Differential amplification profiles in fresh and archived DNA samples

Abstract

The use of mitochondrial DNA in forensic genetics has allowed researchers to answer questions relating to diversity, population scale and movements. Field sampling as well as transportation and storage, can negatively affect the quality of DNA for research. This project aims to investigate the common problem of PCR dropout of the longest mtDNA fragments. Fresh and archived DNA samples were used in multiplexed PCRs amplifying human HVS1, HVS2 and the intervening region. PCR products ranging from 120 to 1100 base pairs (bps) were amplified in different stages. Amplicons were separated using PAGE and stained with SYBR-Gold to identify specific amplification profiles. Over all, dropout of larger products was seen for the oldest samples. Fresher stored samples showed predictable amplification patterns, as expected from recently extracted DNA. Less than 25% of older samples yielded products between 146 and 506 bps. In older samples, the dropout rate for the longest products was 1.5 times that of the shortest. PCR across the HVS1–HVS2 region was significantly hampered in older samples. Follow-up PCRs may have been affected by the abundance of mtDNA in the cell and the prevalence of smaller fragments in degraded DNA. This study suggests that degradation of mtDNA D-loop is progressive and provides strong indication that the process may be confined to specific regions of the molecule.