Teeth as a Post-Mortem DNA Source for Forensic Parentage Verification in Dogs: A Case Report

In post-mortem genetic identification, teeth represent one of the most reliable sources of nuclear DNA when other biological samples are unavailable, owing to their ability to protect genetic material from autolysis, microbial degradation, and environmental insults. The present study describes a forensic veterinary case report involving a Staffordshire Bull Terrier litter in which the appearance of blue-coated offspring from a phenotypically black sire and a blue dam prompted parentage verification. The sire died before formal ante-mortem sampling could be carried out. In accordance with Regulation (EC) No. 1069/2009, Article 15, the carcass was interred in the owner's garden. Before burial, two teeth were collected as the sole biological material available for identification by the official veterinarian, ensuring an unambiguous chain of custody from death certification to laboratory analysis. DNA extraction was performed using two independent protocols: Method A, a silica-membrane-based purification (QIAamp DNA Kit, Qiagen), applied to dental pulp, and Method B, a decalcification-based workflow (T-Bone Ex Kit) followed by DNA extraction using EZ2 Connect Instruments (Qiagen). Quantification of the DNA samples obtained were done by fluorometry. Short Tandem Repeat (STR) genotyping was performed using the Canine Genotypes™ Panel 1.1 by Applied Biosystems (Thermo Fisher Scientific, Waltham, MA, USA) targeting the 18 autosomal ISAG-recommended microsatellite loci. Parentage assignments were carried out against reference STR profiles obtained from peripheral blood of the dam and offspring. The two methods, considering a different biological matrix and different operating conditions, allowed us to obtain the STR profile, but Method B showed quality electropherograms, improving the confidence and interpretation of all the markers analyzed. The differences observed between the two approaches may reflect a combination of factors, including the different dental matrices analysed, the quantity and quality of the starting material, tooth identity and tissue preservation, and differences inherent to the extraction procedures themselves; therefore, in this case, the more complete and clearer STR profile obtained with Method B should be considered a case-specific observation rather than evidence of a direct effect of the extraction method. Overall, regardless of the DNA extraction method used, the results did not exclude the biological paternity of the blue-mantled offspring. Furthermore, this case report provides practical methodological guidance for DNA recovery from canine teeth and supports their use as a reliable source of post-mortem DNA for STR-based parentage testing.