DNA Profiling Techniques Research Articles

A journey into the innovations and expertise of Dubai police and the general department of forensic science and criminology

This manuscript offers an in-depth exploration of the Dubai Police and its General Department of Forensic Science and Criminology, emphasizing their critical role in law enforcement through advanced forensic science and innovative practices. Beginning with a historical overview, it traces the evolution of forensic disciplines from Edmond Locard's foundational principles to Sir Alec Jeffreys' groundbreaking DNA profiling techniques. The manuscript details the structure and functions of specialized sections within the department, including Biology and DNA, Forensic Chemistry, Forensic Toxicology, Firearms and Tool Marks, Questioned Documents, Forensic Explosive, Forensic Fire Investigation, Forensic and Mechanical Engineering, and Nuclear Physics. Key achievements, such as the Biology and DNA Section's processing of thousands of cases and the generation of highly discriminating DNA profiles, are highlighted. The manuscript also addresses the department's challenges, including the rapid evolution of technology and the increasing complexity of crimes. Future plans to enhance capabilities through investment in new technologies, international collaboration, and continuous professional development are outlined. By showcasing the department's commitment to innovation, excellence, and public safety, this manuscript underscores the pivotal contributions of the Dubai Police and its forensic department to crime resolution and community safety. The insights provided aim to foster a deeper understanding of the critical role of forensic science in the criminal justice system.

Indirect DNA Transfer and Forensic Implications: A Literature Review.

Progress in DNA profiling techniques has made it possible to detect even the minimum amount of DNA at a crime scene (i.e., a complete DNA profile can be produced using as little as 100 pg of DNA, equivalent to only 15-20 human cells), leading to new defense strategies. While the evidence of a DNA trace is seldom challenged in court by a defendant's legal team, concerns are often raised about how the DNA was transferred to the location of the crime. This review aims to provide an up-to-date overview of the experimental work carried out focusing on indirect DNA transfer, analyzing each selected paper, the experimental method, the sampling technique, the extraction protocol, and the main results. Scopus and Web of Science databases were used as the search engines, including 49 papers. Based on the results of this review, one of the factors that influence secondary transfer is the amount of DNA shed by different individuals. Another factor is the type and duration of contact between individuals or objects (generally, more intimate or prolonged contact results in more DNA transfer). A third factor is the nature and quality of the DNA source. However, there are exceptions and variations depending on individual characteristics and environmental conditions. Considering that secondary transfer depends on multiple factors that interact with each other in unpredictable ways, it should be considered a complex and dynamic phenomenon that can affect forensic investigation in various ways, for example, placing a subject at a crime scene who has never been there. Correct methods and protocols are required to detect and prevent secondary transfer from compromising forensic evidence, as well as the correct interpretation through Bayesian networks. In this context, the definition of well-designed experimental studies combined with the use of new forensic techniques could improve our knowledge in this challenging field, reinforcing the value of DNA evidence in criminal trials.

DNA profiling in forensic investigation – A review

DNA Profiling is a revolutionary method for individual and relationship analysis, crime investigation, hereditary disorders, etc. It is a universal method used to establish accurate results during the process of forensic investigation. DNA profiling techniques, which are based on repetitive sequences within DNA, have proven to be of paramount importance, albeit the complete utilization of knowledge still remaining unexplored. Even a hair strand, blood drop or even skin flakes can be used to identify DNA sequences. It has a wide range of applications both in forensics and law. Because of the advancement in the field of forensics in the past four decades, DNA evidence now stands as one of the most reliable forms of proof in a court of law. In the following article, the authors explore the main concepts of DNA Profiling, and the techniques which are widely used in forensic laboratories such as RFLP, VNTR, STR, AFLP, mtDNA analysis, Y-chromosome analysis and gender typing.

