Developing mitochondrial dna amplification and sequencing strategy for degraded forensic samples

Analysis of human mitochondrial DNA (mtDNA) is a powerful tool for testing the forensic identity of highly degraded skeletal remains due to its stability and large number of genome copies per cell. Since highly degraded samples contain DNA molecules that are severely restricted in size, several alternatives have been developed to achieve successful results in mtDNA sequencing of these products. Multiplex PCR sets with midi and mini primers were designed to facilitate the sequencing of mtDNA control region in highly degraded skeletal remains and to obtain mtDNA profile. DNA samples from teeth or femur bones buried between 3 and 68 years, of 14 individuals aged between 22-95 years, were used to investigate the effectiveness of midi and mini multiplex PCR sets in severely degraded skeletal remains in this study. To obtain mtDNA profile in DNA samples isolated from 1 g bone powder by partially modified silica-based DNA extraction method, two separate multiplex PCR sets were prepared to produce amplicons of various lengths. One of the multiplex sets is the midiplex set, which allows the amplification of fragments of mtDNA with an average length of 250 bp, and the other is the miniplex set, which allows the amplification of fragments of 100-150 bp lengths, which can be used in further degraded samples. Midiplex set included 2 reactions, miniplex set included 3 reactions. While successful results were obtained for skeletal remains up to 68 years with the mini primer set, successful results were obtained for skeletal remains up to 45 years with the midiplex set. These primer sets can be used in highly degraded DNA samples, especially skeletal remains, in the forensic DNA laboratory. Since the same amount of DNA is used on average in monoplex and multiplex sets, the amount of genomic DNA used for analysis with multiplex sets is reduced and sample loss is minimized. It has also been possible to significantly reduce both labor and cost with multiplex sets when compared to the materials and labor used for monoplex analysis.