Intra‐ and Inter‐Element Variability in Mitochondrial and Nuclear DNA from Fresh and Environmentally Exposed Skeletal Remains,

Successful identification of skeletonized remains often relies upon DNA analyses, frequently focusing on the mid‐diaphysis of weight‐bearing long bones. This study explored intra‐bone DNA variability using bovine and porcine femora, along with calcanei and tali. DNA from fresh and short‐term environmentally exposed bone was extracted utilizing demineralization and standard lysis buffer protocols, and DNA quantity and quality were measured. Overall, femoral epiphyses, metaphyses, and the tarsals had more nuclear and mitochondrial DNA than did the femoral diaphyses. DNA loss was much more rapid in buried bones than in surface exposed bones, while DNA quality differed based on environment, but not bone region/element. The demineralization protocol generated more DNA in some bone regions, while the standard lysis was more effective in others, and neither significantly affected DNA quality. Taken together, these findings reinforce the importance of considering inter‐ and intra‐bone heterogeneity when sampling skeletal material for forensic DNA‐based identifications.     

Dealing with low amounts of degraded DNA: Evaluation of SNP typing of challenging forensic samples by using massive parallel sequencing

A set of eighty-two forensic samples with different levels of degradation, as well five in vitro damaged samples were analyzed by the Precision ID Identity Panel. PCR amplifications were performed with scalar amount of DNA (from 1 ng to 12 pg) and through different number of cycles. A minimum coverage of 50 x was adopted for “locus call”. Very informative profiles (based on about 65–70% of the loci) were obtained even in highly degraded samples when the amount of template range from 0.1 to 1.0 ng. When dealing with low amount of degraded DNAs, no more than half of the loci were typed, and the risk of mistyping (due to drop out phenomena) increased dramatically. The employment of a high number of PCR cycles is discussed.     

Development of a forensic DNA phenotyping panel using massive parallel sequencing

The HIrisPlex-S system, targeting a total of 41 SNPs, allows the simultaneous eye, hair and skin color prediction from DNA. In the present study, we developed a massive parallel sequencing (MPS) multiplex assay in order to genotype all the HIrisPlex-S markers in degraded casework samples. PCR amplicons sizes of target regions were kept below 180 bp, in order to allow analysis of degraded DNA samples. Individuals with known phenotype, artificially degraded DNA samples and a set of 2800M control DNA dilutions were sequenced on a Ion PGM System, in order to evaluate the concordance testing results and the forensic suitability of this 41-plex MPS assay. Full and reliable profiles could be obtained with 0.1 ng of input DNA. The increment of the number of PCR cycles results in improvement of sensitivity or in typing results but an increase of artifacts were also observed.     

Evaluation of five preservation methods for recovery of DNA from bone

The effectiveness of five methods for the preservation of bone were assessed: cell lysis solution (with 1% sodium azide), dehydration/freeze-drying, ethanol (96%), freezing, and room temperature storage. Preserved bone samples were extracted using five optimised extraction methods. These preservation methods were tested for their efficiency for storage of fresh and degraded bone samples for 6 weeks, 6 months and 1 year. Freezing was found to be the best preservation method for longer-term storage of bone samples; this was followed by ethanol (96%), dehydration/freeze-drying, and room temperature storage. Full profiles were obtained from bone samples using all these preservation methods.     

Validation of Reduced Reagent Volumes in the Implementation of the Quantifiler® Trio Quantification Kit

The Quantifiler^® Trio Quantification Kit has been developed to quantify the total amount of amplifiable and human male DNA in samples and to estimate the extent of DNA degradation. To minimize the cost of DNA quantification, we evaluated kit performance using a reduced volume of reagents (1/10‐volume) using DNA samples of varying types and concentrations. Our results demonstrated concordance between the manufacturer's method and the low‐volume method for DNA quantification, DNA degradation index estimation, and human male DNA quantification. We confirmed the practical utility of the low‐volume method with 109 casework samples by evaluating short tandem repeat (STR) profiling success with respect to DNA quantity and quality. We also defined a cutoff value for DNA quantity to ensure reliable STR results. Using a reduced volume of reagents, 10 times more reactions per kit are possible; accordingly, this method reduces the cost of DNA quantification, while maintaining performance.     

