DNA on drugs

The use of illicit drugs is a continuing blight on society. Detecting DNA from individuals involved in the manufacturing and distribution of drugs can provide valuable investigative information or strategic intelligence which, in turn, can be used to disrupt the supply and distribution of illicit drugs. Our study details the transfer, persistence, prevalence, and recovery of human DNA on the exterior of tablets and capsules, as well as within drug powders. Various experiments were conducted to mimic stages in the creation and packaging of tablets and capsules. We showed that the act of brief contact (1–3 s) is sufficient to generate informative DNA profiles that can be uploaded and compared to databases internationally. This work complements chemical drug profiling data by linking seizures to each other and individuals via DNA profiles, providing information to prosecution or intelligence agencies. The generation of DNA information from illicit drug preparations is another tool that can be used in the fight against illicit drug manufacture and distribution.     

Identification and separation of DNA mixtures using peak area information

We introduce a new methodology, based upon probabilistic expert systems, for analysing forensic identification problems involving DNA mixture traces using quantitative peak area information. Peak area is modelled with conditional Gaussian distributions. The expert system can be used for ascertaining whether individuals, whose profiles have been measured, have contributed to the mixture. It can also be used to predict DNA profiles of unknown contributors by separating the mixture into its individual components. The potential of our probabilistic methodology is illustrated on case data examples and compared with alternative approaches. The advantages are that identification and separation issues can be handled in a unified way within a single probabilistic model and the uncertainty associated with the analysis is quantified. Further work, required to bring the methodology to a point where it could be applied to the routine analysis of casework, is discussed.     

Estimating the Number of Contributors to Forensic DNA Mixtures: Does Maximum Likelihood Perform Better Than Maximum Allele Count?

Abstract: Determining the number of contributors to a forensic DNA mixture using maximum allele count is a common practice in many forensic laboratories. In this paper, we compare this method to a maximum likelihood estimator, previously proposed by Egeland et al., that we extend to the cases of multiallelic loci and population subdivision. We compared both methods’ efficiency for identifying mixtures of two to five individuals in the case of uncertainty about the population allele frequencies and partial profiles. The proportion of correctly resolved mixtures was >90% for both estimators for two‐ and three‐person mixtures, while likelihood maximization yielded success rates 2‐ to 15‐fold higher for four‐ and five‐person mixtures. Comparable results were obtained in the cases of uncertain allele frequencies and partial profiles. Our results support the use of the maximum likelihood estimator to report the number of contributors when dealing with complex DNA mixtures.     

DNA profiling from heroin street dose packages

A large amount of heroin street doses are seized and examined for drug content by the Israel police. These are generally wrapped in heat-sealed plastic. Occasionally it is possible to visualize latent fingerprints on the plastic wrap itself, but the small size of the plastic item and the sealing process makes the success rate very low. In this study, the possibility of extracting and profiling DNA from the burnt edge of the plastic wrap was investigated. The idea was based on the assumption that epithelial cells might be trapped during the sealing process. The results show that there are sufficient quantities of DNA deposited at the "amorphic" burnt edges of sealed street doses for DNA profiling to be carried out. A controlled experiment using a known donor was performed. This subject carried out sealing of "street drug" packages and consequent DNA extractions were performed to show that known DNA profiles could be recovered from such packages, as a result of handling by the "packer." "Square-like" burnt edges did not yield DNA profiles, probably because of differences in the sealing process. It was also shown that DNA could be recovered from the plastic wrap itself and not only from the amorphic burnt edges. As heroin dealers and drug users are often involved in other crimes and run-ins with the law, the effective extraction and addition of their DNA profiles from such items of evidence to the newly established DNA database in Israel provides new avenues in the continued fight against crime and drug traffickers.     

