Forensic Molecular Biology Research Articles

Virtual Reality Immersive Simulations for a Forensic Molecular Biology Course—A Quantitative Comparative Study

Molecular biology is a complex, abstract, subject that can be challenging for higher education students to comprehend. The current manuscript describes the design, implementation, and evaluation of two immersive VR simulations of a DNA lab and a crime scene investigation (CSI) for a forensic molecular biology course in the context of the “TESLA” Erasmus+ project. It illustrates the instructional design and technical aspects of the VR simulations’ development. The experimental study employed a comparative quantitative research design. The guiding research questions examined how instructional modalities (online vs. face-to-face) affect learners’ perceptions of VR-based training in higher education and the key factors influencing learners’ intention for their adoption. Forty-six (n = 46) undergraduate students completed a 17-item questionnaire, which served as the main data collection instrument. Results demonstrate that both online and face-to-face VR-based instruction can effectively convey core concepts, thus challenging the traditional notion that face-to-face interaction is inherently superior. Its implications underscore the potential of VR simulations to supplement or even substitute traditional teaching methods, particularly for complex science subjects.

RNA Analysis in Forensic Molecular Biology.

Different types of RNA take on multiple crucial functions in living cells and tissues. Messenger RNA (mRNA) is a temporary molecular carrier of genetic information. Analysis of the composition of all mRNA contained in a cell at a given moment, the so-called transcriptome, enables the determination of the type of cell and its condition, e.g., in pathologically altered states. This review is based on pertinent publications retrieved by a selective literature search. The analysis of differential gene expression has already been used in forensic molecular biology to determine the type of tissue contained in biological specimens. It is also being used in criminal investigations to determine the composition of mixed traces of various bodily fluids and/or organ tissues. The method is limited by degradation of the mRNA molecules through environmental influences. The use of newly developed molecular biological methods such as massive parallel sequencing can expand the information obtainable by this investigative method. Current research also addresses the forensic potential of deriving relevant information about the crime-e.g., its timing, or the condition of the involved persons-from the totality of mRNA species present in the specimens. Forensic RNA analysis can yield a great deal of relevant information. It is likely to be applicable in a much wider variety of forensic situations in the near future.

TrACES of time: Transcriptomic analyses for the contextualization of evidential stains – Identification of RNA markers for estimating time-of-day of bloodstain deposition

Obtaining forensically relevant information beyond who deposited a biological stain on how and under which circumstances it was deposited is a question of increasing importance in forensic molecular biology. In the past few years, several studies have been produced on the potential of gene expression analysis to deliver relevant contextualizing information, e.g. on nature and condition of a stain as well as aspects of stain deposition timing. However, previous attempts to predict the time-of-day of sample deposition were all based on and thus limited by previously described diurnal oscillators. Herein, we newly approached this goal by applying current sequencing technologies and statistical methods to identify novel candidate markers for forensic time-of-day predictions from whole transcriptome analyses. To this purpose, we collected whole blood samples from ten individuals at eight different time points throughout the day, performed whole transcriptome sequencing and applied biostatistical algorithms to identify 81 mRNA markers with significantly differential expression as candidates to predict the time of day. In addition, we performed qPCR analysis to assess the characteristics of a subset of 13 candidate predictors in dried and aged blood stains. While we demonstrated the general possibility of using the selected candidate markers to predict time-of-day of sample deposition, we also observed notable variation between different donors and storage conditions, highlighting the relevance of employing accurate quantification methods in combination with robust normalization procedures.This study’s results are foundational and may be built upon when developing a targeted assay for time-of-day predictions from forensic blood samples in the future.

Introducing the New Editorial Team

Introducing the New Editorial Team

Blood and Saliva Identification through miRNA in Forensic Molecular Biology

A class of non-coding miRNAs has attracted a lot of interest in the field of forensic sciences. miRNA has a relatively small size thus it is quite stable to the external environmental pressures and factors, which makes it very useful in forensic examination when used as a bio marker. At present, many of the specific miRNAs of body fluids can be distinguished by measuring their level of expression. Fresh blood samples and saliva samples were taken from 3 males and 3 females. RNA was extracted using TRIzol method and cDNA synthesis was done. RT reactions were made using SYBR Green master mix for RT-qPCR for quantification and detection of miRNAs. The designed primers were runs against the sample in qPCR and result was seen and compiled. Two miRNA were detected and expressed in both blood and saliva but they failed to differentiate between blood and saliva due to the non-specific primer binding according the methodology used in this study. Hsa-miR20a and Hsa-miR583 have been found to be non-specific for the blood and saliva. No stark difference could be observed on the basis of which we can say that Hsa-miR20a and HsamiR583 were specific for blood and saliva due to non-specific primer binding. Further research work is required in this domain of body fluids identification and differentiation using miRNA markers.

