Development of DNA Methylation Markers to Infer Age, Smoking Status and Body Fluid Types for Forensic Application

In forensic investigations, biological evidences have great potential to place a suspect at the scene of a crime. However, in many cases suspect(s) usually can’t be identified with any local or national databases. In such challenging cases, police need a tool that can provide investigative leads. Thus, different genetic biomarkers including DNA methylation markers have been developed for different phenotypic traits to serve as source of intelligence information to investigators in cases of unknown DNA profiles. In this project, novel sets of DNA methylation markers were developed for the forensic estimation of human age, determining the smoking status and body fluid identification. First, single- and dual-locus age predictors for saliva and blood were developed from CpG sites in KLF14 and SCGN based on pyrosequencing data and using a multivariate linear regression analysis. In saliva, single-locus model in particular was efficient for younger subjects (³40 years) correctly predicting age of 78.9% of the samples with a MAD of 5.1 years in the validation set. Second, a quick and cost effective 4-CpG pyrosequencing assay was built to infer smoking habit using multinomial logistic regression model (MLR). In blood, the model correctly predicted 90.0% of current smokers, 66.7% of former smokers, and 84.9% of never smokers. In addition, the MLR model correctly predicted 86.9% of current smokers, 54.5% of former smokers, and 77.8% of never smokers in saliva. Finally, new set of tissue specific differentially methylated region (tDMRs) were identified for forensic discrimination of body fluids. NMUR2 and UBE2U markers were found to be specific for semen and the two assays developed can be employed using both pyrosequencing or High resolution melt (HRM) analysis. In addition, the developed AHRR marker can discriminate blood stains from other body fluids. These three piece of information may be very valuable source of investigative leads aiming to trace unknown perpetrators who could not be identified using the conventional autosomal DNA typing

Optimization of the use of PrepFiler Express™ Forensic DNA extraction kit lysis buffer discard for mRNA profiling

mRNA profiling for body fluid or tissue type identification is able to provide circumstantial information alongside DNA profiles from biological material found at a crime scene. To solve the challenge of material limitation in body fluid identification, studies have successfully developed mRNA profiles of RNA extracted from lysis buffer discard of DNA-extraction. It would be interesting to examine whether this method is possible for other combinations of DNA- and RNA-isolation kits. Promising results were found for RNA quantity and quality measured in the DNA-extraction lysis buffer discard and DNA-eluate from body fluids extracted with the PrepFiler Express™ DNA Extraction system. The protocols of three RNA-isolation kits were optimized for extraction from Prepfiler™ lysis buffer discard, and the kits were compared in terms of RNA quantity and quality. The mirVana™ kit was found to be most suited for this purpose. Although the ReliaPrep™ RNA Miniprep kit had similar results for quantity and quality, a continuous problem was contamination of genomic DNA. The Direct-zol™ RNA Miniprep kit gave poor results and was not further examined. A preliminary singleplex of body fluid specific mRNA markers and housekeeping genes, using different PCR programs was performed on RNA extracted from pure body fluids, and products were separated by capillary electrophoresis. An overload of the system was observed for most of the body fluid markers. Primer tests should be rerun with less RNA, e.g. RNA extracted from lysis buffer discard instead of pure body fluids, before further optimization

Effects of microwave radiation on the digestion of proteins involved in body fluid identification

Body fluid identification plays an important role in understanding how the events during a crime may have taken place. The presence of a body fluid may help identify an individual who committed a crime, while the type of body fluid present may help investigators determine how a crime occurred. Current body fluid identification techniques are not always conclusive and may only suggest the presence of a body fluid type. A protocol developed by the New York City Office of the Chief Medical Examiner (OCME) established a method that confirms body fluid type through mass spectrometry. To identify marker proteins in a body fluid sample, the proteins in the sample must be digested into their smaller peptide fragments. Currently, this digestion is performed with trypsin in an overnight process that takes at least eight hours to complete. Microwave radiation has been shown to assist in the digestion of the body fluid protein samples, and the time needed for digestion reduced from eight hours to just minutes. The effects of microwave radiation on the digestion of protein samples commonly found in body fluids were examined here and compared to traditional methods. Ideal microwave assisted temperature of protein samples was determined to be 37°C, however no significant differences were observed between microwave assisted digestion and the traditional methods of digestion at different incubation times. Overall, this research shows microwave radiation can be used to assist protein digestion, achieving similar results to traditional digestion methods at 37°C

Current and future trends in the laboratory diagnosis of sexually transmitted infections

Sexually transmitted infections (STIs) continue to exert a considerable public health and social burden globally, particularly for developing countries. Due to the high prevalence of asymptomatic infections and the limitations of symptom-based (syndromic) diagnosis, confirmation of infection using laboratory tools is essential to choose the most appropriate course of treatment and to screen at-risk groups. Numerous laboratory tests and platforms have been developed for gonorrhea, chlamydia, syphilis, trichomoniasis, genital mycoplasmas, herpesviruses, and human papillomavirus. Point-of-care testing is now a possibility, and microfluidic and high-throughput omics technologies promise to revolutionize the diagnosis of STIs. The scope of this paper is to provide an updated overview of the current laboratory diagnostic tools for these infections, highlighting their advantages, limitations, and point-of-care adaptability. The diagnostic applicability of the latest molecular and biochemical approaches is also discussed

DNA Transfer in Forensic Science: Recent Progress towards Meeting Challenges.

