Forensic criminal casework often involves DNA profiling of human postmortem tissues, whereas degradational processes can affect PCR-based Short Tandem Repeat (STR) analysis.

Forensic Science International
Volume 173, Issues 2–3, 20 December 2007, Pages 103–106

The Achilles tendon as a DNA source for STR typing of highly decayed corpses
⦁ A. Roeper, ,
W. Reichert,
R. ⦁ Mattern
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doi:10.1016/j.forsciint.2007.02.004

Abstract
Forensic criminal casework often involves DNA profiling of human postmortem tissues, whereas degradational processes can affect PCR-based Short Tandem Repeat (STR) analysis. Degradation of DNA is observed to vary among different tissues and with time. Therefore, the stability of DNA in Achilles tendon samples is compared to that in muscle and kidney specimens with a variety of postmortem histories. Tissue samples from 28 autopsy cases, including 15 decomposed corpses and a control group of 13 nondecayed corpses were analysed. DNA was isolated using the All-tissue DNA Kit (GEN-IAL, Troisdorf, Germany), quantified by spectrophotometric measurement, amplified by the multiplex PCR genRES MPX-2 (Serac, Bad Homburg, Germany), and analysed on the ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Darmstadt, Germany).
Quantitative analysis of nondecomposed tissues revealed that the recovery of DNA was highest in kidney followed by muscle, whereas Achilles tendon tissue was the poorest source of isolated DNA.
Only small amounts of DNA were present in both kidney and muscle samples from decomposed corpses. However, from decayed Achilles tendon samples twice as much DNA as from nondecayed samples could be isolated on average. These results suggest DNA to be better protected in Achilles tendons. Moreover, postmortem changes in Achilles tendons may even improve DNA isolation.
Keywords
⦁ Short Tandem Repeats (STRs);
⦁ Postmortem tissues;
⦁ Achilles tendon;
⦁ DNA stability

1. Introduction
Short Tandem Repeats (STRs) are an important and widely used tool in forensic casework. Hoff-Olsen et al. [1] could show, that there were no somatic mutations or other potential postmortal changes even when the substrate was heavily decomposed. The degree of postmortem changes of tissues had no effect on the stability of microsatellites and so mistypings did not occur. This is crucial to the safety and reliability of these analyses.
The stability of DNA varies among different tissues, and its degradation continuously proceeds with the time passed since death. Bär et al. [2] and Ludes et al. [3] found good postmortem DNA stability in different tissues by testing VNTRs (variable number of tandem repeats). Hoff-Olsen et al. [1] investigated brain, hair, cartilage, liver, thyroid and blood by STR-analysis but the most suitable tissue for STR analysis could not be established.
As Microsatellite analysis of bone tissue is very time-consuming and complex in this study it is investigated whether the Achilles tendon is a useful material for STR analysis when dealing with the identification of decayed corpses. The Achilles tendon is postulated to be a stable tissue, which is protected against autolysis and putrefaction.
2. Materials and methods
Human muscle, kidney and Achilles tendon specimens were collected from 28 autopsy cases performed at the Institute of Legal Medicine and Traffic Medicine in Heidelberg. The postmortem period of the corpses ranged from about 4 days to 2 years. Of the 28 reported cases 12 were decayed, two lay in the water for a longer period and one was nearly completely skeletonized. Thirteen nondecayed corpses, including three burn victims, represent the control group. The tissue samples were stored at −20 °C.
DNA from tissue samples was extracted using the All-tissue DNA Kit (GEN-IAL, Troisdorf, Germany). Fifty milligrams of each sample – muscle, kidney and Achilles tendon – were used for DNA extraction according to the manufacturer’s standard protocol. Optimal results were obtained with lysis for 1 h at 65 °C. The isolated DNA was dissolved in 50 μl ddH2O. DNA quantitation was performed by spectrophotometric measurement.
One nanogram of spectrophotometrically quantitated DNA was used as template for amplification. PCR was performed using the commercially available PCR multiplex system genRES MPX-2 (Serac, Bad Homburg, Germany). With this MPX-2 Kit Amelogenin and the eight STR-systems Hum TH01, vWA, FGA, D21S11, SE33, D3S1358, D8S1179 and D18S51 can be simultaneously amplified in one reaction. Amplification was carried out in a T3-Thermocycler (Biometra, Göttingen, Germany) under the following conditions: 95 °C for 12 min, then 30 cycles comprising 93 °C 1 min, 59 °C 1 min, 72 °C 90 s followed by a final extension of 45 min at 60 °C.
Electrophoresis was performed on an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Darmstadt, Germany) using a 50 μm i.d. capillary and the polymer POP4.
One microliter of the PCR product was mixed with 12 μl HiDi-formamide and 0.4 μl of the internal standard genRES LS500ROX (Applied Biosystems, Darmstadt, Germany). The mixture was denatured for 3 min at 95 °C, cooled on ice and then placed on the autosampler of the Genetic Analyzer. The ABI Prism 310 Collection Software was used for data acquisition, which were further analysed by the GeneScan Analysis Software 3.7 (Applied Biosystems, Darmstadt, Germany).
3. Results
3.1. Quantitative analysis
The isolation of DNA from tissue samples by the GEN-IAL Kit yields DNA in high purity.Fig. 1 shows the amount of DNA isolated from different tissues of corpses without signs of decomposition. Except for four cases, where no kidney tissue was available the recovery of DNA from kidney was much higher compared to that of muscle. The lowest quantity of DNA was found in Achilles tendon tissue. The amount of isolated DNA ranged from 863 to 2240 ng/mg (average value 1375 ng/mg), from 194 to 480 ng/mg (average value 327 ng/mg), and from 18 to 193 ng/mg (average value 57 ng/mg) for kidney, muscle and Achilles tendon specimens, respectively.

