Pengaruh Suhu dan Konsentrasi EDTA terhadap Inaktivasi DNase I untuk Preservasi DNA Asal Saliva

Abstract

Background: Deoxyribonuclease I (DNase I) catalyzes the endonucleolytic cleavage of double stranded DNA into phosphodinucleotides and phosphooligonucleotides. DNase inactivation is influenced by EDTA, pH, and temperature. EDTA is an indirect inhibitor of DNase I. Purpose: To study the inhibition of DNase activity in saliva and DNase I by temperature and EDTA. Methods: This is a pure experimental study, using post test only with a control group design. Salivary DNA was extracted using the spin column method. DNA degradation assays were performed using 5 μL of salivary supernatant or 2.5 mg/mL of DNase I which were incubated at 20°C, 2 8°C, room temperature, 40°C, and 50°C, and saliva supernatant at EDTA concentration of 0.125 mM, 0.25, 0.5, 1.0, and 2.0 mM was incubated for 60 minutes. EDTA concentrations of 0.125 mM, 0.25, 0.5, 1.0, and 2.0 mM were added with 2.5 mg/mL DNase I and incubated into the DNA sample for 60 minutes. The results of the DNA degradation assay were visualized by agarose gel electrophoresis. Results: The average concentration of salivary DNA was 61.70 μg/mL and the purity was 1.893. Incubation at 20°C, 2 8°C, and room temperature degraded DNA, but at 50°C partially did not degrade DNA. EDTA concentrations of 0.125 mM, 0.25, 0.5, 1.0, and 2 mM with 5 μL of salivary supernatant, some samples showed DNA bands. EDTA concentrations of 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, and 2 mM with 2.5 mg/mL DNase I, some samples showed DNA bands. Conclusion: This study’s most effective temperature and EDTA concentration for inhibiting DNase I activity was 50°C and 2,0 Mm EDTA concentration.