Use of Canine Microsatellite Polymorphisms in Forensic Examinations

Use of Canine Microsatellite Polymorphisms in Forensic Examinations S. Mu¨ller, G. Flekna, M. Mu¨ller, and G. Brem

A forensic case report is presented. Using pooled SSCP analysis of three polymorphic microsatellites one possible candidate was excluded in a time and cost saving way. Microsatellites show abundance, uniform distribution, and a high degree of polymorphism in genomes. Therefore they have become a valuable tool in genome mapping, paternity testing, population studies, and forensic identity testing. The aim of molecular genetic identity testing is to confirm or to exclude a suspected animal by comparing the genotypes. A selected number of microsatellites is necessary to create a DNA fingerprint pattern. This unique profile which no other individual possesses can be re-created any time in order to confirm individual identity. In a given case a dog caused a motorcycle accident with high material damage and personal injury. Before being identified the shocked and injured animal escaped. A small bunch of dog hair was left as evidence on the tire surface. It was collected by a policeman. Two dogs living in the neighborhood were suspected to have caused the accident. Blood samples were taken as comparison material.

Material and Methods

From the Institut fu¨r Tierzucht und Genetik der Veterina¨rmedizinischen Universita¨t Wien (Mu¨ller, Mu¨ller, and Brem) and Ludwig Boltzmann Institut fu ¨ r Immuno-, Zyto- und Molekulargenetische Forschung ( Flekna and Brem), Veterina¨rplatz 1, A-1210 Wien, Austria. This paper was delivered at the International Workshop on Canine Genetics at the College of Veterinary Medicine, Cornell University, Ithaca, New York, July 12–13, 1997. q 1999 The American Genetic Association 90:55–56

The total amount of evidence material was five hair bulbs. DNA was isolated by boiling the samples in the presence of a protein binding matrix ( InstaGeney, Bio Rad). This matrix efficiently absorbs cell lysis products that interfere with the PCR amplification process. After boiling the detritus was spun down. Following the kit’s instructions 20 ml of the supernatant were used in PCR. DNA was not quantitated. Comparison material was blood samples. Leukocytes were isolated from 300 ml EDTA-blood by lysis of the erythrocytes (150 mM ammonium chloride pH 7.4.) and centrifugation (5 min at 30003 g). The leukocyte pellet was resuspended in 100 ml proteinase K lysis buffer (50 mM Tris-HCl

pH 8.0, 100 mM NaCl, 100 mM EDTA pH 8.0, 1% SDS, 0.5 mg/ml proteinase K). After a phenol/chloroform extraction step the DNA was precipitated from the aqueous phase by 2.5 volumes of ethanol; 100 ng DNA were used in the PCR. The PCR reaction was performed in a final volume of 50 ml with 1 3 PCR buffer (16.6 mM ( NH4)2SO4, 67.7 mM Tris-HCl pH 8.8, 0.01% (v/v) Tween 20, 1.5 mM MgCl2), 200 mM dNTP, 1 mM of each primer and 1 U Taq polymerase (BRL, LifeTechnologies). For each sample PCR, reactions for three different loci—AHT107, AHT137 ( Holmes et al. 1995), and VIAS-D10 (Primmer and Matthews 1993)—were carried out. After an initial denaturation (948C, 5 min) PCR was carried out with 1 min denaturation step at 948C, 40 s annealing at 588C and 40 s for polymerization at 728C for 28 cycles in a RoboCyclert (Stratagene). For the SSCP electrophoresis 5–20 ml of the PCR products were denatured at 948C by adding 3 volumes of SSCP loading buffer [90% (v/v) formamide, 20 mM NaOH, 0.05% (v/v) xylene cyanole, 0.05% (v/v) bromphenol blue] and cooled on ice. The three different PCR products were subsequently pooled and electophoresed through a native polyacrylamide gel (12% polyacrylamide/29 : 1; 5% glycerol; 0.5 3 TBE) at room temperature in a HOEFER SE 600 apparatus (gel size 160 mm 3 160 mm). Running time was 2 h. The gel was stained with dimidium bromide.

Results and Discussion In the present case report it was possible to exclude one of the suspected candidates by SSCP-analysis of three polymorphic canine microsatellites—AHT107, AHT137 ( Holmes et al. 1985), and VIASD10 (Primmer and Matthews 1993)—in a

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Figure 1. Lane 1: control dog 1; lane 2: control dog 2; lane 3: suspected candidate 1; lane 4: suspected candidate 2; lane 5: dog involved in the accident; lane 6: ladder.

time and cost saving way. To deal with the problem of very limited sample material, the most effective DNA extraction method had to be selected. For only a few hair bulbs and small amounts of tissue it was advantageous to use an extraction technique without DNA precipitation or any further cleaning step. For SSCP analysis the individual microsatellites were amplified separately but pooled three in one tube before denaturation, thus three microsatellite markers of different fragment size were loaded in one slot. The purpose of choosing comparing SSCP analysis instead of the more normally used microsatellite analysis was the special questioning in the present case. To

56 The Journal of Heredity 1999:90(1)

exclude one of two suspected candidates the comparison of the individual polymorphic pattern is sufficient. In comparison to autoradiographic methods or automated detection of fluorescence-labeled fragments this method is cheap and easy to handle. The case was ready for attesting after a total time expense of 9 h. The results of the experiment are shown in the Figure 1: While the polymorphic microsatellite pattern of the suspected candidate 2 ( lane 4) and of the dog involved in the accident ( lane 5) are identical, the suspected candidate 1 ( lane 3) has a different genotypic pattern for microsatellite AHT137. Thus candidate 1 could be discharged of debt.

An improvement of the method was met by using the combinations of AHT117/ CPH06/AHT127 and/or AHT123/VIASD10/ CPH016, they are clearly definable and do not have additional bands. Because of the merely occasional appearance of forensic cases, fragment size determinations or PIC value calculations were not done.

References Holmes NG, Dickens HF, Parker HL, Binns MM, Mellersh CS, and Sampson J, 1995. Eighteen canine microsatellites. Anim Genet 26:132–133. Primmer CR and Matthews ME, 1993. A canine tetranucleotide repeat polymorphism at the VIAS-D10 locus. Anim Genet 24:332. Corresponding Editor: Kunal Ray