In this study fifty subjects who were willing to participate and fulfilled the inclusion criteria were selected from the dental outpatient department. The thickest or most pronounced portion of dental calculus was identified. Using a sterile probe, pressure was applied on the edge directly adjacent to the tooth enamel until dental calculus break free. Once calculus was removed from the tooth, it was collected gently using tweezers on sterilised gauze pieces and tipped in disposable Eppendorf centrifuge tube vials. For barr body analysis, buccal smear was made by gently scrapping the buccal mucosa two-three times uni-directionally. These swabs were stored in small labelled sterile plastic bags. For DNA estimation and quantification, gel electrophoresis was carried out. For gender determination DNA samples were subjected to Short Tandom Repeat (STR) profiling for identification of sex specific chromosomes/genes. In bar body test of gender determination, buccal smears were analysed histologically for the presence/absence of barr bodies.