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Summary

In medico-legal samples DNA is often broken in pieces. In many cases, only the amplification of short tandem repeated DNA stretches (STRs) which are located in non coding regions allow DNA typing of such degraded materials. To demonstrate the high diversity of biological materials which forensic biologists have to deal with, and to give a sketch on success rates and limits of the method, we describe five cases (minute amount of tissue on barrel, tissue in decay, tumor tissue, sperm after multiple rape, stored urine samples) in which forensic DNA typing was successfully performed by use of the short tandem repeats HUMDHFRP2, HUMD8S306, HUMCD4, HUMF13A1, HUMTHO1, HUMVWA, HUMFES.

1. Introduction

In our DNA typing routine work, biological material coming in (e.g., blood, tissue, hair and bones) has often been exposed to influences like ultraviolet light, humidity, decay, or years of storage in paraffin or denaturing preservatives. Depending on these (mostly unknown) conditions of storage, the DNA is degraded in many cases which means that just low amounts of short length DNA can be processed. For that reason, forensic DNA typing often requires the use of technologies which allows the detection of short, informative loci.

Since 1991, PCR [1-3] has become the major tool for forensic analysis of biological material [4-6]. Because of their high sensitivity and especially due to their short length (around 80-400 bp), short tandem repeats (STRs) are now the first choice for PCR stain analysis [7,8]. Like many other types of repeated DNA sequences, short tandem repeats are located in non coding regions spread over the whole genome [9,10]. Repeated DNA tends to be highly polymorphic and thus can be used for personal identification [11-16].

Due to their shortness, short tandem repeats can often be amplified down to DNA amounts of as little as 50 pg [19] whereas other polymorphisms (e.g. Amplifiable Fragment Lenghts Polymorphisms (AmpFLPs) [5,6,]) need 1 ng DNA at least to be detected by PCR. On the other hand, many short tandem repeats do not show more than 10 alleles in any population [17, 18] which means that the detection of an allele in just one short tandem repeat system in a biological stain cannot be an conclusive proof of identity. Short tandem repeats, however, have successfully been used in innumerable cases and their use is widely accepted by both criminal investigators and/or lawyers in Europe [20-29].

Being confronted with a high diversity of biological materials of which all suffer predominantly from bad DNA quality and low quantity, we wish to illustrate the advantages (high sensitivity, semiautomatic detection) and disadvantages (low discriminating power) of short tandem repeats as the major tool in today's DNA typing routine in Europe.

2. Material and methods
DNA was extracted by use of phenol/chloroforme after lysis in lysis buffer and Proteinase K [35]. DNA was concentrated using Centricon 100 (Amicon) spin colums and washed with 1xTE buffer (0.01 M Tris-HCl pH 8.0, 0.001 M EDTA). Amplification of short tandem repeats was performed using the following parameters: 1 u Taq polymerase (Promega), 0.25 µM primer each, 1x reaction buffer (Promega), 150 µM dNTPs. Temperature cycles were: 1 min 94°C; 1 min 54°C (VWA, FES), or 1 min 55°C (F13A1), or 1 min 60°C (TC11/THO1), or 58°C (multiplex with DHFRP2, CD4, D8S306); 2 min 72°C; 28 to 31 cycles. The PCR products were seperated on polyacrylamide gels and detected by fluorescent laser detection (A.L.F. Sequencer (Pharmacia Biotech)) or by silver staining using standard protocols.

3. Results and discussion

3.1. Shooting of a kidnapper: Suicide or not?

During a sightseeing tour in July 1995 in Cologne, one of the participants kidnapped the bus and shoot down the coach driver and a female tourist. A few hours later, police forced its way into the coach where the kidnapper was found lying dead on the floor. A shot in his breast and another in the head was detected. Adhering to the muzzle of the barrel of the kidnapper's gun, one single hair and minute amounts of blood and tissue were found. Although none of the bullets was available to us, police asked (among other things) if the kidnapper committed suicide. To answer this, we compared the DNA profile of hemolytic blood from all three dead with all tissues found adhering to the barrel. After extraction, the four tetrameric short tandem repeat systems HUMVWA (138-162 bp) [30], HUMTHO1 (179-203 bp) [4], HUMF13A1 (181-235 bp) [31] and HUMFES (222-250 bp) [32] were amplified and PCR products separated electrophoretically by use of vertical PAGE. Visualization was performed either by silver staining (HUMVWA, HUMTHO1, HUMFES) or by semiautomated fluorescence detection (HUMF13A1) using the A.L.F. sequencer (Pharmacia Biotech) [33,34]. The typing results are given in Table 1.