Abstract A19: Integrated single cell analysis reveals co-evolution of malignant B cells and the tumor microenvironment in transformed follicular lymphoma

Abstract Introduction: Histological transformation from follicular lymphoma (FL) to aggressive B-cell lymphoma (tFL) is a disease course altering event linked to poor prognosis for affected patients. From a biological point of view, it is paradigmatic of disease dynamics with distinct clinical stages that project onto genetically and phenotypically divergent states. Aim: By applying a series of high-dimensional single cell (sc)RNA and DNA profiling techniques, we aimed to characterize the clonal and phenotypic evolution of tumor B cells and to reveal dynamic interactions with components of the tumor microenvironment (TME) during transformation. Methods: We included 11 tFL patients with paired FL (tFL-FL) and DLBCL (tFL-DLBCL) timepoint biopsies, and 11 indolent FL controls (with > 6y of follow-up without evidence of progression or transformation). Single cell whole transcriptome (scWTS) and BCR sequencing was performed for all samples and single cell whole genome sequencing (scWGS) for transformation pairs. Results: In each transformation pair, BCR sequencing confirmed the clonal relationship between FL and DLBCL timepoints. Clustering of scRNA data from each pair showed an inverse correlation between transcriptional similarity and time between the two biopsies. Some tFL-FL cells could always be found within the tFL-DLBCL clusters. Therefore, we labeled these cells as presumed “early-DLBCL cells”. Phylogenetic analysis using scWGS data showed distinct FL and DLBCL clones, and “mixed-clones” composed of cells from both timepoints in most pairs. FL cells in DLBCL clones were favored to represent precursor cells of transformation. DLBCL cells in FL clones likely represent residual FL cells after transformation, and could be found in the majority of the pairs. Divergent evolution from FL to DLBCL with specific copy number abnormalities unique to each timepoint was the most common mode of evolution during transformation, and only one pair showed linear evolution. Integrative analysis of scRNA and scDNA data highlighted that samples with the fewest genomic changes showed the least transcriptomic changes and vice versa. Differential expression and gene set enrichment analysis of malignant cells identified “MYC targets V1“ as the main pathway enriched in tFL-DLBCL cells in comparison to tFL-FL cells. Cells from the indolent control FL cases had a significantly lower MYC score than cells from pre-transformed FL. In parallel to the insights into tumor cell evolution, scRNAseq analysis also revealed significant shifts in TME composition, from T cells with a TFH and central memory phenotype in tFL-FL samples, to cells with an exhausted cytotoxic phenotype in tFL-DLBCL samples. Conclusion: Applying high-dimensional scRNA and DNA profiling techniques we identified precursor cell populations of transformation at the genomic and phenotypic level and linked genomic and phenotypic evolution with shifting TME composition in a comprehensive disease evolution model of transformation. Citation Format: Clementine Sarkozy, Shaocheng Wu, Katsuyoshi Takata, Tomohiro Aoki, Susana B Neriah, Katy Milne, Brad Nelson, Andrew Weng, David Scott, Jeffrey W Craig, Christian Steidl, Andrew Roth. Integrated single cell analysis reveals co-evolution of malignant B cells and the tumor microenvironment in transformed follicular lymphoma [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A19.

A Study on DNA Profiling Techniques and Transnational Exchange of DNA Data from Databank

DNA technology has shown to be a valuable investigative tool in the release of innocent people and the identification of those responsible for serious crimes. In the battle against illegal immigration, cross-border crime, and terrorism, the transnational DNA data interchange from national DNA databanks has become a current trend. The data types that can be shared and the system is managed by a national authority are governed by individual national legislation, which determines the scope of the data exchange. Furthermore, one of the most difficult problems in forensic science is DNA profiling, and it is a hotly debated topic. The number of unknowns in a combination raises the computational difficulty of DNA profiling dramatically. To overcome this issue, various approaches have been designed and implemented. As a result, we examine DNA profiling methodologies and tools in this study, focusing on their computational accuracy and performance. Furthermore, this research examines the available data on DNA exchange and comparison across borders. We hope this review provides more ideas for future research to choose efficient profiling techniques.

What’s on the bag? The DNA composition of evidence bags pre- and post-exhibit examination

Current forensic DNA profiling kits and techniques enable the detection of trace amounts of DNA. With advancements in kit sensitivity, there is an increased probability of detecting DNA from contamination. Research into DNA transfer within operational forensic laboratories provides insight into the possible mechanisms that may lead to exhibit contamination. To gain a greater understanding of the potential for evidence bags to act as DNA transfer vectors, the level of DNA accumulating on the exterior of evidence bags during the exhibit examination process was investigated. The exterior of 60 evidence bags were tapelifted before and after the examination of the exhibit inside of the bag resulting in 120 DNA profiles. These DNA profiles were compared to DNA profiles of staff working within the building and samples taken from the exhibit inside the bag. Common DNA profile contributors from each sample were also identified through STRmix™ mixture to mixture analysis. The average DNA quantity and number of profile contributors was higher in samples taken from the bag before exhibit examination than after examination. Fifty six percent of all samples taken identified a match between DNA recovered from the evidence bag and at least one staff member. On 11 bags, a common contributor was identified between the exhibit in the bag and the exhibit package post-examination. In one instance a DNA profile, matching that of a donor, on the exhibit bag before examination was also detected on a sample taken from the exhibit, raising the possibility of outer bag-to-exhibit DNA contamination. This study demonstrates that operational forensic laboratories must consider exhibit packages as a potential source of DNA contamination and evaluate their exhibit handling and storage procedures accordingly.

