Whose blood is it? Application of DEPArray™ technology for the identification of individual/s who contributed blood to a mixed stain.

The interpretation and statistical evaluation of mixed DNA profiles often presents a particular challenge in forensic DNA investigations. Only in specific combinations can single cellular components of a mixture be assigned to one contributor. In this study, the DEPArray™ technology, which enables image-assisted immunofluorescent-sorting of rare single cells using dielectrophoretic (DEP) forces, was applied together with different preliminary tests to identify the individual/s who contributed blood to a given mixture. The technique was successfully applied in two routine casework samples. In order to ascertain how old a stain can be and still be processed successfully, white blood cells from two 10- and one 27-year-old stains were investigated. Depending on the stain's age, the associated DNA degradation level and the number of target cells successfully isolated, the final profile reflects a compromise between the gain of information due to isolation of pure cells of a specific cell type from a single contributor and the loss of discriminatory power due to incomplete profiles caused by DNA degradation.

Comparison of telogen hair analyses: genRES MPX-2SP kit versus genRES MPX-SP1 and genRES MPX-SP2 kits.

STR investigations of telogen hair are invariably difficult due to the small amounts of nuclear DNA and its degradation products. However, in recent years there has been a considerable improvement. This study examined the suitability of a new STR kit with shortened amplicons for the investigation of hair in routine casework. This kit allows the simultaneous amplification of the eight STR-loci D3S1358, VWA, FGA, TH01, SE33, D8S1179, D18S51, and D21S11, and the sex-determining amelogenin system. It was tested against the genRES MPX-SP1 and genRES MPX-SP2 kits. The sensitivity of the new genRES MPX-2SP kit was demonstrated to be inferior to that of the genRES MPX-SP1, but almost equal to that of the genRES MPX-SP2 kit.

Comparative investigation of hair with the genRES(®) MPX-SP1, genRES MPX-SP2, and genRES MPX-2 kits.

In recent years there has been considerable improvement in short-tandem repeat (STR) investigations of hair, which were previously marred by small amounts of nuclear DNA and its degradation. This study examined the suitability of two STR kits with shortened amplicons for the investigation of hairs from routine casework. The overall sucess rate was more than 20%. Furthermore, the usefulness of quantification with real-time ploymerase chain reaction as a screening method was demonstrated.

Sex-specific fluorescent labelling of cells for laser microdissection and DNA profiling.

Sex-specific isolation of cells from mixtures would greatly facilitate forensic casework. Thus, male and female cell mixtures were marked with a fluorescent X/Y-probe CEP X SpectrumOrange/Y SpectrumGreen DNA probe kit for fluorescence in situ hybridization, and single cells were isolated via laser microdissection (LMD). DNA profiling of LMD isolated, hybridized cells showed usable short tandem repeat profiles for at least 20 cells, which are comparable with results from other studies. To simulate casework samples, the method was also optimized for air-dried samples.

[Paternity tests--current methods and legal background]. / Vaterschaftstests: Wie sie funktionieren und worauf es vor Gericht ankommt: Meins oder seins?

Paternity expertises are established on the basis of so-called STR (short-tandem repeat) polymorphisms. As a rule, they are requested by a judge for use as evidence in the clarification of the question of fathershaft in civil court cases. In addition to this, however, they are being requested more and more frequently by private persons, in the majority of cases with the aim of challenging presumptive fathershaft. In this latter case, however, it must be noted that when a minor is involved, the consent of the person who has the care and custody of the child must be available.

Species identification by means of pyrosequencing the mitochondrial 12S rRNA gene.

The aim of this study was to develop a new method for species identification based on the analysis of a very short nucleotide sequence. For this reason, the mitochondrial 12S rRNA gene, together with the new method of pyrosequencing, was used. The detection of only 20 nucleotides, following the sequencing primer within a 149-bp fragment by pyrosequencing, was sufficient to identify the biological origin of the samples by alignment with a reference sequence database. A case example with a piece of skin is presented, and the question whether this piece of skin came from a missing wife or from an animal could be answered.

