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Genetic profiling from challenging samples: Direct PCR of touch DNA
... r touch DNA sampling. This technique involves the use of a sterile swab rubbed on the surface of an object to collect shed skin cells (Alketbi, 2023;Bonsu, Rodie, et al., 2021;Giovanelli et al., 2022;Haase et al., 2019;Kanokwongnuwut et al., 2018Kanokwongnuwut et al., , 2019Liu, 2015;Nolan & Linacre, 2024;Schulte et al., 2023;Seiberle et al., 2022;J. Templeton et al., 2013;J. E. L. Templeton et al., 2015;Verdon et al., 2013;Vickar et al., 2018;Währer et al., 2023). In addition, the double swab technique, which involves using two swabs in sequence (one wet swab followed by a dry swab), is also often used to maximize DNA collection (Alem et al., 2017;da Rocha Marques et al., 2022;Eckstein et al., 2000;Franci ...
... In past studies it has been shown that swabbing techniques are effective (J. Templeton et al., 2013;Verdon et al., 2013), however this review incorporates more recent studies investigating the effectiveness of other sampling techniques such as tape lifting and wet-vacuum sampling (Kanokwongnuwut et al., 2019;Vickar et al., 2018). Besides, this review gives a certain range of reference values which may help forensic practitioners in knowing what to expect from touch DNA analysis by providing a summary of DNA concentration data from different studies. ...
... her surfaces. On non-porous/smooth surfaces, the type of surface most often mentioned in the literature is glass(Alem et al., 2017;Bonsu, Rodie, et al., 2021;Haase et al., 2019;Recipon et al., 2023;Szkuta et al., 2017;J. E. L. Templeton et al., 2015), plastic(Alem et al., 2017;Bonsu, Rodie, et al., 2021;Francisco et al., 2020;Hartless et al., 2019;J. Templeton et al., 2013; ...
Touch DNA analysis has become increasingly important in forensic investigations. This systematic review aims to compare the effectiveness of various sampling techniques on different surface types and examine the concentration of DNA obtained. Following PRISMA guidelines, relevant studies were identified through PubMed, Scopus, and Web of Science databases. Data were extracted using NVivo and synthesized using a narrative approach, while study quality was assessed using the Mixed Methods Appraisal Tool (MMAT). Swabbing, particularly double swabbing, was the most common technique. However, tape lifting was more effective on porous/uneven surfaces, and wet-vacuum sampling outperformed double swabbing on brick. DNA concentrations varied (0.002−0.707 ng/μL) based on surface type and sampling method. Findings provide guidance for forensic practitioners in selecting optimal sampling techniques, refining DNA extraction protocols, and interpreting results. Nevertheless, methodological heterogeneity and laboratory focus limit generalizability. This review offers a comprehensive understanding of factors influencing touch DNA analysis success. Future research should focus on larger, standardized studies, novel sampling techniques, and advanced DNA analysis methods to address challenges associated with degraded or limited touch DNA samples. Continued research is crucial for enhancing the reliability and applicability of touch DNA evidence in forensic investigations.
... KEYWORDS: forensic science, DNA typing, touch DNA, archived latent fingerprints, short tandem repeat typing, adhesive tape, paper substrate DNA analysis was introduced to the criminal justice system in 1986, and since then, there have been several improvements in forensic technology and commercial products allowing for short tandem repeat (STR) multiplexing from minute levels of template DNA, such as 100 pg or lower (1)(2)(3)(4). Due to such sensitivity levels, there has been a greater demand for analysis of touch DNA (mixture of corneocytes and sweat gland secretions) where low levels of DNA are often encountered (1,(4)(5)(6)(7)(8). Several challenges have been discovered with touch DNA evidence samples. ...
... For example, identifying an efficient sampling technique that can remove and collect as much of the DNA-containing biological material present is crucial for low template DNA evidence because any loss of biological material during laboratory analysis could leave too little DNA to produce a successful STR profile. Direct cuttings and swabbings from adhesives for DNA analysis have been successfully reported (18,19) and, outside of the ubiquitously used sterile water (20), several swab diluents, such as SDS and Tween 20, have been recommended for touch DNA collection (2,3,7,15,18,21). Beyond sampling, DNA extraction, purification, and concentration methods can impact the quality and quantity of DNA yields. ...
... Several studies have suggested that the swab diluent used for touch DNA swabbings could have a direct impact on the quality of the resulting STR profile (2,3,7,15,18). Consequently, water, detergents, a set of commercial reagents, and isopropanol were used as diluents to prewet swabs for sampling archived latent fingerprints. ...
DNA profiles have been obtained from fingerprints, but there is limited knowledge regarding DNA analysis from archived latent fingerprints-touch DNA "sandwiched" between adhesive and paper. Thus, this study sought to comparatively analyze a variety of collection and analytical methods in an effort to seek an optimized workflow for this specific sample type. Untreated and treated archived latent fingerprints were utilized to compare different biological sampling techniques, swab diluents, DNA extraction systems, DNA concentration practices, and post-amplification purification methods. Archived latent fingerprints disassembled and sampled via direct cutting, followed by DNA extracted using the QIAamp® DNA Investigator Kit, and concentration with Centri-Sep™ columns increased the odds of obtaining an STR profile. Using the recommended DNA workflow, 9 of the 10 samples provided STR profiles, which included 7-100% of the expected STR alleles and two full profiles. Thus, with carefully selected procedures, archived latent fingerprints can be a viable DNA source for criminal investigations including cold/postconviction cases.
... In addition, the risk of contamination is minimized by side-stepping multiple tube changes and wash steps. Recent studies highlight the benefits of omitting the extraction step, also known as direct PCR, in a forensic context (11)(12)(13)(14)(15). Direct PCR has previously been used to amplify DNA adhering to cotton swabs that were premoistened with water (16). ...
... However, in light of recent advances, new swab types (17) and new swab media (4) can now be considered as alternative methods for DNA recovery. Novel DNA capture methods in conjunction with direct PCR can be used to explore the potential for improvement in information obtained from forensic samples (15). ...
... Samples and substrates used in this study did not reflect all possible scenarios of contact DNA left behind in forensic investigations (e.g., damaged, degraded, and intact cellular DNA). We have previously reported that the direct PCR method can be used to generate full DNA profiles from fingermarks deposited 2 h after individuals washed their hands (15). The direct PCR approach in this study led to significantly higher peak heights of alleles (p = 0.001) compared to the extraction experiment when controlling for DNA mass and substrate (See Table S1). ...
This study reports on the comparison of a standard extraction process with the direct PCR approach of processing low-level DNA swabs typical in forensic investigations. Varying concentrations of control DNA were deposited onto three commonly encountered substrates, brass, plastic, and glass, left to dry, and swabbed using premoistened DNA-free nylon FLOQswabs(™) . Swabs (n = 90) were either processed using the DNA IQ(™) kit or, for direct PCR, swab fibers (~2 mm(2) ) were added directly to the PCR with no prior extraction. A significant increase in the height of the alleles (p < 0.005) was observed when using the direct PCR approach over the extraction methodology when controlling for surface type and mass of DNA deposited. The findings indicate the potential use of direct PCR for increasing the PCR product obtained from low-template DNA samples in addition to minimizing contamination and saving resources.
