DNA methylation is a robust biomarker for forensic identification, particularly in body fluid determination and chronological age inference. Specimens left at crime scenes are often limited and degraded, necessitating highly sensitive and multiplexed strategies for methylation analysis to maximize genetic information output from trace specimen. Bisulfite conversion-based methods are authoritative for DNA methylation profiling, featuring high sensitivity and multiplex compatibility. It selectively deaminates cytosine to uracil while preserving 5-methylcytosine, converting epigenetic difference into sequence variation detectable by nucleic acid amplification. However, an inherent conflict exists between the high sensitivity of amplification and the massive amplicons, leading to a considerable risk of carryover contamination and false positive result. Although the uracil-DNA glycosylase (UDG)-based strategy is widely used to prevent contamination, it is only applicable to natural DNA but bisulfite-converted DNA, as both aerosol and intended template can be hydrolyzed by UDG. Herein, a novel strategy to eliminate carryover contamination based on a nucleic acid damage repair mechanism is developed to satisfy the demand of bisulfite-converted DNA methylation analysis. By incorporating deoxyinosine triphosphate during amplification, amplicons are labeled with hypoxanthine, which can be specifically recognized and cleaved by endonuclease V, while native or uracil-containing templates remain intact. For forensic validation, this anti-aerosol strategy is incorporated into a multiplex real-time quantitative PCR (qPCR) assay for quantifying the methylation level of a semen-specific CpG site. The strategy is fully compatible with qPCR, achieving accurate methylation quantification, while expanding the quantitative dynamic range by significantly delaying the time to threshold of false positive signal. To support rapid on-site forensic testing and expedite case investigation, the strategy is integrated into a multiplex PCR-based lateral flow assay for visual detection. This integration effectively eliminates aerosol contamination and completely suppresses false positives without compromising amplification efficiency, specificity, or sensitivity. To further enhance speed and sensitivity, the system is incorporated into a loop-mediated isothermal amplification (LAMP)-based lateral flow biosensor for DNA methylation detection. Although LAMP is well-suited for analyzing challenging forensic specimens, it is far more susceptible to contamination than PCR, due to its greater sensitivity and higher amplicon yield. With the proposed strategy, carryover contamination exceeding that encountered in real-world forensic scenarios is effectively eliminated, rendering this method competent for practical application. Overall, this anti-carryover contamination strategy overcomes the long-standing contamination bottleneck in forensic epigenetic analysis, exhibiting great potential for practical DNA methylation detection.

Authors

• Huyun Zhou (School of Forensic Medicine, Shanxi Medical University, China)
• Wenjing Hu (School of Forensic Medicine, Shanxi Medical University, China)
• Xiaonan Liu (School of Forensic Medicine, Shanxi Medical University, China)
• Jiangwei Yan (School of Forensic Medicine, Shanxi Medical University, China)