Application of droplet digital PCR method for DNA methylation-based age prediction from saliva |
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| Institution: | 1. Forensic DNA Division, National Forensic Service, Wonju, Gangwon-do, South Korea;2. DNA Analysis Division, National Forensic Service Daejeon Institute, Daejeon, South Korea;3. Genetrigger, Seoul, South Korea;4. Department of Bio-Nanotechnology, Hanyang University, Ansan, Gyeonggi-do, South Korea;5. Department of Forensic Sciences, Graduate School of Sungkyunkwan University, Suwon, Gyeongi-do, South Korea;1. School of Forensic Medicine, Kunming Medical University, Kunming, Yunnan Province, People’s Republic of China;2. Judicial Expertise Center of Kunming Medical University, Kunming, Yunnan Province, People’s Republic of China;1. Department of Forensic Dental Science, Unit of Social Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan;2. Department of Forensic Pathology and Science, Unit of Social Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan;3. Atomic Bomb Disease Institute, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan;1. Forensic DNA Division, National Forensic Service, 10 Ipchun-ro, Wonju-si, Gangwon-do, 26460, South Korea;2. SeaSun Biomaterials, N517, 11-3 Techno 1-ro, Yuseong-gu, Daejeon, 34015, South Korea;3. Technology & Science, 5-6F, 19-12 Daehak-ro, 76 beonan-gil, Yseong-gu, Daejeon, 34183, South Korea;4. Department of Forensic Sciences, Graduate School of Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon, 16419, South Korea;1. Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow, G4 0RE, Scotland, UK;2. Technology and Innovation Centre, Department of Pure & Applied Chemistry, University of Strathclyde, 99 George Street, Glasgow G1 1RD, Scotland, UK;3. Centre for Forensic Science, University of Strathclyde, UK;4. Strathclyde Institute of Pharmacy and Biomedical Sciences (SIPBS) Building, University of Strathclyde, 161 Cathedral Street, Glasgow, G4 0RE, UK;1. XinHua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China;2. Shanghai Children’s Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China;3. MOE-Shanghai Key Laboratory of Children’s Environmental Health, Shanghai, China;4. Department of Genetics, Shanghai-MOST Key Laboratory of Health and Disease Genomics, Chinese National Human Genome Center and Shanghai Academy of Science & Technology, Shanghai, China;5. Department of Pediatric Dentistry, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China |
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| Abstract: | The recent studies reported that DNA methylation markers show changes with age, and expected that the DNA methylation markers can be effectively used for estimation of age in forensic genetics. In this study, we applied droplet digital PCR (ddPCR) method to investigate the DNA methylation pattern in the CpG sites, and we constructed an age prediction model based on the ddPCR method. The ddPCR is capable of highly sensitive quantitation of nucleic acid and detection of sequence variations in gene by separating the sample into large number of partitions and clonally amplifying nucleic acids in each partition. We extracted DNA from saliva samples collected from several age groups. The DNA was bisulfite converted and subjected to ddPCR using specifically designed primers and probes. The methylation ratio of each sample was calculated and correlation between the methylation ratio and the chronological age was analyzed. In the results, methylated DNA ratio at the 4 CpG sites (cg14361627, cg14361627, cg08928145 and cg07547549) showed strong correlation with chronological age. Percent-methylation values at 4 CpG markers and chronological ages of the 76 individuals were analyzed by multiple regression analysis, and we constructed an age prediction model. We observed a strong correlation (Spearman’s rho = 0.922) between predicted and chronological ages of 76 individuals with a MAD from chronological age of 3.3 years. Collectively, the result in this study showed the potential applicability of ddPCR to predict age from saliva. |
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| Keywords: | Droplet Digital PCR DNA methylation Age prediction Saliva Forensic science |
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