Evaluation of an automated liquid hybridization method for DNA quantitation

The AluQuant (Promega Corporation) liquid hybridization DNA quantitation method was evaluated on an automated robotic platform (Biomek 2000, Beckman Coulter, Fullerton, CA) for use in forensic PCR-STR systems. DNA from bloodstains and buccal swabs was extracted by three different methods: Chelex, Qiagen and DNA IQ (Promega). Samples were quantitated using both the Quantiblot and the AluQuant systems. Concordance between methods was determined by comparing the average AluQuant DNA concentrations for samples having matching (binned) Quantiblot values. Studies testing the "accuracy" (STR analysis), precision, sensitivity, and specifies specificity of the AluQuant method were also conducted. The effect of inhibitors (carpet, denim, and suede) was evaluated. The results indicate that the AluQuant quantitation system equals the Quantiblot system in "accuracy", sensitivity, precision, and primate-specificity. While extracts from denim and suede affected (inhibited) both systems minimally, the carpet extracts produced a sharp increase in DNA quantitation values in the AluQuant but not the Quantiblot system. The speed and user-friendliness of the AluQuant system on a robotic platform offer specific advantages to the forensic community.     

The killer outfit and timing: Impact of the fabric and time in body fluid identification and DNA profiling

The present work aimed to study the detection, through lateral flow immunochromatographic (LFI) tests, of saliva samples over time in three different types of fabrics, as well as, the possibility of DNA isolation and characterization from the sample tubes and the cassettes. Fifty microliters of saliva (three samples/time) were deposited in denim, cotton, and polyester. Saliva was identified by SERATEC® Amylase Test and the Crime Scene version SALIVA CS, being able to detect it up to six months of deposition, although with different band intensities. Polyester showed stronger bands than cotton, probably due to its synthetic nature, and denim, as an inked fabric, showed less band intensities. Statistical analyses confirmed significant differences among fabrics, but not over time in the same type of fabric. Total DNA from the sample tubes was successfully recovered, in contrast, from the cassettes, only polyester retrieved amplifiable DNA. These findings indicated that it is possible to recover and identify saliva up to six months after deposition, also obtaining DNA. Future research will be able to expand these results, analyzing the stability of other body fluids, and the sensitivity of lateral flow immunochromatographic tests to detect them.     

Molecular Assay for Screening and Quantifying DNA in Biological Evidence: The Modified Q‐TAT Assay*

Abstract: A method is described for the quantitation of total human and male DNA. Q‐TAT utilizes end‐point, multiplex polymerase chain reaction (PCR) amplification of the amelogenin and SRY loci to quantify DNA and incorporates a cloned nonhuman template to detect PCR inhibition. Standard curves of fluorescence from amelogenin or SRY amplicons were generated from amplification of known amounts of NIST traceable SRM‐female or SRM‐male DNA. Curves showed good linearity up to 500 pg of SRM‐template (R² > 0.99) and reliably estimated total and male DNA content in casework samples. The nonhuman pRL_null template included in each PCR was a sensitive indicator of known PCR inhibitors including EDTA, hemin, blue denim dye, and humic acid. Finally, the SRY amplicon was a sensitive indicator of male DNA and, in mixtures, could reliably estimate male DNA present in an excess of female DNA. The Q‐TAT multiplex is a reliable quantitation method for forensic DNA typing.     

Deoxyribonucleic acid (DNA) analysis by restriction fragment length polymorphisms of blood and other body fluid stains subjected to contamination and environmental insults.

Deoxyribonucleic acid (DNA) restriction fragment length polymorphism (RFLP) profile results were obtained from bloodstains and other body fluid stains subjected to mixture with other body fluids, environmental insults (sunlight and temperature), different substrates (cotton, nylon, blue denim, glass, aluminum, and wood), and contaminants (gasoline, bleach, sodium hydroxide, soil, motor oil, detergent, phosphate salt, glacial acetic acid, and microorganisms). Of the samples that produced profile results, all had profiles that were consistent with those of untreated control samples.     

Developmental Validation of the PrepFiler™ Forensic DNA Extraction Kit for Extraction of Genomic DNA from Biological Samples*

Abstract: The PrepFiler? Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 μL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA^® paper), and touch evidence‐type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol–chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts.     

