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The German capital Berlin originates from the two medieval settlements Berlin and Cölln on either side of the river Spree. Whereas Berlin is world famous, there is very little awareness of former Cölln. From 2007-2009, during excavations of the earliest cemetery of this forgotten medieval town; 3,126 graves were discovered containing the remains of 3,717 individuals. Amongst those graves was an unusual triple burial. This grave was exceptional due to the relative postures of the skeletons and their extensive facial injuries. Here we present genetic and isotope data for this grave. Genetic results confirmed all of them as biological male individuals and ruled out their biological kinship. Combining genetic ancestry information with strontium isotope data we furthermore determined that two of the men most likely originated from the Berlin-Brandenburg region, whereas a more distant origin of the third individual can be debated.

A particularly challenging aspect of forensic casework is the accurate interpretation of samples that contain DNA from more than one contributor, since DNA analysis of specific identification markers often results in ambiguous mixed profiles. Physically separating the various haploid components of a diploid or polyploid DNA sample or of a specimen containing DNA from multiple individuals can resolve this problem. We adapted and combined previously developed methods of haplotype-specific extraction (HSE) to separate and analyze mitochondrial DNA (mtDNA) in samples containing DNA from two individuals. After optimizing conditions and probes, we performed HSE to separate mitochondrial DNA mixtures, which manifest as mixed nucleotide calls at single base positions. We were able to subsequently identify the DNA of the two individuals by sequencing. Based on our findings from this proof-of-concept experiment, this novel assay will be useful for distinguishing among the mtDNA of individuals in mixed DNA samples.

In forensic analysis, the interpretation of DNA mixtures is the subject of ongoing debate and requires expertise knowledge. Haplotype-specific extraction (HSE) is an alternative method that enables the separation of large chromosome fragments or haplotypes by using magnetic beads in conjunction with allele-specific probes. HSE thus allows physical separation of the components of a DNA mixture. Here, we present the first multiplex HSE separation of a Y-chromosomal haplotype consisting of six Yfiler short tandem repeat markers from a mixture of male DNA.

Aim A collaborative exercise with several institutes was organized by the Forensic DNA Service (FDNAS) and the Institute of the Legal Medicine, 2nd Faculty of Medicine, Charles University in Prague, Czech Republic, with the aim to test performance of different laboratories carrying out DNA analysis of relatively old bone samples. Methods Eighteen laboratories participating in the collaborative exercise were asked to perform DNA typing of two samples of bone powder. Two bone samples provided by the National Museum and the Institute of Archaelogy in Prague, Czech Republic, came from archeological excavations and were estimated to be approximately 150 and 400 years old. The methods of genetic characterization including autosomal, gonosomal, and mitochondrial markers was selected solely at the discretion of the participating laboratory. Results Although the participating laboratories used different extraction and amplification strategies, concordant results were obtained from the relatively intact 150 years old bone sample. Typing was more problematic with the analysis of the 400 years old bone sample due to poorer quality. Conclusion The laboratories performing identification DNA analysis of bone and teeth samples should regularly test their ability to correctly perform DNA-based identification on bone samples containing degraded DNA and potential inhibitors and demonstrate that risk of contamination is minimized.

One of the most demanding DNA extractions is from bones and teeth due to the robustness of the material and the relatively low DNA content. The greatest challenge is due to the manifold nature of the material, which is defined by various factors, including age, storage, environmental conditions, and contamination with inhibitors. However, most published protocols do not distinguish between different types or qualities of bone material, but are described as being generally applicable. Our laboratory works with two different extraction methods based on silica membranes or the use of silica beads. We compared the amplification success of the two methods from bone samples with different qualities and in the presence of inhibitors. We found that the DNA extraction using the silica membrane method results an in higher DNA yield but also in a higher risk of co-extracting impurities, which can act as inhibitors. In contrast the silica beads method shows decreased co-extraction of inhibitors but also less DNA yield. Related to our own experiences it has to be considered that each bone material should be reviewed independently regarding the analysis and extraction method. Therefore, the most ambitious task is determining the quality of the bone material, which requires substantial experience.

Current human genome databases for public single nucleotide polymorphisms (SNPs) still contain a substantial fraction of false entries. The main reasons for errors include sequencing or assembly errors, paralogous sequence-, and private variants. In the course of our studies on the Y chromosome, we established a set of internal laboratory guidelines for reliably identifying false SNP entries in databases.

In forensic work, the interpretation of DNA profiles becomes complicated when samples contain more than one contributor because the simultaneous amplification of individual identification markers results in mixed profiles. To overcome this problem, we present haplotype-specific extraction (HSE) as a more straightforward method to analyze a DNA mixture. HSE has been developed to clarify ambiguous HLA alleles by separating diploid samples into their haploid components to facilitate HLA typing. We have started to establish new protocols and strategies to adapt HSE for the separation of male DNA mixtures in forensic analysis. First results have shown an improved enrichment of male DNA from a single contributor. We have also evaluated a new, optimized buffer composition by testing different concentrations of its components. Improved separation of a male DNA mixture is detected using AmpFℓSTR(®) Yfiler short tandem repeat analysis.