Probabilistic genotyping of single cell replicates from complex DNA mixtures recovers higher contributor LRs than standard analysis

DNA mixtures are a common source of crime scene evidence and are often one of the more difficult sources of biological evidence to interpret. With the implementation of probabilistic genotyping (PG), mixture analysis has been revolutionized allowing previously unresolvable mixed profiles to be analyzed and probative genotype information from contributors to be recovered. However, due to allele overlap, artifacts, or low-level minor contributors, genotype information loss inevitably occurs. In order to reduce the potential loss of significant DNA information from donors in complex mixtures, an alternative approach is to physically separate individual cells from mixtures prior to performing DNA typing thus obtaining single source profiles from contributors. In the present work, a simplified micro-manipulation technique combined with enhanced single-cell DNA typing was used to collect one or few cells, referred to as direct single-cell subsampling (DSCS). Using this approach, single and 2-cell subsamples were collected from 2 to 6 person mixtures. Single-cell subsamples resulted in single source DNA profiles while the 2-cell subsamples returned either single source DNA profiles or new mini-mixtures that are less complex than the original mixture due to the presence of fewer contributors. PG (STRmix™) was implemented, after appropriate validation, to analyze the original bulk mixtures, single source cell subsamples, and the 2-cell mini mixture subsamples from the original 2–6-person mixtures. PG further allowed replicate analysis to be employed which, in many instances, resulted in a significant gain of genotype information such that the returned donor likelihood ratios (LRs) were comparable to that seen in their single source reference profiles (i.e., the reciprocal of their random match probabilities). In every mixture, the DSCS approach gave improved results for each donor compared to standard bulk mixture analysis. With the 5- and 6- person complex mixtures, DSCS recovered highly probative LRs (≥10²⁰) from donors that had returned non-probative LRs (<10³) by standard methods.     

A mixed DNA profile controversy revisited

Semaan et al. (J Forensic Res, 2020, 11, 453) discuss a mock case “where eight different individuals [P₁ through P₈] could not be excluded in a mixed DNA analysis. Even though … expert DNA mixture analysis software was used.” Two of these are the true donors. The LRs reported are incorrect due to the incorrect entry of propositions into LRmix Studio. This forced the software to account for most of the alleles as drop-in, resulting in LRs 60–70 orders of magnitude larger than expected. P₁, P₂, P₄, P₅, and P₈ can be manually excluded using peak heights. This has relevance when using LRmix which does not use peak heights. We extend the work using the same two reference genotypes who were the true contributors as Semaan et al. (J Forensic Res, 2020, 11, 453). We simulate three two-donor mixtures with peak heights using these two genotypes and analyze using STRmix?. For the simulated 1:1 mixture, one of the non-donors’ LRs supported him being a contributor when no conditioning was used. When considered in combination with any other potential donors (i.e., with conditioning), this non-donor was correctly eliminated. For the 3:1 mixture, all results correctly supported that the non-donors were not contributors. The low-template 4:1 mixture LRs with no conditioning showed support for all eight profiles as donors. However, the results from pair-wise conditioning showed that only the two ground truth donors had LRs supporting that they were contributors to the mixture. We recommend the use of peak heights and conditioning profiles, as this allows better sensitivity and specificity even when the persons share many alleles.     

An innovative transfer DNA experimental design and qPCR assay: Protocol and pilot study

Forensic “touch” DNA samples are low-quantity samples that are recovered from surfaces that have been touched by single or multiple individuals. These samples can include DNA from primary contributors who directly touched the surface, as well as secondary contributors whose DNA was transferred to the surface through an intermediary. It is difficult to determine the type of transfer, or how often and under what conditions DNA transfer occurs. In this paper, we present an innovative protocol that combines (1) a paired male and female transfer DNA experimental design in which the presence of male DNA indicates secondary transfer and (2) a cost-effective quantitative PCR (qPCR) assay of a sex-specific region in the Amelogenin gene to detect male and female DNA. We evaluate the ability of the Amelogenin qPCR assay to detect low concentrations of male and female DNA in mixed samples. We also test experimental DNA samples using our transfer DNA protocol to differentiate primary and secondary DNA transfer. Male DNA was detected in the majority of known mixed samples, even in samples with 4× more female DNA—this result demonstrates the ability to detect low concentrations of male DNA and the presence of secondary transfer DNA in our experimental design. Primary DNA transfer was detected in 100% of our experimental trials and secondary DNA transfer was detected in 37.5% of trials. Our innovative protocol mimics realistic case scenarios to establish rates of primary and secondary DNA transfer in an inexpensive and simplified manner.     

