The Utilization of the SaLux19-Based Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid and Sensitive Identification of Minute Amounts of a Biological Specimen.

Our research has developed a highly sensitive and simple assay to detect small amounts of animal and human biological material in less than 40 min. The handheld SaLux19 device developed at the Max Planck Institute of Experimental Medicine in Göttingen, Germany, was used to validate our concept. The proposed system uses isothermal amplification of DNA in a rapid assay format. Our results show that the assay can detect Sus scrofa nucleic acids with very high sensitivity and specificity. This detection system has potential for forensic scenarios.

Capillary-Electrophoresis-Based Species Barcoding of Big Cats: CR-mtDNA-Length Polymorphism.

This study aimed to provide an overview of the methodological approach used for the species determination of big cats. The molecular system described herein employs mitochondrial DNA control region (CR-mtDNA)-length polymorphism in combination with highly sensitive and precise capillary electrophoresis. We demonstrated that the described CR-mtDNA barcoding system can be utilized for species determination where the presence of biological material from big cats is expected or used as a confirmatory test alongside Sanger or massive parallel sequencing (MPS). We have also addressed the fact that species barcoding, when based on the analysis of mtDNA targets, can be biased by nuclear inserts of the mitochondrial genome (NUMTs). The CR-mtDNA barcoding system is suitable even for problematic and challenging samples, such as hair. CR-mtDNA-length polymorphisms can also distinguish hybrids from pure breeds.

The Influence of Tanning Chemical Agents on DNA Degradation: A Robust Procedure for the Analysis of Tanned Animal Hide-A Pilot Study.

Illegal wildlife trade is currently on the rise, and it is becoming one of the most lucrative crime sectors. The rarer the species, the higher the demand. Wildlife trade falls under international regulations, such as the CITES convention. Proving that this convention has been violated is a complex process and can be very difficult to do. DNA analysis methods remain (in many cases) the only way to determine whether a certain specimen originated from a protected animal species, a specific individual, or a species in which it is legal to trade. Tanned animal hides are a specific type of specimen. With this type of biological material, obtaining amplifiable DNA is often difficult. This pilot study aimed to map the effect of the chemicals used in the tanning process on the degradation of the DNA yielded from such samples. The DNA was quantified using two different approaches: qPCR and Qubit fluorometry. The degree of DNA fragmentation was assessed by determining the degradation index. The results indicate that reagents containing chromium have the greatest influence on DNA degradation. However, by using the presented protocol, enough amplifiable DNA can be obtained from hides treated with aluminum-based reagents.

Progress in forensic bone DNA analysis: Lessons learned from ancient DNA.

Research on ancient and forensic DNA is related in many ways, and the two fields must deal with similar obstacles. Therefore, communication between these two communities has the potential to improve results in both research fields. Here, we present the insights gained in the ancient DNA community with regard to analyzing DNA from aged skeletal material and the potential use of the developed protocols in forensic work. We discuss the various steps, from choosing samples for DNA extraction to deciding between classical PCR amplification and massively parallel sequencing approaches. Based on the progress made in ancient DNA analyses combined with the requirements of forensic work, we suggest that there is substantial potential for incorporating ancient DNA approaches into forensic protocols, a process that has already begun to a considerable extent. However, taking full advantage of the experiences gained from ancient DNA work will require comparative studies by the forensic DNA community to tailor the methods developed for ancient samples to the specific needs of forensic studies and case work. If successful, in our view, the benefits for both communities would be considerable.

Enhanced interrogation of degraded DNA from human skeletal remains: Increased genetic data recovery using the expanded CODIS loci, multiple sex determination markers, and consensus testing.

Skeletal remains are among the most difficult types of samples encountered in forensic DNA casework and historical investigations due to prolonged exposure to environmental insults. DNA extracted from bone often is degraded, in low quantities, and contains co-purified inhibitors from the surrounding soil and/or burial vault material. When sexually dimorphic skeletal elements are not recovered, determining the sex of a decedent can be challenging. With unidentified human skeletal remains, genetic data often are evaluated in concert with anthropological analyses, as well as other types of metadata, to improve confidence in making associations or for positive identifications. This study evaluated a multi-faceted molecular genetic approach to increasing the amount of data that can be recovered from degraded skeletal remains. Results demonstrate that using a newer-generation multiplex (GlobalFiler™) with an expanded set of highly discriminatory DNA markers - combined with co-amplification of three different sex-determining loci, one additional PCR cycle, and testing multiple cuttings from the same bone or multiple regions within a skeleton - can improve reliability and accuracy in skeletal remains identifications by providing data concordance.

Improved Y-STR typing for disaster victim identification, missing persons investigations, and historical human skeletal remains.