The analysis of biological samples from crime scene for a future human DNA profile confrontation. Effects of presumptive test reagents on the ability to obtain STR profiles for human identification

Because of the increase of evidence of blood stains, that have been washed or cleaned in an attempt to mask the analysis of DNA profiles, there is also an increase in the use of presumptive tests on samples sent to laboratories. Some of the presumptive tests, used to identify blood and semen stains, could potentially affect the recovery of high molecular weight DNA from the samples, or extinguish them, especially those already present in small quantities. After the presumptive tests, often these samples are discarded. This study aimed to examine the possibility of obtaining a DNA profile from samples submitted for presumptive testing and cleaned with bleaches with and without chlorine. Two different protocols were conducted: (a) A unique sample of human blood in natura (5 μL), already typed through the DNA techniques with the genetic profile previously known (control), was distributed onto cotton fabrics and dried at room temperature. Four samples of fabric were macerated in saline solution and Coombs serum and then stored for three months (room temperature and freezer −20 °C). (b) Another sample of human blood, type A, in natura, already typed through the techniques of DNA (control) was used. Aliquots of 200 μL were distributed in: cotton, denim and synthetic fabric. The samples were dried at room temperature for 24 h. The blood stains in those fabrics (cotton, denim and synthetic) were then divided into three groups: unwashed, cleaned with chlorine bleach and cleaned with chlorine bleach and soap powder. The samples were again dried at room temperature for 24 h, before the use of luminol. The DNA were extracted with Chelex 100 and amplified with the Identifiler Kit (Applied Biosystems). The blood stains exposed to saline and Coombs serum had DNA profiles consistent with untreated samples (controls). This result shows that the experts should keep and store the samples treated with saline and Coombs serum for future DNA confrontation when necessary. Also discussed in this paper the pattern of blood stains after washing with bleaching solutions, as well as the quantity of DNA obtained from these samples.     

On the frequency of occurrence of a peculiar polyester fibre type found in blue denim textiles

In a double murder investigation, the victims were found after a prolonged stay in a drainage canal. In spite of the expectations, fibre examination established a multitude of primary and secondary transferred fibres. One of these fibre types was a colourless polyester fibre possessing a blue coloured molten fibre end. These matched one of the types present in the suspect's blue denim trousers. The aim of this study was to verify the rarity of this peculiar fibre type and more precisely its presence in blue denim textiles. Over five hundred different blue jeans textiles were examined and only one of these presented exactly the same type. The comparison involved microscopy, microspectrophotometry in the visible range and Raman spectroscopy. The results indicate this fibre type is extremely rare in a blue jeans fabrics and that “standard” blue denim should not be disregarded in case work.     

Sensitivity of the Luminol Test with Blue Denim

Abstract: An article appearing in this journal in 2000 suggested that the sensitivity of the luminol test performed on denim fabric is usually no greater than at a 1:100 dilution of blood. This study shows that the luminol test may be unambiguously interpreted at substantially greater dilutions of blood. In this study, four different types of denim were tested by spraying a swatch of fabric with a typical formulation of the luminol reagent. Testing was conducted of dilutions of blood up to 1:1000, all of which showed distinct chemiluminescence. Diluted blood was applied to denim material in the form of a random number. A successful test was obtained only when a “blind” observer, i.e., an observer who was uninformed of the number, correctly reported the number.     

The effects of environment and substrata on deoxyribonucleic acid (DNA): the use of casework samples from New York City

This study was designed to analyze the effects of the environment and substrata on the quality of deoxyribonucleic acid (DNA) isolated from evidentiary specimens. The quality of DNA isolated from actual casework specimens was determined by measuring its size by agarose gel electrophoresis. The information obtained could be used to predict the suitability of the DNA in the samples for restriction fragment length polymorphism (RFLP) analysis. The evidentiary specimens chosen for DNA were classified according to substrate (scrapings, plastic bags, synthetics, denim, and carpet) and according to a subjective evaluation of the condition of the stain (soiled, damp, or putrefied) and to its size (small or large). The results show that DNA of sufficient quality and high molecular weight (HMW) can be reliably isolated from bloodstains deposited on evidentiary items which have an unknown environmental history and which have dried onto a variety of substrata. Subsequent RFLP analysis of a selected number of these samples verified that the DNA was suitable for this type of analysis.     

直接扩增法在血痕、肋软骨和唾液检材中的应用

目的采用Identifiler Direct PCR试剂盒直接扩增法进行棉签擦拭血痕、肋软骨和烟蒂唾液斑DNA分型检验,并评价其应用价值。方法收集棉签擦拭血痕、烟蒂各20份,肋软骨10份,采用Identifiler Direct PCR试剂盒进行直接扩增及分型检验,以相同检材采用磁珠法/Chelex-100法提取模板DNA后扩增检验结果作为对照,对两组所得结果进行比较分析。结果棉签擦拭血痕和肋软骨一次检测完整分型率均为100%,分型结果与对照组一致;烟蒂上唾液斑有2份检材第一次未能完整分型,调整方法再次检验后获分型成功。结论实际检案中的棉签血痕、肋软骨和烟上唾液斑,采用直接扩增法检测,方法简单、快速、稳定、检材用量小,可在实际检案中选择使用。     

