Results of the 2003–2004 GEP-ISFG collaborative study on mitochondrial DNA: Focus on the mtDNA profile of a mixed semen-saliva stain

We report here a review of the seventh mitochondrial DNA (mtDNA) exercise undertaken by the Spanish and Portuguese working group (GEP) of the International Society for Forensic Genetics (ISFG) corresponding to the period 2003–2004. Five reference bloodstains from five donors (M1–M5), a mixed stain of saliva and semen (M6), and a hair sample (M7) were submitted to each participating laboratory for nuclear DNA (nDNA; autosomal STR and Y-STR) and mtDNA analysis. Laboratories were asked to investigate the contributors of samples M6 and M7 among the reference donors (M1–M5). A total of 34 laboratories reported total or partial mtDNA sequence data from both, the reference bloodstains (M1–M5) and the hair sample (M7) concluding a match between mtDNA profiles of M5 and M7. Autosomal STR and Y-STR profiling was the preferred strategy to investigate the contributors of the semen/saliva mixture (M6). Nuclear DNA profiles were consistent with a mixture of saliva from the donor (female) of M4 and semen from donor M5, being the semen (XY) profile the dominant component of the mixture. Strikingly, and in contradiction to the nuclear DNA analysis, mtDNA sequencing results yield a more simple result: only the saliva contribution (M4) was detected, either after preferential lysis or after complete DNA digestion. Some labs provided with several explanations for this finding and carried out additional experiments to explain this apparent contradictory result. The results pointed to the existence of different relative amounts of nuclear and mtDNAs in saliva and semen. We conclude that this circumstance could strongly influence the interpretation of the mtDNA evidence in unbalanced mixtures and in consequence lead to false exclusions. During the GEP-ISFG annual conference a validation study was planned to progress in the interpretation of mtDNA from different mixtures.     

Results of the 2003-2004 GEP-ISFG collaborative study on mitochondrial DNA: focus on the mtDNA profile of a mixed semen-saliva stain

We report here a review of the seventh mitochondrial DNA (mtDNA) exercise undertaken by the Spanish and Portuguese working group (GEP) of the International Society for Forensic Genetics (ISFG) corresponding to the period 2003-2004. Five reference bloodstains from five donors (M1-M5), a mixed stain of saliva and semen (M6), and a hair sample (M7) were submitted to each participating laboratory for nuclear DNA (nDNA; autosomal STR and Y-STR) and mtDNA analysis. Laboratories were asked to investigate the contributors of samples M6 and M7 among the reference donors (M1-M5). A total of 34 laboratories reported total or partial mtDNA sequence data from both, the reference bloodstains (M1-M5) and the hair sample (M7) concluding a match between mtDNA profiles of M5 and M7. Autosomal STR and Y-STR profiling was the preferred strategy to investigate the contributors of the semen/saliva mixture (M6). Nuclear DNA profiles were consistent with a mixture of saliva from the donor (female) of M4 and semen from donor M5, being the semen (XY) profile the dominant component of the mixture. Strikingly, and in contradiction to the nuclear DNA analysis, mtDNA sequencing results yield a more simple result: only the saliva contribution (M4) was detected, either after preferential lysis or after complete DNA digestion. Some labs provided with several explanations for this finding and carried out additional experiments to explain this apparent contradictory result. The results pointed to the existence of different relative amounts of nuclear and mtDNAs in saliva and semen. We conclude that this circumstance could strongly influence the interpretation of the mtDNA evidence in unbalanced mixtures and in consequence lead to false exclusions. During the GEP-ISFG annual conference a validation study was planned to progress in the interpretation of mtDNA from different mixtures.     

Assessment of body fluid identification and DNA profiling after exposure to tropical weather conditions

Despite current advances in body fluid identification, there are few studies evaluating the effect of environmental conditions. The present work assessed the detection of body fluids, blood, semen, and saliva, through lateral flow immunochromatographic (LFI) tests, exposed to tropical weather conditions over time, also evaluating the possibility of obtaining STR (short tandem repeat) profiles and identifying mitochondrial DNA (mtDNA) polymorphisms. Blood, semen, saliva samples, and mixtures of these fluids were deposited on polyester clothes and exposed to open-air tropical weather conditions for 1 month. The test versions from LFI (SERATEC®, Germany) Lab and crime scene (CS) used for the detection – one per each body fluid type – demonstrated that it is possible to identify body fluids and their mixtures up to 14 days after deposition. At 30 days, blood and semen were detected but not saliva. Full STR profiles were obtained from 14-day-old blood samples, and partial profiles were obtained from the remaining samples. It was possible to sequence mtDNA in the samples previously analyzed for STR profiling, and haplogroups could be assigned. In conclusion, this study demonstrated for the first time the possibility of body fluid identification and DNA profiling after exposure to tropical weather conditions for 1 month and also demonstrated the value of mtDNA analysis for compromised biological evidence.     