The three shortest short tandem repeats could be amplified in tissue/hair/blood from the barrel whereas the longest system HUMFES could not be amplified. One can clearly see that shortness of short tandem repeats is one of their major advantages against longer polymorphic DNA stretches like Amplifiable Fragment Lenghts Polymorphisms (AmpFLPs) which would definitely not have been detected here. The somewhat lower information content of short tandem repeats may be compensated by simply raising the number of markers tested. In this case the results of three short tandem repeat systems where by far sufficient to exclude both victims. Complete congruity of the DNA typing data obtained from the kidnapper's blood on one hand and the tissues from the barrel on the other hand strongly suggested that the kidnapper committed suicide by a shot out of his pistol which he had pressed on his head. At the same time a sharpshooter must have fired at the kidnapper which caused the shot in the chest.

3.2. Decay combined with dismembering of a corpse

In 1987, 1990 and 1994, several body parts (shanks with feet, thighs, skeletized skull with hair, pelvis, chest etc.) were found on different locations in Cologne. Due to the conditions of storage (in vinyl bags on a free playground and a rubble site, completely sealed plastic barrel), DNA typing of all body parts except of the thighs was possible by amplification, vertical PAGE, and silver staining of the tetrameric short tandem repeat systems HUMTHO1 and HUMVWA. (In the she skull, only the shorter short tandem repeat HUMVWA could be amplified.) Alleles were 6 and 9 for HUMTHO1 and 14 and 17 for HUMVWA (Fig. 1). This allele combination was calculated to be 1:300 in the local population. Single locus probes which were used in 1987 and 1990 did not show any result [23].
Together with morphological examination of the body parts we strongly suggested that all parts belonged together; furthermore, a bloodstain which was found in 1994 on a pillow near to the plastic barrel could be shown to correspond to the DNA profile of the body parts. DNA typing of hair found on a saw blade, on tights and under a kitchen cupboard in 1994 - the caretaker had stored several objects which he had found in 1987 in the suspect's flat - did not lead to any further results yet.
In this case, mainly preservation of tissue due to storage over seven years in the plastic barrel allowed successful amplification of two short tandem repeat systems.

3.3. Skin tumor development after transplantation of apparently nontumorous kidneys

A few months after transplantation of the kidneys from a fresh corpse (no malignancies) to two patients, both patients developed metastatic malignant melanomas. The recipients died of the melanomas. No obvious primary site could be found in both cases.

We were asked to answer if the tumor tissue originated from the apparently healthy donor who died of cerebral hemorrhage. The material we could examine was: (a) fresh metastatic tissue from the first recipient, (b) blood from the first recipient and paraffin slices with both ( c) an embedded metastasis and (d) tumor free tissue (closely neighbored) from the second recipient. Neither tissue nor any other reference material from the donor was available.

Amplification and electrophoretic separation of the short tandem repeat systems HUMVWA, HUMTHO1, and HUMFES showed that the first patient's DNA profile was completely different to the DNA profile of the metastasis (Tab. 1). The DNA profile of the second patient's tumor was a mixture of his genuine DNA profile and the tumor phenotype which was observed in the first patient's tumor. For the mutation of 3 individual loci of the recipient resulting in the donor's alleles at the same time is very unlikely, the profiles of the short tandem repeats and the hypervariable loci D1S80 and ApoB was interpreted to show that both tumors originated from the donor's tissue.

3.4. Multiple rape

In December 1993 a woman reported to having been raped by three men (A, B, and C) in turns without use of condoms near Cologne. The woman assumed that persons A and C ejaculated. On the woman's slip, minimal amounts of sperm were detected microscopically. The slip was stored for one year at -70°C until the blood of three suspects could be extracted.

