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The reproducibility of stem cell research relies on the constant availability of quality-controlled cells. As the quality of human induced pluripotent stem cells (hiPSCs) can deteriorate in the course of a few passages, cell banking is key to achieve consistent results and low batch-to-batch variation. Here, we provide a cost-efficient route to generate master and working cell banks for basic research projects. In addition, we describe minimal protocols for quality assurance including tests for sterility, viability, pluripotency, and genetic integrity. © 2020 The Authors. Basic Protocol 1: Expansion of hiPSCs Basic Protocol 2: Cell banking of hiPSCs Support Protocol 1: Pluripotency assessment by flow cytometry Support Protocol 2: Thawing control: Viability and sterility Support Protocol 3: Potency, viral clearance, and pluripotency: Spontaneous differentiation and qRT-PCR Support Protocol 4: Identity: Short tandem repeat analysis.

Each forensic case is characterized by its own uniqueness. Deficient forensic cases require additional sources of human identifiers to assure the identity. We report on two different cases illustrating the role of teeth in answering challenging forensic questions. The first case involves identification of an adipocere male found in a car submersed in water for approximately 2 years. The second scenario, which involves paternity DNA testing of an exhumed body, was performed approximately 2.8 years post-mortem. The difficulty in anticipating the degradation of the DNA is one of the main obstacles. DNA profiling of dental tissues, DNA quantification by using real-time PCR (PowerQuant™ System/Promega) and a histological dental examination have been performed to address the encountered impediments of adverse post-mortem changes. Our results demonstrate that despite the adverse environmental conditions, a successful STR profile of DNA isolated from the root of teeth can be generated with respect to tooth type and apportion. We conclude that cementocytes are a fruitful source of DNA. Cementum resists DNA degradation in comparison to other tissues with respect to the intra- and inter-individual variation of histological and anatomical structures.

In this study a set of 29 X-chromosomal short tandem repeats (STRs) located within the Xq26 region was evaluated. These STRs were found within the 133.14–133.45 Mb region around the HPRTB locus. Evaluation of the microsatellites was performed with regard to polymorphism, reliable amplification, and low stutter artefacts. DXS10101, DXS10102, and DXS10103 were identified as those X-STRs with highest diversity; i.e. PIC values of 0.7174–0.8933. The locus DXS10101 was the optimal candidate for the integration in the commercial available test system Mentype Argus X-8 PCR amplification kit.

The evaluation of four pairs of tightly linked chromosome X (ChrX) short tandem repeat (STR)s at Xp22, Xq12, Xq26 and Xq28 led to the creation of the Argus X 8 multiplex amplification kit. These eight STRs are distributed as four closely linked pairs over the entire X-chromosome, and for practical reasons, they are assigned to four linkage groups 1-4. To achieve a further considerable enhancement in discrimination power, we suggest to include additional markers. A recent paper referred to the earlier evaluation of STR clusters at Xq12, Xq26 and Xq28, and here we present the pending data of linkage group 1 at Xp22. The newly established STR updates the Xp22 STR cluster which now presents three polymorphic markers: DXS10148 (PIC = 0.8556), DXS10135 (PIC = 0.9093) and DXS 8378 (PIC = 0.6454). Typing of 398 X-chromosomes provided 278 different and 200 unique haplotypes. All the other haplotypes observed appeared with frequencies in the range between 0.005 and 0.015. Considering this STR triple in the context with the three further triple clusters Xq12, Xq26 and Xq28 published earlier, we announced the development of a next generation of a ChrX STR cluster typing kit.

The evaluation of the short tandem repeat (STR) markers DXS10079, DXS10074 and DXS10075 was amended to establish a STR cluster spanning a genetic distance

To test for human population substructure and to investigate human population history we have analysed Y-chromosome diversity using seven microsatellites (Y-STRs) and ten binary markers (Y-SNPs) in samples from eight regionally distributed populations from Poland (n = 913) and 11 from Germany (n = 1,215). Based on data from both Y-chromosome marker systems, which we found to be highly correlated (r = 0.96), and using spatial analysis of the molecular variance (SAMOVA), we revealed statistically significant support for two groups of populations: (1) all Polish populations and (2) all German populations. By means of analysis of the molecular variance (AMOVA) we observed a large and statistically significant proportion of 14% (for Y-SNPs) and 15% (for Y-STRs) of the respective total genetic variation being explained between both countries. The same population differentiation was detected using Monmonier's algorithm, with a resulting genetic border between Poland and Germany that closely resembles the course of the political border between both countries. The observed genetic differentiation was mainly, but not exclusively, due to the frequency distribution of two Y-SNP haplogroups and their associated Y-STR haplotypes: R1a1*, most frequent in Poland, and R1*(xR1a1), most frequent in Germany. We suggest here that the pronounced population differentiation between the two geographically neighbouring countries, Poland and Germany, is the consequence of very recent events in human population history, namely the forced human resettlement of many millions of Germans and Poles during and, especially, shortly after World War II. In addition, our findings have consequences for the forensic application of Y-chromosome markers, strongly supporting the implementation of population substructure into forensic Y chromosome databases, and also for genetic association studies.