© 2015 American Academy of Forensic Sciences.
... The concept of subsampling as a method for front end analysis of evidence could reduce overall sample consumption, and in turn, minimize loss of DNA [9][10][11][12][13][14][15][16][17][18]. DNA loss during initial processing can impact downstream DNA typing success [10][11][12][13][14][15]. ...
... DNA loss during initial processing can impact downstream DNA typing success [10][11][12][13][14][15]. Direct amplification of the microFLOQ® Direct Swab reduces sample manipulation and thus should reduce DNA loss during processing [9][10][11][12][14][15][16][17][18][19]. A previous study by Ambers et al. [8] showed that less sample is consumed to obtain a result from stains on a non-porous surface (i.e., glass) when subsampling with the microFLOQ® Direct Swab. ...
The microFLOQ® Direct Swab was tested by sampling diluted blood, semen, and saliva stains deposited on cotton cloth. DNA typing was performed using the PowerPlex® Fusion 6C System by direct PCR or a modified direct PCR. Direct PCR of swabs sampled the center of a stain, compared to their respective edge samplings, and had higher profile completeness and total relative fluorescent units (RFU) for all dilutions of blood and semen stains tested. The modified direct PCR used template DNA eluted from the swab head using the Casework Direct Kit, Custom and washes either contained 1-thioglycerol (TG) additive or no TG. Modified direct PCR had mixed results for blood, saliva, and semen stains, with semen stains showing significant differences in profile completeness (5% and 1%) and total RFU (neat, 5% and 1%) with the addition of TG to the Casework Direct Reagent. No significant difference was seen in any dilution of blood or saliva stains processed with the modified direct PCR, but profile completeness and total RFU were improved overall compared to stains swabbed with cotton swabs or 4N6FLOQSwabs™. This study supports the hypothesis that the microFLOQ® Direct Swab is able to collect minute amounts of DNA from cotton cloth and may be considered as an alternate pre-screening methodology in forensic biology casework.
... Profiler Plus and NGM STR chemistries have been evaluated for use with direct PCR protocols for both punches and swab cuttings [43]. Linacre et al. evaluated SGM Plus and PowerPlex 16 for direct PCR from fabric, swabs, and trace/touch evidence [44]. ...
... Linacre et al. evaluated SGM Plus and PowerPlex 16 for direct PCR from fabric, swabs, and trace/touch evidence [44]. Complete and partial profiles were generated from both the work produced by Templeton et al. [43] and Linacre et al. [44], with an observed decrease in time required for sample generation due to the elimination of the extraction, quantitation, and normalization steps in the forensic DNA typing process. ...
... Previous research by Templeton et al. [28] compared the performance of foam swabs, cotton swabs, and nylon FLOQSwabs® in collecting trace DNA from fingerprints, with the latter demonstrating the highest DNA yield. This study was expanded to include the use of FLOQSwabs® for touch DNA recovery across various substrates, comparing direct amplification to the standard extraction workflow. ...
This study evaluates the efficacy of the microFLOQ™ Direct Swab for touch DNA recovery from various surfaces within an office environment. Samples were collected using the swab and subjected to direct amplification with the GlobalFiler™ PCR Kit. Results revealed a success rate of 73% within three hours post-sample collection, with profiles ranging from full single to partial mixture. Despite observed artifacts, such as split and shoulder peaks, the swab demonstrated potential for efficient touch DNA recovery. However, further research is needed to assess its performance across diverse surfaces and to address challenges associated with artifact generation. Overall, the microFLOQ™ Direct Swab shows promise as a valuable tool for forensic touch DNA analysis, offering streamlined sample processing and potential advancements in forensic casework
... The 4N6FLOQSwabs® (COPAN, Brescia, Italy) is a nylon-flocked swab (certified human DNA free) that, in theory, because of its design, should outperform cotton and rayon swabs on collection and yield of crime scene DNA. Early studies by Benschop et al. [14] (a forensic study), more recent studies by Viviano et al. [15] (a non-forensic study), and others [16][17][18][19][20] support the proposition that the nylon swab collects and yields more DNA, although some studies have come to different conclusions [21,22]. Differences in performance between studies appear to be due to varying protocols that may not be the one validated by the manufacturer for the nylon swab, or for that matter the compared swabs as well [1,16]. ...
Laboratories and their criminal justice systems are confronted with challenges for implementing new technologies, practices, and policies even when there appears to be demonstrative benefits to operational performance. Impacting decisions are the often higher costs associated with, for example, new technologies, limited current budgets, and making hard decisions on what to sacrifice to take on the seemingly better approach. A prospective cost–benefit analysis (CBA) could help an agency better formulate its strategies and plans and more importantly delineate how a relatively small increase to take on, for example, a new technology can have large impact on the system (e.g., the agency, other agencies, victims and families, and taxpayers). To demonstrate the process and potential value a CBA was performed on the use of an alternate and more expensive swab with reported better DNA yield and being certified human DNA free (i.e., nylon 4N6FLOQSwabs®), versus the traditional less costly swab (i.e., cotton swab). Assumptions are described, potential underestimates and overestimates noted, different values applied (for low and modest to high), and potential benefits (monetary and qualitative) presented. The overall outcome is that the cost of using the more expensive technology pales compared with the potential tangible and intangible benefits. This approach could be a guide for laboratories (and associated criminal justice systems) worldwide to support increased funding, although the costs and benefits may vary locally and for different technologies, practices, and policies. With well-developed CBAs, goals of providing the best services to support the criminal justice system and society can be attained.
... The scratching and tape lift strategies permit a bigger surface territory to be tested rather than the cutting strategy. An expansion in surface zone builds the odds of recuperating more skin cells, which expands the odds of getting a DNA profile (Templeton et al. 2013). As referenced, the scratching/tape lift strategies are ideal in circumstances where the researcher can find zones on the things which are well on the way to contain the culprit's skin cells. ...
Touch DNA examination is regularly used to pick up data from natural materials to help examinations related with criminal offenses, catastrophe casualty recognizable proof and missing individual’s examinations. As the noteworthiness estimation of DNA profiling to scientific examinations has expanded, so too wants to create this data from more modest measures of DNA. Touch DNA tests might be characterized as any example which falls beneath suggested edges at any phase of the investigation, from test discovery through to profile translation, and cannot be characterized by an exact picogram sum. The assortment, DNA extraction, intensification, profiling, and translation of touch DNA tests, tainting, and move issues are additionally quickly examined inside the setting of touch DNA examination.
... Several previous studies have reported increased DNA recovery when applying nylon-flocked swabs instead of cotton swabs for pure DNA [23], microbial sampling [22,24], post-coital vaginal sampling [9], plastic microscope slides [25] and cartridge cases [10]. However, no improvements were seen with nylon-flocked swabs on the surfaces tested in our study. ...
