Genetic identification of biological samples buried in clay soil during summer and winter seasons

The analysis of a biological samples found in crime scenes can be a challenging task. Minute amount of DNA, degraded DNA and the presence of inhibitors in biological samples, can interfere with the achievement of a complete genetic profile.Chelating resin, silica membranes and silica-coated with paramagnetic resin were the techniques used in this study for the isolation and purification of DNA. Our aim was to find out the best DNA extraction method for application in the presence of biological samples buried in clay soil.     

Forensic DNA typing strategy of degraded DNA on discarded cigarette ends using the AmpFℓSTR® Identifiler®, Identifiler® Plus and MiniFiler™ PCR Amplification Kits

DNA left on a forensic sample is often prone to degradation, especially if left to the elements. To maximize the chance of retrieving the most information from such compromised DNA, an appropriate profiling scheme using the available technologies needs to be devised. In this study, a total of 62 cigarette ends collected under different conditions of environmental exposure were employed to test the effectiveness of three DNA amplification kits, namely the Applied Biosystems™ AmpFℓSTR® Identifiler®, Identifiler® Plus and MiniFiler™ PCR Amplification Kits, in the profiling of such compromised DNA. We demonstrated that Identifiler® Plus could substitute Identifiler® to improve the effectiveness of profiling for those inhibited cigarette samples. MiniFiler™, on the other hand, could supplement Identifiler®/Identifiler® Plus profiles and provide additional genetic information to enhance the evidential value of the samples, especially for those that have suffered from DNA degradation to a greater extent. The findings in this work allowed us to propose a DNA profiling strategy as follow: 1) samples yielding complete Identifiler®/Identifiler® Plus profiles require no further testing with MiniFiler™; 2) samples yielding partial single-source profiles to be tested with MiniFiler™ to add genetic information; 3) samples yielding no results are unlikely to yield any results with MiniFiler™.     

A more efficient extraction method of human bone resulting in improved DNA profiling

Eight human bone samples, from a forensic case, were extracted in parallel using our standard protocol with and without PTB in the buffer. Both methods were sometimes inadequate for (complete) STR profiling, while the presence of PTB even decreases the DNA yield.The complete decalcification of the bone extraction residues in an EDTA-solution with SDS recovered sufficient amounts of DNA, which resulted in complete STR profiling for all samples. Complete decalcification without SDS yielded even higher amounts of DNA and also complete STR profiling for all samples.Similar results were obtained for the DNA extraction from a human tooth.     

Assessing the Utility of Soil DNA Extraction Kits for Increasing DNA Yields and Eliminating PCR Inhibitors from Buried Skeletal Remains

DNA identification of human remains is often necessary when decedents are skeletonized; however, poor DNA recovery and polymerase chain reaction (PCR) inhibition are frequently encountered, a situation exacerbated by burial. In this research, the utility of integrating soil DNA isolation kits into buried skeletal DNA analysis was evaluated and compared to a standard human DNA extraction kit and organic extraction. The soil kits successfully extracted skeletal DNA at quantities similar to standard methods, although the two kits tested, which differ mechanistically, were not equivalent. Further, the PCR inhibitors calcium and humic acid were effectively removed using the soil kits, whereas collagen was less so. Finally, concordant control region sequences were obtained from human skeletal remains using all four methods. Based on these comparisons, soil DNA isolation kits, which quickened the extraction process, proved to be a viable extraction technique for skeletal remains that resulted in positive identification of a decedent.     

Rapid direct PCR for ABO blood typing

Many different molecular typing methods have been reported to complement routine serological ABO blood typing in forensics. However, these ABO genotyping methods are often time-consuming and call for an initial DNA isolation step that requires the use of expensive kits or reagents. We report here a rapid direct ABO genotyping method that eliminates the need for DNA extraction from fresh blood, hair, and body fluid stains before PCR. Using a fast PCR instrument and an optimized polymerase, the genotyping method-which employs a multiplex allele-specific primer set for the simultaneous detection of three single-nucleotide polymorphism (SNP) sites (nucleotides 261, 526, and 803)-identifies A, B, O01/O02, O03, and cis-AB01 alleles in around 70 min from sample collection to electropherogram. Not only will this ABO genotyping method be efficiently used in forensic practice for rapid screening of samples before full-blown multilocus short tandem repeat profiling, but it will also demonstrate an example of rapid direct genotyping of SNPs that offers the advantages of time- and cost-efficiency, convenience, and reduced contamination during DNA analysis.     