Inferring the Number of Contributors to Complex DNA Mixtures Using Three Methods: Exploring the Limits of Low‐Template DNA Interpretation

In forensic DNA casework, the interpretation of an evidentiary profile may be dependent upon the assumption on the number of individuals from whom the evidence arose. Three methods of inferring the number of contributors—NOCIt, maximum likelihood estimator, and maximum allele count, were evaluated using 100 test samples consisting of one to five contributors and 0.5–0.016 ng template DNA amplified with Identifiler^® Plus and PowerPlex^® 16 HS. Results indicate that NOCIt was the most accurate method of the three, requiring 0.07 ng template DNA from any one contributor to consistently estimate the true number of contributors. Additionally, NOCIt returned repeatable results for 91% of samples analyzed in quintuplicate, while 50 single‐source standards proved sufficient to calibrate the software. The data indicate that computational methods that employ a quantitative, probabilistic approach provide improved accuracy and additional pertinent information such as the uncertainty associated with the inferred number of contributors.     

Cell free DNA as a component of forensic evidence recovered from touched surfaces

In the course of a criminal investigation, DNA is often recovered from items that have been handled by an individual. Whilst there have been studies investigating the propensity of different individuals to deposit DNA, little is known about the factors involved in the transference of DNA through touch. This investigation seeks to clarify some of the underlying processes involved in DNA transfer, as to better understand the significance of so-called “touch DNA” evidence (tDNA). It was shown that an average yield of 11.5 ng of DNA could be recovered from 1 mL cell-free sweat samples leading to the hypothesis that cell-free nucleic acids (CNAs) of a suitable length for standard DNA profiling are transferred during handling/touching items. A method of standardization of tDNA deposition was developed to overcome the significant sample to sample variability in DNA levels characteristic of tDNA samples. The glass bead method allowed the creation of identical tDNA sample sets, thus permitting direct comparisons to be made in the efficiency of various extraction methods. Extraction methods designed to optimize CNA recovery from touched articles resulted in comparable yields in a general population study, however the methods resulted in a twofold increase in DNA yields from touched items touched by individuals with sweaty hands. These results suggest that the CNA component of touched surfaces should be included to maximize profiling success of tDNA.     

An investigation of two methods of DNA recovery from fired and unfired 9 mm ammunition

Cartridge cases are often recovered from crime scenes involving firearms and, in the United Kingdom (where gun possession is strictly controlled), these are commonly from 9 mm calibre ammunition. The ability to obtain informative DNA profiles from touch DNA on recovered cartridges could have a significant impact on the investigation of that type of offence. However, this avenue may not be routinely considered as investigators in the UK have historically had a low expectation of obtaining useful DNA profiles. This stance may not be unreasonable given that (a) only trace amounts of DNA are likely to have been transferred onto the cartridge cases through handling; and (b) when the cartridge is spent, the potential deterioration of that DNA caused by the act of discharging the weapon.We introduce a novel semi-automatable method using direct lysis for the recovery of DNA from ammunition and compare it with a traditional double-swabbing method (using wet and dry swabs). DNA profiling of the DNA recovered using both methods was carried out using the ESI17 FAST STR system (Promega). This demonstrated a significant increase in DNA recovery using the direct lysis approach, and correspondingly improved STR results.We also investigated the effect on the recovery and profiling of DNA from fired, and unfired, 9 mm cartridges using the direct lysis technique. These results demonstrate that DNA suitable for STR analysis can still be recovered from fired ammunition with only slightly reduced yields compared to unfired ammunition. In these experiments, the handler of the ammunition was most commonly either the sole contributor or the major contributor to the recovered DNA profile.     

Two or three contributors of DNA mixtures? – Some practical considerations

The number of contributors is hard to determine in DNA mixture profiles. Here, we deal with the special but frequent case that either two or three contributors are possible. In fact, it might happen that two contributors can explain the number of alleles seen but that three contributors are necessary if a specific person of interest is to be included in the mixture. Then the likelihood ratio assuming two contributors will be zero while the likelihood ratio for three contributors may be large. We evaluate this situation and offer suggestions on how to arrive at an overall likelihood ratio. To exemplify our line of reasoning we use an example proposed by Biedermann, Taroni and Thompson.     