Efficient sampling with swabs is crucial for optimal forensic DNA analysis. The DNA recovery is determined by the skill of the practitioner and the compatibility between the applied swab and the surface. Here we investigate the impact of swabbing technique and swab type on the DNA yield. Thirteen different swabs from four categories (cotton, flocked nylon, small foam and large foam) provided equal DNA yields for smooth/non-absorbing surfaces. Large foam swabs gave higher DNA recovery for an absorbing wood surface. Factorial design of experiments and ANOVA was applied to study swabbing techniques for cotton swabs. Two key factors for efficient sampling were found to be 1) holding the swab with an approximate 60° angle against the surface and 2) to rotate the swab during sampling. For absorbing wood, it was beneficial to wet the swab heavily. The results of the factorial experiments were used to develop swabbing protocols for different surfaces. When ten experienced practitioners sampled according to these protocols, the DNA yield was increased for ridged plastic (around 1.25 times more DNA) and absorbing wood (2.2-6.2 times more DNA). For window glass, representing a smooth/non-absorbing surface, sampling according to the protocol gave DNA yields equivalent to applying individual sampling techniques. The protocol lowered person-to-person variation for ridged plastic. In conclusion, we have developed instructive protocols for cotton swab sampling on three types of surfaces: smooth/non-absorbing, ridged/non-absorbing and smooth/absorbing. We believe that such swabbing protocols will streamline and simplify the training of new practitioners and improve sampling efficiency for invisible DNA in casework.
... The development of scientific investigation techniques leading to a high success rate of DNA extraction from human tissue samples-including crime scene samples-and improvements in short tandem repeat (STR) amplification have further resulted in more reliable DNA profiling results. In addition, more efficient direct polymerase chain reaction (PCR) amplification techniques for obtaining DNA profiles from samples have been recently developed [4][5][6][7]. The direct PCR method, known as colony PCR, has been widely used for rapid detection of gene cloning and transfection in the field of microbiology since the early 1990s [8,9]. ...
Analytical techniques such as DNA profiling are widely used in various fields, including forensic science, and novel technologies such as direct polymerase chain reaction (PCR) amplification are continuously being developed in order to acquire DNA profiles efficiently. However, non-specific amplification may occur depending on the quality of the crime scene evidence and amplification methods employed. In particular, the ski-slope effect observed in direct PCR amplification has led to inaccurate interpretations of the DNA profile results. In this study, we aimed to reduce the ski-slope effect by using dimethyl sulfoxide (DMSO) in direct PCR. We confirmed that DMSO (3.75%, v/v) increased the amplification yield of large-sized DNA sequences more than that of small-sized ones. Using 50 Korean buccal samples, we further demonstrated that DMSO reduced the ski-slope effect in direct PCR. These results suggest that the experimental method developed in this study is suitable for direct PCR and may help to successfully obtain DNA profiles from various types of evidence at crime scenes.
... Also, the majority of these types of samples involve low template amounts, which have a low success rate for producing a DNA profile (Budowle, Eisenberg, & van Daal, 2009;Schiffner et al., 2005). The use of direct PCR and/or portable instrumentation for these types of case samples could allow for prioritization of the more useful samples and improve the success rate of obtaining profiles ( Templeton et al., 2013). ...
Short tandem repeat (STR) profiling is frequently carried out on biological stains in forensic casework, and the application of messenger RNA (mRNA) body fluid identification is becoming more prevalent. Increasing constraints on analysis time, resources, an increase in the number of samples for analysis, and a large backlog of cases creates significant demand for quicker methods that direct polymerase chain reaction (PCR) can help overcome. Direct PCR bypasses DNA and/or RNA extraction, and in some cases, quantification by adding the sample or substrate punch to the amplification reaction. This method is currently used in some laboratories on reference samples. Direct PCR is also a component of portable devices, which could improve efficiency even further by producing results at the scene, and enabling triaging of samples for further testing. Portable devices are currently used for STR profiling of reference samples but not for mRNA profiling. Various concerns regarding the reliability of the method and equipment (portable devices) must first be addressed before incorporation into forensic casework. This review discusses the limitations and factors that need to be considered for direct PCR to be used for the analysis of casework samples, including being used for mRNA body fluid identification.
This article is categorized under:
• Forensic Biology > Forensic DNA Technologies
• Forensic Biology > Body Fluid Identification
• Forensic Biology > Applications of RNA
Abstract
With advances in technology, direct PCR of DNA and RNA for forensic purposes is now a reality. While early progress has been made, there are still a number of factors that need to be considered. This review describes where direct PCR technology is at and what still requires attention before this technique can become a standard technique in forensic science.
... Alternatively, swabs or other materials can be used to remove touch DNA from a substrate and then the swab head can be introduced directly into the PCR reaction. The successful use of direct amplification for touch DNA recovery has been widely reported from a range of sample types [7][8][9][10][11]. ...
Previous studies reported in the literature demonstrate that a range of sampling vehicles can be used effectively for forensic analysis of human DNA in direct amplification reactions. In this study we compared Copan microFLOQ swabs with a range of alternative sampling vehicles, using touch DNA samples donated by 15 different volunteers. MicroFLOQ swabs performed well, as did 3 mm diameter discs punched from analytical filter paper. The 3 mm discs could be used in a 5 μl PCR volume, increasing sensitivity, and reducing costs when compared with other methods that require a larger PCR volume. Other inert sampling vehicles, such as interdental toothbrushes and toothpicks also gave good results in direct amplification. The study found a large variation in results between the 15 touch DNA donors, demonstrating the importance of validating touch DNA recovery techniques with a large pool of donors.
... Many studies indicated that this technique was a rapid, simple, sensitive, and easy to use technology (Kitpipit et al. 2014b). Previous reports have illustrated the potential benefit of direct amplification from several substrates, such as fibers (Linacre et al. 2010), hair (Kitpipit et al. 2014a;Ottens et al. 2013a, b), blood (Barker et al. 1992;Mercier et al. 1990), and even touch DNA (Liu 2014;Swaran and Welch 2012;Templeton et al. 2013). There were many different pyrolysis liquids or direct PCR buffers for different raw materials, whether from animal, microorganism, or plant tissues, such as blood (Mercier et al. 1990;Sharma et al. 2012;Yang et al. 2007;Zhu et al. 2013), cell (Svec et al. 2013), bacteria (Fuchs-Telka et al. 2017Hasap et al. 2013), fungi (Zhao et al. 2014), meat (Kitpipit et al. 2013a(Kitpipit et al. , 2014bMano et al. 2014), hair, horn, bone, feces and urine (Kitpipit et al. 2013b(Kitpipit et al. , 2014a, and sea food and marine species (Tagliavia et al. 2016). ...