Novel multiplex strategy for DNA methylation-based age prediction from small amounts of DNA via Pyrosequencing

DNA methylation-based age estimation is a promising new tool for forensic molecular biology. There is growing understanding of the best predictive CpG loci and their performance in various sample types. Since forensic samples usually provide only small amounts of DNA, the sensitivity of the method is crucial. Pyrosequencing is one of the most sensitive methods but only capable to analyze different target regions separately. Thus, multiple input DNA samples are required for investigations of different target regions, which is required for all current age estimation models. To overcome this limitation, we developed a novel multiplex strategy for Pyrosequencing, which allows the investigation of different target regions from a single small amount of input DNA. A pre-amplification step was introduced to increase the amount of target-specific template for the subsequent sequencing PCR step. We tested this multiplex strategy for eight target regions including 15 age CpGs associated with the genes of ELOVL2, FHL2, CCDC102B, C1orf132, KLF14, EDARADD, PDE4C and SST. Except for FHL2, all target regions were successfully sequenced with the multiplex strategy and the precision in terms of reproducibility of the measurements was equal to the singleplex strategy. The measured methylation values at the age CpGs displayed borderline significant differences between both analytical strategies for six out of 14 CpG sites whereas both strategies delivered equal methylation values for the remaining eight age CpGs. In total, our results indicate that the multiplex strategy can act as a promising alternative for age estimation studies in cases when only limited amounts of DNA samples are available.

Uncharted waters: Next-generation sequencing and machine learning software allow forensic science to expand into phenotype prediction from DNA samples.

Forensic molecular biology is entering uncharted waters with ethical and legal shallows to navigate. Next‐generation sequencing and other technologies now enable determination of phenotypic characteristics and thereby ethnic and racial profiling from recovered DNA samples from crime scenes, which represents a major new frontier for forensic science. At the same time, two traditional areas of forensic biology—DNA profiling and entomology to establish the time of death—have also made major advances based on these new sequencing technologies. It also has allowed forensic biology to expand into new terrains such as the illegal trafficking of rhinoceros horns and elephant tusks. > MPS has now further improved the accuracy and power of DNA typing Until around a decade ago, the two principle areas of forensic biology were DNA profiling and entomology. In some countries, palynology was also used to analyse pollen and spores so as to track where a suspect or objects from a crime scene had been before the crime. Now, the availability of massively parallel sequencing (MPS) or next‐generation sequencing (NGS) is driving genuinely new developments in forensic molecular biology. Another emerging area is the analysis of epigenetic markers on DNA samples, which can potentially yield valuable information about the age and lifestyle of suspects. “The most notable recent development is the widespread adoption of MPS or NGS for forensic purposes”, said Dennis McNevin from the Forensic Genetics department at the University of Canberra, Australia. “This has been driven by the availability of what I would call ‘forensically mature’ benchtop MPS platforms”. As a single targeted assay can cover the whole genome from field DNA samples, NGS has also enhanced traditional DNA profiling to identify suspects against a database. DNA profiling was first used in 1985 in an immigration test case by its inventor Alec Jeffreys at the University of Leicester, UK …

Investigation of metabolites for estimating blood deposition time

Trace deposition timing reflects a novel concept in forensic molecular biology involving the use of rhythmic biomarkers for estimating the time within a 24-h day/night cycle a human biological sample was left at the crime scene, which in principle allows verifying a sample donor’s alibi. Previously, we introduced two circadian hormones for trace deposition timing and recently demonstrated that messenger RNA (mRNA) biomarkers significantly improve time prediction accuracy. Here, we investigate the suitability of metabolites measured using a targeted metabolomics approach, for trace deposition timing. Analysis of 171 plasma metabolites collected around the clock at 2-h intervals for 36 h from 12 male participants under controlled laboratory conditions identified 56 metabolites showing statistically significant oscillations, with peak times falling into three day/night time categories: morning/noon, afternoon/evening and night/early morning. Time prediction modelling identified 10 independently contributing metabolite biomarkers, which together achieved prediction accuracies expressed as AUC of 0.81, 0.86 and 0.90 for these three time categories respectively. Combining metabolites with previously established hormone and mRNA biomarkers in time prediction modelling resulted in an improved prediction accuracy reaching AUCs of 0.85, 0.89 and 0.96 respectively. The additional impact of metabolite biomarkers, however, was rather minor as the previously established model with melatonin, cortisol and three mRNA biomarkers achieved AUC values of 0.88, 0.88 and 0.95 for the same three time categories respectively. Nevertheless, the selected metabolites could become practically useful in scenarios where RNA marker information is unavailable such as due to RNA degradation. This is the first metabolomics study investigating circulating metabolites for trace deposition timing, and more work is needed to fully establish their usefulness for this forensic purpose.

