The muzzle to target distance -staining inside different parts of the firearm barrel.

Biological traces inside firearm barrels were observed as a result of contact shots to the head. The present study was conducted to investigate the influence of the muzzle to target distance on staining inside the anterior and posterior part of firearm barrels. Ninety-nine shots were fired to so-called reference cubes (10% gelatine, 12 cm edge length, embedded paint-blood-pad) using three current handguns. Shot range was varied from contact to 50 cm distance. High-speed cameras recorded external backspatter. Endoscopic examination assessed visible staining along the barrel. Each two swabbings were gathered from the anterior and the posterior part of the barrel. The first swabs were submitted to quantitative PCR, the second ones to DNA-RNA-co-extraction. Thorough mechanical and chemical cleaning was performed to avoid any contamination which was controlled by negative zero swabs after each cleaning. In single shots up to 50 cm distance, minimal, but DNA-positive sporadic traces were detected inside the barrel in vicinity of the muzzle. Visible complex staining varying in extent was observed in the anterior barrel part for 10 cm or less distance in dependence of the calibre. The posterior part showed detectable traces only after close range shots (< 5 cm). Generally staining inside the barrel decreased from the muzzle to the rear end, which correlated with the yield of DNA. Some contact shots did not cause any staining in the posterior part of the barrel despite massive external backspatter. Blood-specific miRNA was primarily found where DNA was detected. This experience encourages to take a second swab for RNA analysis. The amount of nucleic acids in the barrel at varying muzzle to target distances is subject to large variations between individual shots and therefore appears not suitable for a reliable determination of the shot distance in a particular case on its own. Instead, shot range estimation should also take into account morphology and distribution of traces inside the barrel.

Population genetic analysis of 12 X-chromosomal STRs in a Swiss sample.

X-chromosomal STRs are a powerful tool to assess a broad variety of complex kinship scenarios. We introduce herewith the first Swiss X-STR dataset based on 1198 individuals (592 female, 606 male), characterized with the Qiagen Investigator® Argus X-12 QS multiplex kit. Anomalous allele patterns, allele and haplotype frequencies, and forensic and population genetic parameters are presented. We detected linkage disequilibrium within three out of the four designated linkage groups and no apparent intra-national population substructure. We compared the dataset to a global panel of X-STR datasets and it fits well in the European context, as expected.

Expanding the Swiss autosomal marker set to 32 STRs.

By genotyping 1198 individuals with the Qiagen Investigator® HDplex Kit, we expand the Swiss autosomal STR dataset to 32 loci, providing additional resources for complex kinship cases. We present the first high-quality allele frequency dataset for loci D2S1360, D5S2500, D7S1517, and D10S2325 that will be accessible through the ENFSI reference database STRidER. For loci D3S1744, D4S2366, D6S474, D8S1132, and D21S2055, we provide a first European STRidER dataset.

DNA-free does not mean RNA-free-The unwanted persistence of RNA.

Contact shots to the head often provoke a transfer of biological traces into firearm barrels, which are not visible at endoscopic inspection. STR-PCR can amplify these latent traces and assign them to the victim. Via RNA-DNA-co-extraction also miRNA can be detected, which allow a conclusion to be drawn about the body fluid or tissue. Molecular genetic analysis of experimental stains in firearm barrels requires the guarantee that the barrel is initially free of any nucleic acid. Twelve shots were fired to so-called "reference cubes" (10 % gelatine, 12 cm edge length, embedded paint-blood-pad) using three current handguns: from 20 and 30 cm distance, four at close range (1-2.5 cm) and six contact shots. After endoscopic examination and swabbing of the barrels, a previously described mechanical and chemical cleaning using DNAExitusPlus™ was performed. The inner surface of the barrel was thoroughly wiped off using moistened forensic swabs, which were submitted to RNA-DNA-co-extraction. The combined thorough mechanical cleaning with Ballistol® and the application of DNAExitusPlus™ eliminated any profilable DNA in all samples. However, in 10 of 12 samples RNA concentrations between 0.11 - 0.79 ng/µl were measured. Furthermore, in 9 of 12 samples blood-specific miRNA (miR-451a) was detected. Summarizing, none of the experimentally contaminated barrels was RNA-free despite the performed cleaning procedure. Further investigation showed, that even "professional" cleaning by a gunsmith did not remove RNA.

