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.     

Simplified low-copy-number DNA analysis by post-PCR purification

Frequently, evidentiary items contain an insufficient quantity of DNA to obtain complete or even partial DNA profiles using standard forensic gentotyping techniques. Such low-copy-number (LCN) samples are usually subjected to increased amplification cylces to obtain genetic data. In this study, a 28-cycle polymerase chain reaction (PCR) was used to evaluate various methods of post-PCR purification for their effects on the sensitivity of fluorophore-based allelic detection subsequent to capillary electrophoretic separation. The amplified product was purified using filtration, silica gel membrane, and enzyme mediated hydrolysis purification techniques and evaluated for their effect on fluorescent allelic signal intensity. A purification method was selected and its effect on fluorescent allelic signal intensity was compared with that of the unpurified PCR product. A method of post-PCR purification is described which increases the sensitivity of standard 28-cycle PCR such that profiles from LCN DNA templates (<100 pg DNA) can be obtained. Full DNA profiles were consistently obtained with as little as 20 pg template DNA without increased cycle number. In mock case type samples with dermal ridge fingerprints, genetic profiles were obtained by amplification with 28 cycles followed by post-PCR purification whereas no profiles were obtained without purification of the PCR product. Allele dropout, increased stutter, and sporadic contamination typical of LCN analysis were observed; however, no contamination was observed in negative amplification controls. Post-PCR purification of the PCR product can increase the sensitivity of capillary electrophoresis to such an extent that DNA profiles can be obtained from <100 pg of DNA using 28-cycle amplification.     

A review of low template STR analysis in casework using the DNA SenCE post-PCR purification technique

LGC has developed a method for analysing low-level DNA samples called DNA SenCE (Sensitive Capillary Electrophoresis) based on post-PCR treatment of standard 28-cycle SGMplus PCR product and demonstrated to be equally effective at enhancing profiles as 34-cycle PCR. The method has been validated and accredited and used in casework since July 2007. Inherent in the method is the initial generation of a standard 28-cycle SGMplus profile so a direct comparison of standard and DNA SenCE results for all casework is possible. Here we review DNA SenCE casework, reporting the magnitude of peak enhancement and stochastic effects seen in the DNA SenCE profiles.     

Direct comparison of post-28-cycle PCR purification and modified capillary electrophoresis methods with the 34-cycle “low copy number” (LCN) method for analysis of trace forensic DNA samples

The investigation of samples with low amounts of template DNA remains at the forefront of forensic DNA research and technology as it becomes increasingly important to gain DNA profile information from exceedingly trace levels of DNA. Previous studies have demonstrated that it is possible to obtain short tandem repeat (STR) profiles from <100 pg of template DNA by increasing the number of amplification cycles from 28 to 34, a modification often referred to as “low copy number” or LCN analysis. In this study, we have optimised post-PCR purification techniques applied after only 28 cycles of PCR, as well as using modified capillary electrophoresis injection conditions and have investigated the progressive application of these enhanced approaches. This paper reviews the characteristics of the profiles obtained by these methods compared with those obtained on the same samples after 34-cycle PCR. We observed comparable sensitivity to 34-cycle PCR in terms of the number of profiles with evidence of DNA and the number of allelic peaks per profile and we noted improved peak height and area magnitude with some sample types. Certain parameters reported to be adversely affected in 34-cycle LCN investigations, such as non-donor allele peaks and increased stutter peak ratio, were reduced by this approach. There are a number of advantages for trace samples in progressing from the standard 28-cycle process to the post-PCR processing method as compared to 34-cycle PCR method, including reduced sample consumption, reduced number of PCR amplifications required, and a staged approach to sample processing and profile interpretation.     

