The fate of the chemical warfare agent during DNA extraction

Forensic laboratories do not have the infrastructure to process or store contaminated DNA samples that have been recovered from a crime scene contaminated with chemical or biological warfare agents. Previous research has shown that DNA profiles can be recovered from blood exposed to several chemical warfare agents after the agent has been removed. The fate of four toxic agents, sulfur mustard, sodium 2-fluoroacetate, sarin, and diazinon, in a lysis buffer used in Promega DNA IQ extraction protocol was studied to determine if extraction would render the samples safe. Two independent analytical methods were used per agent, selected from GC-MS, 1H NMR, 19F NMR, (31)P NMR, or LC-ES MS. The methods were validated before use. Determinations were carried out in a semi-quantitative way, by direct comparison to standards. Agent levels in the elution buffer were found to be below the detectable limits for mustard, sarin, sodium 2-fluoroacetate or low (<0.02 mg/mL) for diazinon. Therefore, once extracted these DNA samples could be safely processed in a forensic laboratory.     

Identification and separation of DNA mixtures using peak area information

We introduce a new methodology, based upon probabilistic expert systems, for analysing forensic identification problems involving DNA mixture traces using quantitative peak area information. Peak area is modelled with conditional Gaussian distributions. The expert system can be used for ascertaining whether individuals, whose profiles have been measured, have contributed to the mixture. It can also be used to predict DNA profiles of unknown contributors by separating the mixture into its individual components. The potential of our probabilistic methodology is illustrated on case data examples and compared with alternative approaches. The advantages are that identification and separation issues can be handled in a unified way within a single probabilistic model and the uncertainty associated with the analysis is quantified. Further work, required to bring the methodology to a point where it could be applied to the routine analysis of casework, is discussed.     

Y-STRs in forensic medicine: DNA analysis in semen samples of azoospermic individuals

ABSTRACT: The incidence of rape has increased, especially in metropolitan areas, such as the city of São Paulo. In Brazil, studies about it have shown that the majority of this type of crime is committed by the relatives and persons close to the victim. This has made the crime more difficult to be denounced, as only 10% of the cases are reported to competent police authorities. Usually, cytological exams are carried out in sex crime investigations. The difficulty in showing the presence of spermatozoa is frequent, but it does not exclude the presence of male DNA. The absence of spermatozoa in material collected from rape victims can be due to several factors, including the fact that the agressor suffers from azoospermia. This condition can be the result of a successful vasectomy. As the majority of DNA in the ejaculation sample is from spermatozoa, there is much less DNA to be analyzed. This study presents the application of Y‐STRs (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, and DYS393) in DNA analysis of sperm samples from 105 vasectomized men. The study demonstrated a great variation in DNA concentration. DNA extraction and amplification was possible in all sperm samples even in the absence of spermatozoa. The same profile was observed, for each individual, from DNA extracted from blood, pre‐ and postvasectomy semen samples. The use of markers specific for Y chromosome in sex crime cases, especially in the absence of spermatozoa, is very important, mainly because in most situations there is a small quantity of the agressor's DNA in the medium and a large quantity of the victim's DNA.     

Real-time polymerase chain reaction quantification of canine DNA

The accurate quantification of target DNA is an important step in the short tandem repeat analysis of forensic biological samples. By utilizing quantification data to control the amount of template DNA in the polymerase chain reaction (PCR), forensic scientists can optimize testing and minimize the consumption of limited samples. The ability to identify and quantify target DNA in mixed-species samples is crucial when it may be overwhelmed by nontarget DNA, as in cases of dog attack. We evaluated two quantitative real-time PCR assays for dynamic range, species specificity, and inhibition by humic acid. While both assays proved to be highly sensitive and discriminating, the Melanocortin-1 Receptor (MC1R) gene Taqman assay had the advantages of a shorter run time, greater efficiency, and safer reagents. In its application to forensic casework, the MC1R assay has been advantageous for quantifying dog DNA in a variety of mixed-species samples and facilitating the successful profiling of individual dogs.     

Mitochondrial DNA amplification success rate as a function of hair morphology

This study examines the amplification success rate of mitochondrial DNA from human head hair with respect to their potential for forensic application. Mitochondrial DNA was isolated using a Chelex-based extraction method and amplified using the LINEAR ARRAY duplex PCR system. The particular focus of this study was to characterize the morphological features of human head hair in order to further the understanding of the factors that influence amplification success rate in hair tissue using the LINEAR ARRAY duplex PCR system. 2554 head hairs from 132 individuals representing four population groups were amplified. The hair samples were characterized as follows: 1251 were identified microscopically as telogen hairs and 1303 were classified as hairs without roots (removed before extraction). Amplification success was assessed as a function of several independent variables: morphological characteristics; telogen root versus no root; donor age; scalp origin; use of cosmetic hair treatments; and race of the donor. The results show that a positive correlation exists between amplification success and the presence of a telogen root. Combining the amplification success with either the original or optimized protocol, telogen hairs result in an overall success rate of 77.5% compared with 65% for hairs with no roots. Controlling for telogen hairs, the findings indicate that the overall success rate is independent of cosmetic hair treatments; medulla structure; shaft length, diameter, and volume; and scalp origin. Conversely, the age of the donor, the race of the donor, and hair pigmentation all contribute to a variation in amplification success rate.     