Fig. 1.
DNA quantity of kidney, muscle and Achilles tendon from nondcayed corpses (samples 1–10), samples 11–13 from burn victims.
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The results obtained from tissue samples collected from decomposed corpses are summarised in Fig. 2. In two cases (21 and 28) kidney tissue was not available at autopsy and in one case (23) collection of muscle tissue was not possible. Apart from few cases the recovery of isolated DNA was poor in both kidney and muscle tissue specimens, whereas higher amounts of DNA could be obtained from Achilles tendon specimens in most cases. The range of DNA was from 4 to 1080 ng/mg (average value 217 ng/mg), from 5 to 722 ng/mg (average value 174 ng/mg), and from 6 to 345 ng/mg (average value 100 ng/mg) for kidney, muscle and Achilles tendon, respectively.

Fig. 2.
DNA quantity of kidney, muscle and Achilles tendon from decayed corpses (samples 14–25), samples 26 and 27 from dead bodies which lay in the water for an unknown period, sample 28 is a nearly completely skeletonized corpse.
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3.2. Qualitative analysis
Table 1 shows the results of the STR-profiles obtained from nondecayed and decayed samples. In all cases where decomposition was not present, the entire profiles (Amelogenin and 8 STR-systems) could be established. The amplification quality related to the peak height generated the best results for Achilles tendon followed by kidney and muscle.
Table 1.
STR profiles of nondecayed and decayed samples
n No profile Incomplete profile Complete profile
Nondecayed samples (all tissues) 13 – – 13

Decayed samples
Muscle 14 4a 2 8
Kidney 13 – 1 12
Achilles tendon 15 3 – 12
a
In one sample Amelogenin was successfully amplified.
Table options
Only 8 out of 14 decomposed muscle samples showed a complete profile. In two cases an incomplete one and in four cases no alleles were observed. In the case of 13 decayed kidney tissue samples a complete profile was found in 12 cases whereas one case showed an incomplete profile. No incomplete profile was found in decayed Achilles tendon tissues. A complete profile was obtained from 12 out of 15 Achilles tendon samples, whereas three samples failed to produce a profile. Among the samples where no profile was obtained, two were from highly putrefied corpses and one was nearly completely skeletonized. In general the observed peak height of decayed tissue samples was much lower, except for Achilles tendon specimens where the peak height was comparable to that observed in muscle from nondecayed tissues.
The typing results of the tissues from decomposed corpses are shown in detail in Table 2.
Table 2.
STR analysis performed on tissue samples from 15 decayed corpses
Tissue n1 Amelogenin D3S1358 TH01 vWA FGA D21S11 D18S51 D8S1179 SE33 Total Possible typings

