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Pyrosequencing: A Simple Method for Accurate Genotyping
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Establishment of a Highly Accurate and Sensitive Age Prediction Model for Multiple Body Fluids: Blood, Saliva, and
Xudong Zhao1,2,3,4, Daijing Yu2,3,4, Jingjing Xu2,3,4
1Department of Forensic Medicine, Inner Mongolia Medical University, Hohhot, People's Republic of China.
Electrophoresis
|May 2, 2026
Summary
This study developed a DNA methylation method for accurate age prediction across multiple body fluids, crucial for forensic investigations. The new tool enhances criminal investigations by providing reliable age estimation from various biological samples.
Area of Science:
- Forensic science
- Epigenetics
- Biochemistry
Background:
- DNA methylation-based age prediction is vital for individual identification.
- Current models are limited to single biological fluids, risking errors with mixed samples.
- Accurate age prediction across multiple body fluids is critical for forensic casework.
Purpose of the Study:
- To develop and validate a multi-body fluid age prediction model using DNA methylation.
- To assess the accuracy, sensitivity, and limitations of pyrosequencing for forensic age estimation.
- To provide a reliable tool for age prediction in complex forensic scenarios.
Main Methods:
- Screened age-related methylation sites (cg05940966, cg10501210, cg10528482) using public databases.
- Quantified methylation levels in blood samples via pyrosequencing.
- Applied machine learning algorithms to construct and evaluate age prediction models.
Main Results:
- A multiple linear regression model achieved high accuracy (MAD: 2.7-4.1 years across blood, saliva, semen).
- Pyrosequencing showed high sensitivity, with accurate predictions from trace DNA samples (0.5 ng).
- Inhibitor tolerance was limited by high concentrations of oxidized heme (>1 ng/µL) and humic acid (>2 ng/µL).
Conclusions:
- Developed an accurate and reliable DNA methylation-based age prediction tool for multiple body fluids.
- The method enhances forensic investigations by enabling rapid, fluid-type-independent age estimation.
- This technology improves biological evidence interpretation and supports judicial processes.