Molecular technologies based on PCR have been widely used in many biological analysis fields, especially for genetic analysis and DNA barcoding. In this study, a rapid DNA lysis liquid was formulated without any purification step from fresh and processed meat, suitable for conventional PCR amplification. Three different lysis liquid formulas were designed for selection and further optimization for direct PCR and absorbance spectra; DNA concentration and performance in PCR were used to assess the effect of each formula. The results indicated that the formula containing NaOH, EDTA, SDS, Tween 20, and Tris-HCl achieved the best results, and the optimized formula met the need of practical PCR applications. The protocol provided a rapid lysis buffer for DNA replication from any meat samples. The performance of the final formula resulted in high DNA lysis efficiencies for all the tested meat samples and the PCR amplification efficiencies were similar to isolated DNA template using a commercial kit. The whole process can be completed in 30 min. Therefore, this study provides a simple, alternative, cost-effective fast solution for meat molecular analysis based on DNA.
... Previous studies have investigated methods for collection and direct amplification of touch DNA. Templeton et al. [24] compared the performance of foam swabs, cotton swabs, and nylon FLOQSwabs ® in collecting trace levels of DNA from fingerprints and found that the latter swabs generated the highest DNA yield. The study was expanded using the FLOQSwabs ® to recover touch DNA from a variety of different substrates and then compared the direct amplification approach to the standard (extraction) workflow. ...
Previous studies have shown that nylon flocked swabs outperform traditional fiber swabs in DNA recovery due to their innovative design and lack of internal absorbent core to entrap cellular materials. The microFLOQ(®) Direct swab, a miniaturized version of the 4N6 FLOQSwab(®), has a small swab head that is treated with a lysing agent which allows for direct amplification and DNA profiling from sample collection to final result in less than two hours. Additionally, the microFLOQ(®) system subsamples only a minute portion of a stain and preserves the vast majority of the sample for subsequent testing or re-analysis, if desired. The efficacy of direct amplification of DNA from dilute bloodstains, saliva stains, and touch samples was evaluated using microFLOQ(®) Direct swabs and the GlobalFiler™ Express system. Comparisons were made to traditional methods to assess the robustness of this alternate workflow. Controlled studies with 1:19 and 1:99 dilutions of bloodstains and saliva stains consistently yielded higher STR peak heights than standard methods with 1ng input DNA from the same samples. Touch samples from common items yielded single source and mixed profiles that were consistent with primary users of the objects. With this novel methodology/workflow, no sample loss occurs and therefore more template DNA is available during amplification. This approach may have important implications for analysis of low quantity and/or degraded samples that plague forensic casework.
... Genomic analysis of a small fraction of a microswab that had been used to collect vaginal fluid from under fingernails was carried out using a standard forensic DNA profiling system combined with direct PCR [19,20]. As expected, a mixed male/female typing was obtained (Fig. S2). ...
During a crime, biological material such as blood or vaginal fluid may become smeared on the fingers of the victim or suspect or trapped under their fingernails. The type of trapped fluid is extremely valuable forensic information. Furthermore, if either person touches an object at the crime scene with their ‘contaminated’ finger then a ‘contaminated’ finger mark may be deposited. Such marks have great value as they could identify not only who deposited the mark but also who they touched and which part of the body they touched. Here, we describe preliminary work towards a ‘toolbox’ of techniques based on mass spectrometry (MS) for the identification of biological fluid traces under fingernails or the imaging of them in finger marks. Liquid chromatography-multidimensional MS was effective for the detection of protein biomarkers characteristic of vaginal fluid and blood trapped under fingernails, even after hands had been washed. In regard to examination of finger marks for the presence of biological fluids, the most practical implementation of any technique is to integrate it with, but after, routine crime scene finger mark enhancement has been applied. Here, we demonstrate the usage of matrix-assisted laser desorption ionization-time of flight-MS for the detection and mapping of proteins and peptides from body fluids in finger marks, including marks enhanced using aluminium-containing magnetic powder and then ‘lifted’ with adhesive tape. Hitherto, only small molecules have been detected in enhanced, lifted marks. In a novel development, aluminium in the enhancement powder assisted ionization of small molecules in finger marks to the extent that conventional matrix was not required for MS.
... Direct PCR has the ability to effectively amplify low quantities of control DNA recovered from brass, glass, and plastic substrates (Templeton, et al., 2015). Even this technique could be used to generate full DNA profiles from fingermarks deposited 2 h after individuals washed their hands (Templeton, et al., 2013). It also minimized allele dropout in samples with low PCR yield (Sim, et al., 2013). ...
In crime scenes, scarce traces of biological samples are usually found. Direct polymerase chain reaction (PCR) is a fast, cheap and effective forensic
technique. Its benefits overcome several problems that
usually accompany DNA purification from biological
material. Therefore, the possibility of using direct
PCR versus routine DNA extraction-PCR
amplification for enhancing genotype profiles
obtained from very scarce biological material was
tested. 1, 3, 5 and 10 hairs were collected by pulling
in duplicates from ten different human subjects, and
divided into two groups. The first was subjected to
direct PCR, and the second was processed for DNA
extraction then applied to PCR. Fifteen autosomal loci
of short tandem repeats (STRs) were amplified with
PCR, that are D5S818, D13S317, D7S820, D16S539,
CSF1PO, Penta D, D3S1358, TH01, D21S11,
D18S51, Penta E, , vWA, D8S1179, TPOX, FGA, in
addition to a gender identification marker,
Amelogenin. Loading 1 or 3 hairs directly into the
PCR enhanced fluorescence levels significantly for 9-
13 loci, possibly as a direct result for reduction of
PCR-inhibitors normally present in hairs, including
eumelanin. Increasing the number of hairs led to a
gradual decrease in the scale of fluorescence, till a
complete loss of amplification. The results indicated
that direct PCR without DNA extraction and with
only very few number of hairs (1 or 3) gives better
results than performing an initial, pre- PCR DNA
extraction step, reducing then the costs for samples
processing, and also enhancing the detection
efficiency, as inferred from the enhanced fluorescence
peak heights using direct PCR.
... Direct PCR was first used on forensic relevant samples in 2010, illustrating the potential benefit of using this technique with fibers [1]. Fibers have not been explored in any further detail using this technique since then, with much of the research focusing on other sample types such as hair, fingernails, blood, and trace DNA on various substrates [2][3][4][5][6][7][8][9][10][11]. The presence of PCR inhibitors in fabrics, such as dyes, can represent a challenge when directly amplifying single fibers. ...
We report on the successful use of direct PCR amplification of single fibers from items of worn clothing. Items of clothing were worn throughout the course of a day, with the individual commencing regular activities. Single fibers were taken from the cuff of the clothing at regular intervals and amplified directly. The same areas were subjected to tape-lifting, and also amplified directly for comparison. The NGM™ kit that amplifies 15 STR loci plus amelogenin was used. A total of 35 single fiber samples were processed and analyzed from five items of clothing, with 81 % of samples returning a profile of 14 alleles or more. All tape-lift samples amplified directly produced DNA profiles of 15 alleles or more. The aim was to develop a simple, operational method that could be used routinely in forensic science casework and that has the potential to generate more complete profiles, which would not be detected using standard extraction methods on this type of sample. For ease of implementation, the process also adheres to standard methods with no increase in the cycle number.
