Fidelity of polymerase chain reaction-direct sequencing analysis of damaged forensic samples

Polymerase chain reaction (PCR) direct sequence analysis was performed on aged forensic samples, six or thirteen years old. This method allowed unambiguous genetic typing, but PCR products from such samples showed several artifacts. Control samples generated sequence ambiguities at a frequency of 1 in 567 bases, but the aged samples had an error frequency about 30-fold higher. In order to study the molecular composition of these aged DNA samples, reversed-phase high performance liquid chromatography (HPLC) was performed. Reduced amounts of the four DNA bases were observed and anomalous peaks were found. These peaks were analyzed by ionization mass spectrometry and identified as molecular products of DNA oxidation. The frequency of sequencing artifacts was found to be proportional to the decay of the PCR templates. Although PCR fidelity is a relevant concern in the forensic analysis of damaged samples, our data indicate that the risk of mistyping is circumventable by sequencing both strands and by performing replicate amplifications from the same PCR template.