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.     

Forensic human identification: Investigation into tooth morphotype and DNA extraction methods from teeth

When a body is decomposed, hard tissues such as teeth may provide the only DNA source for human identification. There is currently no consensus as to the best DNA extraction method, and there is a lack of empirical data regarding tooth morphotype and condition that may impact DNA recovery. Therefore, this study sought to investigate which variables significantly improved DNA concentration, integrity and profiling success. A total of 52 human teeth were assessed, representing all tooth morphotypes from three deceased individuals. DNA was extracted using both the QIAamp® DNA Investigator Kit and the phenol-chloroform method. DNA concentration and degradation index were assessed using real time PCR, prior to conventional DNA profiling. Contrary to international guidelines promoting the use of molars, DNA profiling from molars was the least successful, with premolars, followed by canines, performing the best. The presence of fillings reduced the DNA quantity and quality obtained and may explain the poor performance of molars. DNA from the maxillae were significantly less degraded when the QIAamp® was used, although this did not influence DNA profiling success. A significant increase in DNA concentration, integrity and profiling success was observed in diseased teeth (periodontitis) compared to those without disease. This may be due to increased white blood cell presence at the site. There was no significant difference in DNA profiling success between the two DNA extraction methods. However, different teeth yielded failed DNA profiles for each extraction method, suggesting that repeated attempts, using alternative DNA extraction methods, is recommended. The recovery of additional DNA profiling information from degraded samples may help to ultimately reduce the burden of unidentified human remains.     

氨基比林血痕预试验处理血痕后样本的DNA定量

目的：探讨氨基比林血痕预试验处理血痕后样本DNA含量的变化及对STR分型检测的影响。方法10名健康无关个体EDTA抗凝血液制成滤纸血痕,氨基比林血痕预试验检测,按试验后血样干燥保存时间分30 min、1 h、3 h、6 h、12 h、24 h共6个实验组,并采用磁珠法、QIAcube DNA纯化法、Chelex-100法三种方法提取样本DNA,应用荧光定量PCR检测样本DNA含量,PCR-STR荧光技术进行STR分型。结果提取方法相同时,氨基比林血痕预试验后血样随干燥保存时间的延长,样本DNA含量呈逐渐降低的趋势。保存时间相同时,不同DNA提取方法间,样本DNA含量差异也有统计学意义。90.56%样本均可获得16个STR基因座明确分型。结论氨基比林血痕预试验对血痕样本DNA有损伤,24 h内多可获有效STR分型。磁珠法提取样本DNA进行STR分型,效果最好。     

Comparisons of protocols for enhanced DNA profile quality from FTA®-deposited urine samples

Disputes over the identity of a urine sample donor have been reported, and urine authentication by genetic profiling has helped resolved the cases. However, since genotyping of urine is not always required, many drug-testing laboratories may face sample storage issues. Several studies have investigated the use of FTA® cards as a convenient tool for keeping specimen at room temperature for extended periods of time. However, generating complete STR profile from some FTA®-deposited urine samples remains challenging due to low levels of genetic material content, necessitating amendments to the laboratory’s standard protocols. This work therefore aims to evaluate the effects of two DNA template preparation methods, both employing FTA® cards as the storage medium, on the success rates of STR profiling from urine. Specimen from a female volunteer, representing a particularly low-yield sample, was employed. Aliquots of 1 and 2 mL were used as the starting material to evaluate DNA template preparation using the FTA® manufacturer’s protocol for disc purification against elution of DNA from the FTA® using Prepfiler™ Forensic DNA Extraction Kit. AmpFℓSTR™ Identifiler™ Plus PCR Amplification Kit was used to amplify the STR markers, and the PCR products were analysed using Applied Biosystems™ 3500xL Genetic Analyzer. The DNA profile qualities were examined in terms of number of loci detected and peak height balance. Comparisons with the profiles obtained from DNA isolated using QIAamp® DNA Micro Kit from 1 and 2 mL of the same batch of urine were also made. The optimised protocol was then tested on urine samples from three male volunteers. The results showed that the purification of FTA® punches according to the manufacturer’s protocol enabled full DNA profiles to be obtained from both 1 and 2 mL of urine from all samples tested, including male samples. In contrast, no DNA profile could be generated from the DNA eluted with the Prepfiler™ kit. When compared with the more conventional solid-phase DNA extraction method, the profiles generated from the FTA® punches exhibited similar reproducibility and quality to those from the template isolated by the QIAamp® Kit. This work further demonstrated the feasibility of FTA® cards as a tool for specimen storage and DNA template preparation from small volumes of urine for authentication by STR profiling. Full STR profiles could be generated from sample from both sexes without modification of the PCR conditions or injection time.     