n2 %
Muscle 14 11 10 10 8 9 9 8 9 8 82 126 65.08
Kidney 13 13 13 13 13 13 13 13 13 12 116 117 99.15
Achilles tendon 15 12 12 12 12 12 12 12 12 12 108 135 80.00
In one case no muscle tissue was available, in two cases kidney samples were missing. n1: number of cases, n2: maximum number of possible typings of single alleles.
Full-size table
Table options
In muscle and kidney tissue samples, STR-systems with longer alleles dropped out more frequently than those with shorter fragments. Only about 65% of possible alleles could be typed in muscle tissues compared to 80% in Achilles tendon. No dropouts of single alleles were observed in tissue samples of Achilles tendon. In these samples either no alleles could be detected or a complete profile could be established.
In all cases DNA profiles from decomposed corpses were identical irrespective of the source material.
4. Discussion
Specimens of human kidney, muscle and Achilles tendon tissue with a variety of postmortem histories were used in a comparative study of DNA recovery and STR analysis.
There was no strong correlation observed between the time passed since death and the DNA content in the tissues under investigation. A high recovery of DNA could be established from nondecayed tissues whereas in kidney and muscle tissue from decomposed corpses a considerable decrease in DNA quantity was evident. In contrast to this, the DNA content of decomposed Achilles tendon tissues was nearly twice as much as in nondecayed tissues. We assume that this phenomenon is due to autolytic processes which facilitate DNA extraction.
The good quality of DNA from nondecayed samples allowed the typing of a complete STR profile in all cases. All alleles could successfully be typed independent of their length. Decomposed samples from kidney and muscle tissue showed variable DNA degradation resulting in incomplete or no profiles (Table 1). Considering each STR system in detail (Table 2) it is evident that especially in muscle tissue longer fragments drop out more often. Obviously longer fragments are earlier affected by degradation than shorter fragments. For Achilles tendon tissue specimens showing a complete profile even the longest SE33 alleles could be typed without difficulties. This indicates that Achilles tendon is a very stable tissue and less affected by degradation processes in human remains. The quality of STR typing does not strongly correlate with the postmortem period of the samples but it depends on the degree of decomposition. A total drop out of Achilles tendon analysis occurred in three corpses which were heavily decomposed.
In conclusion, Achilles tendon is a very suitable tissue for PCR-based STR analysis in human identification and forensic casework when dealing with heavily decomposed remains.
Acknowledgments
We would like to thank the autopsy technicians Ralf Bartsch, Achim Schmidt and Christian Weigl for collecting tissue material.
References
⦁
[1]
⦁ P. Hoff-Olsen, S. Jacobsen, B. Mevåg, B. Olaisen
⦁ Microsatellite stability in human post-mortem tissues
⦁ Forensic Sci. Int., 119 (2001), pp. 273–278
⦁ [SD-008]
⦁
[2]
⦁ W. Bär, A. Kratzer, M. Mächler, W. Schmid
⦁ Postmortem stability of DNA
⦁ Forensic Sci. Int., 39 (1988), pp. 59–70
⦁ [SD-008]
⦁
[3]
⦁ B. Ludes, H. Pfitzinger, P. Mangin
⦁ DNA fingerprinting from tissues after variable postmortem periods
⦁ J. Forensic Sci., 38 (1993), pp. 686–690
⦁ [SD-008]
Corresponding author. Tel.: +49 6221 56 89 56; fax: +49 6221 56 52 52.
Copyright © 2007 Elsevier Ireland Ltd. All rights reserved.

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