Quantitation of human genomic DNA through amplification of the amelogenin locus

An alternate method for quantitation of human genomic DNA is presented. Quantitative template amplification technology (abbreviated "Q-TAT") estimates the quantity of human DNA present in an extract by comparing fluorescence in X and Y amplicons produced from unknowns with fluorescence in a standard curve amplified from known quantities of reference DNA. Q-TAT utilizes PCR and electrophoresis with fluorescent detection/quantitation, precluding the need for new instrumentation, methodology, or quality assurance associated with slot-blot or real-time PCR. In a comparison study incorporating shared samples, Q-TAT was found to be more sensitive than widely used slot-blot methods but somewhat less sensitive than real-time PCR. Among samples containing DNA concentrations ranging from 100 pg/microL to 2-4 ng/microL, Q-TAT produced DNA concentration estimates that agreed reasonably well with either Quantiblot or real-time PCR. Q-TAT was reproducible with a typical coincidence of variation of about 35%. Quantitation of human DNA in this study involved summing fluorescence in X and Y amplicons in unknowns and quantitation standards. However, analyzing fluorescence in X and Y amplicons individually could allow estimates of male and female DNA present in mixtures to be made. Moreover, since X and Y amplicons exhibit sizes of 210 and 216 bp, respectively, the integrity as well as the concentration of the genomic DNA template can be assessed. Q-TAT represents an alternate method useful for the quantitation of human genomic DNA prior to amplification of STR loci used for identity testing purposes. The method uses existing equipment and procedures in conjunction with a well-characterized DNA standard to produce concentration estimates for unknowns that reliably produce STR profiles suitable for analysis.     

荧光原位杂交技术在法庭科学DNA检验中的应用

目的运用荧光原位杂交技术结合激光显微切割技术,分离法医物证男女混合样本中的男性细胞和女性细胞,并进行DNA分型。方法通过双色荧光原位杂交,Y染色体标记上绿色信号,X染色体标记上红色信号,在荧光显微镜下识别男性细胞和女性细胞,并通过激光显微切割技术分别获得男性细胞和女性细胞进行DNA分型。结果运用荧光原位杂交技术,能够分别标记法医物证男女混合样本中的男性细胞和女性细胞,并通过激光显微切割技术获得各自的DNA分型。结论荧光原位杂交技术结合激光显微切割技术,可应用于法医物证男女混合样本的检验,提高个体识别能力。     

DNA Preparation from Sexual Assault Cases by Selective Degradation of Contaminating DNA from the Victim

Abstract:  The standard method to purify sperm DNA from vaginal swabs taken from rape victims is to selectively digest the victim's epithelial cells to solubilize the victim's DNA, and then separate the soluble DNA from the intact sperm by centrifugation. A different approach to removing the soluble victim's DNA is to selectively degrade it using a nuclease, DNase I. DNase I reduces the amount of soluble DNA by over 1000-fold, while having virtually no effect on the sperm DNA remaining in the sperm head and inaccessible to the enzyme. Nuclease inactivation and sperm lysis then yield a soluble, pure male DNA fraction. An aliquot of soluble DNA is removed prior to nuclease addition to provide the victim's fraction. Vaginal swabs taken at defined time points following consensual sex and taken from rape victims were processed using the nuclease method or the standard method and the nuclease method gave superior short tandem repeat profiles.     

What is she doing here? Klinefelter syndrome in forensic casework

In this case report, we describe a sexual assault incident in which the male victim’s seminal fluid contained no sperm cells, as indicated by sperm cell staining and microscopic screening, and DNA profiling results from the non-sperm cell fraction showed a major/minor DNA mixture that could be interpreted as female and male. DNA profiling of a sample from a disposable drinking cup used by the victim at the crime scene provided a single source profile, and showed a 2:1 imbalance between the heights of the X and Y chromosomes, respectively. The victim’s DNA reference sample showed a similar imbalance of the X and Y chromosomes. These observations suggested that the victim might suffer from Klinefelter syndrome, a genetic disorder related to the sex chromosomes.Here, we describe the first reported use of the QIAGEN Investigator® Argus X-12 kit for characterization of X-chromosomal STR loci to potentially identify a case of Klinefelter syndrome. This commercially available kit is primarily used in forensic laboratories to investigate kinship relations and for paternity testing in alleged father/daughter cases. Results of the X chromosome DNA profiling from the victim’s disposable drinking cup and reference samples revealed two alleles at various X-chromosomal STR loci. Moreover, this kit can also amplify a Y chromosome specific sequence (AMEL-Y), and the results indicated that this sample actually originated from a male. Evidence of two X chromosomes in the victim's DNA suggested that he was likely to have Klinefelter syndrome. In this case report, we propose the use of the QIAGEN Investigator® Argus X-12 kit as a practical forensic tool for the detection of potential genetic syndromes related to the sex chromosomes, which can affect test results and, at times, make them difficult to interpret. We also aim to increase awareness within the forensic science community regarding the existence of genetic anomalies, which should be considered when analyzing DNA profiles.     