"I've never been at the crime scene!" - gloves as carriers for secondary DNA transfer.

Over recent years, DNA profiling techniques have become highly sensitive. Even small amounts of DNA at crime scenes can be analysed leading to new defence strategies. At court, defence lawyers rarely question the existence of a DNA trace (source level) but challenge how the DNA was transferred to the scene (activity level). Nowadays, the most common defence strategy is to claim that somebody else had stolen the defendant's gloves and used them while breaking and entering. In this study we tested this statement. Using gloves made of different material (cloth, leather, rubber) and varying secondary transfer surfaces (wood, metal, glass), we simulated a few of the most likely transfer scenarios that occur during breaking and entering. While we detected the presence of DNA on the outside of 92 of the 98 gloves tested, we observed only one case of secondary transfer in a total of 81 transfer experiments. This data demonstrates that secondary transfer under conditions resembling realistic conditions is a very rare event.

Evaluating the use of hypoxia sensitive markers for body fluid stain age prediction

To augment DNA profiling and body fluid identification techniques efforts are being made to increase the amount of information available from a crime scene stain, which includes efforts to identify externally visible characteristics through phenotypic analysis. A key question surrounding crime scene stains is the length of time between deposition of the stain and its subsequent recovery, in that is the stain recovered related to the incident in question or from a previously deposited stain number of weeks earlier? The inability to answer this fundamental question has a detrimental effect upon the successful completion of a criminal investigation. Once a body fluid leaves the body, the oxygen concentration in the environment changes; therefore, it may be that this change could cause a change in the expression of hypoxia-sensitive biomarkers. Here, a range of bloodstains, liquid saliva and liquid semen samples were collected at 0 days, 7 days, 14 days, 21 days and 28 days of degrading at room temperature (19–22 °C), before undergoing total RNA extraction and cDNA synthesis. Blood was recovered from filter paper with 3 mm2, with saliva and semen being left in their tubes and swabbed at the appropriate times. All samples then underwent quantitative PCR targeting Vascular Endothelial Growth Factor A (VEGFA) and Hypoxia-Inducible Factor 1 Alpha (HIF1A), with B-Actin (ACTB) as a reference gene. A range of linear and quadratic correlation values was obtained from the qPCR data and used to develop a predictive model with a mean absolute deviation (MAD) of 4.2, 2.1, and 5 days for blood, saliva, and semen respectively. Blind testing indicated that a stain age prediction model based upon VEGFA with ACTB as a reference gene could be used on samples up to four weeks old with a margin of error ranging from 2 days through to 5 days. While a sizeable potential time frame exists using this model; this represents a significant step towards the target of having an accurate stain age prediction model.

Identification of nasal mucosa markers for forensic mRNA body fluid determination

Biological fluids are commonly encountered as a form of evidence within forensic science, and can often provide important information relating to events which may have occurred. Over the years, significant advancements have been made with DNA profiling techniques, allowing for links to be made between an individual and cellular material recovered from a crime scene. While this DNA analysis can aid in linking an individual to a crime, it can often be beneficial to also determine the body fluid source of the DNA obtained from the sample in question for case context. One increasing area within the forensic field is the use of mRNA profiling for the identification of body fluids. The analysis of gene expression patterns can give information on cell function, and ultimately the body fluid source of the DNA in a sample. Over time this has led to the development of mRNA reverse transcriptase PCR assays to detect body fluid specific RNA transcripts for casework. During the use of these techniques nasal mucosa has been observed to give rise to false positive results. We report here on the identification of promising markers using RNA sequencing for the detection of nasal mucosa, with the aim to incorporate these markers into existing assays to assist in the identification of nasal mucosa and to assist in the interpretation of possible false positive results.

