Methodological Advances in Mitochondrial DNA Analysis for Forensic Genetics

Mitochondrial DNA (mtDNA) analysis is a fundamental tool in forensic genetics, particularly when biological samples exhibit severe degradation or low nuclear DNA content. Its unique biological characteristics, such as a high copy number per cell, strict matrilineal inheritance, and lack of recombination, enable human identification and reconstruction of maternal lineages in complex contexts, including disaster victim identification, historical cases, and missing persons investigations. This narrative review examines contemporary methodological approaches for investigating the human mitogenome. We discuss recent advancements in extraction and enrichment techniques, emphasizing their efficacy in reducing the interference of nuclear mitochondrial DNA sequences (NUMTs) and enhancing the recovery of informative fragments. Moreover, the shift from traditional Sanger sequencing to Massive Parallel Sequencing (MPS) is examined, as MPS has markedly enhanced the sensitivity and capability of contemporary methods to detect low-frequency heteroplasmies. Additionally, the advent of Third-Generation Sequencing (TGS), exemplified by nanopore platforms, is evaluated, which facilitates the reading of full-length native molecules without the biases introduced by PCR amplification. Despite the interpretive challenges posed by heteroplasmy, contamination, and limitations in population databases, ongoing methodological advances in mitochondrial DNA analysis continue to strengthen its reliability and expand its potential in forensic genetics.