植物DNA检验技术在命案现场重建中的应用策略

植物DNA检验技术是利用植物遗传性状的稳态性对关联植物物证进行检验鉴定的手段。将该技术应用于现场重建,应基于植物物证与犯罪嫌疑人、被害人及其活动环境具有＂重大关联性＂。从命案现场重建的需求上看,应围绕犯罪嫌疑人及其可控物品中附着植物与现场植物的种属同一性判断、被害人尸体（尸块或尸骸）及其随附物品中附着植物与中心现场植物的种属同一性判断、疑似侵害物及其附着植物与嫌疑人行为关联植物的种属同一性判断等三个角度或层次进行检验和综合分析。植物DNA检验技术可阐明物证的时空运行停顿规律,为命案现场重建工作提供一种参考性解决方案。     

Development of an instrument for assessing the quality of forensic evidence and expert testimony from three feature-comparison methods: DNA,voice, and fingerprint analysis

In a context of rising violence and long-lasting impunity, in 2008, Mexico's criminal justice system underwent a radical change from an inquisitorial model to an adversarial one, to make it more effective, transparent, and expeditious. The new system tasked judges with publicly determining the admissibility of forensic evidence, as well as assessing its technical quality and probative value—tasks for which they currently receive little to no training. With the aim of contributing to the consolidation of the adversarial model, a comparative framework—in the form of a checklist—of the analysis of fingerprints, DNA samples, and voice recordings was created. To do so, a review of the academic literature, published reports, and guidelines was performed. The collected data were synthesized and submitted to a panel of Mexican judges, who provided feedback on its adequacy. The framework focuses on the steps on which quality assurance of forensic evidence depends, organized in five discrete stages that span from the collection of samples at the scene of a crime to the presentation of evidence at trial, specifying the main technical criteria experts should state to allow a decision maker to examine its accuracy and reliability. Differences and commonalities among the three methods were identified, particularly in terms of how qualitative and quantitative analyses are performed in each. Besides its potential usefulness as an aid for judicial decision-making, the checklist could be a valuable resource for training programs aimed at judges, as well as quality assurance programs.     

Start-up of the criminal genetic database in Mendoza,Argentina

The usefulness of DNA databases is widely known and demonstrated. After the successful experiences of the UK and the USA the creation of databases increased rapidly around the world. In Latin America the implementation was slower and more problematic, with Chile and Uruguay being the first to implement them. In Argentina the problems were greater and more persistent.Although the lack of legislation or applicable laws is a generalized problem, the most difficult one to overcome was the lack of decision, interest and resources by those responsible at an institutional level.In 2016, Mendoza province modified its database law by creating the “Registro Provincial de Huellas Genéticas Digitalizadas” which allowed the process of construction and consolidation to begin. From January 2017 all prisoners, convicted and imputed of all types of crimes began to be sampled. This made the database to grow rapidly, reaching 13.821 samples in that year.During 2018, in addition to the daily imputed individuals, we began with the sampling from all the Mendoza Police Department, including the Scientific Police that deals with the crime scene. At present the database has a total of 40.652 individuals.In August 2018, the FBI's CODIS system was installed, and later the data loading process began. In 12 months we have reached 87 match or hits of which 46 correspond to sexual assault, 17 to robbery, 16 to homicides and the rest to other cases. Given that within the sexual abuse cases we were able to identify several serial sexual offenders, the 87 hits allowed clarifying 174 criminal cases.These results reaffirm the potential of the databases and gives a light of hope for victims of crime.In this work we present the advances and challenges that we faced in a chronological order.     

Assessing the performance of quantity and quality metrics using the QIAGEN Investigator® Quantiplex® pro RGQ kit

The Quantiplex® Pro RGQ kit quantifies DNA in a sample, supports the detection of mixtures and assesses the extent of DNA degradation based on relative ratios of amplified autosomal and male markers. Data show no significant difference in the accuracy and sensitivity of quantification between this and the Promega PowerQuant® System, both detecting the lowest amount of DNA tested, 4 pg. Laboratory controlled mixed male:female DNA samples together with mock sexual assault samples were quantified across a range of mixture ratios. Analysis software detected mixed DNA samples across all ratios for both quantification kits. Subsequent STR analysis using the Investigator® 24Plex QS Kit was able to corroborate mixture detection down to 1:25 male:female DNA ratios, past which point mixtures appeared identical to single-source female samples. Analysis software also detected laboratory degraded DNA samples, with data showing a positive trend between the Degradation Index (DI) and length of time of sonication. When used on ancient remains the assay was able to triage samples for further analysis, and STR profiles were concordant with DNA quantification results in all instances. STR analyses of laboratory-controlled sensitivity, mixture, and degradation studies supports the quality metric obtained from quantification. These data support the use of the Quantiplex® Pro RGQ kit for sample screening and quantification in forensic casework and ancient DNA studies.     

“UNUSUAL” TISSUES AND SAMPLE COLLECTION STRATEGIES ON EXHUMED BODIES

The choice of soft or hard tissues to be sampled in case of exhumation of corpses for identification purposes or family relationship testing is based on the degradation conditions of the corpse: the more the corpse is degraded, the less DNA is expected to be retrieved from soft tissue. Therefore, the choice of the "best" tissue samples usually falls on teeth and bones in these “difficult” cases, even though the DNA extraction procedure requires time and effort and it can often result in unexpected, negative results.We here present the results of a daily practice survey that shows that it is possible to obtain good results even on DNA extracted from tissues that appear to be less “appealing” to the examiner by performing “simple” corneal/scleral swabs along with cartilage.While DNA extracted from cartilage has been already described, to our knowledge there is no evidence of publications in the scientific literature dealing with cornea/sclera as a source of DNA in the forensic laboratory.The obtained results demonstrate that it may be advisable to consider other tissues which bear the potential of returning good profile results despite not appearing particularly useful and better control of contamination.     