Development of a one-tube extraction and amplification method for DNA analysis of sperm and epithelial cells recovered from forensic samples by laser microdissection

Laser microdissection can be used in forensic casework to isolate specific cell types from mixtures of biological samples. Extraction of DNA from selected cells is still required prior to STR amplification. Because of the relatively pristine nature of the recovered cells, laser microdissection is more sensitive than more traditional methods of DNA analysis, theoretically resulting in DNA profiles from less cellular material. A one-tube extraction and amplification method minimises loss of DNA through liquid transfers and reduces the potential for contamination events occurring. In this paper, the development of a one-tube method for the effective extraction of DNA from laser microdissected sperm and epithelial cells is described. The performance of the in-house method was compared to that of a commercial DNA extraction kit for extraction of DNA from sperm and the downstream compatibility with STR amplification was determined for both sperm and epithelial samples. Full Identifiler™ profiles after 28 amplification cycles were obtained from as few as 15 epithelial cells and 30 sperm.     

Separation of sperm and epithelial cells based on fluorescence-activated cell sorting

The detection and separation of spermatozoa is crucial in the forensic investigation of alleged sexual assault cases. Differential lysis and microscopy-based techniques are conventional methods for the isolation of spermatozoa, though are time-consuming and frequently fail with samples containing an unfavourable sperm/epithelial cell ratio. Successful separation by means of fluorescence-activated cell sorting (FACS) has previously been reported, yet little efforts have been dedicated towards the further improvement or routine implementation of this technique. With this ongoing research, a methodology is being developed to sort sperm from epithelial cells by combining the Sperm Hy-Liter™ staining kit and FACS. Sorted sperm cells are then subjected to a direct lysis and low volume PCR (LV-PCR) protocol. Preliminary results demonstrate the successful separation of both cell types at a sperm/epithelial cell ratio of up to 1:500. The direct lysis and LV-PCR protocol allows to produce full haploid profiles from single sperm cells and mostly full diploid profiles from 10 spermatozoa. These data suggest that the proposed methods are potentially viable for forensic casework, yet additional testing is required for validation purposes.     

A multiplexed system for quantification of human DNA and human male DNA and detection of PCR inhibitors in biological samples

Forensic analysts routinely encounter samples containing DNA mixtures from male and female contributors. To obtain interpretable Short Tandem Repeat (STR) profiles and select the appropriate STR analysis methodology, it is desirable to determine relative quantities of male and female DNA, and detect PCR inhibitors. We describe a multiplex assay for simultaneous quantification of human and human male DNA using the ribonuclease P RNA component H1 (RPPH1) human target and the sex determining region Y (SRY) male-specific target. A synthetic oligonucleotide sequence was co-amplified as an internal PCR control. Standard curves were generated using human male genomic DNA. The SRY and RPPH1 assays demonstrated human specificity with minimal cross-reactivity to DNA from other species. Reproducible DNA concentrations were obtained within a range of 0.023-50 ng/μl. The assay was highly sensitive, detecting as little as 25 pg/μl of human male DNA in the presence of a thousand-fold excess of human female DNA. The ability of the assay to predict PCR inhibition was demonstrated by shifted IPC Ct values in the presence of increasing quantities of hematin and humic acid. We also demonstrate the correlation between the multiplex assay quantification results and the strength of STR profiles generated using the AmpF?STR^®PCR Amplification kits.     

Filtration based DNA preparation for sexual assault cases

Police departments in the United States currently have as many as 500,000 unprocessed swabs taken from rape victims. The standard method for purifying sperm from these swabs is to resuspend first all cells and to digest selectively the excess of the victim's epithelial cells. The intact sperm are then separated from the contaminating solubilized DNA by centrifugation, careful removal of supernatant, and extensive washing of the sperm pellet, all steps that are difficult to automate. Vacuum driven filtration is an alternative method for separating sperm from digested epithelial cells that requires only pipetting steps and can be readily automated in a 96 well format. Sperm DNA is enriched 45-fold using this process and the yield of PCR ready DNA is roughly 20% of the amount originally present on the swab.     