Bones are a valuable source of DNA in forensic, anthropological, and archaeological investigations. There are a number of scenarios in which the only samples available for testing are highly degraded and/or skeletonized. Often it is necessary to perform more than one type of marker analysis on such samples in order to compile sufficient data for identification. Lineage markers, such as Y-STRs and mitochondrial DNA (mtDNA), represent important systems to complement autosomal DNA markers and anthropological metadata in making associations between unidentified remains and living relatives or for characterization of the remains for historical and archaeological studies. In this comparative study, Y-STR typing with both Yfiler™ and Yfiler™ Plus (Thermo Fisher Scientific, Waltham, MA, USA) was performed on a variety of human skeletal remains, including samples from the American Civil War (1861-1865), the late nineteenth century gold rush era in Deadwood, SD, USA (1874-1877), the Seven Years' War (1756-1763), a seventeenth-century archaeological site in Raspenava, Bohemia (Czech Republic), and World War II (1939-1945). The skeletal remains used for this study were recovered from a wide range of environmental conditions and were extracted using several common methods. Regardless of the DNA extraction method used and the age/condition of the remains, 22 out of 24 bone samples yielded a greater number of alleles using the Yfiler™ Plus kit compared to the Yfiler™ kit using the same quantity of input DNA. There was no discernable correlation with the degradation index values for these samples. Overall, the efficacy of the Yfiler™ Plus assay was demonstrated on degraded DNA from skeletal remains. Yfiler™ Plus increases the discriminatory power over the previous generation multiplex due to the larger set of Y-STR markers available for analysis and buffer modifications with the newer version kit. Increased haplotype resolution is provided to infer or refute putative genetic relationships.

Results of a collaborative study on DNA identification of aged bone samples.

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.

Forensic genetic analyses in isolated populations with examples of central European Valachs and Roma.

Isolated populations present a constant threat to the correctness of forensic genetic casework. In this review article we present several examples of how analyzing samples from isolated populations can bias the results of the forensic statistics and analyses. We select our examples from isolated populations from central and southeastern Europe, namely the Valachs and the European Roma. We also provide the reader with general strategies and principles to improve the laboratory practice (best practice) and reporting of samples from supposedly isolated populations. These include reporting the precise population data used for computing the forensic statistics, using the appropriate θ correction factor for calculating allele frequencies, typing ancestry informative markers in samples of unknown or uncertain ethnicity and establishing ethnic-specific forensic databases.

Decontamination by Persteril 36 may affect the reliability of DNA-based detection of biological warfare agents-short communication.

Persteril 36 is a disinfectant with a broad spectrum of antimicrobial activity. Because of its bactericidal, virucidal, fungicidal, and sporicidal effectiveness, it is used as a disinfectant against biological warfare agents in the emergency and army services. In case of an attack with potentially harmful biological agents, a person's gear or afflicted skin is sprayed with a diluted solution of Persteril 36 as a precaution. Subsequently, the remains of the biological agents are analyzed. However, the question remains concerning whether DNA can be successfully analyzed from Persteril 36-treated dead bacterial cells. Spore-forming Bacillus subtilis and Gram-negative Pseudomonas aeruginosa and Xanthomonas campestris were splattered on a camouflage suit and treated with 2 or 0.2 % Persteril 36. After the disinfectant vaporized, the bacterial DNA was extracted and quantified by real-time PCR. A sufficient amount of DNA was recovered for downstream analysis only in the case of spore-forming B. subtilis treated with a 0.2 % solution of Persteril 36. The bacterial DNA was almost completely destroyed in Gram-negative bacteria or after treatment with the more concentrated solution in B. subtilis. This phenomenon can lead to false-negative results during the identification of harmful microorganisms.

Technical note: Efficient removal of a PCR inhibitory agent (vivianite) found on excavated bones.

During a rescue excavation in October 2011, archaeologists discovered a mass grave with 10 individuals. The skeletons should belong to victims of the battle of Reichenberg between the Austrian and Prussian armies on April 21, 1757. Several bones of the skeletons were covered with a blue colored encrustation. Initial DNA analysis failed due to strong inhibition. Chemical analysis of the bluish encrustation indicated the presence of the iron phosphate mineral vivianite (Fe3(PO4)2·(H2O)8). This technical note describes a novel procedure for the removal of this inhibitory substance.

Evaluation of 14 Y-chromosomal short tandem repeat haplotype with focus on DYS449, DYS456, and DYS458: Czech population sample.