Evaluation of Commercial Kits for Dual Extraction of DNA and RNA from Human Body Fluids, ,

STR typing of DNA evidence can identify the donor with a high power of discrimination but cannot identify the tissue origin of a body‐fluid stain. Using RNA to attribute a crime scene stain to a particular tissue may aid in reconstruction efforts. With blood from 10 donors, four DNA and RNA coextraction kits were evaluated by measuring yields and STR and mRNA profiles. T tests indicated some significant differences in kit performance. The Zymo Research ZR‐Duet^? kit performed best based on average DNA (41.4 ng) and mRNA (4.07 ng) yields and was the only kit to provide complete DNA/RNA profiles for all samples. The consistency of this kit was challenged by data from additional blood and saliva donors. Further testing is advised before a superior kit is unequivocally chosen. Stand‐alone DNA or RNA purification generally offers higher yield, but coextraction may still allow successful STR profiling and tissue source identification.     

Application of mtDNA SNP analysis in forensic casework

In this study six forensic cases are presented where the routine analysis of samples for short tandem repeats (STRs) failed. The sequencing of the mitochondrial hypervariable region I (HVR I) also failed. Nevertheless, it was possible to analyse the samples with mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) via SNaPshot technique. The age of the analysed samples ranged from 2 months to 1400 years. Saliva-, blood-, sperm-, hair-, tooth- and bone-samples were investigated. Furthermore the mtDNA SNP analysis of a forensic case sample showing a mixed stain profile is presented. It was possible to discriminate two different haplogroups in this mixed-person stain. If compared to another mtDNA SNP profile that was found in a hair, the discriminating SNPs of the hair were as well found in the mixed-person stain.To disburden the SNP analysis in forensic casework, haplogroup assignment criteria and quality criteria for mtDNA SNaPshot analysis are announced.     

批量陈旧血样DNA的自动化检验

目的建立批量陈旧血样DNA自动化提取检验的方法。方法采用普通磁珠法和本文建立的磁珠法经TECAN Freedom EVO150—8型自动化工作站分别提取540份陈旧血样模板DNA,采用Sinofiler^TM试剂盒进行荧光标记复合扩增。结果采用普通磁珠法和本文所建DNA提取方法,在540份样本中获得全部基因座STR分型的样本分别为217份和488份,检验成功率分别为40．2％和90．3％。结论本文所建方法可显著提高大批量陈旧血样自动化检验成功率。     

Human identification and sex determination of dental pulp, bone marrow and blood stains with a recombinant DNA probe

Recombinant DNA hybridizing specifically to a 300 nucleotide repeat DNA sequence (BLUR8) of human specificity and to human repeat DNA sequence (pHY10) on the Y chromosome was used for human identification and sex determination of degraded DNA samples of blood stains, dental pulp, and bone marrow. This radioactive technique enabled reliable and sensitive human and sex determination from blood stains that were more than 80 years old. Less than 1 piece of 0.5 cm length thread of blood stain was enough for both tests. DNA from relatively fresh dental pulp and bone marrow was clearly identified. The human identification test, which could recognize up to 0.3 ng DNA correctly, was 3 to 5 times more sensitive than the sex determination test.     

PuriTyper~(TM)法医学纯化试剂盒的性能验证

目的验证PuriTyperTM纯化试剂盒各项性能指标和法医学应用价值。方法收集及制备抗凝血液、常见案件检材(唾液、烟头、精液、毛发、指甲、骨骼及组织块)、斑痕样本(血斑、唾液斑、精斑)以及模拟添加抑制剂和模仿自然环境中放置的血斑。采用PuriTyperTM纯化试剂盒提取纯化并进行DNA定量,IdentifilerTM复合扩增试剂盒扩增,产物经ABI 3130遗传分析仪进行检测,Genemapper软件分析结果,对该试剂盒灵敏度、稳定性、重复性、检材适应性进行测试。结果采用该试剂盒提取0.1～40μL血液分别获得0.042～26.45ng/μL的DNA。3种斑痕样本DNA产量平行试验结果稳定。不同类型检材重复检验所获IPC的CT平均值在27.60至28.03之间。常见案件检材所得分型与已知结果均一致。结论 PuriTyperTM纯化试剂盒能够满足法医DNA检验的要求,对法医学实践具有重要的应用价值。     

Screening and identification of tissue-specific methylation for body fluid identification

Identification of body fluid stains can bring important information to crime case. Recent research in epigenome indicates that tissue-specific differentially methylated regions (tDMRs) show different DNA methylation profiles according to the type of cell or tissue, which makes it possible to identify body fluid based on analysis of DNA. This study screened and identified tDMRs from genome for forensic purpose. DNA samples from blood, saliva, semen, and vaginal fluid were analyzed by methylation sensitive represent difference analysis and Sequenom Massarray^® quantitative analysis of methylation. Six blood-specific tDMRs were obtained. Two tDMRs display blood-specific hypomethylation, and four tDMRs show blood-specific hypermethylation. These tDMRs may discriminate blood stain from other body fluids. The result indicated that tDMRs could become potential DNA markers for body fluid identification.     