... Finally, the DNA is effectively archived in a small volume which can be re-accessed if required. The advantages of a direct-to-PCR approach have already been demonstrated for blood [10,11] hairs [12,13], fingernails [14], fabrics [15] and so-called Btouch DNA^ [16,17]. This may be a result of Bcell free^DNA which has been demonstrated in a number of forensically relevant samples [18,19]. ...
Disaster victim identification (DVI) often occurs in remote locations with extremes of temperatures and humidities. Access to mortuary facilities and refrigeration are not always available. An effective and robust DNA sampling and preservation procedure would increase the probability of successful DNA profiling and allow faster repatriation of bodies and body parts. If the act of tissue preservation also released DNA into solution, ready for polymerase chain reaction (PCR), the DVI process could be further streamlined. In this study, we explored the possibility of obtaining DNA profiles without DNA extraction, by adding aliquots of preservative solutions surrounding fresh human muscle and decomposing human muscle and skin tissue samples directly to PCR. The preservatives consisted of two custom preparations and two proprietary solutions. The custom preparations were a salt-saturated solution of dimethyl sulfoxide (DMSO) with ethylenediaminetetraacetic (EDTA) and TENT buffer (Tris, EDTA, NaCl, Tween 20). The proprietary preservatives were DNAgard (Biomatrica(®)) and Tissue Stabilising Kit (DNA Genotek). We obtained full PowerPlex(®) 21 (Promega) and GlobalFiler(®) (Life Technologies) DNA profiles from fresh and decomposed tissue preserved at 35 °C for up to 28 days for all four preservatives. The preservative aliquots removed from the fresh muscle tissue samples had been stored at -80 °C for 4 years, indicating that long-term archival does not diminish the probability of successful DNA typing. Rather, storage at -80 °C seems to reduce PCR inhibition.
... Much of the focus has been on direct amplification from a body fluid stain [6] with an emphasis also on the speed of the DNA typing [7] and the types of DNA polymerase used. Previous reports have illustrated the potential benefit of direct amplification from fibers [5], hair [8,9], and touch DNA [10,11]. The potential to generate a profile from fingernail clippings is dependent on the presence of nucleated cells or cell free DNA present on the surface of the nail from skin secretion or sebum. ...
We report on the successful routine amplification of DNA profiles from small sections of fingernails using direct PCR. The data are from 40 nail clippings from eight donors where approximately 4 mm(2) of nail is added directly to the PCR. The NGM™ kit was used that amplifies 15 STR loci plus amelogenin. No increase in cycle number was used and no enrichment of the PCR products was performed. Full DNA profiles were observed in 17 of the 40 profiles with 21 generating partial DNA profiles. The process omits the DNA extraction process, and hence there is no opportunity to quantify the DNA prior to amplifying the STRs, but by not performing a DNA extraction step, the amount of DNA available for PCR is maximized. Single source DNA profiles were observed in 29 of the 38 profiles obtained. The source of the DNA is assumed to be adhering to the underside of the nail. This simple method offers a significant reduction in time to generate DNA profiles from nail clippings, such as those taken from victims of mass disasters, and should be included into a forensic process relatively easily as it requires no change to manufacturer's instructions for amplification.
... Here we report a procedure that eliminates the need for LCN technology by omitting the extraction process, therefore minimizing the opportunity for sample mix-up or introduction of extraneous DNA into the reaction vessel. If DNA exists as a free molecule, then it should bind readily to positively charged fibers (23). Furthermore, the use of a surfactant such as Triton-X is thought to assist in the binding of DNA to a swab (24) and hence, these two features were used to capture DNA from touched substrates. ...
Criminal investigations would be considerably improved if DNA profiles could be routinely generated from single fingermarks. Here we report a direct DNA profiling method that was able to generate interpretable profiles from 71% of 170 fingermarks. The data are based on fingermarks from all 5 digits of 34 individuals. DNA was obtained from the fingermarks using a swab moistened with Triton-X, and the fibers were added directly to one of two commercial DNA profiling kits. All profiles were obtained without increasing the number of amplification cycles; therefore, our method is ideally suited for adoption by the forensic science community. We indicate the use of the technique in a criminal case in which a DNA profile was generated from a fingermark on tape that was wrapped around a drug seizure. Our direct DNA profiling approach is rapid and able to generate profiles from touched items when current forensic practices have little chance of success.
Cartridge casings made from transition metals can be examined ballistically and also serve as significant evidence by containing touch DNA. However, the success rate of profiles obtained from this type of evidence is generally low. To enhance the success of DNA profiling from suspects' biological evidence, using swabs moistened with chemicals can be beneficial. Swabs are typically moistened with water, whose hypotonic nature disrupts cell integrity, causing the release of DNA. However, water is not the only agent used for moistening swabs; various buffer solutions are also utilized. The ability of swabs to transfer touch DNA depends on the type of buffer solution used. Sodium dodecyl sulfate (SDS), a strong anionic detergent, denatures non-covalently linked secondary and tertiary structures to increase the release of bound DNA. Another buffer solution used for swab moistening is Te+4 buffer, which contains EDTA and Tris. EDTA chelates metal ions, inactivating enzymes that could potentially damage DNA, while Tris adjusts the pH to an optimal level. This study aims to compare the effectiveness of microfibre and cotton swabs moistened with SDS, Te+4 buffer, and water in recovering genetic material from blood and epithelial cells deposited on brass cartridge casings. The study also evaluates the impact of firing on the quality of DNA profiles. Among the profiles obtained, the swabs moistened with SDS produced the highest rate of complete profiles at 44%, while the lowest rate of complete profiles at 22% was observed with swabs moistened with Te+4 buffer. SDS is particularly advantageous over water when used on casings with epithelial cells. Microfibre swabs are more effective in eliminating degradative factors caused by firing, thus enhancing profiling success. Comparisons of the RFU values indicate that casings yield lower values compared to cartridges, supporting the negative impact of the high heat, pressure, and residues generated during firearm discharge.
Touch deoxyribonucleic acid (DNA) refers to the DNA that is left behind from skin cells when a person touches or comes into contact with an object. In crimes where the identification of suspects becomes a challenge, touch DNA has been a proven investigative tool. The present study aims to provide a systematic review of the role of touch DNA in criminal cases which discusses the nature and importance of touch DNA evidence at crime scenes; various phenomena including the transfer and persistence of touched samples; different factors affecting the touch sample deposition and DNA shedding; the best recovery methods and collection of samples from different substrates; and the interpretation of profiles through advanced techniques that identify the suspects. The present study also aims to optimize standard protocols in the laboratories for touched samples appropriate to the substrates that improve the success rate of profiles from touched items.
