Hans-Joachim Weisser

Location and frequency of polymorphic positions in the mtDNA control region of individuals from Germany.

Abstract In order to identify polymorphic positions and to determine their frequency in the human mitochondrial D-loop containing region, the mitochondrial DNA (mtDNA) control region of 200 unrelated individuals from Germany were amplified and directly sequenced. Sequence comparison led to the identification of 190 mitochondrial lineages as defined by 202 variable positions. The most frequently occurring lineage comprised 5 individuals, whereas 186 types of D-loop sequences were observed in only one individual. Of the sequences studied 7% are not unique but show at least one counterpart with an identical haplotype. The majority (61%) of the control regions investigated showed between four and eight nucleotide positions deviating from the reference sequence. The maximum number of deviations observed in a single control region was 18. The majority of the variable positions in the D-loop region (88%) are located within three hypervariable regions. Sequence variations are caused by nucleotide substitutions, insertions or deletions. As compared to insertions and deletions, nucleotide substitutions make up the vast majority of the mutations (90%). We have predominantly found transitions (75%) and a significantly lower frequency of transversions (15%) whereas insertions (6%) as well as deletions (4%) are rather rare. Upon sequencing the mitochondrial control region from 200 German Caucasians the genetic diversity was estimated at 0.99. The probability of two randomly selected individuals from a population having identical mtDNA types is 0.6%.

mtDNA as a tool for identification of human remains

Two cases are presented in order to emphasize the importance of mitochondrial DNA in forensic medicine. The first case involved a charred body which could not be identified by morphological means because of severe destruction of all tissues. The parallel use of PCR methods using genomic DNA and sequencing of the mitochondrial d-loop region produced unequivocal and reproducible results. In the second case, various parts of a highly decomposed body were investigated. The application of standard PCR methods for genomic DNA proved unsuitable to answer the question whether the body parts belonged to the same body. However, when sequencing of mitochondrial DNA segments amplified from tissue and bone samples was performed, clearly interpretable results were obtained.

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.

Mitochondrial heteroplasmy among maternally related individuals

Abstract The second hypervariable segment of the human mtDNA control region contains a homopolymeric tract of cytidines between nucleotides (nt) 303 and 315, interrupted by a thymidine at position 310, according to the Cambridge reference sequence. By direct sequencing, some individuals show blurred sequence chromatograms in this region which are not caused by a sequencing artefact but by high levels of length heteroplasmy. With respect to this length heteroplasmy ten maternally related individuals and two unrelated probands were examined. The relative proportions of length variants in the homopolymeric tract in selected individuals were determined by cloning and sequencing of multiple independent clones. All ten family members examined were heteroplasmic while the proportions of each genotype varied widely in different individuals. The size of a possible mitochondrial bottleneck during embryonic development of the offspring is discussed with respect to the changes in mitochondrial haplotypes within mother-offspring pairs. Our data are consistent with both slow and rapid segregation of mtDNAs between the generations, which would implicate a tight as well as a wide bottleneck. Therefore, a common bottleneck size in all individuals from this lineage seems to be very unlikely.

DNA typing of human remains found in damp environments

DNA typing is often used to determine identity from human remains. The environment to which the material has been exposed, however, is crucial for the success of the investigation. Damp conditions in particular can cause a rapid degradation of DNA, even in bone and teeth, and thus reduce the chances of successful typing. Here, we present the results of investigations performed on four skulls that had been lying in a damp environment for periods ranging from almost 1 year to about 45 years. In none of these cases was DNA typing successful on bone or, where present, on teeth. Where remnants of brain tissue were used, however, complete STR typing was possible in one case, partial STR typing in another, and mtDNA sequencing could be carried out in three cases. These findings suggest that brain tissue is relatively resistant to putrefaction in damp environments and, unlike bone, appears to exhibit a certain degree of protection against DNA degradation.

