Melba S. Ketchum, DVM and T.A. Morton, MT (ASCP)
DNA Diagnostics, Inc., Carthage, TX

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Teeth were obtained from one deceased Tennessee Walking Horse and a mandible from another for the purpose of DNA extraction to provide identification and parentage verification of foals in two families. One animal had been deceased for thirteen months and the other for ten months. Both animals were exhumed and the tissue decomposition was severe. The teeth and mandible were shattered and scrapings were extracted using sodium hydroxide and DTT. DNA from blood samples obtained from the two alleged foals and the other parents were also extracted using sodium hydroxide and DTT.

The extracted DNA was then amplified in a single reaction containing PCR primers for the following ten fluorescently labeled dinucleotide repeats: HTG 4, HTG 6, HTG 7, HMS 1, HMS 3, HMS 6, HMS 7, AHT 4, AHT 5 and VHL 20. The lysates prepared from the blood were amplified for thirty cycles while the samples derived from the teeth and bone were amplified for fifty cycles. PCR conditions were an initial hot start at 95ºC for 10 minutes, then 95ºC for 1 minute, 60ºC for 45 seconds, 74ºC for 1 minute, for a total of 50 cycles. The resulting PCR product was then held at 74ºC for 60 minutes and then cooled to 4ºC. The primers utilized were synthesized with three different colored fluorescent amidite dyes (blue, green, yellow) attached to facilitate sizing the dinucleotide repeats that had a size overlap in their alleles.

The resulting PCR product was analyzed using an ABI 377 DNA Sequencer with a 12 cm, 6% polyacrylamide gel at 1100 volts for 45 minutes. The PCR product was denatured at 95ºC for 2 minutes and then chilled to 4ºC prior to loading. Analysis was obtained using GeneScan software by ABI (Perkin-Elmer) and the Local Southern Method of sizing. An internal lane standard labeled with a red fluorescent dye, GeneScan 350 Rox (Perkin-Elmer), was loaded with each PCR product. Allele sizes for the ten dinucleotide repeats had a potential range of 84 to 182 base pairs.

Amplification was successful on DNA from all six horses with all ten dinucleotide repeats in the multiplex. Both samples from the deceased animals amplified in spite of severe degradation and bacterial contamination. Each of the deceased horses qualified as the parent of their respective offspring. Power of identity of this multiplex is greater than 99.9% in Tennessee Walking Horses.