The effect of NucleoSpin® Forensic Filters on DNA recovery from trace DNA swabs

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 for buccal cells that were 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.     

Developmental Validation of the PrepFiler™ Forensic DNA Extraction Kit for Extraction of Genomic DNA from Biological Samples*

Abstract: The PrepFiler? Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 μL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA^® paper), and touch evidence‐type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol–chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts.     

Extraction of high quality DNA from biological materials and calcified tissues

Calcified tissues, such as bone and tooth, and some other sample types, such as those containing adhesive, present a challenge to standard extraction protocols. We have developed a lysis reagent, BTA™ lysis buffer, which is designed for use with PrepFiler™ Kit reagents. The BTA™ lysis buffer disrupts calcified tissue matrices and achieves effective extraction of DNA from pulverized bone and tooth samples. In addition, the BTA™ lysis buffer mildly but efficiently extracts DNA from challenging substrates like tape, chewing gum, and cigarette butts and, as with bone and tooth, DNA from these lysates is purified using established PrepFiler™ reagent extraction protocols.We successfully extracted DNA from powdered human bone samples, chewed gum and smoked cigarettes using BTA™ lysis buffer. Extraction yields for bone, gum and cigarette samples tested were consistent and reproducible. This extraction method efficiently removed potential PCR inhibitors from all samples tested, and C_T values for the internal PCR control of Quantifiler^® Human DNA Quantification Kit were consistent and within the normal range. The DNA extracted from these samples also provided conclusive profiles that were free of PCR artifacts when amplified using the AmpF?STR^® Identifiler^® PCR Amplification Kit. The protocol is easily adapted for automation.     

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.     

Successful STR and SNP typing of FTA Card samples with low amounts of DNA after DNA extraction using a Qiagen BioRobot EZ1 Workstation

FTA Cards (GE Healthcare) have been used for more than 4 years in Denmark for the collection of buccal cells as reference samples in crime cases. Semi-automated protocols for STR typing of DNA on punches of FTA Cards are routinely used. In average, full STR profiles were generated from approximately 95% of the FTA Cards with a standard punching protocol, while partial or no STR profile were obtained from 5% of the samples. Here, the Qiagen BioRobot^® EZ1 Workstation (Qiagen) and the EZ1 DNA Investigator Kit (Qiagen) was used to extract DNA from 29 FTA Cards from which a complete STR profile was not generated with the standard punching protocol. All 29 samples were successfully typed with the AmpF?STR^® Identifiler™ PCR Amplification Kit (Applied Biosystems) and with the SNPforID 49plex SNP assay. The lowest amount of DNA that resulted in complete STR and SNP profiles was 80 pg. The STR and SNP profiles were identical to those generated from another sample collected from each of the 29 individuals.     

Utility validation of extraction of genomic DNA from hard tissues, bone and nail, using PrepFiler™ Forensic DNA Extraction Kit

STR profiling using hard tissues obtained from a severely decomposed body is sometimes a laborious work. There is now on a market a new DNA extraction kit, PrepFiler™ Forensic DNA Extraction Kit (AppliedBiosystems), and we tested it for missing persons. Postmortem intervals ranged from weeks to several years. Fifteen bone fragments and eleven nails were used in this report. Genomic DNA was quantified by QuantiFiler^® DUO Quantification Kit (AppliedBiosystems), and STRs were analyzed using AmpFlSTR^® Identifiler^® PCR Amplification Kit (AppliedBiosystems). The profiling of 16 STR loci was successful in all nail samples. However, STR profiling was successful in only 6 of 15 bone materials. Nine cases failed to analyze STR polymorphisms using another DNA extraction kit, the QIAamp DNA Mini Kit (QIAGEN). For bone samples, it seems that STR profiling depends on the quality of samples.     

Efficiency of DNA IQ System in recovering semen DNA from cotton swabs

With the aim to asses the efficiency of the DNA IQ System in the recovery of DNA from semen samples, cotton swabs were prepared from 1/5 serial dilutions of semen. Each swab was fractionated in four equivalent quarters and the DNA was further extracted following the differential lysis protocol. The recovered DNA was quantified by means of real time PCR and the average DNA yield was used to compare results. Direct extractions of equivalent aliquots of each semen dilution were used as reference samples. Even though a high percentage of the starting material was lost during the process of transfer to/recover from the solid support, our experimental results demonstrated that the DNA IQ system was able to detect around 10³ sperm cells in the starting material, enabling to obtain a complete DNA profile with AmpFl STR IdentiFiler PCR Amplification Kit (Applied Biosystems).     

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.     

