Dual examinations for identification of urine as being of human origin and for DNA-typing from small stains of human urine

Concurrent methods for identification of urine as being of human origin, and for DNA-typing from small stains of human urine were examined. A urine stain was extracted with phosphate-buffered saline (PBS), and the extract was filtered using a Centricon-100 device. The filtrate was subjected to electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS) for identification of human urine and a DNA-typing sample was obtained by dialfiltration of the residue using a DNA purification kit. After the purified residue was treated with an AmpflSTR Profiler PCR amplification kit, the DNA-types were analyzed by capillary electrophoresis using a Genetic Analyzer. It was possible to identify a urine stain as being of human origin, and complete DNA profiles could be successfully obtained from a urine stain which had been created by 50 microL of female urine. Serial analyses of urine stains found at a crime scene provide effective information for forensic investigation. This method is recommended for stain identification and for DNA-typing from a urine stain.     

Expedited, chemically enhanced sperm cell recovery from cotton swabs for rape kit analysis

This report focuses on the development of a method for chemically induced enhancement of cell elution and recovery from cotton swabs. The method exploits the exclusive use of detergents for intact cell removal, and can be utilized in conjunction with, or to circumvent, conventional differential extraction (DE). Samples treated with Sarkosyl (54.4 +/- 1.8%) and sodium dodecyl sulfate (SDS) (78.5 +/- 0.7%) yielded higher sperm cell recoveries than a conventional DE buffer (39.4 +/- 2.1%). The results indicated that the choice of detergent affected sperm cell yield, with anionic detergents having the greatest effect. Storage time of samples affected the concentration of detergent required for optimal sperm cell recovery, longer times requiring increased detergent concentrations. In addition, the extent of sperm cell lysis by proteinase K digestion was evaluated. The results indicate that the exclusive use of SDS enhances the release of sperm and epithelial cells from a cotton swab as compared with DE buffer, providing for a more effective DNA analysis.     

The utility of whole genome amplification for typing compromised forensic samples

Biological evidence has become invaluable in the crime laboratory; however, it may exist in limited quantity and/or quality. Given this, the ability to amplify total DNA obtained from evidence, in an unbiased manner, would be highly advantageous. Methods for whole genome amplification (WGA) have the potential to fulfill this role, resulting in a virtually unlimited supply of DNA. In the research presented, two WGA methods, improved primer extension preamplification and multiple displacement amplification (MDA), were tested using commercial kits. Control DNA, artificially degraded DNA, and DNA from fresh blood, aged blood, hair shafts, and aged bones underwent WGA, followed by short tandem repeat and mitochondrial DNA analysis. The methods did amplify DNA, but performed poorly on forensically relevant samples; the maximum amplicon size was reduced, and MDA often resulted in extraneous bands following polymerase chain reaction. Taken together, WGA appears to be of limited forensic utility unless the samples are of a very high quality.     

The value of DNA material recovered from crime scenes

Abstract:  DNA material is now collected routinely from crime scenes for a wide range of offenses and its timely processing is acknowledged as a key element to its success in solving crime. An analysis of the processing of approximately 1500 samples of DNA material recovered from the property crime offenses of residential burglary, commercial burglary, and theft of motor vehicle in Northamptonshire, U.K. during 2006 identified saliva and cigarette ends as the main sources of DNA recovered (approximately 63% of samples) with blood, cellular DNA, and chewing gum accounting for the remainder. The conversion of these DNA samples into DNA profiles and then into matches with offender profiles held on the U.K. National DNA database is considered in terms of the ease with which Crime Scene Examiners can recover DNA rich samples of different sources, the location of the DNA at the crime scene, and its mobility. A logistical regression of the DNA material recovered has revealed a number of predictors, other than timeliness, that greatly influence its conversion into a DNA profile. The most significant predictor was found to be Crime Scene Examiner accreditation with offense type and DNA sample condition also being relevant. A similar logistical regression of DNA samples profiled that produced a match with an offender on the U.K. National DNA database showed no significance with any of the predictors considered.     

