Deteksi Gen TOP2A, MC4R, dan NOTCH3 dengan Menggunakan Metode Long PCR pada Saliva Manusia yang Sudah Dipreservasi

Abstract

Background: Technological developments enabled modification of techniques in the field of molecular biology. DNA can be isolated from various biological sources such as blood, saliva, buccal swab, sperm, or biopsy tissue. Blood has the highest concentration of DNA compared to other biological sources. Access to blood collection is an obstacle in obtaining DNA. Saliva is an alternative source of DNA. The shortage of salivary DNA is due to the small amount of DNA and the presence of oral microorganisms. The addition of antimicrobials and DNase inhibitors helps maintaining the stability of salivary DNA in the preservation of human DNA. Preservated DNA must present intact whole DNA that might be detected. In part with Long PCR (> 1 kbp), DNA preserved using combination antimicrobe recently is shorter in routine PCR (<600 bp). Aims: to determine the effect of a combination of antimicrobials and DNase inhibitors on the preservation of human genomic DNA from saliva stored at room temperature for up to 14 days. Methods: using a pure experimental design with a pilot study. Saliva was collected from 10 subjects and divided into treatment groups (K1) with the addition of 1,500 μg/mL gentamicin sulfate, 200 μg/mL clindamycin, 1,000 μg/mL ketoconazole and 1 mM EDTA. The untreated group (K2) was not given any addition. Both DNAs from each group were extracted using the spin column method on different days before and after storage, namely day 0, 3, 7, 10, and 14. DNA concentration and purity were determined by nanophotometre. Target genes (TOPA2A, MC4R, and NOTCH3) were amplified by Long PCR and visualised by 1% agarose gel electrophoresis and ethidium bromide. Results: The salivary DNA purity was 1.90 (1.676-2.085) in average and the concentration was 31.91 ng/μL (5.950-100.30 ng/μL) in average. The presence of target genes TOP2A, MC4R and NOTCH3 detected in the treatment group, while untreated group the target genes cannot be detected on day 7. Conclusion: The combination of antimicrobe and DNase inhibitors with Long PCR methods can preserve human DNA of saliva origin stored at room temperature for up to 14 days.