A new multiplex-PCR comprising autosomal and y-specific STRs and mitochondrial DNA to analyze highly degraded material.

The analysis of short tandem repeats is one of the most powerful tools in forensic genetics. Forensic practice sometimes requires the individualization of samples that may contain only highly degraded nuclear DNA, mitochondrial DNA or PCR inhibitors that hamper DNA amplification. We designed a new multiplex PCR with reduced size amplicons (<200 bp), providing a double sex determination (amelogenin plus two Y-STRs), the detection of two autosomal markers and the amplification of mitochondrial specific fragments from the hypervariable region I (HVI). Additionally, a quality sensor was developed to check for the presence of any PCR inhibitors. The new multiplex PCR shows a reproducible detection threshold down to 25 pg and gives signals even out of highly degraded materials. All signals are reproducible and reliable as it could be shown in comparison to results from commercially available STR multiplex-PCRs. In no case DNA fragments were detectable using any other assay when the quality sensor was not detectable. There was a good correlation between detection of mitochondrial specific fragments in the multiplex-PCR and success of subsequent sequencing of HVI region. The same could be shown for STR analysis: Most samples successfully analyzed in our PCR yielded at least a partial STR profile using a commercial STR kit. We present an assay that allows an easy, reliable, and cost efficient evaluation of DNA sample quality combined with a first rough sample individualization and sex determination suitable for forensic purposes. This assay may help the forensic lab personnel to decide on further sample processing.

An illicit love affair during the Third Reich: who is my grandfather?

More than 60 years after an illicit love affair had occurred between Erika H, wife of a Wehrmacht soldier, and a Polish slave worker during World War II, we could clarify the blood relationships of her daughter Uta. When Erika H had become pregnant both of the men could have fathered the child. Erika H was found guilty of fraternization and imprisoned at Ravensbrück concentration camp. She gave birth to Uta and died there in 1944. Uta survived the war as did Erika's husband Gustav, who accepted Uta as his child. Blood samples from family members were taken and DNA extracted. A panel of 16 short tandem repeat (STR) loci were amplified and separated by capillary electrophoresis and the likelihoods calculated using the MLINK software. The combined genotypes yielded a cumulative likelihood ratio of over 200,000 against paternity of Gustav H. This case serves to illustrate the utility of STR profiles for complex deficiency kinship analysis.

[Verification of private paternity analysis at the Institutes of Legal Medicine in Greifswald, Jena, and Kiel]. / Richtlinienkonforme Uberprüfung von privaten Abstammungsgutachten an den rechtsmedizinischen Instituten in Greifswald, Jena und Kiel.

This investigation presents the retrospective evaluation of paternity testing done as a "second opinion" in the last four years at the Institutes of Legal Medicine in Jena, Greifswald, and Kiel (Germany). All analyses were court-ordered and were preceded by paternity tests of "private" labs. The cases were selected in chronological order without any further exclusion criteria. A total of 59 cases, in which "private" laboratories from all regions of Germany had already performed paternity tests, were evaluated. In all cases, analyses were mainly done by PCR-based STR-typing (8 - 20 STRs). 18 % of the investigated "private" expert opinions showed a false determination of alleles. In two cases, paternity was wrongly confirmed or excluded. The reasons for the mistakes of private laboratories were hard to analyse, since most labs did not provide sufficient information (e.g. alleles, kits and chemicals used) in the written test results. In several cases, not even the typing results were revealed. Furthermore, in paternity testing of "private" labs the identity of the persons examined was usually not assured (e. g. by photo documentation or fingerprints) adding to the problem of insufficient test result reliability.

[Significance of additional RFLP single locus probes analysis after STR based determination of sibship]. / Die Aussagekraft zusäitzlicher RFLP-analysen bei Abstammungsbegutachtung mittels STR-Analyse.