Implementation of Prep-n-Go™ Buffer for DNA extraction from buccal swabs

The efficacy of two extraction methods; room temperature and heat protocols was assessed for buccal swabs using the Prep-n-Go™ Buffer. DNA was extracted from buccal swabs using both extraction methods and their effectiveness to produce good quality DNA profiles was evaluated. Heat protocol was found to yield more DNA, however room temperature protocol produced better quality DNA profiles with fewer artefacts when the samples from both extraction methods were amplified directly without any normalisation with the VeriFiler™ Express PCR Amplification Kit.     

Optimization of DNA recovery from toothbrushes

In human identification, the victim's toothbrush is an invaluable personal item as the deposited cellular material contains DNA from which a reference profile can be produced. The profile obtained then allows direct comparison to be made with the profile from the unidentified body. This study was undertaken to determine the minimum number of bristle bundles that would generate a complete DNA profile. The minimum period of usage for a toothbrush to retain enough cells for genotyping was also investigated. We also tested two commonly used DNA extraction methods: QIAamp^® DNA Mini Kit and Chelex^® 100 to explore the efficiency of these protocols in recovering DNA from toothbrushes. In this experiment, volunteers brushed their teeth for 1, 7, 14, or 30 days. DNA was extracted from 5 and 10 bundles of bristles cut from the collected toothbrushes. The amount of DNA recovered was quantified by quantitative real-time PCR, and DNA genotyping was performed for each sample. Data revealed that QIAamp^® DNA Mini Kit performed better at yielding DNA in terms of purity, quantity, and quality than Chelex^® 100. It was also found that, with a suitable method of recovery, DNA samples from five bundles of bristles from all of the toothbrushes generated complete profiles. Based on the experimental results, a general guideline concerning the appropriate extraction method and the quantity of the starting material for the analysis of DNA from toothbrushes could be suggested.     

Analysis of forensic samples in Banco Nacional de Datos Genéticos

Analysis of forensic samples to evaluate the rate of success for molecular markers: autosomal STRs, Y chromosome, and mitochondrial DNA. Since 2006 to date a total of 390 forensic samples were analyzed: bones, teeth, hairs, swabs, stains and paraffin embedded tissue. Bones and teeth, were pulverized in a Freezer Mill, extracted by chloroform/phenol/isoamyl alcohol method, and then purified with Centricon 100 columns. DNA from paraffin was extracted with QIAmp DNA Mini kit (QIAGEN). Mitochondrial DNA Control Region sequences were determined for regions HV1/HV2. Sequencing was performed using the BigDye^® Terminator v 1.1 Kit and analyzed in ABIPRISM^® 3100 Genetic Analyzer (AB). STRs were amplified using Amp FlSTR Identifiler^®, Minifiler^® and YFiler^® Kit (AB) and analyzed in ABI PRISM^® 3100 Genetic Analyzer and ABI PRISM^® 3130xl Genetic Analyzer (AB). Among forensic samples, bones and teeth analyzed for autosomal STRs, we obtained successful results in all of them. Incomplete typing are represented by loci of higher molecular weight, which demonstrates the poor quality of the sample due to its state of degradation and obtained better results using mini STRs. Successful results in sequencing for mitochondrial HV1 region for all samples analyzed, but in few hair samples we obtained mixed sequences and that represented important difficulties for the analysis. Age of samples and conservation are factors related which affect DNA viability. Autosomal STRs solved all the samples analyzed in our study, but Y chromosome analysis and mitochondrial DNA sequencing are also important and necessary markers in some forensic cases.     

Simplified Direct PCR Method for Reference Buccal Samples Using a Non-FTA Card by Omitting the Pretreatment Step

When using non-FTA cards in commercial multiplex STR kits for direct PCR, pretreatment steps with specific buffers are recommended. Here, we designed a rapid direct PCR method utilizing a non-FTA card, Oral Cell Sampling Kit, by omitting the pretreatment step involving Prep-n-Go™ Buffer, and it showed compatibility with the GlobalFiler™ Express PCR Amplification Kit, GlobalFiler™ PCR Amplification Kit, and PowerPlex^® Fusion system. To optimize the PCR conditions, we tested the method with different final PCR volumes and cycles. Finally, we conducted a performance test using 50 Korean buccal samples and confirmed the high performance of the method, detecting more than 90% of the samples with full profiles when using GlobalFiler™ PCR Amplification Kit and PowerPlex^® Fusion system at 29 cycles in a 10 μL final PCR volume. Thus, we report a simple direct PCR set-up to analyze reference samples collected using a non-FTA card manufactured in Korea.     