At that time, we performed differential lysis [35] of the dried vaginal fluid from the slip to separate male sperm and female epithelial cells. DNA out of the epithelial phase and the sperm phase, respectively, was amplified with the four short tandem repeats HUMVWA, HUMTHO1, HUMF13A1 and HUMFES and analyzed both by vertical PAGE/silver staining or by use of a sequencer. The typing results are shown in Tab. 1.

From the beginning, the number of bands in the DNA profile of the sperm fraction of the mixed stain indicated that two men at least were involved in the rape (Tab. 1). In contrast with the impression of the woman, suspect A seemed to be excluded from being one of the offenders: In the two short tandem repeat systems HUMFES and HUMVWA his DNA profile is not represented in the mixed stain. On the other hand, suspect B who was thought not to have ejaculated had a DNA profile which was in concord to the mixed stain in three systems (HUMFES, HUMVWA, HUMF13A1) but excluded in HUMTHO1 because of one missing allele. In the mixed stain, of all short tandem repeat systems examined just one of person C's alleles each was found. Of no suspect all alleles could be found in the mixed stain. Drop out of alleles could easily be explained by a difference in sperm quantity left by each of the three suspects combined with the over all low amount of sperm cells.

Much more surprisingly, the mixed stain showed four alleles which did not correspond to the DNA profiles of any of the three offenders: Allele 6 in HUMTHO1, allele 11 in HUMFES, and alleles 6 and 8 in HUMF13A1. One had to think about the possibility that more men were involved in the rape, or that the women's portrayal of the event was not completely right, or that remnants of the woman's friend's sperm were present on the slip. The appearance of allele 16 in HUMVWA might also represent a so-called slippage band which is a typical PCR artifact in the short tandem repeat system HUMVWA and which can make interpretation of mixed stains difficult [36].

Because of the many unknown factors we went without a complex statistic evaluation and reported to the court that no clear identification was possible. We were aware of the fact that this opinion means that we did not deny that the accused men might have been the offenders.
Under normal circumstances, all three suspects would have been excluded because of several of their alleles missing in the mixed stain. We decided not to do so because of the low amount of sperm cells detected and the possible non random spreading of the cells over the slip.

3.5. Urine samples in doping cases: who's urine is tested?

The very strict rules of the International Olympic Committee allow doping analysis of any athlete's urine (at any time during the Games( [37]. Without prior announcement, 75 mL of athlete's urine at least are collected after a competition; closed bottles then are sent to one of the few laboratories licensed for doping analysis. In quite many cases, athletes manage to exchange their urine loaded with doping substances against (clean( urine by use of imaginative tricks. If not, their defenders since recently claim that clean urine of the athlete was exchanged against another - loaded - urine sample by anyone in the laboratory.
DNA analysis in urine was published to be possible just in a few cases until now [38-41]. Any approach to individualize urine by protein polymorphisms or by DNA typing had severe limitations until now [39,42-47]. In urine samples from doping controls, the low amount of cells and DNA rapidly decreases, maybe because of the bottles being sometimes opened and being contaminated in between. A 6 month storage of 20 mL control urine at 4°C for example led to a decrease from 20-40 ng DNA (males) and 400-800 ng (females) at the beginning down to 1-2 ng (males) and 10-20 ng (females) [41]. However, the variation is very high, and further measurements showed that 0 to 600 ng DNA can be extracted out of 15 mL urine [48]. In 93% of randomly chosen, stored urine probes from doping controls we found a pH lower than 7.0 (pH 5.0-6.9) which we suppose to promote hydrolysis of the DNA backbone. Furthermore, an unknown contaminant seems to severely inhibit PCR. This has to be managed by addition of bovine serum albumin in the PCR mastermix which we suppose to buffer the contaminant.

To allow a quick and reliable screening of high numbers of stored urine samples, we developed a multiplex PCR reaction which combines the three highly sensitive, non overlapping short tandem repeats HUMDHFRP2, HUMD8S306 and HUMCD4 (Fig. 2) [19,48-51]. Amplification and detection of the fluorescent alleles after a vertical denaturing electrophoresis with 12 cm separation distance (A.L.F. Sequencer, Pharmacia Biotech) now allows individualization of urine probes on a single day. The mean exclusion chance of this triplex system was calculated to be 89,1% [48] which allows a good estimation whether a urine sample comes from a certain donor or from another person out of a limited number of alternative donors. (For routine case work, further short tandem repeat profiling is necessary.)