Laser microdissection separation of pure spermatozoa from epithelial cells for short tandem repeat analysis

Short tandem repeat (STR) analysis is a valuable tool in identifying the source of biological stains, particularly from the investigation of sexual assault crimes. Difficulties in analysis arise primarily in the interpretation of mixed genotypes when cell separation of the sexual assailant's sperm from the victim's cells is incomplete. The forensic community continues to seek improvements in cell separation methods from mixtures for DNA typing. The feasibility of applying laser microdissection (LMD) technology to precisely separate sexual assault cell mixtures by visual inspection coupled with laser dissection was assessed through three experiments. First, various histological stains were evaluated for use with LMD and DNA analysis. Second, different DNA isolation methods were evaluated on LMD-collected cells. Finally, STR analysis was performed on LMD-separated sperm cells from mixtures of semen and female buccal epithelial cells. The results indicated (a) hematoxylin/eosin staining performed best in its ability to differentiate sperm and epithelial cells while exhibiting the least negative effect on further downstream analysis; (b) both QIAamp and Lyse-N-Go methods were useful for recovery of DNA from LMD-collected sperm cells; and (c) LMD separation provided clear STR profiles of the male donor with the absence of any additional alleles from the female donor. This report describes an efficient, low-manipulation LMD method for the efficient separation of spermatozoa from two-donor sperm/epithelial cell mixtures.     

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.     

Higher Capillary Electrophoresis Injection Settings as an Efficient Approach to Increase the Sensitivity of STR Typing

Abstract:  Evidentiary traces may contain low quantities of DNA, and regularly incomplete short tandem repeat (STR) profiles are obtained. In this study, higher capillary electrophoresis injection settings were used to efficiently improve incomplete STR profiles generated from low-level DNA samples under standard polymerase chain reaction (PCR) conditions. The method involves capillary electrophoresis with higher injection voltage and extended injection time. STR peak heights increased six-fold. Inherent to the analysis of low-level DNA samples, we observed stochastic amplification artifacts, mainly in the form of allele dropout and heterozygous peak imbalance. Increased stutter ratios and allele drop-in were rarely seen. Upon STR typing of 10:1 admixed samples, the profile of the major component did not become overloaded when using higher injection settings as was observed upon elevated cycling. Thereby an improved profile of the minor component was obtained. For low-level DNA casework samples, we adhere to independent replication of the PCR amplification and boosted capillary electrophoresis.     

The application of miniplex primer sets in the analysis of degraded DNA from human skeletal remains

A new set of multiplexed PCR primers has been applied to the analysis of human skeletal remains to determine their efficacy in analyzing degraded DNA. These primer sets, known as Miniplexes, produce shorter amplicons (50-280 base pairs (bp)) than standard short tandem repeat (STR) kits, but still utilize the 13 CODIS STR loci, providing results that are searchable on national DNA databases. In this study, a set of 31 different human remains were exposed to a variety of environmental conditions, extracted, and amplified with commercial and Miniplex DNA typing kits. The amplification efficiency of the Miniplex sets was then compared with the Promega PowerPlex 16 system. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only 16% of the samples generated full profiles with the Powerplex 16 kit. Complete profiles were obtained for 11 of the 12 Miniplex loci with amplicon sizes less than 200 bp. These data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for anthropological and forensic analysis of degraded DNA from human skeletal remains.     

The Use of Adhesive Tape for Recovery of DNA from Crime Scene Items

Abstract: The selection of the appropriate method of collection of biological material from crime scene items can be crucial to obtaining a DNA profile. The three techniques commonly used for sampling items are: cutting, swabbing, and taping. The tape sampling technique offers an advantage, in that it enables the collection of a potentially highly informative source of DNA, shed epithelial cells, from selected areas on crime scene items (the inside fingers of a glove, for instance). Furthermore, surface collection of biological material by taping reduces co‐sampling of known PCR inhibitors such as clothing dyes. The correct choice of tape for crime scene item sampling is important. Not all tapes are suitable for biological trace evidence collection as well as DNA extraction. We report on one tape that met both these criteria. Three different cases are presented which demonstrate the usefulness of adhesive tape sampling of crime items. Finally, the advantages of the tape collection technique are discussed and guidelines for preferred areas of tape sampling on various casework items are presented.     

The Use of Mitochondrial DNA Single Nucleotide Polymorphisms to Assist in the Resolution of Three Challenging Forensic Cases

Abstract:  Mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) in an 11-plex assay were typed in three missing person cases involving highly degraded human remains. Unlike the traditional forensic approach to analyzing mtDNA which focuses on sequencing portions of the noncoding Control Region, this assay targets discriminatory SNPs that reside principally in the coding region. In two of the cases, the SNP typing successfully excluded one of two reference families that could not be excluded on the basis of mtDNA hypervariable region sequencing alone, and resulted in the final resolution of both decades-old cases. In a third case, SNP typing confirmed the sorting and reassociation of multiple commingled skeletal elements. The application of a specific mtDNA SNP assay in these cases demonstrates its utility in distinguishing samples when the most common Caucasian hypervariable region type is encountered in forensic casework.     

