Use of Embryos Extracted from Individual Cannabis sativa Seeds for Genetic Studies and Forensic Applications

Legal limits on the psychoactive tetrahydrocannabinol (THC) content in Cannabis sativa plants have complicated genetic and forensic studies in this species. However, Cannabis seeds present very low THC levels. We developed a method for embryo extraction from seeds and an improved protocol for DNA extraction and tested this method in four hemp and six marijuana varieties. This embryo extraction method enabled the recovery of diploid embryos from individual seeds. An improved DNA extraction protocol (CTAB3) was used to obtain DNA from individual embryos at a concentration and quality similar to DNA extracted from leaves. DNA extracted from embryos was used for SSR molecular characterization in individuals from the 10 varieties. A unique molecular profile for each individual was obtained, and a clear differentiation between hemp and marijuana varieties was observed. The combined embryo extraction–DNA extraction methodology and the new highly polymorphic SSR markers facilitate genetic and forensic studies in Cannabis.     

Stability of Palatal Rugae as a Forensic Marker in Orthodontically Treated Cases

The palatal rugae have been used as a reference landmark and identification marker by orthodontists and forensic analysts. However, the reliability of palatal rugae as a forensic marker remains questionable once an individual is subjected to orthodontic treatment. This study aimed at evaluating the changes in the rugae pattern after nonextraction, extraction, and maxillary expansion orthodontic treatment. The lengths and shapes of palatal rugae were evaluated on the pretreatment and post‐treatment dental casts of 168 subjects using the Thomas and Kotze classification. Extraction treatment significantly reduced the second and third rugae lengths (p < 0.05), whereas the third rugae length was significantly increased after palatal expansion (p < 0.05). The shape of rugae remained consistent in all the study groups which may be used as a reliable forensic marker in subjects undergoing orthodontic treatment. However, the use of the lengths of palatal rugae in forensic odontology must be made with caution.     

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.     

Sampling and recovery of ignitable liquid residues (ILRs) from fire debris using capillary microextraction of volatiles (CMV) for on-site analysis

A new, fast, and ultra-sensitive headspace sampling method using the Capillary Microextraction of Volatiles (CMV) device is demonstrated for the analysis of ignitable liquid residues (ILRs) in fire debris. This headspace sampling method involves the use of a heated can (60°C) to aid in the recovery of volatile organic compounds (VOCs) from medium and heavy petroleum distillates. Our group has previously reported the utility of CMV to extract gasoline at ambient temperature in less than 5 min in the field. This work evaluates the recovery and analysis of low mass loadings (tens of ng) of VOCs from charcoal lighter fluid, kerosene, and diesel fuel. Nonane, decane, undecane, tridecane, tetradecane, and pentadecane were selected for evaluation of recovery to represent these ILR classes. The face-down heated can headspace sampling technique was compared to the previously reported, non-heated, paper cup headspace sampling technique. Mass recovery improvements of 50%–200% for five of the six target compounds in diesel fuel were achieved compared to the non-heated sampling method. The average relative standard deviation (reported as % RSD) between the replicate trials decreased from an average of 28% to 6% when using the heated can method. Ignitable liquids were spiked onto burned debris in a live burn exercise and sampled using the heated can and paper cup headspace sampling techniques. The heated sampling technique reported here, for the first time, demonstrates an effective extraction method that when coupled to a portable GC–MS instrument allows for a sampling and analysis protocol in the field in less than 30 min.     

Validation of the InnoXtract™ DNA extraction method for bone,teeth, and rootless hair

Forensic casework samples often include human hairs, teeth, and bones. Hairs with roots are routinely processed for DNA analysis, while rootless hairs are either not tested or processed using mitochondrial DNA. Bones and teeth are submitted for human remains identifications for missing persons and mass disaster cases. DNA extraction from these low templates and degraded samples is challenging. The new InnoXtract DNA extraction method utilizes magnetic beads that are optimized to bind small DNA fragments, as small as 100 base pairs, to purify high-yield DNA from compromised samples. This validation study evaluates InnoXtract's ability to obtain amplifiable DNA from samples such as rootless hairs and skeletal remains. Studies performed include sensitivity, stability, repeatability, reproducibility, non-probative samples, and comparison to standard organic extractions. Sensitivity studies demonstrate average yield recoveries ranging from 53% to 100% and 73% to 85% for the InnoXtract hair and bone methods, respectively. Studies demonstrate consistent results across a range of sample types, such as insulted and un-insulted bone and teeth, as well as hair shafts from donors of various ages, gender, race, and hair characteristics. The InnoXtract bone method outperformed organic extraction. The method was successfully automated on a MagMAX™ Express-96, with recoveries over 70% relative to the manual version. InnoXtract has the potential as an automated high-throughput, high-yield bone extraction method with 6 h of total extraction time for up to 96 samples. The validation study results demonstrate that the InnoXtract kits produce high-yield and high-quality DNA from compromised bone, teeth, and hair shaft samples.     