Evaluating the use of hypoxia sensitive markers for body fluid stain age prediction

In order to augment DNA profiling and body fluid identification techniques, efforts are being made to increase the amount of information available from a crime scene stain, which includes efforts to identify externally visible characteristics through phenotypic analysis. A key question surrounding crime scene stains is the length of time between deposition of the stain and its subsequent recovery, in that is the stain recovered related to the incident in question or from a previously deposited stain number of weeks earlier? The inability to answer this fundamental question has a detrimental effect upon the successful completion of a criminal investigation. Once a body fluid leaves the body, the oxygen concentration in the environment changes, therefore it may be that this change could cause a change in expression of hypoxia-sensitive biomarkers. Here, a range of blood, saliva and semen samples were collected at 0 days, 7 days, 14 days, 21 days and 28 days of degrading at room temperature, before undergoing total RNA extraction and cDNA synthesis. All samples then underwent quantitative PCR targeting Vascular Endothelial Growth Factor A (VEGFA) and Hypoxia-Inducible Factor 1 Alpha (HIF1A), with B-Actin (ACTB) as a reference gene. A range of linear and quadratic correlation values was obtained from the qPCR data and used to develop a predictive model with a mean absolute deviation (MAD) of 5.3, 2.0, and 4.3 days for blood, saliva, and semen respectively. Blind testing indicated that a stain age prediction model based upon VEGFA with ACTB as a reference gene could be used on stains up four weeks old with a margin of error ranging from 2 days through to 5 days. Whilst a large potential time frame exists using this model, this represents a major step towards the target of having an accurate stain age prediction model.

Differentially methylated CpG regions analyzed by PCR-high resolution melting for monozygotic twin pair discrimination

Discrimination between monozygotic (MZ) twins is a forensic limitation when using conventional DNA profiling techniques for human identification. Recent works based on epigenetics seem to open a new way to solve this issue due to methylation status of MZ twins change during their lifetime. Methylation analysis through BeadChip platforms allows the study up to 850 K CpG sites revealing that numerous differential methylation regions exist between MZ twins. However, this methodology is difficult to implement in forensic laboratories. On the contrary, PCR-HRM (High Resolution Melting) technology is one of the easiest methods for analyzing DNA methylation and it has been capable to discriminate between MZ twins. The purpose of this study is to contribute with new differential methylation regions in MZ twins to those that have been previously studied through PCR-HRM. Here, we have selected 6 CpG regions located at the ITGA2B, ASPA, PDE4C, ZIC5, USP11 and NOP14 loci that have shown methylation status variation during lifetime. The study has been carried out from saliva-derived DNA of 18 MZ twin pairs. The most discriminating regions were those located at ITGA2B, ASPA and ZIC5 loci showing significant within-pair differences in 44.4% of the cases. Non evidences of relation between age and significant differences between MZ twins were found, although the 50% of MZ twin pairs were discrimnated in the oldest age range (59–66 years old). These results support the use of these regions to increase the number of epigenetics age-related markers available to discriminate between MZ twins in a pair by PCR-HRM in forensic laboratories.

Microbial diversity changes with rhizosphere and hydrocarbons in contrasting soils

In the ecotoxicological assessment of petroleum hydrocarbon-contaminated soil, microbial community profile is important aspect due to their involvement in soil functions. However, soil physicochemical properties and the inhabiting plants could dictate the microbial composition. A question remains unanswered is, how an integrated approach may be utilized to account for various contrasting soil properties, plant types (reference vs. native) and the nature of the hydrocarbon contamination. In this study, we utilized bacterial DNA profiling techniques to investigate the relationship between soil properties, contaminant and plant species. Results identified that Proteobacteria and Actinobacteria were the most abundant bacteria of the 45 phyla identified in the hydrocarbon-contaminated soil. The bulk and rhizosphere microbiome showed that the contaminated soil originally had quite distinct bacterial communities compared to the artificially contaminated soil (mine soil = 95 genera vs. other soils = 2–29 genera). In these cases, not significantly but the native plant slightly increased bacterial diversity and relative abundance in the same soils. Also, within each site, the bacterial community was significantly altered with the hydrocarbon concentration. In this instance, the influence of the contaminant was strong and also with the soil pH and organic matter. These results would significantly contribute to the novel insights on the molecular technique-based hydrocarbon toxicity assessment and the development of the further integrative approach with other microbial community and their metabolic profile in the contaminated sites.

