Forensic tracing of horse identities using urine samples and DNA markers
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abstract
The possibility to carry out DNA testing using horse urine samples collected for doping control was tested using nine microsatellite markers and one mitochondrial D-loop single strand conformation polymorphism (SSCP). Genomic DNA for polymerase chain reaction (PCR) analysis was prepared from different volumes of urine (5, 40 and 80 l urine per reaction) using a simple protocol. Consistent genetic data were obtained from urine and blood samples except in a few cases where PCR analysis of the smallest urine volume per preparation only revealed one alleie in heterozygotes. In these cases serial reactions showed that the two alleles were randomly amplified, probably because of a very low number of initial target molecules. In most cases the amount of PCR product increased with increasing volume per preparation but three samples showed a drastic reduction of PCR product. For these samples ultrafiltration of the most concentrated preparation dramatically improved the results and enabled genotyping with all markers. Our results show that the methods used are highly valuable for tracing identities from appropriately stored horse urine. The total probability of identity by chance for unrelated Standardbred horses using this set of markers was estimated at 6 10 -8 .