Forensic DNA Applications: An Interdisciplinary Perspective – a new book in forensic science

To the Editor: Forensic DNA Applications: An Interdisciplinary Perspective (2014; Edited by Dragan Primorac and Moses Schanfield. CRC Press, Taylor Francis Group. 23 Chapters, 621 pages) is intended for different types of audiences interested in forensic medicine. It could be both a textbook for forensic molecular biology students and a reference book for practitioners of forensic molecular biology. Moreover it is expected to be used by lawyers and judges dealing with civil and criminal cases involving DNA technology. The book covers the ever-growing field of forensic DNA testing, and an effort has been made to present state of the art knowledge on each topic at the time of submission (March 2013). The book was developed as an outgrowth of the Sixth ISABS Conference on Human Genome Project Based Applications in Forensic Science, Anthropology and Individualized Medicine, bi-annual educational conference of the International Society of Applied Biological Sciences (ISABS) held in Split, Croatia, June 1-5, 2009. It should also be noted that the Croatian Medical Journal (CMJ) is the official publication of ISABS and thus far ISABS has published 9 thematic issues of the CMJ, each issue related to one of the conferences. It was felt at these conferences that there was a need for a new book that would be used as a textbook but also as a reference book for people working in the field of forensic molecular biology as well as individuals investigating and adjudicating cases involving DNA evidence, regardless if they are civil or criminal cases. The approach is international, and therefore the information given could not be considered relevant to analysts only working in the US or Europe. Moreover as the title states the approach is “interdisciplinary.” The majority of authors are speakers and participants at the ISABS bi-annual meeting (1). The next meeting will take place on June 22-26, 2015 in Bol, island of Brac, Croatia. This will be a new opportunity to present to the CMJ new submissions related to the leading trends in forensic science. In the book, there are many texts on forensic molecular biology, some authored by a single author, others by a team of experts. This particular volume is unique in the sense that the 51 authors that provided the 23 chapters are experts in their particular areas, meaning that they work in the areas that they are writing about (Table 1-​-4).4). This edition has 4 sections and 23 chapters, representing all aspects of forensic DNA methodology, ethics, law, and policy. This book encompasses the period before DNA testing was done through the present and therefore can provide a unique perspective on the history and practice of forensic DNA testing. The authors come from Australia, Bosnia and Herzegovina, Croatia, the Czech Republic, Finland, the Netherlands, Slovenia, and the United State of America. This book is important because it brings together all of the current areas of interest and research in a single up-to-date volume (2). Table 1 SECTION 1. GENERAL BACKGROUND AND METHODOLOGICAL CONCEPTS (Mitchell M. Holland, Theresa Caragine, Section Editors) Table 4 SECTION 4. LAW, ETHICS AND POLICY (Fredrick Bieber, David H. Kaye, Section Editors)

A novel approach to forensic molecular biology education and training: it’s impact on the criminal justice system

The managers of crime laboratories face significant hurdles when preparing new hires to become productive members of the laboratory. New hires require six months of training/experience in the crime laboratory before becoming a productive member of the Biology (DNA) section. To address this deficiency in forensic DNA education, a novel forensic education curriculum was developed and tested for three consecutive years in the forensic science program at Fayetteville State University, Fayetteville, NC. The curriculum used a CTS proficiency kit, which is the same kit used to validate the proficiency of forensic scientists in crime laboratories in the US. A cost benefit analysis suggests that training students in a classroom instead of in a crime laboratory provides both direct savings to the laboratory and significant societal savings as more DNA profiles are entered into the database. The societal benefit from the combined reduction in the amount of training in a crime laboratory and increasing the number of DNA database profiles entered into a database suggests a societal saving of $8.28 million for each of these months of reduced training.