The Y-chromosomal haplotype and haplogroup distribution of modern Switzerland still reflects the alpine divide as a geographical barrier for human migration.

A sample of 606 Swiss individuals has been characterized for 27 Y-STR and 34 Y-SNPs, defining major European haplogroups. For the first time, a subsample from the southernmost part of Switzerland, the Italian speaking canton Ticino, has been included. The data reveals significant intra-national differences in the distribution of haplogroups R1b-U106, R1b-U152, I1 and J2a north and south of the alpine divide, with R1b-U152 being the most frequent haplogroup among all Swiss subpopulations, reaching 26 % in average and 53 % in the Ticino sample. In addition, a high percentage of haplogroup E1b1b-M35 in Eastern Switzerland corresponds well with data reported from Western Austria. In general, we detected a low level of differentiation between the subgroups north of the alpine divide. The dataset also revealed a variety of microvariants. Some of them were previously known to be associated with particular haplogroups. However, we discovered one microvariant in DYS533 that seems to be closely associated with haplogroup I2-P215 (xM223). This association had not yet been reported to date. The concordance study with two STR-kits suggests that the DYS533 microvariant is due to an InDel in the flanking regions of the marker. One individual carried a large deletion, frequently detected in people of East Asian ancestry, encompassing the amelogenin locus. To our knowledge, this is the first time that such a deletion has been observed within European haplogroup R1b-U152. This is the first comprehensive Y chromosomal dataset for Switzerland, demonstrating significant population substructure due to an intra-national geographical barrier.

A "forensic biobank" to establish comprehensive genetic frequency data for Switzerland.

Comparatively little knowledge is available about the genetic structure of the Swiss population. Missing allele frequency data for some markers frequently used in forensics and paternity / kinship testing is just one practical aspect of this lack of genetic data. Therefore, in an attempt to fill this gap, we established a biobank of 1198 Swiss blood samples, systematically collected throughout the whole country. For the first time, this collection contains a population sample from the southernmost Italian speaking canton of Ticino. In this article, we share the experiences gained with the sampling procedure. Furthermore we present autosomal allele frequencies for 23 loci and a concordance check between the two multiplex PCR kits GlobalFiler® and PowerPlex® Fusion 6C. Statistical evaluation of the data revealed only small genetic differences among regional subpopulations and among language subgroups. The autosomal allele frequencies can therefore be used for forensics and paternity / kinship testing as a valid nationwide dataset. Additionally, the informative value of the three Y-STR markers included in the PowerPlex® Fusion 6C kit was assessed. We could demonstrate that the 3-loci-haplotype can be very informative, with an average haplotype frequency in the relevant Western European metapopulation of 0.026.

DNA recovery from gelatin fingerprint lifters by direct proteolytic digestion.

Fingerprints are a valuable source for DNA profiling in forensic investigations. In practice, the fingerprints are routinely visualized first by powder staining and then often transferred to tapes or gelatin lifters for storage or examination. If at all, fingerprints are usually sampled for DNA in a second step. To target the DNA sampling in an optimal way, it is essential to know how much of the DNA in the sample remains in place and how much is transferred to the lifter. In the present study we addressed this question analyzing 16 pairs of thumb prints and revealed that more than 80% of the DNA from a fingerprint is transferred to the gelatin lifter. Therefore, subsequent DNA sampling of the stored gelatin lifters appears more promising than recovery of the residual DNA from the original fingerprint. Furthermore, as a proof of principle, we developed a protocol for the direct extraction of DNA from gelatin fingerprint lifters by proteolytic digestion of the gelatin matrix followed by organic extraction. We show that DNA recovery from gelatin lifters by this direct extraction protocol is more efficient compared to swabbing the lifter followed by standard magnetic bead extraction of swabs. However, given the more elaborate protocol for direct extraction, we would still recommend the swab technique as the method of choice for forensic routine work.

Touch DNA sampling with SceneSafe Fast™ minitapes.

