Highly efficient and automated extraction of DNA from human remains using a modified EZ1 protocol

Bones and teeth often represent the only sources of DNA available for identifying human remains. DNA in bones and teeth is generally better preserved than that in soft tissues because of the presence of hard connective tissue with a high level of calcium. Because of the extensive mineralisation, the choice of an efficient DNA extraction procedure is important to minimise the sampling of a high level of minerals and to remove polymerase chain reaction (PCR) inhibitors. Some protocols are available for DNA extraction from bones and teeth as part of the Qiagen EZ1 DNA Investigator Kit using the EZ1 Advanced XL automated purification platform. To improve the efficiency of DNA extraction from skeletal remains, the present study focuses on a modification to these already available protocols. In this study, different bones and teeth collected between 1 and 50 years after death were subjected to DNA extraction using the standard EZ1 protocol, a supplementary protocol, and a modified protocol. The modified approach included a decalcification step, whereas the Qiagen protocols worked directly on non-decalcified powder. In all three procedures, 150 mg samples were used for DNA extraction. We evaluated the quantity of DNA recovered from samples, the presence of any PCR inhibitors co-extracted, the level of DNA degradation, the quality of short tandem repeat (STR) profiles, and the reproducibility of the modified procedure. When compared with the other protocols, the modified protocol resulted in the best recovery of DNA that was free of PCR inhibitors. Additionally, the STR profiles were reliable and of high quality. In our opinion, the decalcification step increases DNA recovery by softening tissues, which allows lysis solutions to act more effectively. Furthermore, the use of two lysis solutions and the variation added to the EZ1 purification step allow for DNA recovery with quality and quantity superior to those of the previously available Qiagen-based protocols. These findings may be helpful solutions to the problems commonly encountered when dealing with difficult samples, such as bones and teeth.

Key points

• Bones and teeth often represent the only sources of DNA for identifying human remains.
• The choice of an efficient DNA extraction procedure is important for maximizing DNA recovery and removing PCR inhibitors.
• This study focuses on modifications to the previously available Qiagen-based protocols.
• The modified protocol enabled the best recovery of DNA, and both quality and quantity were superior to those of the previously available Qiagen-based protocols.
• The STR profiles obtained from samples extracted using the modified protocol were reliable and of high quality.

     

Digital reconstruction of fragmented tooth remains in forensic context

Forensic odontology majorly focuses on the identification of victims through the analyses of oral and para-oral structures.Exposure to high temperatures and trauma can occur in mass disasters and may lead to the fracturing and fragmentation of teeth.These fragments may become very fragile and easily damaged while handling.Conventional methodologies such as the use of transparent nail polish,hair spray,cyanoacrylate or adhesives have been used to stabilise the fragmented pieces.This study introduces a new and innovative digital technique that utilises three-dimensional surface scanning(3DSS)and rapid prototyping techniques to reconstruct fractured portions of the teeth.The results of qualitative congruency analysis suggest that over all variance of morphological error(0.0526±0.05)mm.These results imply that the reconstructed 3D model can be used for various morphometric analyses.     

Determination of barbiturates in hair samples by using a validated UHPLC-HRMS method: application in investigation of drug-facilitated sexual assault

In recent years,benzodiazepines and benzodiazepine-like drugs are the most common substances associated with drug-facilitated sexual assaults(DFSA);however,barbiturates are also detected occasionally.Segmental hair analysis provides useful information on the historic pattern of drug use,enabling differentiation between single exposure in DFSA cases and chronic use.However,sensitive and specific methods for barbiturate analysis in hair samples are needed.Herein,we present an ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry(UHPLC-HRMS)method for qualitative and quantitative determination of seven barbiturates in hair samples.Firstly,a hair strand was decontaminated and then freeze-milled in liquid nitrogen.Next,50mg of powdered hair was extracted with methanol in an ultrasonic bath for 10 min in the presence of 10 ng phenobarbital-d5.The supernatant was dried under nitrogen gas,and the pellet was dissolved in 100 μL mobile phase.Afterwards,10 μL of the suspension was injected into the UHPLC-HRMS system.The present method involved two UHPLC conditions for determination of barbiturates(I)and identification of the structural isomers amobarbital and pentobarbital(II).This method showed satisfactory linearity in a range of 0.02–20.00 ng/mg for UHPLC conditions I and II,both with a high determination coefficient(0.9991–0.9999).The selectivity,intra-and interday precision,accuracy and matrix effect of the method were acceptable.Next,the validated method was applied to investigate an authentic DFSA case.Hair samples(black,approximate 25cm long)were collected 3 months after the assault,and the proximal segments(0–5 cm from the root;each segment was 1 cm long)were analysed.Amobarbital was detected at a concentration of相似文献   

Effects of storage time on DNA profiling success from archived latent fingerprint samples using an optimised workflow

Due to recent improvements in forensic DNA testing kit sensitivity,there has been an increased demand in the criminal justice community to revisit past convictions or cold cases.Some of these cases have little biological evidence other than touch DNA in the form of archived latent fingerprint lift cards.In this study,a previously developed optimised workflow for this sample type was tested on aged fingerprints to determine if improved short tandem repeat(STR)profiles could be obtained.Two-year-old samples processed with the optimised workflow produced an average of approximately five more STR alleles per profile over the traditional method.The optimised workflow also produced detectable alleles in samples aged out to 28 years.Of the methods tested,the optimised workflow resulted in the most informative profiles from evidence samples more representative of the forensic need.This workflow is recommended for use with archived latent fingerprint samples,regardless of the archival time.     