Blind Image Steganalysis of JPEG images using feature extraction through the process of dilation

The detection of stego images, used as a carrier for secret messages for nefarious activities, forms the basis for Blind Image Steganalysis. The main issue in Blind Steganalysis is the non-availability of knowledge about the Steganographic technique applied to the image. Feature extraction approaches best suited for Blind Steganalysis, either dealt with only a few features or single domain of an image. Moreover, these approaches lead to low detection percentage. The main objective of this paper is to improve the detection percentage. In this paper, the focus is on Blind Steganalysis of JPEG images through the process of dilation that includes splitting of given image into RGB components followed by transformation of each component into three domains, viz., frequency, spatial, and wavelet. Extracted features from each domain are given to the Support Vector Machine (SVM) classifier that classified the image as steg or clean. The proposed process of dilation was tested by experiments with varying embedded text sizes and varying number of extracted features on the trained SVM classifier. Overall Success Rate (OSR) was chosen as the performance metric of the proposed solution and is found to be effective, compared with existing solutions, in detecting higher percentage of steg images.     

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。     

Comparison of three DNA extraction methods for three different types of fired and unfired ammunition

When handling ammunition for gun loading, epithelial cells from the hands can become adhered to the metal surface, and this trace is a potential source of DNA. This work aimed to compare the efficiency of three DNA extraction methods from fired cartridge cases from three different types of firearms: a 12-gauge shotgun, a point 40 S&W pistol, and a 7.62 mm rifle. Nine volunteers were involved in this study handling 42 pieces of ammunition overall. The unfired ammunition was handled by a known good donor, and we used this data for comparison. DNA profiling was carried out with EZ1 DNA Investigator Kit for EZ1 Advanced XL automated DNA extraction, QIAmp DNA Investigator kit for a non-automated silica-based membrane column method, and direct lysis protocol for a non-automated in-house one. Samples were collected with 0.5 × 0.5 cm pieces of FTA filter paper moistened with distilled water. Quantiplex Pro RGQ kit and Fusion Powerplex 6C were used for genotyping samples. QIAmp DNA Investigator method resulted in the best number of alleles recovered for both conditions tested, both unfired and fired ammunitions: 77 % vs. 19.3 %, followed by the automated extraction (28.6 % vs. 4.3 %) and lysis protocol (0 % vs. 3.9 %). Degradation data from fired cartridge cases were 27 % for column method, 50 % for lysis protocol, and 87 % for EZ1 kit. Kruskal-Wallis test for mean DNA concentration from these samples returned p < 0.05, and Dunn’s multiple comparison test indicated a significant difference between calibers 0.40 S&W and 12-gauge shotgun from lyses protocol method. We did not detect any other significant differences on the test. The 12-gauge shotgun cartridge cases resulted in a high number of alleles overall (56.8 %). The numerous steps for DNA extraction and purification in the column method may explain its better performance. Although the results obtained indicate that all methods be used for DNA extraction from this type of evidence, the silica-based membrane column method appears to be more efficient.     

DNA IQ^TM SYSTEM在疑难指甲DNA检验中的应用

目的探索DNA IQTMSYSTEM在疑难指甲DNA提取中的应用。方法 15份疑难指甲采用Chelex方法检验没有成功获得STR分型图谱,采用DNA IQTMSYSTEM提取法并纯化,采用Identifiler PLUS试剂盒进行复合扩增,产物经ABI3130XL型DNA基因分析仪检测。结果成功获得15例疑难指甲的STR基因座DNA分型。结论 DNA IQTMSYSTEM方法能快速、有效提取疑难指甲DNA进行STR分型。     

A comparative study of two extraction methods routinely used for DNA recovery from simulated post coital samples

In sexual assault cases DNA profiling of spermatozoa can be of critical importance. Most methods use differential extraction of the spermatozoa to separate it from the female component. Here we have compared two commercially available differential extraction methods, the QIAamp^® DNA mini kit (Qiagen) and Differex™ with the DNA IQ^® System (Promega). Simulated postcoital samples were prepared using buccal cells from a female donor and spermatozoa from three male donors. A dilution series ranging from neat semen to a 1:1500 dilution (semen:dH₂O) was prepared and mixed with an equal volume of saliva from a female donor. Extraction efficiency was assessed using DNA concentration measured with NanoDrop 2000 and Quantifiler^® Human DNA Quantification Kit and the profile count of full, partial and mixed DNA profiles generated using SGM Plus and PowerPlex^® ESI 17. Statistical analysis was carried out using Randomisation in R, which is a robust model making no assumption of the distribution of data. Based on the amount of DNA extracted and the types of profiles no significant difference in the performance of the two extraction kits was seen. However, the processing time taken with the Differex™ System was about half than that of the QIAamp^® DNA mini kit and involved fewer liquid transfers.