An Optimized DNA Analysis Workflow for the Sampling,Extraction, and Concentration of DNA obtained from 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.     

Collection and direct amplification methods using the GlobalFiler™ kit for DNA recovered from common pipe bomb substrates

When analyzing DNA from exploded pipe bombs, quantities are often in trace amounts, making DNA typing extremely difficult. Amplifying minute amounts of DNA can cause stochastic effects resulting in partial or uninterpretable profiles. Therefore, the initial DNA collection from “touch” evidence must be optimized to maximize the amount of DNA available for analysis.This proof-of-concept study evaluated two different swab types with two direct amplification strategies to identify the most effective method for recovering DNA from common pipe bomb substrates. PVC and steel pipes, electrical tape, and copper wire spiked with epithelial cells were swabbed with cotton or microFLOQ® Direct Swabs and amplified directly or via a pre-treatment prior to STR amplification.Not only was the microFLOQ® Direct Swab protocol the quickest method with the least risk of contamination, but in combination with direct amplification, the microFLOQ® Direct Swabs also generated the most complete STR profiles.     

Direct amplification of STRs from blood or buccal cell samples

Forensic databasing laboratories routinely analyze blood or buccal cell samples deposited on FTA^® paper. Prior to PCR amplification of the STRs, the FTA^® samples must undergo multi-step sample purification protocols to remove the PCR inhibitors present within the sample and from the FTA^® paper. The multi-step sample purification protocols are laborious, time-consuming and increase the potential for sample cross-contamination.To eliminate the need for DNA purification, we conducted studies to optimize the PCR buffer and thermal cycling parameters to allow for direct amplification of STRs from blood or buccal samples on FTA^® paper. We evaluated the effect of various factors on the DNA profile including: FTA^® disc size, blood sample load variation, and buffer formulation. The new STR assay enables the direct amplification of DNA from single source samples on FTA^® discs without sample purification. The new STR assay improves the workflow by eliminating tedious steps and minimizing sample handling. Furthermore, the new STR assay reduces cost by eliminating the need for purification reagents and expensive robots.     

DNA IQ磁珠法结合Maxwell~（TM） 16自动仪提取接触DNA

目的研究DNA IQ磁珠法结合MaxwellTM 16自动仪对接触DNA提取的应用价值。方法 151份案件接触DNA检材95℃裂解后,采用DNA IQ磁珠法结合MaxwellTM 16自动仪提取DNA,然后进行DNA定量和STR分型检测,统计各种类型的接触DNA含量I、PC CT值和STR分型成功率。结果 151份案件接触DNA检材中,除果核平均DNA获得量为9.51ng以外,其它接触检材的平均DNA获得量均大于10ng,烟蒂检验成功率最高为93%,果核检验成功率较低,为60%。所有DNA样品的IPC CT值均在27左右,纯度高。结论大部分接触DNA检材采用DNA IQ磁珠法结合MaxwellTM 16自动仪可提取到足以进行STR分型的DNA。     

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.     

Autosomal short tandem repeat analysis of ancient DNA by coupled use of mini- and conventional STR kits

Abstract: Multiplex autosomal short tandem repeat (STR) genotyping enables researchers to obtain genetic information from ancient human samples. In this study, we tested newly developed AmpF?STR^® MiniFiler? kit for autosomal STR analysis of ancient DNA (aDNA), using human femurs (n = 8) collected from medieval Korean tombs. After extracting aDNA from the bones, autosomal STR analyses were repeated for each sample using the AmpF?STR^® MiniFiler? and Identifiler? kits. Whereas only 21.87% of larger‐sized loci profiles could be obtained with the Identifiler? kit, 75% of the same loci profiles were determined by MiniFiler? kit analysis. This very successful amplification of large‐sized STR markers from highly degraded aDNA suggests that the MiniFiler? kit could be a useful complement to conventional STR kit analysis of ancient samples.     