Autosomal STR typing alone seems to be no sufficient tool for resolving deficiency cases (e.g. cases of questioned paternity or half-sibships). Therefore, we investigated whether the additional analysis of RFLP single locus probes can improve the solution of such complicated kinship cases. We analyzed 207 children and men from 101 families using the AmpFlSTRIdentifiler multiplex PCR kit and three RFLP single locus probes. A comparison between each child and all unrelated men resulted in 11,023 man / child pairs. Less than three excluding STRs were found in 125 child / unrelated man pairs (1.13%). Additional analysis of RFLP results reduced the number of ambiguous cases to 35. Half-sibling pairs were simulated using STR results from 20 cases with high paternity probabilities (group 1) and relatively low paternity probabilities (group 2). Using a commercially available computer program we calculated probabilities for 778 half-sibling pairs. In 35 pairs (4.49%) half-sibling probabilities over 90.0% could be calculated. Additional investigation of RFLP single locus probes did not lead to a more reliable evaluation of these results. The combined investigation of autosomal STRs and RFLP single locus probes can satisfactorily solve deficient paternities but does not contribute to the solution of questioned half-sibships.

[Problems of assessing sibship probabilities by means of genetic analysis]. / Zur Problematik der Beurteilung von Geschwisterwahrscheinlichkeiten mittels genetischer Analyse.

Nowadays, kinship studies based only on STR analysis are a very common practice. Apart from regular paternity cases, there is a rising number of cases in which the type of sibship between two persons has to be determined. Very often the parents or further relatives are unavailable for testing, so that the probability e. g. for a half-sibship has to be calculated as a deficiency situation. 50 unrelated persons (Group 1), 50 full siblings (Group 2), and 50 half-siblings (Group 3) were genotyped using the 15 STRs comprised by the AmpFlSTR Identifiler multiplex PCR-kit to investigate the reliability of common statistics programs and the indicative value of the calculated probabilities. Using a commercially available statistics program, we calculated probabilities for the following hypotheses: 1. The persons investigated are full siblings; 2. the persons investigated are half-siblings; 3. the persons investigated are unrelated. After STR analysis, half-sibling probabilities between 0.03 %-97.56% for group 1, between 0.01%-78.46% for group 2, and between 24.28%-90.18% for group 3 were calculated. In addition, the authors present two complicated deficiency cases which could be resolved only by extensive DNA typing using X- and Y-chromosomal loci, respectively, as well as the investigation of further relatives. The results of this study suggest that typing of autosomal STRs alone is no sufficient tool for resolving cases of possible half-sibship, even if probabilities of more than 90.0% are obtained. Due to the results of our study we suggest the implementation of mandatory standards for the investigation and interpretation of complicated kinship cases.

Fast and simple DNA extraction from saliva and sperm cells obtained from the skin or isolated from swabs.

The forensic scientist often has to cope with problematic samples from the crime scene due to their minute size and thus the low amount of extractable DNA. The retrieval of DNA from swabs taken from the surface of the skin, for example, in cases of strangulation, can be especially difficult. We systematically investigated swabs taken from the skin (to obtain a genetic profile from the victim and also from a possible offender) and from sperm cell containing swabs using two extraction kits: the Invisorb forensic and the Invisorb spin swab kit (both Invitek, Germany). DNA quality and quantity were tested on ethidium bromide containing agarose gels and in a highly sensitive duplex-PCR, which amplifies fragments specific for mitochondrial and nuclear DNA. Absolute quantification was done using real time PCR. Samples, which were positive in the duplex-PCR, were also employed to genetic fingerprinting using the Powerplex ES and the AmpFlSTRIdentifiler(TM) kits. Our study shows that the easy-to-use Invisorb spin swab kit is very suitable for DNA isolation from swabs taken from the skin and also from sperm cells. Retrieval of cells from the skin with swabs moistened in extraction buffer, not in distilled water, led to a significant higher DNA yield.

The problem of single parent/child paternity analysis--practical results involving 336 children and 348 unrelated men.