Towards molecular autopsies: Development of a FFPE tissue DNA extraction workflow

Sudden unexpected death (SUD) is a devastating event and forms a substantial proportion of the cases investigated at forensic mortuaries each year. Despite post-mortem investigations, the cause of death may remain undetermined. There is potential for these unresolved cases to benefit from retrospective molecular autopsies for investigation into genetic mutations which may have contributed towards death. Often, formalin fixed paraffin embedded tissues (FFPET) are the only archival sources of DNA available for retrospective analyses. However, extracting usable DNA from FFPET is challenging as current methods yield poor quality and quantity DNA. Thus, this study aimed to optimise DNA recovery from FFPET by investigating several variables within the DNA extraction workflow, including the selection of tissue type, number and thickness of tissue sections, deparaffinisation method, and DNA extraction kit. The quantity and quality of DNA recovered were assessed using spectrophotometry, real time PCR, digital capillary electrophoresis and DNA profiling. This study was the first to implement a nuclei quantification using microscopy to guide the selection of the best tissue type to use for DNA analysis. The use of a greater number of thinner tissue sections (100 sections, each 1 μm) significantly improved DNA concentration, purity and fragment length. Additionally, the combination of Deparaffinization Solution with the QIAamp® DNA FFPE Tissue Kit proved most favourable with a median DNA yield of 320 ng and 55% of DNA fragments greater than 400 bp. Isolated DNA was of single source, indicating no contamination in the workflow, and FFPET blocks that were stored for up to 3.5 years did not significantly affect DNA degradation (p = 0.1764). These results are especially informative for designing library preparation and sequencing workflows for determining cause of death in unresolved SUD cases.     

Internal validation of reduced PCR reaction volume of the Qiagen Investigator® Argus X-12 QS Kit from blood samples on FTA cards

The onus of proof in criminal cases is beyond any reasonable doubt, and the issue on the lack of complete internal validation data can be manipulated when it comes to justifying the validity and reliability of the X-chromosomal short tandem repeats analysis for court representation. Therefore, this research evaluated the efficiency of the optimized 60% reduced volumes for polymerase chain reaction (PCR) amplification using the Qiagen Investigator® Argus X-12 QS Kit, as well as the capillary electrophoresis (CE) sample preparation for blood samples on Flinder's Technology Associates (FTA) cards. Good-quality DNA profile (3000–12,000 RFU) from the purified blood sample on FTA card (1.2 mm) were obtained using the optimized PCR (10.0 μL of PCR reaction volume and 21 cycles) and CE (9.0 μL Hi-Di™ Formamide and 0.3 μL DNA Size Standard 550 [BTO] and 27 s injection time) conditions. The analytical and stochastic thresholds were 100 and 200 RFU, respectively. Hence, the internal validation data supported the use of the optimized 60% reduced PCR amplification reaction volume of the Qiagen Investigator® Argus X-12 QS Kit as well as the CE sample preparation for producing reliable DNA profiles that comply with the quality assurance standards for forensic DNA testing laboratories, while optimizing the analytical cost.     

From clean spaces to crime scenes: Exploring trace DNA recovery from titania-coated self-cleaning substrates

Titanium dioxide (titania, TiO₂) is frequently used as a coating for a variety of self-cleaning products, such as antifogging vehicle mirrors, ceramic tiles, and glass windows because of its distinct physiochemical features. When exposed to light TiO₂ causes photocatalytic decomposition of organic contaminants, potentially compromising DNA integrity. The impact of TiO₂-coated commercial glasses, Bioclean® and SaniTise™, on trace DNA persistence, recovery, and profiling was investigated. DNA in saliva and touch samples deposited on self-cleaning glass slides exposed to indoor fluorescent light for up to seven days was more degraded than control samples indicating some degree of fluorescent light-induced photocatalytic activity of the self-cleaning surfaces. When exposed to sunlight, DNA yields from saliva and touch samples deposited on the titania-coated substrates decreased rapidly, with a corresponding increase in DNA degradation. After three days no DNA samples applied to self-cleaning glass and exposed to natural sunlight yielded STR profiles. These results suggest that the photocatalytic activation of TiO₂ is the likely mechanism of action underlying the extreme DNA degradation on the Bioclean® and SaniTise™ glasses. Consequently, rapid sample collection and use may be warranted in casework scenarios involving TiO₂-coated materials.     

Identification and Individualization of Lophophora using DNA Analysis of the trnL/trnF Region and rbcL Gene

Lophophora williamsii (peyote) is a small, spineless, greenish‐blue cactus found in Mexico and the southwestern United States. Ingestion of the cactus can result in hallucinations due to its content of mescaline. In the United States, L. williamsii is classified as a Schedule I controlled substance. In this study, we use DNA analysis of the chloroplast trnL/trnF region and chloroplast rbcL gene to identify the individuals of Lophophora. Using the rbcL gene, Lophophora specimens could be distinguished from outgroups, but species within the genus could not be distinguished. The trnL/trnF region split the Lophophora genus into several groups based on the length and substructure of an AT‐rich segment of the sequence. Our results indicate that the genetic variability at the trnL/trnF locus is greater than previously recognized. Although DNA structures at the trnL/trnF region and rbcL gene do not align with the classification of Lophophora species, they can be used to aid in forensic analysis.