Reduction of Powerplex® Y23 Reaction Volume for Genotyping Buccal Cell Samples on FTATM Cards

PowerPlex^® Y23 is a novel kit for Y‐STR typing that includes new highly discriminating loci. The Israel DNA Database laboratory has recently adopted it for routine Y‐STR analysis. This study examined PCR amplification from 1.2‐mm FTA punch in reduced volumes of 5 and 10 μL. Direct amplification and washing of the FTA punches were examined in different PCR cycle numbers. One short robotically performed wash was found to improve the quality and the percent of profiles obtained. The optimal PCR cycle number was determined for 5 and 10 μL reaction volumes. The percent of obtained profiles, color balance, and reproducibility were examined. High‐quality profiles were achieved in 90% and 88% of the samples amplified in 5 and 10 μL, respectively, in the first attempt. Volume reduction to 5 μL has a vast economic impact especially for DNA database laboratories.     

STR Profiles from DNA Samples with "Undetected" or Low Quantifiler™ Results

Abstract:  Screening methods capable of identifying DNA samples that will not yield short tandem repeat (STR) profiles are desired. In the past, quantitation methods have not been sensitive enough for this purpose. In this study, low level DNA samples were used to assess whether Quantifiler™ has a minimum quantitation value below which STR profiles would consistently fail to be detected. Buccal swabs were obtained and the DNA extracted, quantified, and serially diluted to concentrations ranging from 0.002 to 0.250 ng/μL. Samples were analyzed once with Quantifiler™, followed by Profiler Plus™ amplification and capillary electrophoresis analysis. An absolute minimum value below which STR results were unobtainable could not be defined. From the 96 low level samples tested, STR loci (including one full profile) were successfully amplified and detected from 27% of the samples "undetected" by Quantifiler™. However, no STR alleles were detected in 73% of these "undetected" samples, indicating that Quantifiler™ data may be useful for predicting STR typing success.     

Development of STR profiles from firearms and fired cartridge cases

Fired cartridge cases are a common type of evidence found at crime scenes. However, due to the high chamber temperatures and touch nature of this evidence, DNA testing is not commonly sought because it is believed DNA is only present in low levels, whether it is due to initial low levels of DNA and/or DNA degradation from the heat or inhibition of the PCR reaction. Moreover, very few laboratories report STR typing success with fired cases. This study focused on obtaining STR profiles from fired cartridge cases using the AmpFℓSTR^® MiniFiler™ kit, which is designed to amplify DNA from low level, inhibited, and degraded samples. Comparisons to other STR amplification kits were also conducted. In attempt to simulate casework, random individuals loaded cartridges into a firearm. DNA was recovered from the fired cartridge cases using the double swab technique and extracted using an automated large volume DNA IQ™ method. Initially, testing focused on known shedders handling cartridges for 30 s prior to firing. A significantly greater number of alleles was obtained following amplification with the MiniFiler™ kit versus the PowerPlex^® 16 BIO kit. No alleles were observed using the Identifiler^® kit. In an attempt to better simulate casework, a random selection of laboratory personnel handled shotshells for as long as needed to load and fire the weapon. In this mock sample study, the MiniFiler™ kit successfully amplified an average of 22% of expected alleles from DNA recovered from shotshell cases versus the PowerPlex^® 16 BIO kit where an average of 7% of alleles were observed. However, the total number of alleles obtained from the two kits was not significantly different. The quality of the DNA obtained from fired cases was studied with evidence of inhibition in at least 11% of shotshell case samples. After swabbing the head and the hull of three shotshell cases separately, a significantly greater number of alleles was obtained from the hull as opposed to the head of the fired shotshell case. In addition, after firing, various internal firearm surfaces were swabbed, including the chamber of barrel, ejection port, and breechface, in an attempt to obtain amplifiable DNA. DNA was obtained from the chamber of the barrel and was amplifiable using the MiniFiler™ kit, although mixtures were obtained with extensive drop-in and drop-out making this analysis unlikely to aid an investigation.     

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™.     