To achieve optimal results in the forensic analysis of trace DNA, choosing the right collection technique is crucial. Three common approaches are currently well-established for DNA retrieval from items of clothing, notably cutting, swabbing and tape-lifting. The latter two are non-destructive and therefore preferable on items of value. Even though the most recently established technique of DNA retrieval by adhesive tapes is widely used since quite some years now, little information has been published so far on how well it performs compared to other methods. Even more important, when it comes to choosing the right DNA extraction method for forensic lifting-tapes, the available information one can rely on as a forensic geneticist is quite scarce. In our study we compared the two widely used, commercially available and automation suitable magnetic bead-based extraction methods "iPrep Forensic Kit" and "PrepFiler Express BTA™ Kit" to conventional organic solvent extraction. The results demonstrate that DNA extraction from standardized saliva samples applied to SceneSafe Fast™ minitapes is most efficient with phenol-chloroform. We also provide evidence that SceneSafe Fast™ minitapes perform better than wet cotton swabs in the sampling of touch DNA from cotton fabric. Applying the tape only once in every spot on the tissue is thereby sufficient for a considerably better collection performance of the tapes compared to swabbing.

Electrostatic sampling of trace DNA from clothing.

During acts of physical aggression, offenders frequently come into contact with clothes of the victim, thereby leaving traces of DNA-bearing biological material on the garments. Since tape-lifting and swabbing, the currently established methods for non-destructive trace DNA sampling from clothing, both have their shortcomings in collection efficiency and handling, we thought about a new collection method for these challenging samples. Testing two readily available electrostatic devices for their potential to sample biological material from garments made of different fabrics, we found one of them, the electrostatic dust print lifter (DPL), to perform comparable to well-established sampling with wet cotton swabs. In simulated aggression scenarios, we had the same success rate for the establishment of single aggressor profiles, suitable for database submission, with both the DPL and wet swabbing. However, we lost a substantial amount of information with electrostatic sampling, since almost no mixed aggressor-victim profiles suitable for database entry could be established, compared to conventional swabbing. This study serves as a proof of principle for electrostatic DNA sampling from items of clothing. The technique still requires optimization before it might be used in real casework. But we are confident that in the future it could be an efficient and convenient contribution to the toolbox of forensic practitioners.

About DNA databasing and investigative genetic analysis of externally visible characteristics: A public survey.

During the last decade, DNA profiling and the use of DNA databases have become two of the most employed instruments of police investigations. This very rapid establishment of forensic genetics is yet far from being complete. In the last few years novel types of analyses have been presented to describe phenotypically a possible perpetrator. We conducted the present study among German speaking Swiss residents for two main reasons: firstly, we aimed at getting an impression of the public awareness and acceptance of the Swiss DNA database and the perception of a hypothetical DNA database containing all Swiss residents. Secondly, we wanted to get a broader picture of how people that are not working in the field of forensic genetics think about legal permission to establish phenotypic descriptions of alleged criminals by genetic means. Even though a significant number of study participants did not even know about the existence of the Swiss DNA database, its acceptance appears to be very high. Generally our results suggest that the current forensic use of DNA profiling is considered highly trustworthy. However, the acceptance of a hypothetical universal database would be only as low as about 30% among the 284 respondents to our study, mostly because people are concerned about the security of their genetic data, their privacy or a possible risk of abuse of such a database. Concerning the genetic analysis of externally visible characteristics and biogeographical ancestry, we discover a high degree of acceptance. The acceptance decreases slightly when precise characteristics are presented to the participants in detail. About half of the respondents would be in favor of the moderate use of physical traits analyses only for serious crimes threatening life, health or sexual integrity. The possible risk of discrimination and reinforcement of racism, as discussed by scholars from anthropology, bioethics, law, philosophy and sociology, is mentioned less frequently by the study participants than we would have expected. A national DNA database and the widespread use of DNA analyses for police and justice have an impact on the entire society. Therefore the concerns of lay persons from the respective population should be heard and considered. The aims of this study were to draw a broader picture of the public opinion on DNA databasing and to contribute to the debate about the possible future use of genetics to reveal phenotypic characteristics. Our data might provide an additional perspective for experts involved in regulatory or legislative processes.

Trypanosoma congolense procyclins: unmasking cryptic major surface glycoproteins in procyclic forms.