Von allen bekannten einzelnen DNA-Polymorphismen zeigt das Y-Chromosom die starkste Assoziation zwischen dem allelischen Status multipler gekoppelter Loci (Haplotyp) und der Herkunftspopulation des typisierten mannlichen Individuums. Es ist daher moglich und in ausgewahlten Fallen geubte forensische Praxis, eine Herkunftsbestimmung mannlicher DNA aus Spuren oder Geweben mithilfe der kombinierten Untersuchung von „Y single nucleotide polymorphisms“ (Y-SNP) und „Y short tandem repeats“ (Y-STR) durchzufuhren. Insbesondere bei nichtidentifizierten Leichen wird diese Untersuchungsmethode angewendet, aber auch bei unaufgeklarten schweren Straftaten wird eine Eingrenzung des Kreises der als Tater infrage kommenden Personen mithilfe der Y-chromosomalen Analyse eingesetzt. Der vorliegende Beitrag soll zum Verstandnis der Moglichkeiten und Grenzen der geografischen Herkunftsbestimmung mithilfe der molekularbiologischen Diagnostik beitragen.

Berlin originated from the two twin cities Berlin and Colln, which both were founded at the beginning of the 13th century. However the real date of their foundation as well as the origin of the first settlers is still unknown. On the Berlin site the historic city center is still visible in the Nikolaiviertel, but the medieval origin of Colln disappeared almost completely. In 2007 a large scale excavation, which comprised an area of about 1700m 2 of the historical center of the St. Peters church, recovers the remains of Colln's first citizens and span a period of 500 years of medieval population. Here we present the first genetic analysis of a fivefold children's burial from excavations in Berlin. The genetic data unveiled next to ancestry and eye color data also the kinship and the gender of the five individuals. Together with the archeological context the new gained information help to shed more light on the possible reasons for this burial.

The yeast protein kinases Sat4/Hal4 and Hal5 are required for the plasma membrane stability of the K+ transporter Trk1 and some amino acid and glucose permeases. The transcriptomic analysis presented here indicates alterations in the general control of the metabolism of both nitrogen and carbon. Accordingly, we observed reduced uptake of methionine and leucine in the hal4 hal5 mutant. This decrease correlates with activation of the Gcn2-Gcn4 pathway, as measured by expression of the lacZ gene under the control of the GCN4 promoter. However, with the exception of methionine biosynthetic genes, few amino acid biosynthetic genes are induced in the hal4 hal5 mutant, whereas several genes involved in amino acid catabolism are repressed. Concerning glucose metabolism, we found that this mutant exhibits derepression of respiratory genes in the presence of glucose, leading to an increased activity of mitochondrial enzymes, as measured by succinate dehydrogenase (SDH) activity. In addition, the reduced glucose consumption in the hal4 hal5 mutant correlates with a more acidic intracellular pH and with low activity of the plasma membrane H+-ATPase. As a compensatory mechanism for the low glycolytic rate, the hal4 hal5 mutant overexpresses the HXT4 highaffinity glucose transporter and the hexokinase genes. These results indicate that the hal4 hal5 mutant presents defects in the general control of nitrogen and carbon metabolism, which correlate with reduced transport of amino acids and glucose, respectively. A more acidic intracellular pH may contribute to some defects of this mutant. © 2010, American Society for Microbiology. All Rights Reserved., Este trabajo ha sido financiado por las subvenciones BFU2005-06388-C04-01/BMC del Ministerio de Educación y Ciencia (Madrid), BFU2008-04188-C03-02 y BFU2008-04188-C03-01 del Ministerio de Ciencia e Innovación (Madrid), y Prometeo/2010/038 (Generalitat Valenciana).

Allele-specific extension reactions (ASERs) use 3′ terminus-specific primers for the selective extension of completely annealed matches by polymerase. The ability of the polymerase to extend non-specific 3′ terminal mismatches leads to a failure of the reaction, a process that is only partly understood and predictable, and often requires time-consuming assay design. In our studies we investigated haplotype-specific extraction (HSE) for the separation of male DNA mixtures. HSE is an ASER and provides the ability to distinguish between diploid chromosomes from one or more individuals. Here, we show that the success of HSE and allele-specific extension depend strongly on the concentration difference between complete match and 3′ terminal mismatch. Using the oligonucleotide-modeling platform Visual Omp, we demonstrated the dependency of the discrimination power of the polymerase on match- and mismatch-target hybridization between different probe lengths. Therefore, the probe specificity in HSE could be predicted by performing a relative comparison of different probe designs with their simulated differences between the duplex concentration of target-probe match and mismatches. We tested this new model for probe design in more than 300 HSE reactions with 137 different probes and obtained an accordance of 88%.