The nucleic acid revolution continues - will forensic biology become forensic molecular biology?

Molecular biology has evolved far beyond that which could have been predicted at the time DNA identity testing was established. Indeed we should now perhaps be referring to “forensic molecular biology.” Aside from DNA’s established role in identifying the “who” in crime investigations, other developments in medical and developmental molecular biology are now ripe for application to forensic challenges. The impact of DNA methylation and other post-fertilization DNA modifications, plus the emerging role of small RNAs in the control of gene expression, is re-writing our understanding of human biology. It is apparent that these emerging technologies will expand forensic molecular biology to allow for inferences about “when” a crime took place and “what” took place. However, just as the introduction of DNA identity testing engendered many challenges, so the expansion of molecular biology into these domains will raise again the issues of scientific validity, interpretation, probative value, and infringement of personal liberties. This Commentary ponders some of these emerging issues, and presents some ideas on how they will affect the conduct of forensic molecular biology in the foreseeable future.

PCR-Techniken in der forensischen Molekularbiologie

The analysis of nucleic acids in forensic casework and research is the main objective of the discipline of forensic molecular biology. Biological evidence from crime scenes frequently contains only trace amounts of DNA or RNA. Therefore, PCR based methods permitting analysis of very limited samples are of central importance. Routinely performed techniques include quantitative PCR for nucleic acid quantification, STR multiplex PCR for DNA profiling and RT-PCR for body fluid identification.

The National DNA Data Bank of Canada: a Quebecer perspective

The Canadian National DNA Database was created in 1998 and first used in the mid-2000. Under management by the RCMP, the National DNA Data Bank of Canada offers each year satisfactory reported statistics for its use and efficiency. Built on two indexes (convicted offenders and crime scene indexes), the database not only provides increasing matches to offenders or linked traces to the various police forces of the nation, but offers a memory repository for cold cases. Despite these achievements, the data bank is now facing new challenges that will inevitably defy the way the database is currently used. These arise from the increasing power of detection of DNA traces, the diversity of demands from police investigators and the growth of the bank itself. Examples of new requirements from the database now include familial searches, low-copy-number analyses and the correct interpretation of mixed samples. This paper aims to develop on the original way set in Québec to address some of these challenges. Nevertheless, analytic and technological advances will inevitably lead to the introduction of new technologies in forensic laboratories, such as single cell sequencing, phenotyping, and proteomics. Furthermore, it will not only request a new holistic/global approach of the forensic molecular biology sciences (through academia and a more investigative role in the laboratory), but also new legal developments. Far from being exhaustive, this paper highlights some of the current use of the database, its potential for the future, and opportunity to expand as a result of recent technological developments in molecular biology, including, but not limited to DNA identification.

State of the Art in Forensic Investigation of Sudden Cardiac Death

The sudden death of a young person is a devastating event for both the family and community. Over the last decade, significant advances have been made in understanding both the clinical and genetic basis of sudden cardiac death. Many of the causes of sudden death are due to genetic heart disorders, which can lead to both structural (eg, hypertrophic cardiomyopathy) and arrhythmogenic abnormalities (eg, familial long QT syndrome, Brugada syndrome). Most commonly, sudden cardiac death can be the first presentation of an underlying heart problem, leaving the family at a loss as to why an otherwise healthy young person has died. Not only is this a tragic event for those involved, but it also presents a great challenge to the forensic pathologist involved in the management of the surviving family members. Evaluation of families requires a multidisciplinary approach, which should include cardiologists, a clinical geneticist, a genetic counselor, and the forensic pathologist directly involved in the sudden death case. This multifaceted cardiac genetic service is crucial in the evaluation and management of the clinical, genetic, psychological, and social complexities observed in families in which there has been a young sudden cardiac death. The present study will address the spectrum of structural substrates of cardiac sudden death with particular emphasis given to the possible role of forensic molecular biology techniques in identifying subtle or even merely functional disorders accounting for electrical instability.

Manfred Kayser

Manfred Kayser

Review of: An Introduction to Forensic DNA Analysis, Second Edition

Abstract An Introduction to Forensic DNA Analysis by Rudin and Inman is an update of the 1997 first edition of the same name by Inman and Rudin. Many methods used by forensic scientists endure for decades without undergoing substantial change, so it is rare indeed when a field is transformed so quickly that a thorough text update is needed after only a few years. Books on forensic molecular biology could be updated quarterly.