Assessing the potential of bacterial DNA profiling for forensic soil comparisons

A pilot study was undertaken to evaluate DNA profiling of the bacterial community in soil as an alternative to geological methods for forensic soil comparisons. Soil samples from three different ecosystems were compared, and the variation within and between ecologically different sites was determined by using terminal restriction fragment (TRF) analysis of 16S ribosomal DNA. Comparison of TRF profiles revealed that samples from within a specific ecosystem (e.g., a field) showed a significantly higher similarity to each other than to those from another ecosystem (e.g., a forest). In addition, some profile features were unique to specific ecosystems. These features may allow the determination of characteristic profiles that will facilitate identification of ecologically different sites, so that a given sample collected from a suspect could be identified as originating from, for example, a field, rather than a forest. The implications of these preliminary findings for forensic investigations are discussed.     

HLA-DRB1基因分型芯片的法医物证学应用价值研究

目的对HLA-DRB1基因分型芯片在个体识别中的应用价值进行研究。方法根据HLA-DRB1基因座不同等位基因的独特序列设计探针,制成分型芯片。将待测样品DNA用末端标记了CY5的引物进行PCR扩增,产物与芯片进行杂交,根据杂交产生的荧光信号值确定样品在HLA-DRB1位点的基因型。将这一方法应用于561份样本的HLA-DRB1基因分型,根据基因型分布统计分析其法医学应用价值。同时,进行了家系调查和方法灵敏度分析,并应用于部分案例。结果利用微量检材,HLA-DRB1基因芯片可检测DRB1位点等位基因26个,基因型的分布符合Hardy-Weinberg平衡定律,该位点的观察杂合度(Ho)为0.888,期望杂合度(He)为0.902,多态信息含量(PIC)为0.893,平均非父排除率(PE)为0.801。家系调查和案例运用的结果表明,HLA-DRB1位点等位基因由亲代向子代的传递符合孟德尔遗传定律。结论HLA-DRB1为高度多态位点,其基因分型芯片可在亲子鉴定和个体识别中发挥重要作用。     

两种方法提取汗潜手印DNA的比较研究

目的比较M48和DNeasy○R plant Mini两种方法提取汗潜手印DNA的优劣。方法用M48和DNeasy○Rplant Mini两种方法分别提取16对汗潜手印DNA,并进行DNA定量,比较定量结果。结果 M48法明显比plant Mini法提取到的DNA量多（配对t检验：α=0.05,t=3.45,γ=15,0.002     

A comparison of DNA collection and retrieval from two swab types (cotton and nylon flocked swab) when processed using three QIAGEN extraction methods

The Metropolitan Police Service currently uses cotton swabs to retrieve DNA for forensic profiling. Recently, a new nylon flocked swab type has become available from Copan (MicroRheologics, Brescia, Italy) that it is claimed, offers increased sample recovery and release yields. If true, the flocked swab may have important applications in DNA evidence retrieval. This study examines the DNA retrieval capability of cotton and nylon flocked swabs when extracted using three common extraction platforms (QIAcube, BioRobot EZ1 and manually processed QIAamp DNA investigator kit). Results indicate that both swab types are capable of recovering high percentages of DNA (>50%); however, the extraction platform selected was shown to have a significant effect upon DNA retrieval. Across all experiments, the cotton swab combined with the spin-column extractions was shown to be most effective, with the nylon swab and BioRobot EZ1 combination being the least effective. These findings illustrate the importance of extraction method selection.     

The Recovery and Analysis of Mitochondrial DNA from Exploded Pipe Bombs*

Abstract: Improvised explosive devices (IEDs) represent one of the most common modes of arbitrarily injuring or killing human beings. Because of the heat generated by, and destruction to, an IED postconflagration, most methods for identifying who assembled the device are ineffective. In the research presented, steel pipe bombs were mock‐assembled by volunteers, and the bombs detonated under controlled conditions. The resultant shrapnel was collected and swabbed for residual cellular material. Mitochondrial DNA profiles were generated and compared blind to the pool of individuals who assembled the bombs. Assemblers were correctly identified 50% of the time, while another 19% could be placed into a group of three individuals with shared haplotypes. Only one bomb was assigned incorrectly. In some instances a contaminating profile (mixture) was also observed. Taken together, the results speak to the extreme sensitivity the methods have for identifying those who assemble IEDs, along with precautions needed when collecting and processing such evidence.     

