The Extraction and Recovery Efficiency of Pure DNA for Different Types of Swabs

The extraction and recovery efficiency of swabs used to collect evidence at crime scenes is relatively low (typically <50%) for bacterial spores and body fluids. Cell‐free deoxyribonucleic acid (DNA) is an interesting alternative compared to whole cells as a source for forensic analysis, but extraction and recovery from swabs has not been tested before using pure DNA. In this study cotton, foam, nylon flocked, polyester and rayon swabs are investigated in order to collect pure DNA isolated from saliva samples. The morphology and absorption capacity of swabs is studied. Extraction and recovery efficiencies are determined and compared to the maximum theoretical efficiency. The results indicate that a substantial part of DNA is not extracted from the swab and some types of swab seem to bind effectively with DNA. The efficiency of the different types of swab never exceeds 50%. The nylon flocked 4N6FLOQSwab used for buccal sampling performs the best.     

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.     

Swabs as DNA Collection Devices for Sampling Different Biological Materials from Different Substrates

Currently, there is a variety of swabs for collection of biological evidence from crime scenes, but their comparative efficiency is unknown. Here, we report the results of an investigation into the efficiency of different swab types to collect blood, saliva and touch DNA from a range of substrates. The efficiency of extracting blood and saliva from each swab type was also tested. Some swabs were significantly more effective than others for sampling biological materials from different substrates. Swabs with the highest sampling efficiency, however, often did not have the highest extraction efficiency. Observations were recorded regarding practicality of each swab in a variety of situations. Our study demonstrates that selection of sampling device impacts greatly upon successful collection and extraction of DNA. We present guidelines to assist in evaluation of swab choice.     

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.     

Automated extraction of DNA from reference samples from various types of biological materials on the Qiagen BioRobot EZ1 Workstation

We have validated and implemented a protocol for DNA extraction from various types of biological materials using a Qiagen BioRobot EZ1 Workstation. The sample materials included whole blood, blood from deceased, buccal cells on Omni swabs and FTA Cards, blood on FTA Cards and cotton swabs, and muscle biopsies. The DNA extraction was validated according to EN/ISO 17025 for the STR kits AmpF?STR^® Identifiler^® and AmpF?STR^® Yfiler^® (Applied Biosystems). Of 298 samples extracted, 11 (4%) did not yield acceptable results. In conclusion, we have demonstrated that extraction of DNA from various types of biological material can be performed quickly and without the use of hazardous chemicals, and that the DNA may be successfully STR typed according to the requirements of forensic genetic investigations accredited according to EN/ISO 17025.     

A simple automated instrument for DNA extraction in forensic casework

The Qiagen BioRobot EZ1 is a small, rapid, and reliable automated DNA extraction instrument capable of extracting DNA from up to six samples in as few as 20 min using magnetic bead technology. The San Diego Police Department Crime Laboratory has validated the BioRobot EZ1 for the DNA extraction of evidence and reference samples in forensic casework. The BioRobot EZ1 was evaluated for use on a variety of different evidence sample types including blood, saliva, and semen evidence. The performance of the BioRobot EZ1 with regard to DNA recovery and potential cross-contamination was also assessed. DNA yields obtained with the BioRobot EZ1 were comparable to those from organic extraction. The BioRobot EZ1 was effective at removing PCR inhibitors, which often co-purify with DNA in organic extractions. The incorporation of the BioRobot EZ1 into forensic casework has streamlined the DNA analysis process by reducing the need for labor-intensive phenol-chloroform extractions.     

Optimization of DNA extraction from low-yield and degraded samples using the BioRobot EZ1 and BioRobot M48

Robotic extraction of DNA from dilutions of blood and semen using either the BioRobots EZ1 or BioRobots M48 consistently produced lower recoveries than standard organic extractions of the same samples. In an effort to increase the efficiency of robotically extracted DNA, glycogen and carrier RNA were added following cell lysis. The addition of glycogen, postlysis, resulted in no improvement in DNA recovery with the BioRobot EZ1. However, when carrier RNA was added to the cell lysate of limited and degraded samples extracted on the EZ1 or the M48, DNA recoveries dramatically increased four- to 20-fold. DNA yields obtained by robotic extraction in the presence of carrier RNA were as high, or higher, as those obtained by organic extraction lacking carrier RNA, while experiments that utilized carrier RNA in both types of extractions showed increased sensitivity for both methods. Furthermore, carrier RNA substantially increased the recovery of fragmented DNA with the EZ1.     

