Improved MtDNA sequence analysis of forensic remains using a "mini-primer set" amplification strategy

Mitochondrial DNA (mtDNA) analysis of highly degraded skeletal remains is o ften used for forensic identification due largely to the high genome copy n umber per cell. Literature from the "ancient DNA'' field has shown that hig hly degraded samples contain populations of intact DNA molecules that are s everely restricted in size (1-4). Hand et al. have demonstrated the targeti ng and preferential amplification of authentic human DNA sequences with sma ll amplicon products of 150 bp or less (1,2). Given this understanding of a ncient DNA preservation and amplification, we report an improved approach t o forensic mtDNA analysis of hypervariable regions 1 and 2 (HV1/HV2) in hig hly degraded specimens. This "mini-primer set" (MPS) amplification strategy consists of four overlapping products that span each of the HV regions and range from 126 to 170 bp, with an average size of 141 bp. For this study, 1 1 extracts representing a range of sample quality were prepared from nonp robative forensic specimens. We demonstrate a significant increase in MPS a mplification success when compared to testing methods using similar to 250 bp amplicons. Further, 16 of 17 independent amplifications previously "unre ported" due to mixed sequences provided potentially reportable sequence dat a from a single, authentic template with MPS testing.