Touch DNA analysis has emerged as a powerful tool in forensic science for the identification and profiling of individuals based on the DNA left behind through direct contact with surfaces. This chapter is on touch DNA analysis, focusing on its principles, methodologies, applications, and challenges. Touch DNA refers to the cellular material transferred from the skin to objects during routine human activities, such as handling, grasping, or touching. Touch DNA analysis plays a crucial role in criminal investigations, allowing forensic scientists to link individuals to crime scenes, objects, or victims, even in the absence of visible biological evidence. Overall, touch DNA analysis represents a valuable forensic tool for identifying perpetrators, exonerating the innocent, and contributing to the administration of justice in criminal proceedings.
Since the initial introduction of forensic DNA analysis in the 1980s, advancements have been made within the forensic biology community that have improved the success rate of obtaining DNA profiles. Fingerprints that were originally intended for latent examination could be a potential source of DNA. Archived latent fingerprints contain touch DNA between an adhesive barrier of tape and a paper substrate. Collect a DNA sample by separating the tape and paper material, then cut each substrate into small pieces (approximately 3 mm × 3 mm). Extract DNA samples using the QIAamp® DNA Investigator Kit (QIAGEN®), a silica-column based method, and follow the manufacturer's protocol for "paper and other similar materials." Pair it with the Centri-Sep™ spin column (Thermo Fisher Scientific) concentration method to optimize the biological workflow for DNA profiling.
Crime laboratories have been faced with large casework backlogs due to lengthy processing times, limited resources and scientists, and rising crime rates. Evidence related to sexual assault crimes, specifically sexual assault kits (SAKs), heavily contribute to the reported backlogs. Although more sensitive, faster chemistries and automated techniques have been implemented over the years, the traditional STR workflow remains relatively unchanged. Enhanced workflows such as direct PCR and Rapid DNA have the potential to streamline the processing of forensic evidence items including those commonly submitted in SAKs, but the FBI QAS guidelines restrict CODIS‐approved labs from implementing these methods for forensic samples. Recent studies have shown decreased turnaround times and improved or comparable profiling success with both approaches. However, review of the literature shows a lack of in‐depth research comparing traditional DNA workflows to faster and more sensitive direct PCR and/or Rapid DNA approaches for evidentiary samples, especially for SAKs. By providing the forensic science and criminal justice communities with the strengths and limitations of direct PCR and Rapid DNA methods, stakeholders and policy makers may be better informed.
Forensic DNA profiling is a globally accepted method for human identification, however, obtaining full DNA profiles from trace DNA can be challenging. The optimal recovery of DNA from trace DNA swabs is therefore crucial. Methods for extracting DNA from swabs often make use of a spin basket combined with a centrifugation step, to enhance the release of cells from the swab prior to DNA extraction. The NucleoSpin® Forensic Filter (Macherey-Nagel, Düren) is a type of spin basket, but it has not been thoroughly assessed on trace DNA samples. This study aimed to assess if the inclusion of the NucleoSpin® Forensic Filter significantly improved DNA recovery and DNA profiling success from cotton and flocked swabs used to collect trace DNA and buccal cells (control). Buccal cells and trace DNA samples were collected from 25 volunteers using each swab type (cotton and flocked) in duplicate. DNA was extracted from the samples using the NucleoSpin® DNA Forensic kit, one set with, and the other set without, NucleoSpin® Forensic Filters. DNA concentration was assessed using real time PCR, and DNA profiling was done using the PowerPlex® ESX 16 system. The inclusion of the NucleoSpin® Forensic Filters significantly improved DNA concentration in buccal cells collected using flocked swabs (p = 0.035). However, no significant differences were noted for trace DNA samples for either swab type. There was also no significant difference in DNA profiling success when NucleoSpin® Forensic Filters were used, regardless of swab and sample type. These results may be helpful for laboratories that are considering the NucleoSpin® Forensic Filters in the DNA extraction workflow, particularly for trace DNA samples.
As the use of improvised explosive devices (IEDs) in a broad spectrum of offences continues, it is vital that research is performed to assess the capabilities of the forensic DNA profiling technology currently available to provide information as to potential perpetrators. This work investigates some of the most important gaps in our understanding surrounding the poor success rates in DNA profiling obtained through the sampling of touch DNA on post-detonation IED samples. It has been previously suggested that the use of Diamond™ Nucleic Acid Dye may fix cells to a surface, therefore reducing the effect of an experimental process to remove or damage those cells. This was found not to be the case for samples undergoing a detonation as there was no difference in the resultant post-detonation profiles between the stained samples, stained prior to detonation, and unstained samples. The comparison of data from previously performed research, within an enclosed explosives chamber, to real-world outdoor detonation events in a rural and dusty environment was investigated. It was found that there was a significant difference between the environments for the aluminium but not for the battery or electrical tape substrates indicating that environment has the potential to influence STR success through the introduction of PCR inhibitors; humic acid within rural natural dust was introduced here. No difference was observed in cell loss due to the detonation between environments and the dirt within the PCR was higher in the ‘outdoor’ samples. The effect on cellular retention and damage due to the sample’s distance from the charge has been thoroughly investigated through incremental 100 mm exposure. Distance from the charge was found to affect every metric analysed; these being the cell loss from samples, the number of alleles amplified in resultant direct PCR profiles, and the total RFU of the subsequent profiles. These data outline the importance of this work allowing results to be assessed and triage decisions be made accordingly.
The analysis of wood, PVC pipe, a mobile phone with rubber buttons, a SIM card, and a circuit board showed that none of these samples at 400 mm from the charge caused substrate specific PCR inhibition. On-site collection teams do not need to triage collection based on these sample types as there was no significant difference observed in their ability to return DNA profiling data. Surface area and inhibitor presence are key variables to consider when determining STR processing workflow for post-detonation samples as for samples with larger surface areas within the outdoor environment PCR post-extraction is preferential to direct PCR.
Due to its proficiency to provide the most discriminating results for forensic applications, medical research and anthropological studies, multiplex PCR based STR analysis has been established as the most efficient technique in the forensic DNA analysis. Several multiplex amplification kits based on 4, 5 and 6 dyes chemistry are commercially available and used in forensic DNA typing across the globe. These multiplex PCR systems are routinely used for amplification of multiple STR loci (Autosomal, Y and/or X STR’s) in the DNA extracted from various biological samples. In the routine forensic DNA testing, DNA profile obtained is compared with the DNA profile of the reference sample, which takes a certain turnaround time and employs costly lab resources. Successive development in forensic DNA typing have resulted in advent of improved multiplex kits which have reduced the effective analysis time, cost and minimized the number of steps required in comparison to conventional forensic DNA typing. Specialized direct amplification compatible multiplex kits are also available nowadays. These kits are relatively costlier but still require few pre-processing steps, which does not make them worth the hefty cost. Herein, this study, we have used non-direct multiplex STR kits to assess their efficacy for direct amplification. In the present study, 103 saliva samples were directly amplified without any pre-treatment of the samples using thirteen non-direct multiplex kits (4 dyes, 5 dyes and 6 dyes chemistry based) for forensic DNA typing. Here, we report a validated direct PCR amplification protocol from the reference saliva samples by omitting DNA extraction and quantification steps, which resulted in 80% reduction of the turnaround time. The developed protocol is cost effective, time efficient and it does not compromise with the quality of DNA profiles. To the best of our knowledge, this is the first report for direct amplification of DNA with the most commonly used non-direct multiplex STR kits without any pre-treatment of the sample. Complete DNA profiles matching all the essential quality parameters were obtained successfully from all the tested samples.
