The German Stain Commission: recommendations for the interpretation of mixed stains.

In the course of forensic DNA analysis, the interpretation of DNA profiles of mixed stains, i.e. cell material from more than a single donor, has become increasingly more important. The German Stain Commission, a joint commission of Institutes of Forensic Science and Legal Medicine, has therefore developed guidelines aiming to harmonize the evaluation of mixed stains in German criminal cases.

DYS STR analysis with epithelial cells in a rape case.

We report here the application of Y-chromosomal DNA analysis in a rape case, which occurred in Stuttgart, Germany. Microscopic examination of the victim's vaginal swabs and her underwear showed no sperm cells. DNA was extracted from vaginal and epithelial cells and analysed with the autosomal systems SE33, THO1 (singleplex) and with the multiplex Profiler Plus (Applied Biosystems, Foster City, USA). The results of these autosomal STR analysis gave no hint at a mixed sample and failed to identify a male profile. DYS STR analysis with the systems DYS391, DYS392, DYS393, DYS19 and DYS389 I/II showed the same characteristic features as the suspect. We used this incomplete haplotype to search in the Y-STR Haplotype Reference Database via Internet. In a Caucasian population sample of 3589 minimal haplotypes we found 71 matches. The suspect confessed the crime and was finally condemned to 4 years imprisonment.

Frequency data for the STR loci HumFibra (FGA) and HumACTBP2 (SE33) in a population of Germans and Turks from South-West Germany.

Population studies were carried out on German and Turkish individuals from South-West Germany using the short tandem repeat (STR) systems HumFibra (n = 138 Turkish and 1161 German individuals) and HumACTBP2 (n = 202 Turkish and 1338 German individuals). After electrophoresis 19 alleles could be identified for HumFibra and 55 for HumACTBP2. No significant deviations from Hardy-Weinberg equilibrium were observed.

Frequency distribution of D1S80 alleles in the German population.

The locus D1S80 is a very useful genetic marker system for forensic DNA analysis. It consists of a variable number of tandem repeats (VNTR) and can be analyzed by the polymerase chain reaction (PCR). As accurate data about the distribution of the alleles is one of the most important prerequisites for the application in forensic biology we studied the allele distribution in the German population.

[Measuring precision in determining the length of restriction fragments within the scope of DNA analysis]. / Untersuchungen zur Messpräzision bei der Grössenbestimmung von Restriktions-Fragmentlängen im Rahmen der DNA-Analyse.

Because of the measurement error that is known from the length determination of restriction fragments during DNA analysis, it is necessary to determine the precision by which identical DNA can be evaluated. Besides the collection of frequency data from a certain population this is one of the basic prerequisites to calculate the likelihood ratio of an established polymorphism. By multiple measurement of restriction fragments from different persons indicated by the probes MS1, MS31, MS43A, g3 and YNH24 on different blots the within-laboratory variation was determined in the complete separation area from 1 to 19 kb. The values were compared to procedures used by other laboratories.

ABO and Lewis typing of secretion stains on nitrocellulose membranes using a new dot-blot-ELISA technique.

A new method for ABO and Lewis typing of body fluids is described. It combines the advantages of a good antigen binding to nitrocellulose membranes, the need of only very small amounts of stain material and the high sensitivity of an enzyme-linked immunosorbent assay for antigen detection. This is of special interest because conventional ABO and Lewis typing of secretion stains need relatively large stain dimensions. The method is very easy to handle, does not need any expensive equipment and gives a permanent record. Furthermore the high sensitivity offers the possibility of analyzing even sweat and urine stains without the need of concentrating these extracts.

Subtyping of group specific component (GC) in micro-bloodstains and semen stains by isoelectric focusing in ultrathin immobilized pH gradient gels followed by enzyme immunodetection.

A powerful method for group specific component (GC) subtyping with good resolution of GC bands by isoelectric focusing on ultrathin immobilized pH gradients is described. After separation, GC detection is achieved using a highly sensitive alkaline phosphatase conjugated enzyme-immuno system. The efficiency of the method in forensic case work for subtyping GC from diluted bloodstain extracts, blood micro-stains and semen stains is demonstrated. Furthermore, GC subtyping and selective detection of human GC provides the evidence for human origin of the stain. This is important when analyzing microstains with limited stain consumption.

Subtyping of phosphoglucomutase 1 (PGM1) in microstains of different biological materials with subsequent species differentiation without further stain material consumption.

