Development and evaluation of an optimized Ancient DNA extraction method for femoral diaphyses and simulated heat-treated teeth.

Extracting endogenous DNA from highly degraded hard tissue samples, such as bones and teeth, is crucial yet challenging for forensic human identification. Existing forensic methods and commercial kits for DNA extraction from bones and teeth often inefficient for old, poorly preserved, chemically treated, or heat-exposed samples. Ancient DNA (aDNA) extraction technology is highly effective in recovering degraded, short DNA fragments and has been successfully used to isolate DNA from skeletal remains and sediments dating back to tens of thousands of years. However, its direct application to individual identification and short tandem repeat (STR) analysis may require further adaptation to meet specific forensic requirements. Therefore, this study was conducted to optimize aDNA extraction techniques for developing a method tailored for highly degraded forensic samples. We developed a forensic aDNA-based extraction (FADE) method by refining the aDNA extraction protocol through optimization of lysis and purification conditions. The effectiveness of the developed method was validated using 8 forensic case samples (femoral diaphyses) and 20 simulated heat-treated teeth. For samples subjected to prolonged heat treatment, the FADE method significantly increased peak heights, with improvements of 30 % and 45 % in the 30- and 40-min heat-treatment groups, respectively, and yielded a greater number of amplified loci and alleles. The FADE method developed in this study is well-suited for forensic applications involving highly degraded femoral diaphyses and simulated heat-treated teeth, significantly enhancing the success rates of DNA analysis and providing an effective technical solution for complex forensic DNA extraction.