AIM: To evaluate the novel triplex polymerase chain reaction (PCR) assay for the analysis of polymorphic Y-chromosomal short tandem repeat loci (Y-STR). METHODS: A total of 14 Y-STR loci was analyzed. Allele frequencies for 3 tetrameric Y-STR loci (DYS449, DYS456, and DYS458) and extended haplotype loci typed by Y-PLEXTM 12 system were investigated in a sample of 50 unrelated healthy Czech male donors. We computed the relevant intra-population statistic parameters for our data (gene diversity, average gene diversity over loci, and mean number of pairwise differences) and compared our sample set with other Central European populations using RST pairwise genetic distance. RESULTS: We focused on the comparison of genetic diversity between the Y-STR extended haplotype loci and that of the 3 additional loci, and on the benefit of using DYS449, DYS456, and DYS458 in forensic and population genetics applications. Total gene diversity in our sample set was 0.998367 when using all 14 loci. Our data analysis revealed very high genetic diversity at DYS449 locus (0.876735), which surpasses even the diversity at DYS385a/b (0.819592). Population comparison showed no difference between Czech, Bavarian, Austrian, and Saxon sample set. A minor difference was found between Czech and Polish sample set. CONCLUSION: Typing of 3 Y-chromosomal microsatellite polymorphisms may provide a useful complement to already established sets of Y-STRs.

Kinship and Y-chromosome analysis of 7th century human remains: novel DNA extraction and typing procedure for ancient material.

AIM: To develop novel DNA extraction and typing procedure for DNA identification of the 7th century human remains, determine the familiar relationship between the individuals, estimate the Y-chromosome haplogroup, and compare the Y-chromosome haplotype with the contemporary populations. METHODS: DNA from preserved femur samples was extracted using the modified silica-based extraction technique. Polymerase chain reaction amplification was performed using human identification kits MiniFiler, Identifiler, and Y-filer and also laboratory-developed and validated Y-chromosome short tandem repeat (STR) pentaplexes with short amplicons. RESULTS: For 244A, 244B, 244C samples, full autosomal DNA profiles (15 STR markers and Amelogenin) and for 244D, 244E, 244F samples, MiniFiler profiles were produced. Y-chromosome haplotypes consisting of up to 24 STR markers were determined and used to predict the Y-chromosome haplogroups and compare the resulting haplotypes with the current population. Samples 244A, 244B, 244C, and 244D belong to Y-chromosome haplogroup R1b and the samples 244E and 244F to haplogroup G2a. Comparison of ancient haplotypes with the current population yielded numerous close matches with genetic distance below 2. CONCLUSION: Application of forensic genetics in archaeology enables retrieving new types of information and helps in data interpretation. The number of successfully typed autosomal and Y-STR loci from ancient specimens in this study is one of the largest published so far for aged samples.

Highly effective DNA extraction method for nuclear short tandem repeat testing of skeletal remains from mass graves.

AIM: To quantitatively compare a silica extraction method with a commonly used phenol/chloroform extraction method for DNA analysis of specimens exhumed from mass graves. METHODS: DNA was extracted from twenty randomly chosen femur samples, using the International Commission on Missing Persons (ICMP) silica method, based on Qiagen Blood Maxi Kit, and compared with the DNA extracted by the standard phenol/chloroform-based method. The efficacy of extraction methods was compared by real time polymerase chain reaction (PCR) to measure DNA quantity and the presence of inhibitors and by amplification with the PowerPlex 16 (PP16) multiplex nuclear short tandem repeat (STR) kit. RESULTS: DNA quantification results showed that the silica-based method extracted on average 1.94 ng of DNA per gram of bone (range 0.25-9.58 ng/g), compared with only 0.68 ng/g by the organic method extracted (range 0.0016-4.4880 ng/g). Inhibition tests showed that there were on average significantly lower levels of PCR inhibitors in DNA isolated by the organic method. When amplified with PP16, all samples extracted by silica-based method produced 16 full loci profiles, while only 75% of the DNA extracts obtained by organic technique amplified 16 loci profiles. CONCLUSIONS: The silica-based extraction method showed better results in nuclear STR typing from degraded bone samples than a commonly used phenol/chloroform method.

STR analysis of artificially degraded DNA-results of a collaborative European exercise.

Degradation of human DNA extracted from forensic stains is, in most cases, the result of a natural process due to the exposure of the stain samples to the environment. Experiences with degraded DNA from casework samples show that every sample may exhibit different properties in this respect, and that it is difficult to systematically assess the performance of routinely used typing systems for the analysis of degraded DNA samples. Using a batch of artificially degraded DNA with an average fragment size of approx. 200 bp a collaborative exercise was carried out among 38 forensic laboratories from 17 European countries. The results were assessed according to correct allele detection, peak height and balance as well as the occurrence of artefacts. A number of common problems were identified based on these results such as strong peak imbalance in heterozygous genotypes for the larger short tandem repeat (STR) fragments after increased PCR cycle numbers, artefact signals and allelic drop-out. Based on the observations, strategies are discussed to overcome these problems. The strategies include careful balancing of the amount of template DNA and the PCR cycle numbers, the reaction volume and the amount of Taq polymerase. Furthermore, a careful evaluation of the results of the fragment analysis and of automated allele calling is necessary to identify the correct alleles and avoid artefacts.