The gene for Klebsiella bacteriophage K11 RNA polymerase: Sequence and comparison with the homologous genes of phages T7, T3, and SP6

SummaryWe determined the nucleotide sequence of gene 1 of Klebsiella phage K11, which is a member of the T7 group of phages. The largest open reading frame corresponds to a polypeptide with 906 amino acids and a molecular weight of 100383 daltons. The deduced amino acid sequence of this polypeptide shows 71% homology to the T7 RNA polymerase (the product of T7 gene 1), 72% homology to the T3 RNA polymerase and 27% homology to the SP6 RNA polymerase. Divergent evolution was clearly most pronounced in the amino-terminal portion.

Transfer of biological traces in cases of hanging and ligature strangulation

In hanging and ligature strangulation, the noose mostly causes a mark or groove which is formed partly by compression of the skin and partly by abrasion with loss of the upper epidermal layers. The horny scales abraded from the neck may be transferred to the strangulation device or to the interposed textiles where they are sometimes visible at stereomicroscopic examination or even to the naked eye as silver-grey particles. The morphologic features of the epidermal transfer due to hanging and ligature strangulation is demonstrated by 14 case examples. The biological traces may be sufficient for comparative DNA typing by means of PCR-based methods. In 9 out of the 14 cases, genomic DNA typing was successful. Analysis of mtDNA succeeded in another two cases, although genomic DNA could not be detected. Beside the accumulation of solid epidermic particles the paper describes deposition of serous and fatty tissue fluid at the ligature (mainly adjacent to skin ridges).

Recovery of Latent Fingerprints and DNA on Human Skin

Abstract:  The project “Latent Fingerprints and DNA on Human Skin” was the first systematic research in Europe dealing with detection of fingerprints and DNA left by offenders on the skin of corpses. One thousand samples gave results that allow general statements on the materials and methods used. The tests were carried out according to a uniform trial structure. Fingerprints were deposited by natural donors on corpses. The latent fingerprints were treated with magnetic powder or black fingerprint powder. Afterward, they were lifted with silicone casting material (Isomark®) or gelatine foil. All lifts were swabbed to recover DNA. It was possible to visualize comparable and identifiable fingerprints on the skin of corpses (16%). In the same categories, magnetic powder (18.4%) yielded better results than black fingerprint powder (13.6%). The number of comparable and identifiable fingerprints decreased on the lifts (12.7%). Isomark® (14.9%) was the better lifting material in comparison with gelatine foil (10.1%). In one‐third of the samples, DNA could be extracted from the powdered and lifted latents. Black fingerprint powder delivered the better result with a rate of 2.2% for full DNA profiles and profiles useful for exclusion in comparison with 1.8% for the magnetic powder traces. Isomark® (3.1%) yielded better results than gelatine foil (0.6%).

DNA profiling of bloodstains on linen pretreated with remedies used for cleaning and maintaining clothes

SummaryLinen was pretreated with 20 remedies for cleaning and maintaining clothes and 20 μl blood was applied on each sample and dried. Restriction enzyme digest of bloodstain DNA was irregularly inhibited by highly concentrated residues of 2 detergents and a stainremover and colour-brightener. An additional dialysis step to purify DNA (Gill 1987) reliably prevented disturbance. High molecular DNA was obtained in every case and bandshifts were not observed.ZusammenfassungLeinenstoff wurde mit 20 Mitteln, die zur Wäschereinigung und -pflege verwendet werden, in normaler und sehr hoher Konzentration behandelt, absichtlich unzureichend oder nicht gewaschen, getrocknet und mit 20 μL Blut versetz. Hohe Rückstandskonzentrationen von zwei Waschmitteln und eines Aufhellers führten unregelmäßig zu partieller und totaler Inhibition der Restriktionsspaltung. Durch einen Dialyseschritt bei der Reinigung der DNA (Gill 1987) konnten die Störungen zuverlässig verhindert werden. Hochmolekulare DNA konnte in jedem Fall gewonnen werden, Bandshifts traten nicht auf.

Extraction of DNA from Coagulated Blood Samples

In forensic medicine, blood samples available for case work are coagulated predominantly. Following routine manuals, yield of DNA is irregular and sometimes low.