A rapid wire-based sampling method for DNA profiling

Abstract: This paper reports the results of a commission to develop a field deployable rapid short tandem repeat (STR)‐based DNA profiling system to enable discrimination between tissues derived from a small number of individuals. Speed was achieved by truncation of sample preparation and field deployability by use of an Agilent 2100 Bioanalyser^TM. Human blood and tissues were stabbed with heated stainless steel wire and the resulting sample dehydrated with isopropanol prior to direct addition to a PCR. Choice of a polymerase tolerant of tissue residues and cycles of amplification appropriate for the amount of template expected yielded useful profiles with a custom‐designed quintuplex primer set suitable for use with the Bioanalyser^TM. Samples stored on wires remained amplifiable for months, allowing their transportation unrefrigerated from remote locations to a laboratory for analysis using AmpFlSTR^® Profiler Plus^® without further processing. The field system meets the requirements for discrimination of samples from small sets and retains access to full STR profiling when required.     

Multiplex short tandem repeat amplification of low template DNA samples with the addition of proofreading enzymes

Abstract:  With <100 pg of template DNA, routine short tandem repeat (STR) analysis often fails, resulting in no or partial profiles and increased stochastic effects. To overcome this, some have investigated preamplification methods that include the addition of proofreading enzymes to the PCR cocktail. This project sought to determine whether adding proofreading polymerases directly in the STR amplification mixture would improve the reaction when little template DNA is available. Platinum Taq High Fidelity and GeneAmp High Fidelity were tested in Profiler Plus? STR reactions alone and in combination with AmpliTaq^® Gold. All reactions included the additional step of a post‐PCR purification step. With both pristine low template DNA and casework samples, the addition of these polymerases resulted in comparable or no improvement in the STR amplification signal. Further, stochastic effects and artifacts were observed equally across all enzyme conditions. Based on these studies, the addition of these proofreading enzymes to a multiplex STR amplification is not recommended for low template DNA work.     

人体接触检材前处理方式对磁珠法提取DNA效果的影响

目的比较3种常见的接触检材前处理方式对磁珠法提取DNA效果的影响。方法收集烟蒂、牙刷、纱线手套各10份;分别采用95℃、70℃直接裂解和TNE、SDS、PK预消化方式进行前处理,再用磁珠法提取纯化DNA,并进行DNA定量,统计提取的接触DNA量和IPC CT值;同时用Sinofiler复合扩增系统进行STR分型检测。结果 3种方法前处理后用磁珠提取的DNA纯度均较高I,PC CT值在26.63～27.19之间。用预消化法获得的DNA量高于裂解法,而95℃裂解与70℃裂解方法提取的DNA量无显著性差异。STR扩增检测结果亦表明,采用预消化法处理的样品STR分型成功率高于裂解法9,5℃与70℃裂解方法处理的样品STR分型成功率无显著性差异。结论人体接触检材采用预消化磁珠法提取DNA,有助于提高STR检验成功率。     

DNA Typer~(TM)15 plus直扩试剂盒在DNA数据库建设中的应用

目的测试DNA TyperTM15 plus直扩试剂盒的技术性能指标,评价其在DNA数据库建设中的应用价值。方法采用DNA TyperTM15 plus试剂盒,并使用IdentifilerTM和DNA TyperTM15试剂盒进行比较,设定不同体系和引物量、不同退火温度和循环次数以进行方法验证;设定不同模板量标准品、不同比例混合样本,取猪、狗、兔等动物的血液样品,血痕、骨骼、唾液斑等常见检材样本以及不同建库样本,以验证试剂盒灵敏度、特异性、稳定性以及混合样本、常见检材及建库样本的检测能力。结果直扩试剂盒分型结果准确,重复性好,灵敏度可达0.125ng,不同批次间试剂检测结果稳定,对不同检材有很好的适应性。10μL扩增体系时FTA卡和加强型血液采集卡取样直径应为0.5mm,而血滤纸、血液采集卡样本和经典型血液采集卡取样直径应为1.0mm。结论 DNA TyperTM15 plus直扩试剂盒的性能可以满足DNA数据库建设及检案的需要,可在相关实验中选择使用。     