Digoxigenin labelling and laser capture microdissection of male cells.

Laser capture microdissection (LMD) is a relatively new technique for the isolation of single cells. The application in forensic investigations has become more and more widespread, especially to select spermatozoa out of mixtures with vaginal cells. In particular in cases with low numbers of sperm it could be profitable to isolate all male cells (e.g. sperm and male epithelial cells) instead of focussing on the sperm only. Therefore, the specific labelling and detection of the male cells in a male/female cell mixture is necessary. In order to label all cells carrying a Y-chromosome we used a digoxigenin labelled chromosome Y hybridisation probe (Q Biogen). The stained cells were isolated with the SL microCut LMD system from Molecular Machines & Industries AG (MMI). At least ten diploid male cells were required to obtain a partial STR profile, with 20 cells, a full profile could be obtained.

Ninhydrin treatment as a screening method for the suitability of swabs taken from contact stains for DNA analysis.

More and more swabs containing unknown traces of biological material are submitted for forensic DNA analysis. Most of the samples are swabs taken from handled items such as tools, weapons and handles etc. Therefore, we tried to develop a screening method in order to focus the investigation on samples containing biomolecules, such as amino acids which might be associated with nucleic acids. A total of 285 swabs taken from various items collected during crime scene investigations were treated with ninhydrin which leads to a purple colour for samples containing amino acids. Of the swabs 158 were classified as ninhydrin positive and 76% of these samples yielded DNA profiles that fulfil the criteria for inclusion in the German national DNA database (profile frequency greater than 1 in 100,000) or in DNA mixtures which could at least be compared with suspects. In comparison only 9% of the 127 samples shown to be ninhydrin negative, revealed a usable DNA profile. Consequently, ninhydrin treatment was found to be an effective screening method which resulted in an increase in the rate of successfully typed samples and subsequently in a reduction of the costs due to the lower number of samples that needed to be typed.

Evaluation and application of the AmpF/STR profiler plus PCR amplification kit in a Bavarian population sample.

Allele frequencies for the nine tetrameric STR loci D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820 were determined in a population sample of 155 unrelated Bavarians using the AmpF/STR Profiler Plus PCR amplification kit. No deviations from the Hardy-Weinberg equilibrium were observed. The influence of the PCR cycle number as well as the template DNA concentration on the performance of the kit was studied. DNA concentrations lower than 75 pg DNA per 25 microliters reaction volume resulted in allelic drop-out.

Online reference database of European Y-chromosomal short tandem repeat (STR) haplotypes.

The reference database of highly informative Y-chromosomal short tandem repeat (STR) haplotypes (YHRD), available online at http://ystr.charite.de, represents the largest collection of male-specific genetic profiles currently available for European populations. By September 2000, YHRD contained 4688 9-locus (so-called "minimal") haplotypes, 40% of which have been extended further to include two additional loci. Establishment of YHRD has been facilitated by the joint efforts of 31 forensic and anthropological institutions. All contributing laboratories have agreed to standardize their Y-STR haplotyping protocols and to participate in a quality assurance exercise prior to the inclusion of any data. In view of its collaborative character, and in order to put YHRD to its intended use, viz. the support of forensic caseworkers in their routine decision-making process, the database has been made publicly available via the Internet in February 2000. Online searches for complete or partial Y-STR haplotypes from evidentiary or non-probative material can be performed on a non-commercial basis, and yield observed haplotype counts as well as extrapolated population frequency estimates. In addition, the YHRD website provides information about the quality control test, genotyping protocols, haplotype formats and informativity, population genetic analysis, literature references, and a list of contact addresses of the contributing laboratories.

Identification of the skeletal remains of Martin Bormann by mtDNA analysis.