Liquid Biopsy-Analysis of Circulating Tumor DNA (ctDNA) in Bladder Cancer.

Recent advances in DNA profiling techniques have enabled sensitive detection of tumor-associated genomic aberrations in peripheral blood. This type of minimally-invasive molecular interrogation has the potential to guide subsequent treatment selection. The potential utility of ctDNA in bladder cancer (BC) is bolstered by the high somatic mutation rate, meaning that very small numbers of genes or target regions can be informative. First reports indicate that analysis of ctDNA may represent a sensitive method for disease surveillance in patients with different stages of BC. Moreover, recent evidence suggests that ctDNA analysis reveals previously unknown genomic alterations in metastatic patients. Since some of these gene alterations represent therapeutic targets, ctDNA analysis provides an attractive tool to guide individualized therapy in BC.

Bioactive Natural Products from Plants and Biotechnological Approaches for their Production

Bioactive natural products are economically important as drugs, fragrances, pigments, food additives and pesticides. The biotechnological tools are important to select, multiply, improve and analyze medicinal plants for production of such products. The utilization of medicinal plant cells for the production of natural or recombinant compounds of commercial interest has gained increasing attention over the past decades. Plant tissue culture systems are possible source of valuable medicinal compounds, fragrances and colorants, which cannot be produced by microbial cells or chemical synthesis. In vitro production of bioactive natural products in plant cell suspension culture has been reported from various medicinal plants and bioreactors are the key step towards commercial production. Genetic transformation is a powerful tool for enhancing the productivity of novel products; especially by Agrobacterium tumefacians . Combinatorial biosynthesis is another approach in the generation of novel natural products and for the production of rare and expensive natural products. Recent advances in the molecular biology, enzymology and bioreactor technology of plant cell culture suggest that these systems may become a viable source of important secondary metabolites. Genetic fingerprinting could be a powerful tool in the field of medicinal plants to be used for correct germplasm identification. In addition, when linked to emerging tools such as metabolomics and proteomics, providing fingerprints of the plant’s metabolites or protein composition, it gives data on phenotypic variation, caused by growth conditions or environmental factors, and also yield data on the genes involved in the biosynthesis. DNA profiling techniques like DNA microarrays serve as suitable high throughput tools for the simultaneous analysis of multiple genes and analysis of gene expression that becomes necessary for providing clues about regulatory mechanisms, biochemical pathways and broader cellular functions. New and powerful tools in functional genomics can be used in combination with metabolomics to elucidate biosynthetic pathways of natural products.

Complex DNA mixture analysis: Report of two cases

During the last years, DNA profiling techniques have become more sensitive and mixtures with multiple contributors occur often. Nevertheless, interpretation of mixture profiles is the most complex task in the forensic field. The ISFG published recommendations for the interpretation of mixed DNA profiles that may have allelic drop-out or drop-in. The DNA commission of the ISFG has supported the use of likelihood ratios (LRs) as preferable to other methods. LR can be calculated by probabilistic programs, using semi and fully continuous (probabilistic) methods. These methods increase the efficiency of forensic laboratories and improve the consistency and transparency of the reported results. LRmix is an open-source system dedicated to the interpretation of forensic DNA profiles with a particular focus on complex DNA mixtures. This software is programmed on a LR model and facilitates the calculation of LR for complex mixtures, with partial profiles with known and unknown contributors. Here, we report the re-analysis of two old complex cases processed in our laboratory in the last years. In both cases, we used the software LRmix Studio with interesting consequences: in one case we solved the crime, in the other one, the results allowed the case to be reopened.

Genetic and epigenetic changes in somatic hybrid introgression lines between wheat and tall wheatgrass.

Broad phenotypic variations were induced in derivatives of an asymmetric somatic hybridization of bread wheat (Triticum aestivum) and tall wheatgrass (Thinopyrum ponticum Podp); however, how these variations occurred was unknown. We explored the nature of these variations by cytogenetic assays and DNA profiling techniques to characterize six genetically stable somatic introgression lines. Karyotyping results show the six lines similar to their wheat parent, but GISH analysis identified the presence of a number of short introgressed tall wheatgrass chromatin segments. DNA profiling revealed many genetic and epigenetic differences, including sequences deletions, altered regulation of gene expression, changed patterns of cytosine methylation, and the reactivation of retrotransposons. Phenotypic variations appear to result from altered repetitive sequences combined with the epigenetic regulation of gene expression and/or retrotransposon transposition. The extent of genetic and epigenetic variation due to the maintenance of parent wheat cells in tissue culture was assessed and shown to be considerably lower than had been induced in the introgression lines. Asymmetric somatic hybridization provides appropriate material to explore the nature of the genetic and epigenetic variations induced by genomic shock.

