Despite the significant progress of DNA profiling in forensic science, the extraction of DNA from challenging samples remains a formidable obstacle in forensic laboratories. Particularly, hard tissues (bones, teeth, hair, and nails), formalin-fixed tissues, and contaminated samples pose considerable difficulties. DNA extraction from such samples is often complicated, resulting in scanty, degraded, and contaminated DNA. Moreover, the presence of inhibitors from the surrounding environment further hinders DNA quantification and amplification, presenting additional challenges. This review article delves into the molecular basis of these challenges and examines the mechanistic principles underlying standard DNA extraction protocols. To overcome these obstacles, skilled efforts and additional pre-processing techniques are generally required before organic or silica column-based DNA extraction. Such techniques may involve scraping the waste, sample cleaning with detergents, disinfecting, demineralization of bones and teeth, long proteolytic enzyme treatment, in some cases, harsh methods like hot alkali treatment, tailored to the specific sample type. By addressing these challenges and understanding the molecular intricacies involved in DNA extraction, forensic scientists can improve the reliability and success rate of DNA analysis from difficult forensic samples. This review serves as a comprehensive resource for understanding the complexities of DNA extraction and offers potential solutions for recovering the DNA from highly challenging samples.
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