 15.9 ± 6.2 years. DNA extraction yielded ~ 40 ng/μl of concentrated DNA, and each DNA sample had more than 1500 bp of DNA length. The purity ranged between 1.8 and 2.0. DNA sequencing analysis did not reveal any mutations in exons 5 and 6 of RUNX2. Conclusion: This study did not reveal any mutations in exons 5 and 6 of RUNX2 in non-syndromic patients with supernumerary tooth. Clinical relevance: Analysis of mutations in RUNX2 is important to enhance the understanding of tooth development in humans. [ABSTRACT FROM AUTHOR]