Micromethods for subtyping of phosphoglucomutase 1 (PGM 1) in small amounts of biological stain material are described, using an applicator for highly diluted stain extracts. With the aid of this applicator strip blood and semen micro-stains as well as single hair-roots could be extracted by electroelution directly on the PGM1 isoelectric focusing gel. Species differentiation was also possible either by radial immunodiffusion using the extract remaining in the applicator strip after isoelectric focusing or by interpretation of the PGM pattern itself.

Electrotransfer of proteins after isoelectric focusing (with or without urea) on fabric-reinforced, immobilized pH gradient gels.

In the present article a procedure is described which combines the horizontal isoelectric focusing (IEF) of proteins on fabric-reinforced polyacrylamide gels with the subsequent electrophoretic transfer of the proteins to nitrocellulose or Immobilon. The application of a carrier material that is permeable for current and molecules and that serves as a physical support of the IEF gel is one of the central prerequisites for the method to work. Moreover, it is important to fix the pH gradient topographically by the use of Immobilines mainly in order to avoid distortion of the protein pattern during the electrotransfer (Western blot). The Western blot can be performed either in the submerse or in the so-called "semi-dry" blotting system. Our procedure is compatible with IEF protocols employing buffer systems with or without urea. The efficacy of our method is demonstrated by the IEF and Western blotting of several known marker proteins.

Isoelectric focusing of group specific component in semen and vaginal stains on ultrathin immobilized pH gradient gels.

Subtyping of the group specific component in secretions of semen and vaginal fluids is impossible with conventional detection systems. By means of a highly sensitive alkaline-phosphatase secondary antibody system the group specific component can reliably be detected in semen stains. Results with vaginal swabs were inconsistent and in saliva stains Gc activity could not be detected.

[Occurrence of pydixaminephosphate oxidase and pyridoxal kinase in Gram-negative and Gram-positive bacteria]. / Vorkommen von Pyridoxaminphosphat-Oxidase and Pyridoxal-Kinase in Gram-negativen und Gram-positiven Bakterien.

Pyridoxamine-phosphate oxidase and pyridoxal kinase were detected in Gram-negative bacteria. In Gram-positive bacteria, generally only pyridoxal kinase is detectable.

[Vitamin B 6 biosynthesis in Bacillus subtilis]. / Vitamin-B6-Biosynthese in Bacillus subtilis.

1) A vitamin-B6-producing mutant, BA 1, was selected by treatment of Bacillus subtilis with N-methyl-N'-nitro-N-nitrosoguanidine. Using gradient plates supplemented with the vitamin B6 antagonist isonicotinohydrazide, three mutants of BA 1 were isolated, which excrete 2-5 mg of vitamin B6/l of growth medium. 2) Mutation of the three vitamin-B6-producing strains BA 1, BA 11 and L 71 led to the isolation of 49 vitamin-B6 deficient mutants. All mutants are able to grow with pyridoxine, pyridoxal, pyridoxamine, and even with 4'-deoxypyridoxine. Glycolaldehyde or nicotinic acid do not support growth of the mutants. Some of these vitamin-B6-deficient mutants can also grow in the absence of vitamin B6, providing isoleucine is present. Others show a growth stimulation, when isoleucine is added to a medium containing a vitamin B6 compound. Isoleucine can be replaced by 3-methyl-2-oxovalerate. Cross-feeding experiments indicated a division of the mutants into two groups. Using chromatographic methods, substances which support growth of the mutants were purified, but have not yet been identified. Following the addition of 4'-deoxypyridoxine, 4'-deoxypyridoxine 5'-phosphate was isolated from the growth medium of a vitamin B6-deficient mutant. 3) Threonine dehydratase, transaminase B and transaminase C from wild-type Bacillus subtilis were compared with the enzymes from vitamin-B6-producing strains and with the enzymes from vitamin-B6-deficient mutants. Both the vitamin-B6-producing and the vitamin B6-deficient mutants show higher specific activities than wild type. In the mutant strains no multivalent repression of the threonine dehydratase and transminase B by isoleucine, leucine and valine could be demonstrated. Leucine dehydrogenase, the first enzyme of the isoleucine catabolic pathway, is constitutively produced in the vitamin-B6-producing and in the vitamin-B6-deficient mutants. In the vitamin-B6-deficient mutants, there is a correlation between growth yield in the presence of isoleucine and the specific activity of leucine dehydrogenase. In the crude extract of Bacillus subtilis no pyridoxamine-phosphate oxidase activity could be demonstrated, whereas pyridoxal kinase was readily detectable.