Developmental validation of the PowerPlex ESX 16 and PowerPlex ESX 17 Systems

We describe the developmental validation study performed on the PowerPlex^® ESX 16 (European Standard Extended 16) and the PowerPlex^® ESX 17 Systems, part of a suite of four new DNA profiling kits developed by Promega in response to the ENFSI and EDNAP groups’ call for new STR multiplexes for Europe. The PowerPlex^® ESX 16 System combines the 11 loci compatible with the UK National DNA Database, contained within the AmpFlSTR^® SGM Plus^® PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to incorporate these five new loci as mini- and midi-STRs while maintaining the loci found in the AmpFlSTR^® SGM Plus^® kit as standard size. The PowerPlex^® ESX 17 System amplifies the same loci as the PowerPlex^® ESX 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpFlSTR^® SGM Plus^® kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex^® ESX 16 and ESX 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5 pg of a fully heterozygous single source DNA template. In mixture analysis, a range of 52-95% of unique minor contributor alleles was observed at 19:1 mixture ratios where only 25 pg of the minor component was present. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of information obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors.     

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.     

A comparison of three automated DNA purification methods in Forensic casework

Manual Chelex^®-100 and organic extractions (phenol/chloroform) are used as routine methods at the Swedish National Laboratory of Forensic Science, SKL. The aim of this study was to find an automated DNA purification system to replace the organic method. The following methods were evaluated and compared to each other and to the organic method used routinely; BioRobot^® EZ1 with EZ1 DNA Investigator Kit and Card (Qiagen), iPrep™ Purification Instrument with iPrep™ ChargeSwitch^® Forensic Kit and Card (Invitrogen), Magnatrix™ 1200 Workstation with the Magnatrix™ gDNA Blood Kit Forensic and two different protocols; Forensic protocol A and B (Magnetic Biosolutions). Blood on fats, cotton swabs, moist snuff, paper towels and leather, post-mortem blood and muscle tissue were extracted with the different methods. DNA concentration and quality of the electropherograms were examined. Individual comparisons between the four extraction methods showed that iPrep™ and Magnatrix™ 1200 gave significantly lower mean quantities compared to BioRobot^® EZ1 and the organic extraction method (p < 0.05). There were no significant differences between the latter two. BioRobot^® EZ1 generated the best results and is in the process of being validated for routine analysis at SKL.     

A production system to generate reference genetic profiles from buccal swab cells on FTA cards

To generate the large quantity of genetic profiles that continuously feeds the French Reference Sample Database (F.N.A.E.G.), a secure and robust process is required. A complete automated production chain has been developed by Hamilton Robotics and the French Police Scientifique in Lyon to produce genetic profiles from buccal swab cells on FTA^® cards. The activities have been divided between Pre- and Post-PCR. The samples on FTA^® cards are punched directly into PCR plates. The plates are then transferred onto the Hamilton Microlab^® STAR liquid handling instruments. Air displacement pipetting coupled with Hamilton's CO-RE Technology allows a highly secure and contamination free pipetting for FTA^® wash and STR^® Identifiler^® PCR reaction set-up. Plates are sealed and transferred to the Post-PCR system for pooling into 384 format and denaturation. Throughout the process steps a complete sample tracking is performed and sample information is transferred to the LIMS system. This poster describes in detail this production system with an actual throughput of 40,000 samples/month, which is in production since 2006.     

Adaptation and evaluation of the PrepFiler™ DNA extraction technology in an automated forensic DNA analysis process with emphasis on DNA yield, inhibitor removal and contamination security

Within the initial step of the forensic DNA analysis process, the DNA extraction efficiency and especially the removal of potential PCR inhibitors is crucial for subsequent steps, e.g. quantification by real-time PCR and amplification of short tandem repeats (STRs). The protocol of the PrepFiler™ Forensic DNA Extraction Kit was optimized for the application on a Tecan liquid handling workstation Freedom EVO^® 150. This modified application of the PrepFiler™ technology was compared with respect to DNA yield, sensitivity and the ability to remove potential PCR inhibitors to an established routine method working on the same liquid handling workstation based on ChargeSwitch^® Technology (CST) from Invitrogen.     

Identification and isolation of male cells using fluorescence in situ hybridisation and laser microdissection, for use in the investigation of sexual assault

In cases of sexual assault involving an azoospermic assailant, vaginal swabs taken from the victim may fail to provide an autosomal DNA profile with which to search a suspect database, as the signal from any male cells present would be masked by that from the overwhelming number of female cells collected on the swab. Here, we describe a method of visually identifying diploid male cells in such samples using fluorescence in situ hybridisation, and selectively harvesting them by means of laser microdissection. This combination of techniques was tested on 26 post-coital vaginal swabs taken at a range of times after intercourse; the collected cells were then subjected to a simple lysis procedure and DNA was amplified using the AmpFlSTR^® SGMPlus^® multiplex under low copy number conditions. Useful DNA profiles were generated from samples taken up to 24 h after intercourse.     

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.     

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.