4. Concluding remarks

In many forensic and clinical cases, cells and DNA in the minute amounts of material available are degraded. To individualize the samples, it is often necessary to use short DNA polymorphisms which are likely to be intact even in damaged DNA stretches. Because of their shortness and low detection limit (often down to 50 pg DNA), short tandem repeats (STRs) are a very good target for DNA typing of forensic specimens. Due to their structural similarity, simultaneous amplification of the loci (multiplex PCR) followed by semiautomatic detection on a sequencer is possible and allows to individualize most materials within a single day.

In contrast to short tandem repeat systems with a lower discriminating power, complex short tandem repeat systems like HUMSE33, HUMFIBRA and HUMD21S11 [8,52,53,54] with many alleles have a higher information content and thus might be the type of DNA polymorphisms which will mainly be used in future. However, because of their high number of alleles, those systems need to be electrophoretically resolved down to one base pair each.
In some short tandem repeat systems (e.g. HUMVWA), weak additional bands occur which are the result of slippage of the Taq polymerase during amplification [36]. Usually, in tetrameric short tandem repeats these so-called slippage bands are four base pairs shorter than the genuine alleles. In dealing with mixed samples, slippage bands sometimes pose a problem for interpretation.

Because of the high sensitivity of short tandem repeats one might expect an increased susceptibility for contamination artifacts, e.g. caused by DNA of the laboratory personnel. Such contaminations were never observed, however.

As one can see from the cases outlined above, DNA degradation sometimes pushes us to the limits of our possibilities. Furthermore, damaged DNA templates (very old bones, hair shafts) and minute amounts of cells (urine, sometimes sperm) occasionally lead to drop outs of single or - in the worst case - all alleles, and sometimes we also get irreproducible typing results. In contrast to such extreme examples, DNA typing of short tandem repeats leads to clear results in far the most cases. This means that now even denatured, old and degraded DNA is accessible for individualization.

Acknowledgments
Dr. Mechthild Prinz (OCME, NY) and Carmen Broicher (Inst. f. For. Med., Univ. Cologne) ran many of the gels on which we report here. Prof. Dr. Michael Staak, director of the Institute for Forensic Medicine, University of Cologne, supported and encouraged the authors in any possible way. Financial support for M.B. was given by the Bundesinstitut für Sportwissenschaften, Köln.

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Table 1: Overview over the typing results in three forensic cases (see text). Brackets indicate weak bands on the gels.
Figure 1 Electrophoretical separation of HUMTHO1 (that is TC11, left) and HUMVWA (right) amplification products. Lane (2),(3),(4),(5) resp. (8),(9),(10),(11): shanks, part of a rib (barrel), muscle (barrel), bloodstain. (1),(6): Allelic ladder HUMTHO: 5,6,7,8,9,9.3,11. (7),(12): Allelic ladder HUMVWA: 13,14,15,16,17,18,19,20,21 (from top to bottom). Additional bands interpretated as due to low quality of DNA: (3),(4),(10).

Figure 2 Electrophoretic separation of a DNA sample out of stored urine on a A.L.F. sequencer (Pharmacia Biotech, detection of fluorescent labelled primers by single wave laser, separation distance of used "short plates": 12 cm). Numbers on the right edge indicate lane numbers of the gel; codes on bottom indicate short tandem repeat system designation. Note that the additional band in lane 8 at D8S306 is outside the range of regular alleles.

Table 1 Overview over the DNA profiles in cases 3.1., 3.3. and 3.4.. Kidnapping--Tumor--Rape--Tissues from barrel--Kidnapper--(blood of corpse)--Coach driver--(blood of corpse)--Woman--(blood of corpse)--First recipient's DNA profile (blood)--First recipient's tumor--Second recipient's DNA profile (tumor tissue, paraffin embedded)--Second recipient's DNA profile (normal tissue, paraffin embedded)--Epithelial fraction--(female; dried & stored vaginal fluid-- Sperm fraction--(male; dried & stored vaginal fluid)--Suspect A--(fresh blood)--Suspect B--(fresh blood)--Suspect C--(fresh blood)