A cocaine body packer with normal abdominal plain radiograms. Value of drug detection in urine and contrast study of the bowel

Drug packets are usually detected by ordinary abdominal radiographs, which is of major importance in the apprehension and prosecution of drug body packers, but false negatives may occur. We report the case of a cocaine body packer who had used the prograde route and in whom the initial abdominal plain films were normal. The diagnosis of cocaine body packing was assessed by seldom-described complementary methods of detection, including drug detection in the urine and contrast study of the bowel.     

How to avoid driving DNA caseworkers crazy: CaseSolver,an expert system to investigate complex crime scenes

DNA analyses can be used for both investigative (crime scene-focused), or evaluative (suspect-focused) reporting. Investigative, DNA-led exploration of serious crimes always involves the comparison of hundreds of biological samples submitted by the authorities for analysis. Crime stain comparisons include both evidence to evidence profiles and reference to evidence profiles. When many complex DNA results (mixtures, low template LT-DNA samples) are involved in the investigation of a crime, the manual comparison of DNA profiles is very time-consuming and prone to manual errors. In addition, if the person of interest is a minor contributor, the classical approach of performing searches of national DNA databases is problematic because it is realistically restricted to clear major contributors and the occurrence of masking and drop-out means that there will not be a definitive DNA profile to perform the search with.CaseSolver is an open source expert system that automates analysis of complex cases. It does this by three sequential steps: a) simple allele comparison b) likelihood ratio (LR) based on a qualitative model (forensim) c) LR based on a quantitative model (EuroForMix). The software generates a list of potential match candidates, ranked according to the LRs, which can be exported as a report. The software can also identify contributors from small or large databases (e.g., staff database or 1 mill. individuals). In addition, an informative graphical network plot is generated that easily identifies contributors in common to multiple stains. Here we describe recent improvements made to the software in version v1.5.0, made in response to user requirements during intensive casework usage.     

DNA on the inside door handles of entrance doors

DNA from door handles on entry doors could provide a clue as to who last left the scene. However, after years of extensive research on DNA transfer and persistence it can be considered common knowledge that general claims like "the last who touched leaves the most DNA" do not hold true. But who's DNA do we find on door handles that are usually used several times per day by the inhabitants? To assess this question, we sampled inside door handles from real-life burglaries and at the same time collected reference samples from all the inhabitants, to determine if we can detect any (major) profiles from non-inhabitants. We also searched to evaluate how often we detect DNA from the person who last touched the door handle as a (major) contributor. Only small amounts of DNA were recovered from the handles, originating most often, but not always, from inhabitants or even the last inhabitant touching the handle.     

The propensity of individuals to deposit DNA and secondary transfer of low level DNA from individuals to inert surfaces

We have shown that there is a difference between individuals in their tendency to deposit DNA on an item when it is touched. While a good DNA shedder may leave behind a full DNA profile immediately after hand washing, poor DNA shedders may only do so when their hands have not been washed for a period of 6h. We have also demonstrated that transfer of DNA from one individual (A) to another (B) and subsequently to an object is possible under specific laboratory conditions using the AMPFISTR SGM Plus multiplex at both 28 and 34 PCR cycles. This is a form of secondary transfer. If a 30 min or 1h delay was introduced before contact of individual B with the object then at 34 cycles a mixture of profiles from both individuals was recovered. We have also determined that the quantity and quality of DNA profiles recovered is dependent upon the particular individuals involved in the transfer process. The findings reported here are preliminary and further investigations are underway in order to further add to understanding of the issues of DNA transfer and persistence.     