Blowfly larvae found on human corpses are important for the estimation of the postmortem interval (PMI) and other questions of forensic relevance. Some of these species are difficult to differentiate morphologically, therefore a molecular method was elaborated for species identification. Specific fragments of the COI and COII region of the mitochondrial DNA (mtDNA) were amplified followed by digestion with different restriction enzymes. Using a 1.3 kb fragment, identification of Lucilia sericata, Calliphora vicina and Calliphora vomitoria was possible by digestion with only one restriction enzyme using either DraI or HinfI. Furthermore, we sequenced 349 bp (a part of the COI and COII regions) from the same three species and found 34 nucleotide distinctions between C. vicina and L. sericata, 30 between C. vomitoria and L. sericata and 15 between the two Calliphora species. These results aid in quick identification of species used for estimation of PMI.

Sequencing of mtDNA is an advanced method for the individualisation of traces. Disadvantages of this method are expensive and time-consuming analysis and evaluation procedures as well as the necessary stock of population-genetic data which is still insufficient. Central European institutes of forensic medicine from Germany, Austria, and Switzerland have been working together since the beginning of 1998 to establish a mtDNA database. The aim is to build up a large stock of forensically established data and provide population-genetic data for frequency investigations, which will serve as a basis for expert opinions and scientific research. Good data quality is ensured by using original sequences only. Ring tests, which have been conducted to enhance analytical reliability, revealed a high correspondence rate of the analytical results obtained by the individual member institutes. Today 1410 sequences are available for comparison, of which 1285 sequences in the HV1 and HV2 regions cover the full ranges from 16051 to 16365 and from 73 to 340 (according to Anderson). The major part is formed by Central European sequences comprising 1256 data sets from Germany, Austria, and Switzerland. Today the database contains sequences from a total of 12 European, six African and three Asian countries including 100 sequences from Japan. This paper is aimed at discussing the individualisation potentials of mtDNA as well as the possibilities and limits of ethnic differentiation by means of pairwise sequence differences on the basis of the data stock available.

The aim of this study was to develop an optimal strategy for dealing with contact stains in order to get a complete DNA-profile. Different methods of collecting material suitable for DNA typing and different methods for the extraction of DNA were compared. The results obtained will be described in detail.

A population study on six miniSTR loci (D10S1248, D14S1434, D22S1045, D1S1677, D2S441 and D4S2364) was performed on Northern German unrelated individuals. Allele frequencies and the usual forensic statistical parameters were defined. Additionally, the six loci were tested on DNA samples from mobiles phones, gear shifts plus steering wheels to test their robustness and sensitivity.

A large number of short tandem repeat (STR) markers spanning the entire human X chromosome have been described and established for use in forensic genetic testing. Due to their particular mode of inheritance, X-STRs often allow easy and informative haplotyping in kinship analyses. Moreover, some X-STRs are known to be tightly linked so that, in combination, they constitute even more complex genetic markers than each STR taken individually. As a consequence, X-STRs have proven particularly powerful in solving complex cases of disputed blood relatedness. However, valid quantification of the evidence provided by X-STR genotypes in the form of likelihood ratios requires that the recombination rates between markers are exactly known. In a collaborative family study, we used X-STR genotype data from 401 two- and three-generation families to derive valid estimates of the recombination rates between 12 forensic markers widely used in forensic testing, namely DXS10148, DXS10135, DXS8378 (together constituting linkage group I), DXS7132, DXS10079, DXS10074 (linkage group II), DXS10103, HPRTB, DXS10101 (linkage group III), DXS10146, DXS10134 and DXS7423 (linkage group IV). Our study is the first to simultaneously allow for mutation and recombination in the underlying likelihood calculations, thereby obviating the bias-prone practice of excluding ambiguous transmission events from further consideration. The statistical analysis confirms that linkage groups I and II are transmitted independently from one another whereas linkage groups II, III and IV are characterised by inter-group recombination fractions that are notably smaller than 50%. Evidence was also found for recombination within all four linkage groups, with recombination fraction estimates ranging as high as 2% in the case of DXS10146 and DXS10134.

We propose that clusters of closely linked markers, which segregate as stable haplotypes, provide a high potential to solve complex kinship cases. It is known that the X-chromosomal centromere region shows an extremely low degree of recombination. Hence, we focused our interest on the region between 56 and 64 Mb distant from the Xp telomere and considered 6 STRs which are now registered in the Genome Data Base as DXS10161, DXS10159, DXS10162, DXS10163, DXS10164, and DXS10165. All of these markers show a tetranucleotide or pentanucleotide structure and exhibit high or medium polymorphic information content. As a peculiarity, DXS10163 is a combination of a pentanucleotide STR and an 18 bp INDEL polymorphism. We report here the primer sequences, the repeat structures, the allele distributions and parameters of forensic interest for a German population sample.