In a certain amount of paternity investigations, only DNA from child and alleged father is analyzed, thus increasing the possibility of false paternity inclusions. The aim of this study was to determine how many wrong paternity inclusions could be detected in a rather small geographical area comparing empirical results from 336 children and 348 men (13-15 STRs were investigated per person). This comparison between each child and all unrelated men (i.e. all putative fathers from the other cases) with an especially designed computer program resulted in 116,004 man/child pairs. Less than three excluding STRs were found in 1666 child/unrelated man pairs (1.44% of the comparisons). At least one unrelated man with only two or less STR mismatches could be determined for 322 children (95.8% of all investigated children). In 26 comparisons no STR mismatches between a child and an unrelated man were detected, thus at least one and up to three "second father(s)" under 350 men could be found for 23 children, if the mother is excluded. Paternity probabilities between 95.475% and 99.996% were calculated. Our results underline the difficulties in motherless paternity cases using only STR analysis and advise great precaution in assigning verbal predicates such as "paternity proven" in those investigations.

Possible pitfalls in motherless paternity analysis with related putative fathers.

Nowadays, more and more paternity cases are carried out investigating only child and putative father, mostly for economical or private reasons. Usually, reliable results can be obtained and the putative father can be included or ruled out with a high certainty. Considerable problems might arise when a relative of the biological father is investigated as being the putative father. In this study, we investigated 164 persons from 27 families creating artificial deficiency cases using the AmpFlSTRIdentifiler kit, which amplifies 15 STRs simultaneously. We analyzed 93 child/biological father pairs and the corresponding uncles, respectively the brothers of the biological fathers. The average paternity probability for the biological father was 99.9699% (paternity index (PI): 3321.26); only in three cases the results were under 99.9%. In five out of 125 child/uncle pairs no STR mismatches were found and paternity probabilities between 99.9726% (PI 3652) and 99.9970% (PI 33,545) were calculated. The average number of excluding loci was 3.4, but in 31.2% of the cases only zero, one or two mismatches were found. When both putative fathers were genetically typed, the biological father usually had a statistically higher paternity probability. Nevertheless, the differences between probabilities for father and uncle were only small. These results show that a reliable investigation of deficiency cases (i.e. child and putative father) seems to be more difficult than generally assumed. Especially in cases with an unknown familiar background and/or when investigating foreigners for immigration purposes, the laboratory expert should include the mother, increase the number of investigated loci or include a second method such as RFLP-analysis, some serological systems or typing of X-chromosome specific STRs to further ascertain the results.

Genetic analysis of modern and historical burned human remains.

Burning of corpses is a well-known funeral procedure that has been performed for a long time in many cultures. Nowadays more and more corpses are burned in crematories and buried in urns, often for practical and financial reasons. In some scientific, criminal or civil cases even after cremation there is the need of genetic investigations for identification or paternity testing. Furthermore, burned remains are the only remains left in North Europe from 1200 BC to 500 AD. This makes genetic investigation of those materials interesting for anthropological reasons. We present on one hand a systematic investigation of 10 corpses before and after the cremation and on the other hand the analysis of seven historical remains representing the bronze age. We chose the ground bone powder and the less destroyed bone parts respectively and employed a slightly modified commercially available DNA extraction method. The presence of human nuclear and mitochondrial DNA was tested by a simple but highly sensitive Duplex-PCR. DNA quantification was done using real time PCR, and genetic typing was tried out using the AmpFISTR Identifiler Multiplex Kit, followed by an automatic analysis on an AbiPrism310.

[Paternity analysis in deficiency cases with related putative fathers: simulation of a deficiency analysis in 27 families]. / Abstammungsbegutachtungen in Defizienzfällen mit verwandten Putativvätern: eine simulierte Analyse in 27 Familien.

During the last few years, the number of privately ordered paternity investigations has increased considerably. Probably due to financial reasons in more and more cases only the putative father and the child are investigated. Additionally, very often only one method, such as STR analysis, is employed. This raises the question whether such a reduced analysis leads to reliable and clear results when investigating cases with related putative fathers. We investigated 165 individuals from 27 families using the AmpFlSTRIdentifiler multiplex PCR and calculated the paternity probabilities of the children to their biological fathers, uncles, grand fathers and other relatives. In more than 30% less than three exclusions between child and relative were detected. In five cases no exclusions were found between child and uncle, always leading to paternity probabilities >99.9%. These results show that the calculation of high probabilities (>99.9%) does not necessarily lead to the accurate conclusion of fatherhood. In many of our cases misleadingly the brother of the real father or another close relative would have been declared to be the biological father.