单细胞分离荧光原位杂交法用于男女混合血DNA分型

目的采用单细胞分离荧光原位杂交法精确分离混合血样中男性和女性细胞并进行分型检验。方法收集男、女性血,按照男∶女为1∶5、1∶10、1∶20制备混合血样,加入0.075mol/L KCl 600μL,轻混、放置30min后加入150μL固定液离心留沉淀涂片,利用Vysis 30-161050试剂盒进行荧光原位杂交,并用PALM激光显微捕获系统分离出男、女性细胞,使用Identifiler试剂盒复合扩增并进行检测。结果捕获8个血细胞即可得到完整的DNA分型,且随着细胞数目的增多,检出率逐渐提高而等位基因丢失率逐渐降低。10个血细胞的检出率最高,为93.75%。5μL男性血液与本实验各比例女性血混合用本文方法检验均可获得男性分型。案例混合血斑经检验获得单一男性和女性分型。结论单细胞分离荧光原位杂交法可用于男女混合血样本中DNA分型检验。     

单细胞检验技术用于混合上皮细胞的分离和检验

目的建立单细胞显微捕获联合低体积扩增技术,用于混合上皮细胞检材分离检验。方法取5名男性口腔上皮细胞拭子浸泡液30μL,分别滴加到5份含同一女性皮肤表皮细胞拭子上,制成5份混合上皮细胞样本为实验组,同时制备同样的5份样本为对照组。实验组样本采用显微捕获单个口腔上皮细胞,并使用低体积扩增技术进行扩增;对照组用M48纯化试剂盒提取DNA,Identifiler试剂盒复合扩增,扩增体系为10μL。所有产物均用ABI 3130遗传分析仪进行STR分型。结果 5份实验组样本均得到男性STR分型结果,5份对照组样本则均仅得到混合分型结果。将该方法应用于1例强奸杀人案例检验,取得了满意效果。结论单细胞显微捕获联合低体积扩增技术可用于混合上皮细胞样本的分离检验。     

Developmental Validation of the Quantifiler® Duo DNA Quantification Kit for Simultaneous Quantification of Total Human and Human Male DNA and Detection of PCR Inhibitors in Biological Samples*

Abstract: The Quantifiler^® Duo DNA Quantification kit enables simultaneous quantification of human DNA and human male DNA as well as detection of inhibitors of PCR in a single real-time PCR well. Pooled human male genomic DNA is used to generate standard curves for both human (ribonuclease P RNA component H1) and human male (sex determining region Y) specific targets. A shift in the cycle threshold (C_T) values for the internal positive control monitors the presence of PCR inhibitors in a sample. The assay is human specific and exhibits a high dynamic range from 0.023 to 50 ng/μL. In addition, the multiplex assay can detect as little as 25 pg/μL of human male DNA in the presence of a 1000-fold excess of human female DNA. The multiplex assay provides assessment of the DNA extract and guidance for the selection of the appropriate AmpFℓSTR^® Amplification Kit to obtain interpretable short tandem repeat profiles.     

A time course study on STR profiles derived from human bone, muscle, and bone marrow.

The use of the polymerase chain reaction (PCR) to define deoxyribonucleic acid (DNA) types at several loci was investigated. PCR was used to amplify nine short tandem repeat (STR) loci along with the amelogenin locus on the X and Y chromosomes using the AmpF/STR Profiler Plus PCR amplification kit (Perkin Elmer). Rib bones were collected from 12 individuals. Five cm portions were buried at a depth of approximately 30 cm and 5 cm portions were left on the surface of the ground. Samples were exposed to the environment for periods of time ranging from two weeks to 17 months. Dried blood standards were prepared for use as reference standards for each rib sample. Bone, muscle, and bone marrow were collected from each sample. DNA from each tissue type was extracted. Complete profile results were obtained from the surface bone samples out to an exposure time of 17 months. None of the muscle or bone marrow samples produced complete profile results beyond eight weeks. All DNA typing results from complete or incomplete profiles were consistent with DNA typing results of the corresponding blood standard. Results suggest that using the AmpF/STR Profiler Plus PCR Amplification Kit is a valid way to establish the DNA profile of tissue types from human remains.     