Concurrent methods for identification of urine as being of human origin, and for DNA-typing from small stains of human urine were examined. A urine stain was extracted with phosphate-buffered saline (PBS), and the extract was filtered using a Centricon-100 device. The filtrate was subjected to electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS) for identification of human urine and a DNA-typing sample was obtained by dialfiltration of the residue using a DNA purification kit. After the purified residue was treated with an AmpflSTR® Profiler™ PCR amplification kit, the DNA-types were analyzed by capillary electrophoresis using a Genetic Analyzer. It was possible to identify a urine stain as being of human origin, and complete DNA profiles could be successfully obtained from a urine stain which had been created by 50 μL of female urine. Serial analyses of urine stains found at a crime scene provide effective information for forensic investigation. This method is recommended for stain identification and for DNA-typing from a urine stain.

The evaluation of four pairs of X-chromosomal short tandem repeats (STRs), i.e. DXS10135-DXS8378, DXS7132-DXS10074, HPRTB-DXS10101 and DXS7423-DXS10134 was carried out using the Argus X-8 Multiplex amplification kit. These eight STRs are distributed as four closely linked pairs over the entire X-chromosome (ChrX), and for practical reasons they are assigned to four linkage groups 1-4. The genetic distance within the STR pairs is assumed to be

Intron 40 of the von Willebrand factor (vWF) gene exhibits a highly variable region of about 0.65 kb, which contains 5 juxtaposed STRs. We sequenced 0.65 kb amplicons from 68 chromosomes and found 2 frequent indel polymorphisms and 5 SNPs. The 68 chromosomes investigated here presented a total of 47 different haplotypes. Regarding the SNP allele distribution in our sample, we arranged our results of the vWF intron 40 into a system of 3 haplotypes, i.e. haplotypes a, b and c. Our review may be valuable in further optimising vWF typing in forensic applications and in avoiding pitfalls. Further attempts to develop sophisticated techniques may soon enable haplotyping using autosomale STR clusters.

For several years Y-chromosomal microsatellites (short tandem repeats, STRs) have been well established in forensic practice. In this context, the genetic characteristics of the Y chromosome (i.e. its paternal inheritance and lack of recombination) render STRs particularly powerful. However, genetic differences between male populations appear to be larger for Y-STRs than for autosomal STRs, a fact that is most likely due to the higher sensitivity of Y-chromosomal lineages to genetic drift (Forensic Sci Int 118 (2001) 153). The assessment of probabilities for matches between haplotyped male persons or traces/persons requires the typing of a large number of haplotypes in the appropriate reference populations. The haplotype data of a large number of European as well as South and North American populations have been collected and are continuously published online (Y-STR Haplotype Reference Database--YHRD; http://www.ystr.org). The most recent multicentric effort has led to the establishment of an Asian YHRD (http://www.ystr.org/asia) which has been available since January 2002. All databases are maintained and curated at the Institute of Legal Medicine, Humboldt-University, Berlin and will soon be fused to a global repository including populations from all continents.

Monoclonal antibodies against purified rat liver 4-hydroxybiphenyl UDP-glucuronosyl-transferase were developed using the hybridoma technology. In immunoblot analysis the antibodies specifically reacted with purified 4-hydroxybiphenyl-UDPGT but not with other purified UDPGT enzyme fractions. One single band was detected with microsomes of rat liver and small intestine but not with microsomes of kidney and lung. The reactive protein was also found in dog and human liver microsomes. It could be shown that there was no increase of immunoreactive protein after pretreatment with phenobarbital or 5,6-benzoflavone. This supports the hypothesis that more than one 4-hydroxybiphenyl-UDPGT exist in rat liver which are differently inducible.

In the collaborative exercise GEDNAP IV one EDTA blood sample (2 ml) and 5 bloodstains (0.5 ml on cotton) were investigated and in GEDNAP V, a total of 8 bloodstains (0.5 ml on cotton), including 2 mixed bloodstains. DNA typing was carried out using the RFLP systems YNH24/Hinf I and MS43a/Hinf I and the PCR systems HLA DQ alpha, D1S80, ApoB and YNZ22. In both exercises approximately 20 laboratories obtained results using the RFLP systems. Of the PCR systems, D1S80 was the most commonly used (14 labs in GEDNAP IV; 18 labs in GEDNAP V). The interlaboratory standard deviation for YNH24 in both exercises was approx. 0.6%, for MS43a 0.7-2.2% (GEDNAP IV) and 0.4-1.4% (GEDNAP V), depending on the fragment size. The fragment size calculation performed in each laboratory yielded a standard deviation twice that obtained when the fragment size calculation was performed centrally (IfR, Münster). In GEDNAP III, a system-specific corridor was developed to define the limits of deviation; this was modified for the present study by combining the fragment size ranges of YNH24 and MS43a. In both studies a subgroup of laboratories was involved in preliminary exercises using three PCR VNTRs and the system HLA DQ alpha. Owing to the substantial variation in experience of the participating laboratories with PCR typing the results obtained in these two studies do not fulfil the basic quality criteria of the GEDNAP studies.