DNA Purification Cell Lysis and Wash Step Modifications for Low-Template DNA Sample Processing,

As DNA technology becomes increasingly sensitive, forensic laboratories are receiving more low-template DNA samples. These samples, already low in DNA content, become even more challenging to process as the available DNA becomes further reduced during the extraction step. In this study, two extraction modifications were tested to determine if the cause of DNA loss could be identified and mitigated. A double lysis technique was used to test for DNA loss in the sample collection substrate, and lysate eluates were re-extracted to determine DNA loss from inefficient binding to the silica column. Both modifications showed DNA was lost at these steps. However, resulting STR profiles from these samples had fewer peaks and lower peak heights when compared to samples processed with no extraction modifications. Overall, the potential benefits of adding these extraction modifications for low-template DNA sample processing are not enough to justify the risk associated with additional manipulation.     

Optimization and validation of a fully automated silica-coated magnetic beads purification technology in forensics

Automated procedures for forensic DNA analyses are essential not only for large-throughput sample preparation, but are also needed to avoid errors during routine sample preparation. The most critical stage in PCR-based forensic analysis is DNA isolation, which should yield as much highly purified DNA as possible. The extraction method used consists of pre-treatment of stains and samples, cell lysis using chaotropic reagents, binding of the DNA to silica-coated magnetic particles, followed by elution of the DNA. Our work focuses mainly on sample preparation, obtaining the maximum possible amount of biological material from forensic samples, and the following cell lysis, to create a simple standardized lysis protocol suitable for nearly all forensic material. After optimization and validation, the M-48 BioRobot((R)) workstation has been used for more than 20,000 routine lab samples. There has been no evidence of cross contamination. Resulting DNA from as small as three nuclear cells yield reliable complete STR amplification profiles. The DNA remains stable after 2 years of storage.     

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.     

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

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

Elevated and similar urinary testosterone/epitestosterone ratio in all samples of a competition testing: suspicion of a manipulation

The case of seven urine samples collected for anti-doping purposes during a cycling stage race with moderately elevated testosterone and epitestosterone ratio (T/E) is reported. The very low probability of having all seven urine samples with such similar elevated T/E ratio (from 3.2 to 4.7) was very suspicious. Different pattern classification tools were tested to categorize the most similar steroid profiles, but none of the models enabled a clear classification of the different urine samples. Subsequently, genetic profiling of all urine samples was performed and demonstrated that three of the seven samples were collected from the same cyclist. Finally, the International Federation confirmed DNA profiling results. This suggests that urinary steroid data using several methodologies are not appropriate for identification purposes and to an extent not unique to individuals.     

Gamma Irradiation as a Biological Decontaminant and Its Effect on Common Fingermark Detection Techniques and DNA Profiling

Abstract:  The use of disease-causing organisms and their toxins against the civilian population has defined bioterrorism and opened forensic science up to the challenges of processing contaminated evidence. This study sought to determine the use of gamma irradiation as an effective biological decontaminant and its effect on the recovery of latent fingermarks from both porous and nonporous items. Test items were contaminated with viable spores marked with latent prints and then decontaminated using a cobalt 60 gamma irradiator. Fingermark detection was the focus with standard methods including 1,2-indanedione, ninhydrin, diazafluoren-9-one, and physical developer used during this study. DNA recovery using 20% Chelex extraction and quantitative real-time polymerase chain reaction was also explored. Gamma irradiation proved effective as a bacterial decontaminant with D -values ranging from 458 to 500 Gy for nonporous items and 797–808 Gy for porous ones. The results demonstrated the successful recovery of latent marks and DNA establishing gamma irradiation as a viable decontamination option.     