Nondestructive Biological Evidence Collection with Alternative Swabs and Adhesive Lifters

In forensic science, biological material is typically collected from evidence via wet/dry double swabbing with cotton swabs, which is effective but can visibly damage an item's surface. When an item's appearance must be maintained, dry swabbing and tape‐lifting may be employed as collection techniques that are visually nondestructive to substrates' surfaces. This study examined the efficacy of alternative swab matrices and adhesive lifters when collecting blood and fingerprints from glass, painted drywall, 100% cotton, and copy paper. Data were evaluated by determining the percent profile and quality score for each STR profile generated. Hydraflock^® swabs, BVDA Gellifters^®, and Scenesafe FAST? tape performed as well as or better than cotton swabs when collecting fingerprints from painted drywall and 100% cotton. Collection success was also dependent on the type of biological material sampled and the substrate on which it was deposited. These results demonstrated that alternative swabs and adhesive lifters can be effective for nondestructive DNA collection from various substrates.     

Evaluation of the use of freezer mill to improve DNA retrieval from dried cotton swabs

This investigation evaluated the use of a freezer mill to improve retrieval of DNA from dried cotton swabs (using 100 μl saliva) compared to uncrushed swabs and whole saliva by measuring DNA yield and profile average peak height. Three treatments were tested; short, medium (as for a bone sample) and extended. The samples subjected to the freezer mill had the powder that remained on the freezer mill components collected (using a water and agitation method). All freezer mill samples returned a lower average DNA yield than either uncrushed swabs or whole saliva. The powder from the crushed swabs comprised an average of 35% of the total DNA yield, whereas the powder from the components comprised 65%. Allele drop-out was observed in samples exposed to extended treatments. Both short and medium treatments provided significantly higher peak heights than uncrushed cotton swabs with equal quantities of DNA (P < 0.05). Using a freezer mill on dried cotton swabs does not increase the DNA yield. This investigation suggests collecting powder from the freezer mill components will increase DNA yield, especially from trace samples.     

Application of BioRobot M48 to forensic DNA extraction

The development of a nucleic acid extraction method based on magnetic separation has opened up possibilities forl automation of DNA extraction. The BioRobot M48 is one of robotic stations applicable to automated DNA extraction in forensics. However, each new method should be thoroughly validated before application to routine casework. Our aim was to compare the effectiveness of the currently utilized organic/Microcon 100 based extraction procedure and magnetic extraction with BioRobot M48. The DNA concentration of DNA extracts obtained from different kinds of typical forensic material was evaluated followed by amplification with the SGM Plus or Identifiler kit and capillary electrophoresis using ABI 3100 Avant. We can conclude that BioRobot M48 is a very effective instrument for DNA extraction from most specimens and can be successfully applied in forensic laboratories.     

Contribution to the Development of Guidelines in the Analysis of Biological Evidence in Sexual Assault Investigations

This investigation intends to study materials and techniques used for biological evidence collection in sexual assault cases and is divided into two stages: in stage one, methods for biological evidence collection (the single swab (including three variants) and the “double swab technique”) were compared; in stage two, swabs’ component material was compared. The sampling was composed of 42 heterosexual couples who provided mock samples. The collection methods in which the whole swab is covered by evidence presented significantly better outcomes (p < 0.001), such as the “double swab technique.” Additionally, nylon swabs proved to present significantly better features regarding the capacity of sample elution, providing significantly higher amounts of DNA (p ≤ 0.034). This study provides guidelines for better collection of biological evidence regarding the collection method using a swab and the proper swab material to utilize.     

The use of DNA stabilizing solution to enable room temperature storage and transportation of buccal and trace sample swabs

It is proposed that a DNA stabilizing solution (DNA Genotek Inc.) designed to preserve DNA in saliva samples at room temperature can be extrapolated to the storage of swab heads. The aim of this study was to evaluate the effectiveness of the solution for the preservation of reference swabs (buccal) and trace samples (facial swabs). To this end, the solution was used during a twin-site DNA transfer project assessing background levels of carer DNA present in children. Tubes containing 400 μl of solution were used to store and transport swab heads. At the laboratory, samples were extracted using the QIAamp DNA Mini Kit (Qiagen), quantified using the Quantifiler Duo Kit and profiled using the AmpF?STR^® SGM Plus^® PCR Amplification Kit (both Applied Biosystems). Twenty-eight PCR cycles were applied to all samples. Thirty-four cycles or a longer electrophoresis injection time was applied to trace samples where necessary. All Reference swabs produced high quantities of DNA and full DNA profiles after 28 cycles. Profile morphology indicated good quality DNA with no degradation. Of the trace samples, sufficient profiles were achieved to study the transfer of carer DNA making the solution fit for continued use in this project. DNA stabilizing solution enables the storage and transportation of swabs without freezing. This is convenient, reduces transportation costs and enables instant analysis of samples upon arrival at the laboratory. This is a useful alternative for a multi-site research project as well as a reliable storage tool for use in remote areas.     