Imagine a burglary scene, a ransacked house, and a missing locker full of the family fortune. A crime scene investigator arrives and starts spraying an aerosolized chemical on several spots and observes as these make the fingerprints visible in stark contrast from the surface. A swab is then taken from these spots and sent to the laboratory where they not only get a DNA profile but also are able to determine how the perpetrator looks. The photo is thus developed and is circulated among local law enforcement and the suspects are lined up. The fingerprint and DNA confirm the identity and the perpetrator is convicted. Though, all this might seem futuristic or fictitious currently, it is safe to say that forensic science is undergoing several advancements daily that are pushing the limits of current technology. Be it chemical identification, personal identification, or sensing equipment to be used at the scene of the crime, the domain has seen several advances such as touch DNA, molecular photofitting, geotagging by isotopic detection, sweat analysis for toxicants, and performing autopsy without incisions. These techniques are relying on researchers all around the globe for development. Some of these techniques are discussed in this chapter. It is expected that these techniques will soon observe a reliable and standardized approach to establish a validated protocol to be widely used and be accepted in the courtroom, as these smoothens the scientific investigation and helps a case reach a quick verdict.
The use of a fluorescent dye to visualize cellular material on surfaces offers a targeted sampling approach for locating touch DNA on casework items. However, the current application of such dye is not feasible for examination of relatively large items. As a result, development of an efficient dye application system is required to translate this approach into practice. Here, the spray pattern (area covered, intensity, and evenness) of 15 different commercial spray devices was examined visually using food coloring. From this, five devices were selected to apply Diamond Nucleic Acid Dye (DD) to three substrates (glass slide, plastic sheet, and brown packing tape) seeded with saliva and touch DNA. The cellular material was visualized using the Dino-lite Microscope and Polilight. The inhibitory effects of DD afforded by each spray device were examined using Identifiler Plus® DNA profiling kit and a DNA input of 800 pg. The two most promising devices were further tested on a range of mock casework items seeded with touch DNA. The results presented demonstrate the feasibility of a spray system to apply DD to large surfaces and subsequently detect cellular material at both micro and macroscale. Specifically, the data suggest that a pressurized continuous-spray system is favorable and that droplet size influences the intensity of fluorescence and surface coverage. Furthermore, this study indicates that full STR profiles can be obtained following spraying with DD solution, even with excessive application, which is essential for the widespread use of these devices in casework.
Bombing accounts for the largest share of terrorist incidents worldwide. Most involve an improvised explosive device (IED): a bomb made from household items. Touch DNA may be left on parts of an IED during assembly. However, an IED conflagration degrades DNA, and there has never been a way to locate where touch DNA may remain. To solve this problem, we combined the use of fluorescent dye to locate latent DNA and direct PCR to improve STR profiles of DNA obtained from IEDs. Six fluorescent DNA-binding dyes were evaluated at various concentrations for the purpose of staining latent DNA. SYBR® Green I and Diamond™ Nucleic Acid dye were able to visualize touch DNA on IED substrates. Inhibition studies with extracted DNA and touch DNA using both dyes revealed that Diamond™ dye inhibited direct STR amplification, while SYBR® Green I did not. Stability studies at three temperatures showed optimum performance of SYBR® Green I up to 24 h after formulation. As such, only SYBR® Green I was further used to develop a “visualized-direct PCR” method. Using the conventional approach and the novel “visualized-direct PCR” approach in a single-blind investigation of mock IED evidence, the “visualized-direct PCR” approach had a 98.6% chance of obtaining more alleles (95% highest density interval (HDI): 0.7 to 10.0 alleles). A decrease in non-donor’s alleles (mixed profiles) was also observed. The developed approach has the potential to revolutionize the process of STR typing from touch DNA.
Direct-to-PCR methodology adds samples directly to PCR tubes offering gains in efficiency and sensitivity. The approach has been applied to a variety of biological sources including blood, saliva, tissue, hair and nail. We added various preservative solutions to a range of biological samples to leech DNA into solution, whilst preserving at room temperature. Tubes containing ‘free DNA’ then followed automated workflows for amplification and capillary electrophoresis. Routine FASS-automated workflows (including DNA extraction and quantification) were compared with published direct-to-PCR methodology and automated amplification of an aliquot of preservative solution. Applying preservative solutions to ~30-year-old blood stains stored at room temperature resulted in recovery of a larger quantity of DNA and more alleles (using PowerPlex 21) when compared with routine automated typing. Trials were extended to blood, saliva, hair and nail, mimicking ante-mortem samples collected in a disaster victim identification effort. Despite slightly lower allelic recovery, the faster processing times, lower costs and storage potential offers advantages for the processing of ante-mortem samples.
The use in courtrooms of forensic DNA typing results from presumably touched or handled items is increasing as the sensitivity of detection techniques improves. Research investigating how much DNA can be recovered from handled items, whether trace DNA can be detected under certain scenarios including varying degrees of indirect transfer, and factors which may influence these results is summarized here. Fundamentally, our current understanding of the cellular content of touch deposits and the origins of the potential trace DNA therein is extremely limited. Possible origins include anucleate corneocytes, fragmentary cells/nuclei, nucleated epithelial cells from hands, transferred nucleated cells, and cell-free DNA. Here we review the existing evidence for each possible source and consider remaining knowledge gaps regarding forensically relevant touch depositions.
DNA profiling has emerged as the gold standard for the identification of victims in mass disaster events providing an ability to identify victims, reassociate remains and provide investigative leads at a relatively low cost, and with a high degree of discrimination. For the majority of samples, DNA-based identification can be achieved in a fast, streamlined and high-throughput manner. However, a large number of remains will be extremely compromised, characteristic of mass disasters. Advances in technology and in the field of forensic biology have increased the options for the collection, sampling, preservation and processing of samples for DNA profiling. Furthermore, recent developments now allow a vast array of new genetic markers and genotyping techniques to extract as much genetic information from a sample as possible, ensuring that identification is not only accurate but also possible where material is degraded, or limited. Where historically DNA profiling has involved comparison with ante mortem samples or relatives, now DNA profiling can direct investigators towards putative victims or relatives, for comparison through the determination of externally visible characteristics, or biogeographical ancestry. This paper reviews the current and emerging tools available for maximising the recovery of genetic information from post mortem samples in a disaster victim identification context.
