Forensic DNA Division, National Forensic Service, Wonju, Gangwon-do, South Korea.
DNA Analysis Division, National Forensic Service Daejeon Institute, Daejeon, South Korea.
Leg Med (Tokyo). 2022 Feb;54:101992. doi: 10.1016/j.legalmed.2021.101992. Epub 2021 Nov 17.
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.
最近的研究报告称，DNA 甲基化标记物随年龄发生变化，并预计这些 DNA 甲基化标记物可有效地用于法医遗传学中的年龄估计。在这项研究中，我们应用液滴数字 PCR（ddPCR）方法研究 CpG 位点的 DNA 甲基化模式，并基于 ddPCR 方法构建了年龄预测模型。ddPCR 能够通过将样品分成大量分区并在每个分区中克隆扩增核酸来高度灵敏地定量核酸和检测基因中的序列变异。我们从几个年龄组的唾液样本中提取 DNA。将 DNA 进行亚硫酸氢盐转化，并使用专门设计的引物和探针进行 ddPCR。计算每个样本的甲基化比率，并分析甲基化比率与实际年龄之间的相关性。结果表明，4 个 CpG 位点（cg14361627、cg14361627、cg08928145 和 cg07547549）的甲基化 DNA 比率与实际年龄具有很强的相关性。通过多元回归分析对 4 个 CpG 标记的百分比甲基化值和 76 个人的实际年龄进行分析，并构建了年龄预测模型。我们观察到，76 个人的预测年龄与实际年龄之间存在很强的相关性（Spearman's rho=0.922），平均偏差为 3.3 岁。总之，本研究的结果表明 ddPCR 具有从唾液中预测年龄的潜在适用性。