Contrary to statements of an eye-witness who reported that Martin Bormann, the second most powerful man in the Third Reich, died on 2 May 1945 in Berlin, rumours persisted over the years that he had escaped from Germany after World War II. In 1972, skeletal remains were found during construction work, and by investigating the teeth and the bones experts concluded that they were from Bormann. Nevertheless, new rumours arose and in order to end this speculation we were commissioned to identify the skeletal remains by mitochondrial DNA analysis. The comparison of the sequence of HV1 and HV2 from the skeletal remains and a living maternal relative of Martin Bormann revealed no differences and this sequence was not found in 1,500 Caucasoid reference sequences. Based on this investigation, we support the hypothesis that the skeletal remains are those of Martin Bormann.

Is it possible to differentiate mtDNA by means of HVIII in samples that cannot be distinguished by sequencing the HVI and HVII regions?

The mitochondrial control region includes three so-called hypervariable (HV) regions, in which the polymorphic positions show a particularly high frequency. According to a population study of 200 unrelated individuals from Germany, HVI (positions 16,024-16,365, according to Anderson) showed 88 variable positions in a total length of 342 bp (26%) and HVII (positions 73-340) displayed 65 mutable sites in 268 bp (24%). HVIII (positions 438-574) exhibited a slightly lower variability, with 25 polymorphic sites within 137 bp (18%), but contrasted clearly with the background, which showed variability rates of only 7% (positions 16,366-16,569, 1-72) and 3% (positions 341-437), respectively. At present, the displacement (D)-loop database in Magdeburg comprises 904 sequences of the mitochondrial HVI region and HVII region from Germans, Austrians and Swiss. By means of this material, the extent to which the mtDNA sequences that do not differ in the HVI and HVII regions can be differentiated by additionally sequencing HVIII was investigated.

A new method for the evaluation of matches in non-recombining genomes: application to Y-chromosomal short tandem repeat (STR) haplotypes in European males.

A 9-locus microsatellite framework (minimal haplotype), previously developed for forensic purposes so as to facilitate stain analysis, personal identification and kinship testing, has been adopted for the establishment of a large reference database of male European Y-chromosomal haplotypes. The extent of population stratification pertaining to this database, an issue crucial for its practical forensic application, was assessed through analysis of molecular variance (AMOVA) of the 20 regional samples included. Despite the notion of some significant haplotype frequency differences, which were found to correlate with known demographic and historic features of Europeans, AMOVA generally revealed a high level of genetic homogeneity among the populations analyzed. Owing to their high diversity, however, accurate frequency estimation is difficult for Y-STR haplotypes when realistic (i.e. moderately sized) datasets are being used. As expected, strong pair-wise and higher order allelic associations were found to exist between all markers studied, implying that haplotype frequencies cannot be estimated as products of allele frequencies. A new extrapolation method was therefore developed which treats haplotype frequencies as random variables and generates estimates of the underlying distribution functions on the basis of closely related haplotypes. This approach, termed frequency 'surveying', is based upon standard population genetics theory and can in principle be applied to any combination of markers located on the Y-chromosome or in the mitochondrial genome. Application of the method to the quality assured reference Y-STR haplotype database described herein will prove very useful for the evaluation of positive trace-donor matches in forensic casework.

Y-chromosomal STR haplotypes in a population sample from Bavaria.

The seven Y-chromosomal STRs DYS 19, DYS385 I/II, DYS389 I/II, DYS390, DYS391, DYS392 and DYS393 were amplified using two multiplex PCRs. The optimization of the PCR conditions led to reliable and sensitive systems. Co-amplification of the amelogenin locus was possible in both multiplex systems. In a population sample of 151 Bavarian males, a gene diversity of 0.99 was observed. Sensitivity studies revealed a detection limit of 50 pg DNA per 25 microl reaction volume. PCR experiments with combinations of male/male and male/female DNA showed that in male/male mixtures, the minor component could be detected up to a ratio of 1:15, whereas in male/female mixtures the male component could be found in a higher ratio up to 1:60.