联苯胺预试验处理血痕后样本DNA定量的研究

目的探讨联苯胺血痕预试验处理后样本DNA含量的变化及对STR分型检测的影响。方法选取10名无关个体EDTA抗凝血液制成滤纸血痕,保存干燥时间分0．5h、1h、3h、6h、12h、24h六个实验组,并采用磁珠提取法、QIAcubeDNA提取纯化法、chelex-100提取法提取样本DNA,应用RT-PCR定量技术检测样本DNA含量,同时应用PCR-STR技术和Idfiler-plus试剂盒检测相应样本STR分型。结果联苯胺血痕预试验后处理样本,随保存时间的延长,其样本DNA含量显示逐渐降低的趋势。回归线性对数分析显示,磁珠提取法：Y=-0．40871n（x）＋0．7044R0—0．7633;QIAcubeDNA提取纯化法：Y=-0．23931n（x）＋0．4764R0—0．8715;chelex-100提取法：Y=-0．11781n（x）＋0．2302R2=0．9571。不同DNA提取方法对同一保存时间的联苯胺血痕预试验试剂处理样本DNA含量间差异有极显著性,P〈O．01。结论联苯胺血痕预试验后对后续STR分型影响较大,联苯胺血痕预实验后的血痕不能继续进行STR分型检测。     

New optimized DNA extraction protocol for fingerprints deposited on a special self-adhesive security seal and other latent samples used for human identification

Abstract: Obtaining complete short tandem repeat (STR) profiles from fingerprints containing minimal amounts of DNA, using standard extraction techniques, can be difficult. The aim of this study was to evaluate a new kit, Fingerprint DNA Finder (FDF Kit), recently launched for the extraction of DNA and STR profiling from fingerprints placed on a special device known as Self‐Adhesive Security Seal Sticker^® and other latent fingerprints on forensic evidentiary material like metallic guns. The DNA extraction system is based on a reversal of the silica principle, and all the potential inhibiting substances are retained on the surface of a special adsorbent, while nucleic acids are not bound and remain in solution dramatically improving DNA recovery. DNA yield was quite variable among the samples tested, rendering in most of the cases (>90%) complete STR profiles, free of PCR inhibitors, and devoid of artifacts. Even samples with DNA amount below 100 pg could be successfully analyzed.     

STR和性别位点PCR产物的荧光自动检测

运用荧光染料标记引物、变性聚丙烯酰胺凝胶电泳 (denaturingpolyacrylamidegelelec trophoresis,denaturingPAGE)结合荧光法检测6个STR位点 (vWA31A、TH01、F13A1、FES、TPOX、CSF1PO)及1个性别鉴定位点(Amelogenin) ,就荧光自动检测的重复性、准确性、分辩力等方面进行了研究 ,建立了6个STR位点等位基因判定视窗及用荧光DNA测序仪进行STR分析的方法 ,实现电脑自动判读结果。结果表明 ,本文所述STR和性别鉴定的自动检测方法可靠、分辨力高 (可达1bp) ,所得数据有助于荧光自动DNA分型软件系统的数据积累。     

A review of the current understanding of burned bone as a source of DNA for human identification

Identification of incinerated human remains may rely on genetic analysis of burned bone which can prove far more challenging than fresh tissues. Severe thermal insult results in the destruction or denaturation of DNA in soft tissues, however genetic material may be preserved in the skeletal tissues. Considerations for DNA retrieval from these samples include low levels of exogenous DNA, the dense, mineralised nature of bone, and the presence of contamination, and qPCR inhibitors. This review collates current knowledge in three areas relating to optimising DNA recovery from burned bone: 1) impact of burning on bone and subsequent effects on sample collection, 2) difficulties of preparing burned samples for DNA extraction, and 3) protocols for bone decalcification and DNA extraction. Bone decalcification and various DNA extraction protocols have been tested and optimised for ancient bone, suggesting that prolonged EDTA (Ethylenediaminetetraacetic acid) demineralisation followed by solid-phased silica-based extraction techniques provide the greatest DNA yield. However, there is significantly less literature exploring the optimal protocol for incinerated bones. Although burned bone, like ancient and diagenetic bone, can be considered “low-copy”, the taphonomic processes occurring are likely different. As techniques developed for ancient samples are tailored to deal with bone that has been altered in a particular way, it is important to understand if burned bone undergoes similar or different changes. Currently the effects of burning on bone and the DNA within it is not fully understood. Future research should focus on increasing our understanding of the effects of heat on bone and on comparing the outcome of various DNA extraction protocols for these tissues.