Manfred Kayser

Manfred Kayser

Comments on the review of low copy number testing

Dear Sir,A challenge to the reliability of low copy number (LCN)DNA profiling in the trial of Sean Hoey in Belfast CrownCourt in Northern Ireland (R v Hoey [2007] NICC 49, 20December, 2007) prompted the UK’s new Forensic ScienceRegulator (Andrew Rennison) to commission a review oflow template DNA profiling techniques. That review [2],conducted by Professor Brian Caddy (with the assistance ofDr. Adrian Linacre and Dr. Graham Taylor) was released on12 April, 2008 and concluded that LCN DNA profiling is“robust” and “fit for purpose.” Yet, the review accepts thatthe evidence presented in Sean Hoey’s trial was insufficientto establish the validity of the technique. It also enumerates21 recommendations for specific improvements that shouldbe undertaken to improve the methodology, including suchbasic steps as the development of a consensus on theinterpretation of test results and efforts to establish “bestpractices” for interpretation.We believe the conclusions of the review are inconsistentwith its recommendations in a number of respects. Forexample, it is difficult to see how a forensic techniquecould be deemed adequately validated for use in thecourtroom when there is not yet a consensus on how itsresults should be interpreted. The review thus raisesimportant issues about what it means for a forensic sciencetechnique to be validated. It also establishes grounds forconcern about the way that LCN DNA test results havebeen interpreted in earlier cases.We are concerned that the review team relied only oninput regarding the merits of LCN approaches fromorganizations that are dedicated to promoting its use bylaw enforcement. Consultation with known critics of thetechnique (or even a review of their published works)would have provided the reviewers with a broaderperspective of what work remains to be done before theapproach can become generally accepted within theinternational scientific community. There are in fact thingsabout LCN approaches upon which the reviewers andcritics do agree. For instance, caution that “[p]ublicizing thepotential of the application of LCN typing withoutdescribing its limitations may cause misunderstanding” [1]which is consistent with the review’s recommendations 1,3, and 13. But given the conclusion that “[t]he methodcannot be used for exculpatory purposes” [1], the review’sultimate conclusion that LCN testing is “fit for purpose”leaves the important but unanswered question of “what isthat purpose?”

Short tandem repeat (STRs) and sex specific Amelogenin analysis of blood samples from neurosurgical female transfused patients

Blood transfusion and its effects on DNA profiles of an individual is an important issue in the forensic context. In the present study, the effects of unfiltered whole male blood transfusion in seven female subjects were investigated by PCR-based assays on serial post-transfusion blood samples. PCR analysis of specific short tandem repeat (STR) primer pair to CSF1PO, TPOX, TH01, F13A01, FESFPS, vWA and sex specific Amelogenin loci for banding pattern of alleles generated from the pre- and post-transfusion of seven female recipients and their corresponding donors were studied. Pre- and post-transfusion DNA profiles of all the seven female recipients were found to be consistent with no evidence of the male and female donor genetic material. It is concluded that currently used PCR-based STRs and sex specific Amelogenin loci DNA profiling techniques in forensic science are reliable and informative for paternity and identity purposes in situations of transfusion of 1 or 2 U of unfiltered whole male and female blood (>72 h old) to female recipient up to 24 h post-transfusion.

Attribution of DNA profiles to body fluid stains

Ultra-sensitive DNA profiling techniques, which now enable a profile to be obtained from a single cell, are greatly advantageous to the forensic community where biological material is often limited. However, it is not always possible to infer the cellular origin of any resulting profile. Here, we assess the likelihood of generating a DNA profile by the standard processing of visible stains that originates from extraneous cells on the substrate rather than the body fluid. Stain size and quality, substrate material and an individual's propensity to deposit DNA were all proven to be significant factors. It was demonstrated that an association between DNA profile and body fluid is not implicit, even where a positive presumptive test is obtained.