Population substructure can significantly affect reliability of a DNA-led process of identification of mass fatality victims

Aiming to evaluate the effects of population substructure on the reliability of a DNA correspondence in the process of human identification, we used the model of "in silico" constructed populations with and without substructure. Effects of population substructure were evaluated at the level of locus heterozygosity, Hardy-Weinberg equilibrium and mini-haplotype distribution. Inbreeding in a subpopulation of 100 individuals through 10 generations did not significantly alter the level of heterozygosity and Hardy-Weinberg equilibrium. However, analysis of mini-haplotype distribution revealed a significant homogenization in separated subpopulations. Average observed mini-haplotype frequency (f(o)) increased to threefold from expected values (f(e)), and the number of mini-haplotypes with f(o)/f(e) above 10 increased over sixfold, suggesting that the effects of population substructure on calculated likelihood ratios (LR) might be larger than previously estimated. In most criminal cases, this would not represent a problem, whereas for identifications in large-scale mass fatality events, population substructure might considerably increase the risk of false identification.     

Comparative Analysis of the Effects of Heat on the PCR‐Amplification of Various Sized DNA Fragments Extracted from Sus Scrofa Molars*

Abstract: This study examined the effects of heat on the amplification of DNA from the dental pulp of Sus scrofa molars and investigated the protection afforded to the pulp tissue by the dental enamel, alveolar process, and soft tissue of the head. Segments of defleshed maxilla and mandible encasing the first molar (n = 60) were subject to a range of temperatures for 15 min. Dental pulps were retrieved. Amplifications using three‐primer and four‐primer multiplexes showed no degradation of the largest fragment following exposure to 450°C. Amplifications in the three‐primer multiplex (283 bp) were successful following exposure to 525°C in maxillary samples only. This study revealed the enamel density of maxillary molars to be greater than mandibular molars in Sus scrofa. Following incineration of intact heads for 15 min (n = 10) and 1 h (n = 4) at an average temperature of 625°C, amplifications of the largest fragment (450 bp) were successful from both maxillary and mandibular teeth.     

Recovery of Environmental Human DNA by Insects*,†

Abstract: We tested the hypotheses that foraging insects can acquire human DNA from the environment and that insect‐delivered human DNA is of sufficient quantity and quality to permit standard forensic analyses. Houseflies, German cockroaches, and camel crickets were exposed to dusty surfaces and then assayed for human mitochondrial and nuclear loci by conventional and qPCR, and multiplex STR amplification. Over two experiments, 100% of insect groups and 94% of dust controls tested positive for human DNA. Of 177 individuals, 33–67% tested positive and 13 yielded quantifiable human DNA (mean = 0.022 ± 0.006 ng; mean dust control = 2.448 ± 0.960 ng); four had at least one positive allele call for one or more locus; eight others showed multiple peaks at some loci. Results imply that application to routine forensic casework is limited given current detection methodology yet demonstrate the potential use of insects as environmental samplers for human DNA.     

DNase-Ⅰ纯化结合碱性裂解法提取混合斑精子DNA

目的研究脱氧核糖核酸酶I(DNase-I)纯化结合碱性裂解法提取混合斑精子DNA的方法在法医学中的应用。方法收集79份性犯罪案件混合斑检材,分别用DNase-I纯化结合碱性裂解法和差异裂解法提取精子DNA,采用STR荧光标记复合扩增体系进行16个STR基因座分型,并比较检验结果。结果应用DNase-I纯化结合碱性裂解法提取精子DNA,64例检材分型成功;应用差异裂解法提取精子DNA,57例检材分型成功;两种方法比较结果存在显著性差异(P=0.039),DNase-1纯化结合碱性裂解法提取精子DNA的STR分型成功率更高,成本低廉。结论DNase-I纯化结合碱性裂解法提取混合斑精子DNA可提高检验成功率,操作简便,快速,易于自动化,适于法医学个体识别鉴定。     

浓缩DNA法结合miniSTR分型技术检验微量DNA

目的优化低拷贝数DNA STR分型方法。方法对采用磁珠法或Chelex-100法提取DNA,Identi-filer试剂盒扩增,未获得分型结果的日常检案检材,采用物理浓缩法或过柱浓缩法浓缩DNA,采用miniFilerTM试剂盒再次扩增分型。结果 127例检材中,47例磁珠法提取DNA未获得分型的样品,分型成功率为36%;80例Chelex-100法提取DNA未获分型的样品,分型成功率为30%。结论采用浓缩法和miniFilerTM试剂盒,可以提高日常检案中低拷贝数检材的STR检验分型成功率。