Contributions of anatomy to forensic sex estimation: focus on head and neck bones

This study sought to provide an up-to-date review of the importance of anatomy to human identification,focusing on the usefulness of anatomical knowledge about the head and neck bones and teeth to sex estimation in routine forensic anthropology methods.A detailed search of osteology applications in forensic sex estimation was conducted through the electronic databases for the 10 years prior to July 2020.Relevant articles and classic literature on the subject were gathered and outlined in this review.Among the available literature,several metric analyses showed accuracy superior to 80%in sexual diagnosis.Angles measured from the inclination of glabellae and analysis of the external frontal bone surface through three-dimensional computer-aided design emerge as reliable cranial indexes for sex estimation.in the mandible,the condylar and coronoid height,bigonial width,and condylion-gonion distance express significant sexual dimorphism.Measurements of the canine are the best option for sex estimation using teeth,as well as the thickness of the dentine or enamel of incisors.The axis vertebra surpasses other neck bones for sex estimation due to its atypical shape and the presence of the odontoid process.Metric analyses based on anatomy can provide reliable accuracy in sexual diagnosis.Adequate training and anatomical knowledge can reduce bias and interobserver differences,and the use of three-dimensional models and computed tomography images can enhance the accuracy of these methods for sex estimation.However,every method should be validated before being applied to a different population.     

脑组织与其相关分析推断死亡时间的研究进展

推断死后间隔时间(postmortem interval,PMI)一直是法医学鉴定中需要解决的重要问题之一.可用于推断PMI的尸体检材有很多种,脑组织因其独特的解剖学位置与生理功能,是推断PMI的重要检材之一.本文从三个方面将根据脑组织推断PMI的方法进行综述,包括了通过脑组织的温度、形态特点等尸体现象推断PMI;通过...     

电击伤后大鼠心肌TIMP-1表达的实验研究

目的探讨心脏电击伤后心肌金属蛋白酶组织抑制物（TIMP-1）随时间变化的规律及其法医学意义。方法220V低压电电击复制大鼠电击伤模型，采用免疫组化方法和图像分析技术，观测电击伤后不同时间点心肌TIMP-1免疫组化反应的表达情况及其面积积分光密度，并对所测数据进行统计学分析。结果电击伤后心肌TIMP-1阳性细胞在伤后0．5h内开始增加，24～72h达到高峰，随后逐渐减少，至12d时恢复到正常水平。结论电击伤后心脏TIMP-1阳性表达细胞数呈现一定的时序性变化，可望将其用作推断电击伤后经过时间的指标之一。     

Genetic polymorphisms and phylogenetic analyses of the Ü-Tsang Tibetan from Lhasa based on 30 slowly and moderately mutated Y-STR loci

As a result of the expansion of old Tibet on the Qinghai-Tibet Plateau, Tibetans diverged into three main branches, Ü-Tsang, Amdo, and Kham Tibetan. Ü-Tsang Tibetans are geographically distributed across the wide central and western portions of the Qinghai-Tibet Plateau while Lhasa is the central gathering place for Tibetan culture. The AGCU Y30, a 6-dye fluorescence kit including 30 slowly and moderately mutated Y-STR loci, has been validated for its stability and sensitivity in different biomaterials and diverse Chinese populations (Han and other minorities), and widely used in the practical work of forensic science. However, the 30 Y-STR profiling of Tibetan, especially for Ü-Tsang Tibetan, were insufficient. We utilized the AGCU Y30 to genotype 577 Ü-Tsang Tibetan unrelated males from Lhasa in the Tibet Autonomous Region of China to fill up the full and accurate Y-STR profiles. A total of 552 haplotypes were observed, 536 (97.10%) of which were unique. One hundred and ninety-four alleles were observed at 26 single copy loci and the allelic frequencies ranged from 0.0017 to 0.8180. For the two multi-copy loci DYS385a/b and DYS527a/b, 64 and 36 allelic combinations were observed, respectively. The gene diversity (GD) values ranged from 0.3079 at DYS391 to 0.9142 at DYS385a/b and the overall haplotype diversity (HD) was 0.9998, and its discrimination capacity (DC) was 0.9567. The population genetic analyses demonstrated that Lhasa Ü-Tsang Tibetan had close relationships with other Tibetan populations from Tibet and Qinghai, especially with Ü-Tsang Tibetan. From the perspective of Y haplogroups, the admixture of the southward Qiang people with dominant haplogroup O-M122 and the northward migrations of the initial settlers of East Asia with haplogroup D-M175 hinted the Sino-Tibetan homologous, thus, we could not ignore the gene flows with other Sino-Tibetan populations, especially for Han Chinese, to characterize the forensic genetic landscape of Tibetan.     

Mitochondrial genome sequencing with short overlapping amplicons on MiSeq FGx system

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