A dedicated automated system for extraction, quantification and STR amplification of forensic evidence samples

Forensic DNA analysis is a multi-step process involving extraction of DNA, quantification of human DNA in the extract, amplification using multiplex STR systems, separation of products, and data analysis. The backlog of forensic casework is increasing worldwide. Automation is one significant way to alleviate the bottleneck of sample processing in forensic labs. The HID EVOlution™ Combination System described here is a robust, reliable sample processing platform, easily adapted to forensic laboratory workflows. Using a variety of forensic sample types including: blood stained FTA paper, cotton fabric and denim, dried blood spiked with known PCR inhibitors, saliva on cotton swabs, and semen stains, we found that yields of human DNA and STR profiles obtained with AmpFlSTR^® Idenitfiler^® kits were complete, highly reproducible, and equivalent to results obtained using the manual PrepFiler™ reagent extraction method. Automated operation was clean, and no cross-contamination was detected between extraction blanks and interspersed high DNA content samples.     

Recovery of human DNA profiles from poached deer remains: A feasibility study

Poaching is a crime that occurs worldwide and can be extremely difficult to investigate and prosecute due to the nature of the evidence available. If a species is protected by international legislation such as the Convention on International Trade in Endangered Species of Wild Fauna and Flora then simply possessing any part of that species is illegal. Previous studies have focused on the identification of endangered species in cases of potential poaching. Difficulties arise if the poached animal is not endangered. Species such as deer have hunting seasons whereby they can legally be hunted however poaching is the illegal take of deer, irrespective of season. Therefore, identification of deer alone has little probative value as samples could have originated from legal hunting activities in season. After a deer is hunted it is usual to remove the innards, head and lower limbs. The limbs are removed through manual force and represent a potential source of human touch DNA.We investigate the potential to recover and profile human autosomal DNA from poached deer remains. Samples from the legs of ten culled deer were obtained (40 in total) using minitapes. DNA from samples was extracted, quantified and amplified to determine if it would be possible to recover human STR profiles.Low quantification data led to the use of an extended PCR cycling protocol of 34 cycles. Samples from seven deer amplified, however some samples were excluded from further analysis due to ‘drop in’ alleles or the low level of successfully amplified loci. Samples from five deer could be further analysed and gave match probabilities ranging from 6.37 × 10^{− 3} to 9.53 × 10^{− 11}.This study demonstrates the potential of recovering human touch DNA from poached animal remains. There is the potential for this test to be used in relation to other species of poached remains or other types of wildlife crimes. This is the first time, to our knowledge, that human STR profiling has been successfully applied to touch DNA in regards to simulated wildlife crime.     

Direct STR amplification from whole blood and blood- or saliva-spotted FTA without DNA purification

The DNA purification step has been thought to be essential for typing of STR DNA. However, this process is time-consuming, and there is a risk of unexpected cross-contamination during purification. We report a new method for direct short tandem repeat (STR) amplification using a newly developed direct PCR buffer, AnyDirect, which can amplify STR loci from whole blood and blood- or saliva-spotted FTA cards without DNA purification. The autosomal and Y chromosomal STR loci were analyzed for whole blood and blood or saliva spots of random individuals, followed by comparison of the results with those of corresponding purified DNA. The results from whole blood and blood spots showed perfect concordance with those from purified DNA without allele or locus drop-out. However, in the case of saliva spots, no amplification or locus drop-out was observed in some of the samples, which offers a topic for further study. Additionally, some commercial hot-start DNA polymerases other than AmpliTaq Gold DNA polymerase were also found to be compatible with this buffer system. Therefore, this direct PCR buffer was demonstrated to be useful for fast forensic DNA analysis or criminal DNA databases for which there is no need to store DNA samples.     