海事安全目标导向型公约:生成发展与我国应对

基于海事安全风险管控从"经验公式""二元并存"向"技术标准""一元主导"的转变,海事安全公约未来发展方向将由"描述型"转为"目标导向型"。海事安全目标导向型公约的创制技术包括分层立法框架、条款背离和等效替代等方面,从而对相关法律规范的表述模式、相关法律关系的具体面向和各缔约国履约责任的逻辑路径等带来深刻影响。作为正在从海事大国迈向海事强国的我国来说,应对海事安全目标导向型公约及其创制技术的发展,需要进一步提升船舶建造技术标准规范创制水平,积极抢占标准战略竞争的制高点;更新海事安全法律规范的创制理念,构建更具灵活性、主动性的立法模式;完善海事履约审核体系和各构成要素,确保我国的海事安全目标导向型公约履行义务的全面实现。     

A novel FTA™ elute card collection method that improves direct DNA amplification from bloodstained concrete

Concrete is a common construction material found in residential and commercial buildings, bridges and parking lots that is a composite matrix containing aggregate held together with cement. The porous nature of concrete can make the collection and genotyping of biological fluids, such as blood, challenging. Forensic evidence can become embedded within the matrix, potentially reducing the amount of DNA available for analysis. In forensic science, “direct” amplification refers to a genotyping method that amplifies a DNA profile directly from a sample without DNA extraction, saving time and money. We investigated a novel application of Whatman? FTA? Elute cards in their ability to directly amplify PowerPlex® Fusion and Y23 profiles from minute amounts of blood that had been deposited on different concrete structures. In comparison to traditional collection methods, directly profiling blood stained construction materials using FTA? Elute cards increased the percentage loci amplified and significantly improved both allele peak height and peak height ratio while reducing allelic drop-out. FTA? Elute cards can provide a reliable, inexpensive and superior alternative to traditional methods.     

样品成份对“咖啡环”法DNA检测的影响

目的调查样品中常见成份对"咖啡环"法DNA检测的影响。方法将含有不同浓度SDS、Na Cl等的DNA-EB滴加到载玻片上,室温自然蒸发,用凝胶成像系统观察荧光沉积形貌(Deposition Pattern,DP)、计算积分光密度(Integrated Optical Density,IOD),分析量效关系。结果低浓度非DNA成份存在时,DP仍以环状沉积为主,但有年轮环、放射纹等成份特异性细微差异。浓度高时,DP因成份而异,可为环+散在光点型(Na Cl)、中央光斑+弱小环型(SDS)、同心或年轮状多环型(Triton X-100)、环+斑点型(H^+)等。非DNA成份对DNA含量估计有不同程度影响,但估计值多数在0.5~2倍以内。结论样品成份对DNA的DP和IOD均有影响,但不影响DNA含量的粗略估计,且能提供样品纯度和残留物信息。     

An Optimized DNA Analysis Workflow for the Sampling,Extraction, and Concentration of DNA obtained from Archived Latent Fingerprints

DNA profiles have been obtained from fingerprints, but there is limited knowledge regarding DNA analysis from archived latent fingerprints—touch DNA “sandwiched” between adhesive and paper. Thus, this study sought to comparatively analyze a variety of collection and analytical methods in an effort to seek an optimized workflow for this specific sample type. Untreated and treated archived latent fingerprints were utilized to compare different biological sampling techniques, swab diluents, DNA extraction systems, DNA concentration practices, and post‐amplification purification methods. Archived latent fingerprints disassembled and sampled via direct cutting, followed by DNA extracted using the QIAamp® DNA Investigator Kit, and concentration with Centri‐Sep? columns increased the odds of obtaining an STR profile. Using the recommended DNA workflow, 9 of the 10 samples provided STR profiles, which included 7–100% of the expected STR alleles and two full profiles. Thus, with carefully selected procedures, archived latent fingerprints can be a viable DNA source for criminal investigations including cold/postconviction cases.