Chemical Enhancement Techniques of Bloodstain Patterns and DNA Recovery After Fire Exposure*

Abstract: It is common in forensic casework to encounter situations where the suspect has set a fire to cover up or destroy possible evidence. While bloodstain pattern interpretation, chemical enhancement of blood, and recovery of deoxyribonucleic acid (DNA) from bloodstains is well documented in the literature, very little information is known about the effects of heat or fire on these types of examinations. In this study, a variety of known types of bloodstain patterns were created in a four‐room structure containing typical household objects and furnishings. The structure was allowed to burn to flashover and then it was extinguished by firefighters using water. Once the structure cooled over night, the interior was examined using a bright light. The bloodstains were evaluated to see if the heat or fire had caused any changes to the patterns that would inhibit interpretation. Bloodstain patterns remained visible and intact inside the structure and on furnishings unless the surface that held the blood was totally burned away. Additionally, a variety of chemical techniques were utilized to better visualize the patterns and determine the possible presence of blood after the fire. The soot from the fire formed a physical barrier that initially interfered with chemical enhancement of blood. However, when the soot was removed using water or alcohol, the chemicals used, fluorescein, luminol, Bluestar^®, and Hemastix^®, performed adequately in most of the tests. Prior to DNA testing, the combined phenolphthalein/tetramethyl benzidine presumptive test for the presence of blood was conducted in the laboratory on samples recovered from the structure in an effort to assess the effectiveness of using this type of testing as a screening tool. Test results demonstrated that reliance on obtaining a positive presumptive result for blood before proceeding with DNA testing could result in the failure to obtain useful typing results. Finally, two DNA recovery methods (swabbing the stain plus cutting or scraping the stain) were attempted to evaluate their performance in recovering samples in an arson investigation. Recovery of DNA was more successful in some instances with the swabbing method, and in other instances with the cutting/scraping method. Therefore, it is recommended that both methods be used. For the most part, the recovered DNA seemed to be unaffected by the heat, until the temperature was 800°C or greater. At this temperature, no DNA profiles were obtained.     

Development of a radial gel diffusion technique for the identification of urea in urine stains

A radial gel diffusion method utilizing urease and bromthymol blue has been developed for urine stain identification. Urea, present in urine in relatively high concentrations, can be detected from urine stain extracts. This technique provides both qualitative and quantitative results, and is sensitive enough to detect 0.078 micrograms/microliter of urea.     

染色混合精斑涂片DNA检验方法的研究

建立了染色混合精斑涂片DNA的提取方法,通过两步扩增的技术与垂直聚丙烯酰胺凝胶电泳及银染色法,成功检测了3个STR位点.结果显示,所有染色涂片DNA分型均与对照血样一致,苏木素-伊红(HE)染色比酸性复红-美蓝(Baecchi)染色对DNA的降解作用要小;涂片灵敏度检验可达到少于10个精子,且室温保存一年以内的涂片其灵敏度光显著差异.该方法简单、快速、可靠,为染色混合精斑涂片的PCR分析提供了新方法.     

Identification in blood stains through DNA typing with C4 and HLA-DR probes

A restriction fragment length polymorphism analysis using double digestion of DNA preparations with XbaI and BglII restriction enzymes and hybridization with C4 and HLA-DR probes is described. The typing conditions selected reveal extensive individual variation in both C4 and DR gene regions. In our panel of 46 unrelated individuals, 37 different phenotypic patterns were recognized when both probes were used, and preliminary discriminative power values of 0.865 and 0.914 were calculated for C4 and DR beta, respectively. The probability of a chance match using both systems is probably about 1.5.10(-2). The potential of this method for individual identification of blood stains was demonstrated on DNA prepared from 6-month-old dried blood stains from seven panel individuals. The seven individuals were all identified when comparing stain DNA patterns with panel control patterns. No RFLP pattern changes were observed following storage of blood stains. Based on these experiments with C4 and DR beta DNA typing under laboratory conditions, it is concluded that DNA typing with such probes may become a powerful tool in future stain identification analyses.