Extended PCR conditions to reduce drop-out frequencies in low template STR typing including unequal mixtures

Forensic laboratories employ various approaches to obtain short tandem repeat (STR) profiles from minimal traces (<100 pg DNA input). Most approaches aim to sensitize DNA profiling by increasing the amplification level by a higher cycle number or enlarging the amount of PCR products analyzed during capillary electrophoresis. These methods have limitations when unequal mixtures are genotyped, since the major component will be over-amplified or over-loaded. This study explores an alternative strategy for improved detection of the minor components in low template (LT) DNA typing that may be better suited for the detection of the minor component in mixtures. The strategy increases the PCR amplification efficiency by extending the primer annealing time several folds. When the AmpF?STR^® Identifiler^® amplification parameters are changed to an annealing time of 20 min during all 28 cycles, the drop-out frequency is reduced for both pristine DNA and single or multiple donor mock case work samples. In addition, increased peak heights and slightly more drop-ins are observed while the heterozygous peak balance remains similar as with the conventional Identifiler protocol. By this extended protocol, full DNA profiles were obtained from only 12 sperm heads (which corresponds to 36 pg of DNA) that were collected by laser micro dissection. Notwithstanding the improved detection, allele drop-outs do persist, albeit in lower frequencies. Thus a LT interpretation strategy such as deducing consensus profiles from multiple independent amplifications is appropriate. The use of extended PCR conditions represents a general approach to improve detection of unequal mixtures as shown using four commercially available kits (AmpF?STR^® Identifiler, SEfiler Plus, NGM and Yfiler). The extended PCR protocol seems to amplify more of the molecules in LT samples during PCR, which results in a lower drop-out frequency.     

Collecting and Analyzing DNA Evidence from Fingernails: A Comparative Study,,

Forensic practitioners and crime laboratories regularly collect and analyze fingernail evidence; however, the best techniques for processing such evidence have not been established. In this study, numerous aspects of fingernail evidence processing—collection of exogenous cells, transportation, purification of DNA, and STR analysis—were analyzed using fingernails harboring applied blood or epithelial cells from scratchings. Autosomal STR mixtures resulted when fingernails were soaked or swabbed, while scrapings rarely generated mixtures but exhibited allelic dropout. Y‐STRs yielded single source profiles, with scrapings again showing dropout. A silica‐based kit extraction recovered significantly more exogenous DNA than did organic extraction, neither of which was affected by nail polish. Swabbing nails in succession resulted in some cross‐contamination from exogenous material, while transporting nails together did not, although there was loss of exogenous cells. Optimized nail processing produced complete Y‐STR profiles of male volunteers from female fingernails following scratchings.     

Identification of a novel polymorphism in the X-chromosome region homologous to the DYS456 locus

During an extensive multipopulation study with Y-short tandem repeat (STR) loci, amplified using the AmpFlSTR Yfiler PCR amplification kit, amplification of a 71 bp fragment was observed in 2.32% of the male samples analyzed (N = 3141). By direct sequencing of this fragment, it was determined that the primer binding sequences were identical to those of the DYS456 locus. A T to G single-nucleotide polymorphism (SNP) enabled amplification of the 71 bp fragment. The SNP is located within an X-Y homologous region at Xq21.31 and was observed with the highest frequency within the African American and Sub-Saharan African populations in our study. Presence of SNP on the X chromosome did not interfere with the reliability of typing the DYS456 locus and the other Y-STR loci typeable using the AmpFlSTR Yfiler PCR amplification kit. Full profiles in a mixture of male:female at 1:4000 were obtained using the current configuration of the AmpFlSTR Yfiler kit even in the presence of female DNA containing the G variant.     

STR Genotyping from a Dry‐Cleaned Skirt in a Sexual Assault Case

In this sexual assault case, the standard preliminary semen examinations could not confirm physically or biochemically whether the accused's semen had stained the victim's skirt because the skirt had been dry‐cleaned for stain removal and had been worn for more than a year after the assault. Fortunately, however, a photograph taken just after the assault was found in the court records that showed white stains on the checkered skirt. The locations of the stains were estimated based on the checkered pattern of the fabric, and microscopic examination using Baecchi's staining revealed the presence of spermatozoa. Further analysis indicated the male DNA profile generated from the sperm cells was consistent with the suspect's DNA using three multiplex STR typing systems for a total of 21 autosomal and 17 Y chromosomal short tandem repeats (STRs). Ultimately, the result of the DNA profile played a very useful role as additional evidence.