DNA extraction (Normal samples)
DNA was extracted from blood and tissue samples using QIAamp® Tissue Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturers' handbook. Isolated DNA was diluted in 50 μL HPLC-H2O and used for further analyses.
DNA extraction (Trace material e.g. bones)
Bone samples were roughly ground with a pestle and mortar, then finely powdered in a Retsch mill. Bone powder (0.3 g) was incubated in 1.5 mL of 0.5 M EDTA (pH 8.3) for 20 h while rotating. The suspension was centrifuged for 4 min at 4000 rpm. The supernatant was transferred to a fresh tube or to an automated nucleic acids extraction system (Nucleic Acid Extractor 341A, Applied Biosystems) and 1.6 mL sterile distilled water (Ampuwa, Fresenius) was added. As the extraction procedure was automated the volumes of reagents dispensed may have varied between runs. Five hundred microliters of Proteinase K was added and the mixture incubated for 1 h at 58°C with shaking. Three milliliters of phenol/chloroform/isoamyl alcohol (25:24:1, pH 7.5–8.0) were added and the mixture was further incubated at room temperature for 6 min while shaking. The phases were allowed to separate by incubating at room temperature for 8 min without shaking and the organic phase and interphase, if present, were discarded. Chloroform (4.5 mL, 100%) was added to the aqueous phase and the mixture incubated for 6 min at room temperature while shaking. The phases were again allowed to separate by incubating at room temperature for 8 min without shaking and the organic phase and interphase, if present, were discarded. Ninety microliters of sodium acetate (pH 4.5) and 3.2 mL of 100% isopropanol were added followed by incubation for 2 min with shaking. Five microliters of Glasmilk (Dianova) were added and the suspension was shaken for another 10 min. To obtain a pellet, the solution was filtered through Precipitette filters (Applied Biosystems) or centrifuged for 3 min at 5000 rpm. The pellet was washed with 80% ethanol and eluted into 50 μL sterile distilled water (Ampuwa, Fresenius). Five to ten microliters of extract were used for PCR amplification or the extract was stored at -20°C. Glasmilk was not removed prior to amplification.
For RFLP analysis, a 195 bp long PCR fragment was amplified from mitochondrial cytochrome b region. The following primer pair was used for amplification, CB7u (5'-GCGTACGCAATCTTACGATCAA-3') and CB7l (5'-CTGGCCTCCAATTCATGTGAG-3').
The PCR was carried out in a total volume of 50 μL consisting of 10 ng DNA, 60 mM KCl; 12 mM Tris-HCl; 2.5 mM MgCl2; 150 μM dNTPs; 0,18 μM of each Primer and 2 U AmpliTaq Gold (PE Applied Biosystems). Cycling conditions included a denaturation step at 95°C for 3 minutes, followed by 32 cycles of 95°C for 1 minute, primer annealing at 54°C for 1 minute and elongation at 72°C for 1 minute in a thermocycler (Hybaid).
The 195 bp fragment was digested with TSP509 (New England Biolabs) for two hours at 65°C. The resulting fragments were separated by gelelectrophoresis in a 2.5 % agarose gel.
MtDNA Dloop PCR sequencing reaction
A single fragment of the mitochondrial DNA Dloop region was amplified using primers Dloopu (5'-AAATGTAAAACGACGACGGCCAGTAATCCCAATAACTCAACAC-3') and Dloopll (5'-AAACAGGAAACAGCTATGACCACTCATCTAGGCATTTTC-3').
Amplifications were performed in a final volume of 20 μL in 10 × PCR buffer (15 mM MgCl2, pH 8.3) and Q-solution, 100 μM for each dNTP, with 1 M Taq Polymerase and 10 pmol of each primer. Four microlitres of the DNA-extract were added to the PCR mix. The amplification was carried out with initial denaturation at 95°C for 10 min, followed by 35 cycles of one denaturation step at 94°C for 40 sec, primer annealing at 52°C for 40 sec and primer extension at 72°C for 45 sec in a Hybaid thermocycler. PCR-products were purified using the QIAEX II Gel Extraction Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturers' instructions. Sequencing was performed using ABI-Prism™ Dye Kit V3 (Applied Biosystems) in a 10 μL volume containing 2 μL purified PCR-product and 5 pmol of primer. Sequencing reactions underwent 27 cycles of 30 sec at 94°C, 30 sec at 50°C and 3 min at 60°C in a Techne thermocycler. The dye terminators were removed by sephadex-G45 column purification (Millipore). Sequencing reactions were electrophoresed for 2 h on an ABI Prism® 3100 genetic analyzer (Applied Biosystems) according to the manufacturers' instructions.
In order to test the specifity of the technique the following numbers of specimens were tested:
7 unrelated samples from cattle: Holstein Frisian, Charolais, Limousin, Angus
7 unrelated samples from sheep
7 unrelated samples from goat
7 unrelated samples from deer collected from Lower Saxony and North Hesse
7 unrelated samples from roe deer collected from Lower Saxony
Regarding closely related species, we analyzed mouflon DNA (Ovis aries musimon) and observed approximately 100% sequence identity compared with Ovis aries. Furthermore, we investigated different cattle breeds and we could not find any sequence differences within the tested cytochrome b DNA-fragment.