Efficiency of Casework Direct Kit for extraction of touch DNA samples obtained from cars steering wheels

Analysis of STR profiles obtained from touch DNA has been very useful to the elucidation of crimes. Extraction method may be determinant for the recovery of genetic material collected from different surfaces. Vehicle theft is one of the most common crimes in São Paulo city, Brazil, but collection of biological traces in car steering wheels is not considered, because of the belief that profiles generated won’t be able to identify the thief, only the owner. This study aimed to analyze the efficacy of extraction methods for obtaining DNA profiles in samples collected from steering wheels. Eight criminal acts were simulated with 2 different individuals each (mixture of victim and thief), in duplicate, in order to compare two extraction methods: DNA IQ™ and Casework Direct Kit (both Promega Corporation). Genetic material was collected by double swab method and quantified by Quantifiler™Trio (ThermoFisher Scientific). Amplification was conducted with PowerPlex® Fusion System (Promega). It was possible to obtain STR profiles for all experiments. The mixtures were compared with reference profiles to evaluated how many alleles of each donor were observed. Samples extracted with Casework Direct Kit obtained STR profiles with higher averages of alleles for primary and secondary donors (88.7% and 59.9%, respectively) than those extracted with DNA IQ™ (60.4% and 38.1%, respectively). This could be explained by the differences established in the protocols of both methods, since DNA IQ™ is based on successive washes and can result in loss of DNA, whereas Casework Direct Kit minimizes this problem. We concluded that Casework Direct Kit was more efficient for processing touch DNA samples than DNA IQ™.     

The risk of body packing: a case of a fatal cocaine overdose

The process of swallowing or inserting illegal packets of drugs for the purpose of evading law enforcement officers carries risks other than criminal charges. It can be fatal. Individuals engaged in such activities are called "Body Packers" or "Mules". The most frequent cause of the death among body packers is acute drug intoxication due to rupture of the package(s) within the gastrointestinal tract. We present the first documented case of a body packer that died from cocaine intoxication following the rupture of packets of cocaine in Western Pennsylvania.     

Analysis of DNA profiles extracted from degraded samples from archival of formalin fixed tissue included in paraffin (FFTIP) and hairs

The possibility of studying DNA extracted from archival of formalin fixed tissue included in paraffin (FFTIP) enables valuable retrospective investigations. However, according to some authors it is difficult to obtain genomic DNA of good quality, since the process of fixation often results in fragmentation of DNA. In order to evaluate the quality and quantity of DNA extracted, necropsy samples of FFTIP (spleen/lung) and hairs, with or without bulbs, were analyzed using three methods of extraction (QIAamp DNA mini, QIAamp DNA micro-kit and phenol–chloroform followed by microcon YM-30). The amount of DNA recovered was quantified by spectrophotometer. The β-actin, amelogenin gene and the profiles of STR were analyzed. Based on experimental results, a general guideline concerning the appropriate extraction method according to the tissue and the quantity of the starting material for the analysis of DNA from FFTIP and hairs could be suggested.     

Trace DNA success rates relating to volume crime offences

In this study, 252 trace DNA samples (from handled surfaces) from 201 burglary, robbery and drugs cases were compiled to assess success rates and to interpret the value of trace DNA evidence in volume crime investigations. The average amount of DNA recovered from the trace DNA samples collected was 1.7 ng. Full or major (12 or more alleles) profiles were recovered from 14% of samples. Samples from firearms and burglary points of entry were the least successful. Mixtures were recovered from 21% of samples, presenting a case for the collection of more elimination profiles to enable more samples to be used for database purposes. The research highlighted the difficulties in collecting data relating to the success rates of samples. Computerised automation of this process would be extremely beneficial in the assistance of policy development, method application, training, and investigative usefulness.     

Evaluation and quantification of nuclear DNA from human telogen hairs

Nuclear DNA was extracted from human telogen hairs from 60 individuals. Six to nine hairs from each individual were individually extracted. The amount of DNA recovered from each individual varied greatly, and most samples yielded a quantity of 550 pg or less per hair. A selective extraction buffer was used to remove epithelial cell DNA and the amount of exogenous DNA was determined. DNA was also quantified by real time PCR using three different sized amplicons targeting an Alu sequence. The results were used to determine the state of degradation of the extracted DNA. Different quantities of sample (<100 pg, 100-500 pg, >500 pg) were amplified with the Miniplex kits to determine the minimum DNA template required for successful amplification. DNA recovered from hair showed degradation; however, partial profiles were obtained for those samples containing at least 60 pg using MiniSTRs.