In the tsetse fly, the protozoan parasite Trypanosoma congolense is covered by a dense layer of glycosylphosphatidylinositol (GPI)-anchored molecules. These include a protease-resistant surface molecule (PRS), which is expressed by procyclic forms early in infection, and a glutamic acid- and alanine-rich protein (GARP), which appears at later stages. Since neither of these surface antigens is expressed at intermediate stages, we investigated whether a GPI-anchored protein of 50 to 58 kDa, previously detected in procyclic culture forms, might constitute the coat of these parasites. We therefore partially purified the protein from T. congolense Kilifi procyclic forms, obtained an N-terminal amino acid sequence, and identified its gene. Detailed analyses showed that the mature protein consists almost exclusively of 13 heptapeptide repeats (EPGENGT). The protein is densely N glycosylated, with up to 13 high-mannose oligosaccharides ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) linked to the peptide repeats. The lipid moiety of the glycosylphosphatidylinositol is composed of sn-1-stearoyl-2-lyso-glycerol-3-HPO(4)-1-(2-O-acyl)-d-myo-inositol. Heavily glycosylated proteins with similar repeats were subsequently identified in T. congolense Savannah procyclic forms. Collectively, this group of proteins was named T. congolense procyclins to reflect their relationship to the EP and GPEET procyclins of T. brucei. Using an antiserum raised against the EPGENGT repeat, we show that T. congolense procyclins are expressed continuously in the fly midgut and thus form the surface coat of cells that are negative for both PRS and GARP.

Phylogenetic analysis of clinical mumps virus isolates from vaccinated and non-vaccinated patients with mumps during an outbreak, Switzerland 1998-2000.

During the past decade mumps outbreaks have occurred in several European countries with universal vaccination programs probably due to poor efficacy of the Rubini vaccine strain. However, the evolution of vaccine escape mutants has also been considered. A phylogenetic analysis was undertaken on 69 clinical mumps isolates obtained from 39 vaccinated and 22 non-vaccinated mumps cases (and six cases with unknown vaccination status) during an outbreak in 1998-2000. Two major strain clusters (SWI-H, SWI-C) with two subgroups each (SWI-H1/2, SWI-C1/2) were identified, which belonged to genotypes C and H. No association between viral clusters and vaccination status or a specific vaccine strain (Jeryl-Lynn or Rubini) was found. Cluster SWI-C1 occurred more frequently in the Western part of Switzerland (P < 0.001). Isolates causing complicated disease tended to cluster more frequently with SWI-H1 (P = 0.11). Wild-type strains homologous or similar to the Rubini vaccine strain (isolated in Switzerland in 1974) were no longer circulating. Therefore, there was no evidence for vaccine escape mutants. Strain redistribution may have occurred during the past decades. Continuous monitoring of circulating mumps virus populations is needed.

Low-level resistance to rifampin in Streptococcus pneumoniae.

Rifampin is recommended for combination therapy of meningitis due to beta-lactam-resistant Streptococcus pneumoniae. High-level rifampin resistance (MIC, > or =4 mg/liter) has been mapped to point mutations in clusters I and III of rpoB of the pneumococcus. The molecular basis of low-level resistance (MICs, > or =0.5 and <4 mg/liter) was analyzed. Spontaneous mutants of clinical pneumococcal isolates were selected on Columbia sheep blood agar plates containing rifampin at 0.5, 4, 10, or 50 mg/liter. Low-level resistance could be assigned to mutations in cluster II (I(545)N, I(545)L). Sensitive (MIC, <0.048 mg/liter) wild-type strains acquired low-level resistance at a rate approximately 10 times higher than that at which they acquired high-level resistance (average mutation frequencies, 2.4 x 10(-7) for low-level resistance versus 2.9 x 10(-8) for high-level resistance [P < 0.0001]). In second-step experiments, the frequencies of mutations from low- to high-level resistance were over 10 times higher than the frequencies of mutations from susceptibility to high-level resistance (average mutation frequencies, 7.2 x 10(-7) versus 5.0 x 10(-8) [P < 0.001]). Mutants with low-level resistance were stable upon passage. Sequencing of a clinical isolate with low-level resistance (MIC, 0.5 mg/liter) revealed a Q(150)R mutation upstream of cluster I. The frequencies of mutations to high-level resistance for this strain were even higher than the rates observed for the in vitro mutants. Therefore, a resistance-mediating mutation located outside clusters I, II, and III has been described for the first time in the pneumococcus. In vitro low-level rifampin resistance in S. pneumoniae could be mapped to cluster II of rpoB. Mutants of pneumococcus with low-level resistance may be selected in vivo during therapy in tissue compartments with low antibiotic concentrations and play a role in the development of resistance.