DNA evidence sample processing typically involves DNA extraction, quantification, and STR amplification; however, DNA loss can occur at both the DNA extraction and quantification steps, which is not ideal for forensic evidence containing low levels of DNA. Direct PCR amplification of forensic unknown samples has been suggested as a means to circumvent extraction and quantification, thereby retaining the DNA typically lost during those procedures. Direct PCR amplification is a method in which a sample is added directly to an amplification reaction without being subjected to prior DNA extraction, purification, or quantification. It allows for maximum quantities of DNA to be targeted, minimizes opportunities for error and contamination, and reduces the time and monetary resources required to process samples, although data analysis may take longer as the increased DNA detection sensitivity of direct PCR may lead to more instances of complex mixtures. ISO 17025 accredited laboratories have successfully implemented direct PCR for limited purposes (e.g., high-throughput databanking analysis), and recent studies indicate that direct PCR can be an effective method for processing low-yield evidence samples. Despite its benefits, direct PCR has yet to be widely implemented across laboratories for the processing of evidentiary items. While forensic DNA laboratories are always interested in new methods that will maximize the quantity and quality of genetic information obtained from evidentiary items, there is often a lag between the advent of useful methodologies and their integration into laboratories. Delayed implementation of direct PCR of evidentiary items can be attributed to a variety of factors, including regulatory guidelines that prevent laboratories from omitting the quantification step when processing forensic unknown samples, as is the case in the United States, and, more broadly, a reluctance to validate a technique that is not widely used for evidence samples. The advantages of direct PCR of forensic evidentiary samples justify a re-examination of the factors that have delayed widespread implementation of this method and of the evidence supporting its use. In this review, the current and potential future uses of direct PCR in forensic DNA laboratories are summarized.
The HomyGene19+14Y System (HG19+14Y) is a PCR-based amplification kit that enables typing of 18 autosomal short tandem repeat (STR) loci (i.e., CSF1PO, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, Penta E, TPOX, TH01, vWA), 14 widely used Y chromosome STR (Y-STR) loci (Y_GATA_H4, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS438, DYS439, DYS456, DYS458, DYS635), and amelogenin. This multiplex system was designed for the simultaneous analysis of amelogenin-Y allele mutation, single-source searches, kinship (including familial searching), mixture profiles, international data sharing, and other forensic applications. In this study, the multiplex system was validated for sensitivity, specificity, DNA mixtures, stability, precision, stutter, reproducibility, parallel tests, PCR-based conditions, and population analysis according to the Scientific Working Group on DNA Analysis Methods (SWGDAM) developmental validation guidelines. A total of 212 alleles were detected for the 18 autosomal STR loci among 528 Guangdong Han individuals, and 431 haplotypes were found for 14 Y-STRs among 452 unrelated males. The combined match probability (CMP) of the HG19+14Y System was calculated as 2.39 × 10⁻²⁹. All the validation results showed that the HG19+14Y System would be a robust, reliable, highly polymorphic, and informative forensic kit.
We report on successful amplification of canine STR DNA profiles from single dog hairs. Dog hairs are commonly found on clothing or items of interest in forensic casework and may be crucial associative evidence if linked to an individual dog. We used direct amplification from these hairs to increase the DNA yield of the sample, as well as greatly reducing analysis time. Hairs from different somatic regions were used from several different dog breeds to amplify a selection of eight loci from the validated DogFiler multiplex. Naturally shed canine hairs were processed, with a mix of coarse topcoat (guard) hairs and thinner soft undercoat hairs. Multiple sections of single hairs were amplified in 5 mm segments to determine the viability of DNA recovery from the shaft of the hair. Single guard hairs were cut into 5 mm sections and added directly into a PCR tube. Undercoat hairs, which are very fine, were amplified together in a single tube (approximately 10 small hairs). Coarse hairs were found to be the most successful in producing full DNA profiles at all eight loci, matching the corresponding reference profile for that dog. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
We report on successful amplification of DNA profiles from a single hair. Direct amplification was used on the root tip of both anagen and telogen hairs using a kit to amplify 15 STR loci. All 30 anagen hairs tested from five different people gave full DNA profiles after 29 cycles with no allelic drop-in or heterozygous imbalance. Six of the 30 telogen hairs tested resulted in a full DNA profile, and a further four telogen hair samples tested produced a DNA profile of five or more complete loci that could be up-loaded to the National DNA Database (Australia). A full DNA profile was also obtained from the shaft of an anagen hair. Current practice for many laboratories is that a single hair may not be subjected to DNA testing as there is little chance of success, hence this 100 % success rate from anagen hairs is a significant advancement. A full DNA profile was obtained from a 5 year-old single hair illustrating the success when using direct PCR rather than attempting an extraction prior to the amplification step. The process described deliberately uses current DNA profiling methods with no increase in cycle number, such that the methodology can be incorporated readily into operational practice. For the first time in the field of human identification, single hairs can be analyzed with confidence that a meaningful DNA profile will be generated and the data accepted by the criminal justice system.
Retrieval of DNA from touched objects, for the purpose of generating genetic profiles, can be improved. The amount of DNA deposited varies depending on parameters that include; the individual, the area of contact, the history of previous touches, the material being touched, moisture levels and the presence of fingerprint powder. Much of what is deposited is not retrieved from an object by a single swabbing. This can be improved by employing multiple swabbing techniques. Furthermore, much of the DNA that is collected by a swab is not retrieved from the swab during the extraction phase. Quantiblot™ quantitation results of the retrieved DNA are not accurate and should be used as a guide only, for further analyses. Further improvement in the collection and extraction techniques of trace amounts of sample should be sought to increase the chance of providing probative value to criminal investigations.
DNA profiles can be obtained from fabrics where a person has made direct contact with clothing. A standard approach is to cut out a section of the fabric and then use a commercially available method to extract and isolate the DNA. Alternative methods to isolate DNA include the use of adhesive tape to remove traces of cellular material from the fabric prior to extraction. We report on a process to obtain full DNA profiles using direct amplification from a range of fabrics. The absence of an extraction step both reduces the opportunity for contamination and reduces the loss of DNA during the extraction process, increasing the sensitivity of the process of generating a DNA profile. The process does not require the use of commercially available extraction kits thus reducing the cost of generating a DNA profile from trace amounts of starting material. The results are in part dependent upon the nature of the fabric used to which the DNA has been transferred.
A comparison between swab types, using control DNA deposited on a plastic slide, prior to swabbing, PCR and STR DNA profiling. Fig. 2. Example of an STR DNA profile generated, using direct PCR, from a fingerprint deposited on a plastic slide prior to swabbing and processing (29 cycles of PCR
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Fig. 1. A comparison between swab types, using control DNA deposited on a plastic slide, prior to swabbing, PCR and STR DNA profiling. Fig. 2. Example of an STR DNA profile generated, using direct PCR, from a fingerprint deposited on a plastic slide prior to swabbing and processing (29 cycles of PCR, using NGM TM STR kit (ABI)).
- J M Butler
- Dna Forensic
- Typing
J.M. Butler, Forensic DNA Typing: Biology, Technology, and Genetics of STR
Markers, 2nd ed., Elsevier Academic Press, New York, 2005.
- J Templeton
J. Templeton et al. / Forensic Science International: Genetics Supplement Series xxx (2013) xxx-xxx
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