Advantage of ForensiX Swabs in Retrieving and Preserving Biological Fluids

This study compares two novel swabs (forensiX) with a standard cotton swab (EUROTUBE) for the collection of saliva stains on glass slide for STR analysis. ForensiX collection tubes are a standard cotton swab in an “active drying” tube, where swab sample is soon dried by its innovative tube surface of the wall. The other is forensiX Nylon Flocked Swab. The study is two phases: The first “phase” assesses swab types regarding to retrieve ability of saliva. The second “phase” compares the drying ability of each swab to assess how crime samples would fare when left in storage. The main result showed that “active drying” is effective to store swabbed sample. The forensiX swabs generally are effective for higher (twofold to fourfold) DNA yield compared to delta lab swab (around 750 pg and 250 pg from 0.5 μL of saliva), respectively. These findings demonstrate the importance of drying performance in the preservation of DNA and swab selection.     

Evaluation of direct PCR for routine DNA profiling of non-decomposed deceased individuals

Forensic DNA profiling is a standard method used in the attempt to identify deceased individuals. In routine investigations, and if available, the preferred sample type is usually blood. However, this requires the invasive re-opening of the body, days or weeks after the autopsy, which is undesirable in resource-constrained mortuary settings. Motivated by the ease of sampling as well as reduced health and safety risks, this study aimed to establish the success rate of generating a full DNA profile on first attempt from buccal swab lysates using a direct PCR approach. Buccal swab samples were collected from 100 unidentified deceased males, and were subjected to direct DNA profiling with use of the Promega PowerPlex® Y23 Kit. At the time of sample collection, these individuals had been stored for between 1 and 887 days. This study shows that full DNA profiles were initially obtained from 73% of samples, which constitutes the first empirical data pertaining to first time success rates of direct PCR from post-mortem buccal lysates. Further investigation of partial and failed DNA profiles using real-time PCR showed that samples did not contain PCR inhibitors, DNA was not degraded, but DNA concentration was particularly low. Repeating DNA profiling with increased lysate input and extra PCR cycles yielded an additional six full DNA profiles, resulting in an overall success rate of 79%. Overall, DNA profile success rate was not associated with the duration of storage (p = 0.387). Lastly, massively parallel sequencing with the ForenSeq™ Signature DNA Prep kit provided more informative profiles for three additional samples. These results indicate that blood should therefore remain the sample of choice in a post-mortem setting, yet buccal lysates hold potential to be optimised further, which may ease the human identification workflow.     

Extended PCR conditions to reduce drop-out frequencies in low template STR typing including unequal mixtures

Forensic laboratories employ various approaches to obtain short tandem repeat (STR) profiles from minimal traces (<100 pg DNA input). Most approaches aim to sensitize DNA profiling by increasing the amplification level by a higher cycle number or enlarging the amount of PCR products analyzed during capillary electrophoresis. These methods have limitations when unequal mixtures are genotyped, since the major component will be over-amplified or over-loaded. This study explores an alternative strategy for improved detection of the minor components in low template (LT) DNA typing that may be better suited for the detection of the minor component in mixtures. The strategy increases the PCR amplification efficiency by extending the primer annealing time several folds. When the AmpF?STR^® Identifiler^® amplification parameters are changed to an annealing time of 20 min during all 28 cycles, the drop-out frequency is reduced for both pristine DNA and single or multiple donor mock case work samples. In addition, increased peak heights and slightly more drop-ins are observed while the heterozygous peak balance remains similar as with the conventional Identifiler protocol. By this extended protocol, full DNA profiles were obtained from only 12 sperm heads (which corresponds to 36 pg of DNA) that were collected by laser micro dissection. Notwithstanding the improved detection, allele drop-outs do persist, albeit in lower frequencies. Thus a LT interpretation strategy such as deducing consensus profiles from multiple independent amplifications is appropriate. The use of extended PCR conditions represents a general approach to improve detection of unequal mixtures as shown using four commercially available kits (AmpF?STR^® Identifiler, SEfiler Plus, NGM and Yfiler). The extended PCR protocol seems to amplify more of the molecules in LT samples during PCR, which results in a lower drop-out frequency.