Understanding the factors that may impact the transfer, persistence, prevalence and recovery of DNA (DNA-TPPR), and the availability of data to assign probabilities to DNA quantities and profile types being obtained given particular scenarios and circumstances, is paramount when performing, and giving guidance on, evaluations of DNA findings given activity level propositions (activity level evaluations). In late 2018 and early 2019, three major reviews were published on aspects of DNA-TPPR, with each advocating the need for further research and other actions to support the conduct of DNA-related activity level evaluations. Here, we look at how challenges are being met, primarily by providing a synopsis of DNA-TPPR-related articles published since the conduct of these reviews and briefly exploring some of the actions taken by industry stakeholders towards addressing identified gaps. Much has been carried out in recent years, and efforts continue, to meet the challenges to continually improve the capacity of forensic experts to provide the guidance sought by the judiciary with respect to the transfer of DNA

Selection of highly specific and sensitive mRNA biomarkers for the identification of blood

In the present work, we have evaluated eight reportedly blood-specific mRNA markers (HBB, HBA, ALAS2, CD3G, ANK1, PBGD, SPTB, AQP9) in an attempt to determine the most suitable ones for use in forensic applications based on their sensitivities, specificities and performance with casework samples. While varying levels of expression were observed, all markers were relatively sensitive requiring as little as 1 ng of RNA input into the reverse transcription (RT) reaction. In singleplex reactions, seven of the eight analyzed blood markers (all except AQP9) demonstrated a high degree of specificity for blood. In multiplex reactions, non-reproducible cross-reactivity was observed for several of the mRNA markers, which was reduced and, in most cases, eliminated when less input total RNA was used. Additionally, some cross-reactivity was observed with tissue and animal samples. Despite differences in the observed sensitivity and specificity of the blood markers examined in this study, a number of the candidates appear to be suitable for inclusion in appropriately validated multiplex mRNA-based body fluid identification systems

Targeted-Ion Mass Spectrometry for the Identification of Forensically Relevant Biological Fluids and Samples from Sexual Assault Evidence

Forensic practitioners have long sought efficient and reliable means for identifying those samples that are best suited for successful genetic profiling. Traditional serological screening methodologies rely upon enzyme activity and antibody-based serological tests. These tests can be consumptive, laborious and costly while reliance on antibody-based serological testing can be prone to error. Positive results resulting from non-target biological fluids, the potential for cross- reactivity and non-specific binding events yield merely presumptive results. This has led forensic biologists to omit serological testing, at least in the case of sexual assault kit samples, in favor of Y-Screen assays. While these Y-Screen approaches achieve rapid screening of samples for the presence of a detectible male DNA, they do not provide any serological information and therefore lack critical investigative/biological context. A more sensitive and accurate technology for the confirmatory identification of biological fluids would greatly bolster the weight of serological evidence presented in court and assist with more informed sample prioritization. A particularly promising approach combines high- specificity protein biomarkers with a target-ion mass spectrometry. Applying absolute quantitation of protein targets in the biomarker panel will enable forensic practitioners to make fuller use of serological information in their decision making on downstream analyses in order to improve the successful analysis of challenging sexual assault samples. This research demonstrated the prevalence of false-positive results associated with antibody-based serological methods, developed and validated a multiplex targeted-ion mass spectrometry-based assay for the identification of six forensically relevant biological fluids, demonstrated improved sensitivity and specificity of mass-spectrometry based body fluid identification as compared to traditional techniques, developed a modified assay for seminal fluid compatible with sexual assault kit evidence and evaluated the relationship between quantitative levels of target seminal fluid peptides and the ability to generate Y-STR profiles. These results provide the forensic and criminal justice communities with a powerful tool to aid in the criminal investigation of violent crimes

Development and Testing of a Rapid Multiplex Assay for the Identification of Biological Stains

While DNA profiling makes it possible to individualize biological stains, the identification of the stain itself can present forensic serologists with a significant challenge. Current antibody- and enzyme activity-based assays used by forensic practitioners for biological stain identification yield only presumptive results. Positive results with non-target body fluids or cross-reactivity with non-human sources has also been well documented. Some tests can consume unacceptable quantities of precious evidence while for some body fluids (vaginal fluid and menstrual blood) there are simply no available tests at all. This research presented here aims to develop and rigorously test a fast, accurate, and sensitive multiplex assay for the simultaneous identification of saliva, semen, urine, peripheral blood, menstrual blood, and vaginal secretions. This research is based on the following three research phases: 1. Biomarker Identification – Utilizing multidimensional protein separation technologies, bioinformatics tool, and tandem mass spectrometry a database of fluid-specific candidate markers will be developed for each body fluid. 2. Biomarker Verification – Using the most promising candidates from Phase 1 a targeted multiplex assay will be developed on a Quadruple Time-of-Flight mass spectrometer to verify the specificity of these candidate biomarkers with single source laboratory samples as well as single and mixed source casework type samples. 3. Prototype Validation – Development and testing of a case working laboratory prototype assay on a triple quadrupole mass spectrometer using single and mixed source casework-type samples. This work has the potential to significantly improve the accuracy and sensitivity of forensic serological testing. It will provide practitioners with greatly improved tests for saliva and seminal fluid while also enabling the identification of vaginal secretions for which there are currently no accurate tests. The multiplex design will eliminate the need to perform separate tests on an unknown stain. In short, the successful completion and implementation of this research will provide the forensic community with a powerful tool to aid in criminal investigations