Recovery and STR amplification of DNA from RFLP membranes

Restriction fragment length polymorphism (RFLP) techniques were utilized in the forensic DNA community until the mid 1990s when less labor-intensive polymerase chain reaction short tandem repeat (PCR STR) techniques became available. During the transition from RFLP technology to PCR-based STR platforms, a method for comparing RFLP profiles to STR profiles was not developed. While the preferred approach for applying new technology to old cases would be to analyze the original biological stain, this is not always possible. For unsolved cases that previously underwent RFLP analysis, the only DNA remaining may be restriction cut and bound to nylon membranes. These studies investigate several methods for obtaining STR profiles from membrane bound DNA, including removal of bound DNA with bases, acids, detergents, various chemicals, and conventional cell extraction solutions. Direct multiplex STR amplification of template in the membrane-bound state was also explored. A partial STR profile was obtained from DNA that was recovered from an archived membrane using conventional extraction buffer components, indicating promise for recovering useful STR information from RFLP membranes that have been maintained in long-term frozen storage.     

Chelex法和两种磁珠法提取接触DNA效果的比较

目的比较Chelex法、DNA IQ磁珠法、EQ国产磁珠法对接触DNA的提取效果。方法将稀释为10ng、100ng的标准品DNA,分别采用Chelex法、DNA IQ磁珠法、EQ国产磁珠法处理;对30例烟蒂和30例牙刷分别采用Chelex法、DNA IQ磁珠法和EQ国产磁珠法提取DNA,然后进行PCR定量和STR检测。结果Chelex法对DNA的提取无损失,DNA IQ磁珠法、EQ国产磁珠法对DNA的提取均有不同程度的损失;烟蒂、牙刷等检材采用Chelex法提取的接触DNA量和IPC CT值显著高于IQ磁珠法、EQ国产磁珠法,但STR检验成功率却低于IQ磁珠法、EQ国产磁珠法。2种磁珠法提取的DNA量、IPC CT值和STR检验成功率无显著性差异。结论污染轻、杂质少的接触DNA检材,用Chelex法提取最为方便快捷;IQ磁珠法、EQ国产磁珠法更适合污染接触DNA检材的提取及自动化操作。     

DNA Typing for the Identification of Old Skeletal Remains from Korean War Victims*

Abstract: The identification of missing casualties of the Korean War (1950–1953) has been performed using mitochondrial DNA (mtDNA) profiles, but recent advances in DNA extraction techniques and approaches using smaller amplicons have significantly increased the possibility of obtaining DNA profiles from highly degraded skeletal remains. Therefore, 21 skeletal remains of Korean War victims and 24 samples from biological relatives of the supposed victims were selected based on circumstantial evidence and/or mtDNA‐matching results and were analyzed to confirm the alleged relationship. Cumulative likelihood ratios were obtained from autosomal short tandem repeat, Y‐chromosomal STR, and mtDNA‐genotyping results, and mainly confirmed the alleged relationship with values over 10⁵. The present analysis emphasizes the value of mini‐ and Y‐STR systems as well as an efficient DNA extraction method in DNA testing for the identification of old skeletal remains.     

Effects of Cyanoacrylate Fuming,Time After Recovery,and Location of Biological Material on the Recovery and Analysis of DNA from Post‐Blast Pipe Bomb Fragments*

Abstract: This study investigated the effects of time, cyanoacrylate fuming, and location of the biological material on DNA analysis of post‐blast pipe bomb fragments. Multiple aliquots of a cell suspension (prepared by soaking buccal swabs in water) were deposited on components of the devices prior to assembly. The pipe bombs were then deflagrated and the fragments recovered. Fragments from half of the devices were cyanoacrylate fumed. The cell spots on the fragments were swabbed and polymerase chain reaction/short tandem repeat analysis was performed 1 week and 3 months after deflagration. A significant decrease in the amount of DNA recovered was observed between samples collected and analyzed within 1 week compared with the samples collected and analyzed 3 months after deflagration. Cyanoacrylate fuming did not have a measurable effect on the success of the DNA analysis at either time point. Greater quantities of DNA were recovered from the pipe nipples than the end caps. Undeflagrated controls showed that the majority (>95%) of the DNA deposited on the devices was not recovered at a week or 3 months.     

Investigative studies into the recovery of DNA from improvised explosive device containers

Apprehending those who utilize improvised explosive devices (IEDs) is a national priority owing to their use both domestically and abroad. IEDs are often concealed in bags, boxes, or backpacks to prevent their detection. Given this, the goal of the research presented was to identify IED handlers through postblast DNA recovery from IED containers. Study participants were asked to use backpacks for 11 days, after which they served as containers for pipe bombs. Eleven postdeflagration backpack regions likely to be handled were swabbed and analyzed via mini-short tandem repeats (miniSTRs) and alleles were called blind. An experimental consensus method was examined in which profiles from all regions were considered, to help identify spurious drop-in/out. Results were correct for all loci, except one that remained ambiguous. The results show that recovering DNA from IED containers is a viable approach for aiding in the identification of those who may have been involved in an IED event.