亚硫酸氢盐
焦测序
DNA甲基化
亚硫酸氢盐测序
CpG站点
甲基化
表观遗传学
DNA
生物
口腔黏膜测试
聚合酶链反应
基因组DNA
分子生物学
人口
遗传学
医学
基因
基因表达
环境卫生
作者
Masakazu Shiga,Masaru Asari,Yuta Takahashi,Shotaro Isozaki,Chisato Hoshina,Kanae Mori,Ryo Namba,Kunio Okuda,Keiko Shimizu
标识
DOI:10.1016/j.legalmed.2023.102336
摘要
DNA methylation modifications are known to influence epigenetic phenomena and have been a focus of forensic science research for some time. Degraded DNA after bisulfite treatment is widely used in DNA methylation analysis. In this study, we analyzed methylation levels at 12 CpG sites of four selected genomic regions by pyrosequencing after bisulfite treatment. DNA was extracted from buccal swab samples collected from 102 Japanese individuals who were 21–77 years old. We also developed a simple method to quantify the degradation levels of bisulfite-converted DNA by real-time PCR, and evaluated the effect of DNA degradation on age estimation. We found that the methylation levels and chronological ages were highly correlated in the four selected regions, and the mean absolute deviation (MAD) between chronological and estimated ages was low at 3.88 years. These results indicated that pyrosequencing analysis at the 12 CpGs was useful for age estimation in the Japanese population. To develop a sensitive quantification method, we analyzed the amplification efficiency of short and long fragments from 10 regions by real-time PCR. The amplification efficiency was highest for CCDC102B, and the degradation levels of bisulfite-converted DNA for the 102 samples were categorized as moderately or heavily degraded. For the younger age groups (20–49 years), the MADs were lower for moderately degraded DNA than they were for heavily degraded DNA. This finding indicates that degradation levels affected the accuracy of age estimation in most of the samples; the exception was the samples from the 50–77 years age group.
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