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.     

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).     

Expedited, chemically enhanced sperm cell recovery from cotton swabs for rape kit analysis

This report focuses on the development of a method for chemically induced enhancement of cell elution and recovery from cotton swabs. The method exploits the exclusive use of detergents for intact cell removal, and can be utilized in conjunction with, or to circumvent, conventional differential extraction (DE). Samples treated with Sarkosyl (54.4 +/- 1.8%) and sodium dodecyl sulfate (SDS) (78.5 +/- 0.7%) yielded higher sperm cell recoveries than a conventional DE buffer (39.4 +/- 2.1%). The results indicated that the choice of detergent affected sperm cell yield, with anionic detergents having the greatest effect. Storage time of samples affected the concentration of detergent required for optimal sperm cell recovery, longer times requiring increased detergent concentrations. In addition, the extent of sperm cell lysis by proteinase K digestion was evaluated. The results indicate that the exclusive use of SDS enhances the release of sperm and epithelial cells from a cotton swab as compared with DE buffer, providing for a more effective DNA analysis.     

法医学DNA提取试剂盒优化及适用性评价

目的用磁性纳米磁珠和自主设计的试剂体系及提取流程,来建立一种基因组DNA的快速提取优化方案。方法利用自主研制的法医DNA提取纯化试剂盒设计实验方案对陈旧棉签血样进行DNA提取,用紫外分光光度计分析定量,通过对实验结果进行分析比较进一步优化自主研发的法医学DNA提取试剂盒。用优化之后的试剂盒提取各种不同的疑难捡材,探索本试剂盒的适用性。结果通过优化试验条件,同样获得了各种检材理想的DNA提取效果,却大大降低了DNA样本的提取成本。结论经优化过的国产磁珠DNA提取试剂盒完全可以应用与法医DNA检测中。     

A Comparison of Common Swabbing Materials for the Recovery of Organic and Inorganic Explosive Residues

The efficiency of solvent based extraction methods used to remove explosive residues from four different swab types was investigated. Known amounts of organic and inorganic residues were spiked onto a swab surface with acetonitrile or ethanol:water combined with ultrasonication or physical manipulation used to extract the residues from each swab. The efficiency of each procedure was then calculated using liquid chromatography‐ultraviolet detection for organic residues and ion chromatography for inorganic residues. Results indicated that acetonitrile combined with physical agitation proved to be the most efficient method; returning analyte recoveries c. 95% for both alcohol based swabs and cotton balls. Inorganic residues were efficiently extracted using ethanol:water, while the use of acetonitrile followed by water significantly reduced the recovery of inorganic residues. Swab storage conditions were then investigated with results indicating decreased storage temperatures are required to retain the more volatile explosives.     

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.     

Development of a rapid, 96-well alkaline based differential DNA extraction method for sexual assault evidence

We present a rapid alkaline lysis procedure for the extraction of DNA from sexual assault evidence that generates purified sperm fraction extracts that yield STR typing results similar to those obtained from the traditional organic/dithiothreitol differential extraction. Specifically, a sodium hydroxide based differential extraction method has been developed in a single-tube format and further optimized in a 96-well format. The method yields purified extracts from a small sample set (∼2-6 swabs) in approximately 2 h and from a larger sample set (up to 96 swabs) in approximately 4 h. While conventional differential extraction methods require vigorous sample manipulation to remove the spermatozoa from the substrate, the method described here exploits the propensity of sperm to adhere to a substrate and does not require any manipulation of the substrate after it is sampled. For swabs, sample handling is minimized by employing a process where the tip of the swab, including the shaft, is transferred to the appropriate vessel eliminating the need for potentially hazardous scalpels to separate the swab material from the shaft. The absence of multiple handling steps allows the process to be semi-automated, however the procedure as described here does not require use of a robotic system. This method may provide forensic laboratories a cost-effective tool for the eradication of backlogs of sexual assault evidence, and more timely service to their client agencies. In addition, we have demonstrated that a modification of the procedure can be used to retrieve residual sperm-cell DNA from previously extracted swabs.