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.     

Screening Biological Stains with qPCR versus Lateral Flow Immunochromatographic Test Strips: A Quantitative Comparison using Analytical Figures of Merit

Biological fluid identification is an important facet of evidence examination in forensic laboratories worldwide. While identifying bodily fluids may provide insight into which downstream DNA methods to employ, these screening techniques consume a vital portion of the available evidence, are usually qualitative, and rely on visual interpretation. In contrast, qPCR yields information regarding the amount and proportion of amplifiable genetic material. In this study, dilution series of either semen or male saliva were prepared in either buffer or female blood. The samples were subjected to both lateral flow immunochromatographic test strips and qPCR analysis. Analytical figures of merit—including sensitivity, minimum distinguishable signal (MDS) and limit of detection (LOD)—were calculated and compared between methods. By applying the theory of the propagation of random errors, LODs were determined to be 0.05 μL of saliva for the RSID? Saliva cards, 0.03 μL of saliva for Quantifiler^® Duo, and 0.001 μL of semen for Quantifiler^® Duo. In conclusion, quantitative PCR was deemed a viable and effective screening method for subsequent DNA profiling due to its stability in different matrices, sensitivity, and low limits of detection.     

Personal identification using Y-chromosomal short tandem repeats from bodily fluids mixed with semen

The male-specific, human Y-chromosomal short tandem repeats (Y-STRs) are very useful in forensic analysis. The authors report a sexual crime case in which the direct Y-STR haplotype analysis of several mixtures of various bodily fluids including semen was very effective for identifying the perpetrator of the crime. The typing of three Y-STRs (DYS19, DYS389II, and DYS390) could be detected from the mixed DNA of sperm and female cells in the victim's vagina, vaginal orifice, and anus. These haplotypes originated from one man and matched those of the suspect. Accordingly, the combination of direct extraction of DNA and Y-STR haplotype analysis is considered to be very useful for mixtures of bodily fluids, including semen or other male cells.     

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.     

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。     

Detection of microRNAs in DNA Extractions for Forensic Biological Source Identification

Molecular‐based approaches for biological source identification are of great interest in the forensic community because of a lack of sensitivity and specificity in current methods. MicroRNAs (miRNAs) have been considered due to their robust nature and tissue specificity; however, analysis requires a separate RNA extraction, requiring an additional step in the forensic analysis workflow. The purpose of this study was to evaluate miRNA detection in blood, semen, and saliva using DNA extraction methods commonly utilized for forensic casework. RT‐qPCR analysis revealed that the tested miRNAs were consistently detectable across most tested DNA extraction methods, but detection was significantly reduced compared to RNA extracts in some biological fluids. DNase treatment was not necessary to achieve miRNA‐specific results. A previously developed miRNA panel for forensic body fluid identification was evaluated using DNA extracts, and largely demonstrated concordance with results from samples deriving from RNA extracts of semen, blood, and saliva.     

D20S161和D8S384两个基因座在法医学中的应用

评估D2 0S16 1和D8S384两个基因座在法医学中的应用价值。用自制的D2 0S16 1和D8S384两个DNA分型试剂盒 ,对人血、人精液、人唾液、动物血、人血与动物血的混合检材和人血痕、人精液斑、人唾液斑、动物血痕、人血与动物血的混合斑痕检材 ,以及陈旧血痕检材进行检测分型 ,并用这两个基因座PCR引物序列与DNA数据库进行联网对比分析。自制的D2 0S16 1和D8S384两个DNA分型试剂盒能对人血、人精液、人唾液、人血与动物血的混合检材分型 ,而动物血没有PCR产物 ;自制的D2 0S16 1和D8S384两个DNA分型试剂盒能对人血痕、人精斑、人唾液斑和人血与动物血的混合斑痕检材正确分型 ,而动物血痕没有PCR产物 ;斑痕检材分型结果与对应体液检材分型结果无差异 ;5 0份陈旧血痕检材全部获得阳性分型结果。DNA数据库联网比较提示 ,D2 0S16 1和D8S384基因座引物除了能与各自的模板序列发生特异性扩增外 ,理论上不能与DNA数据库中 6 0 6 36 4种已知序列产生PCR产物。D2 0S16 1和D8S384两个基因座具有高度的种属特异性 ,抗污染能力强 ,不易受降解的影响 ,是解决法医现场生物检材个人识别和亲子鉴定的理想手段     

Isolation of deoxyribonucleic acid (DNA) from saliva and forensic science samples containing saliva.

Saliva and saliva-stained materials were examined as potential sources of deoxyribonucleic acid (DNA) for DNA analysis and identity testing. In this paper, the authors demonstrate that DNA was isolated and DNA banding patterns suitable for DNA typing were obtained from fresh saliva and various saliva-stained materials, such as envelopes, buccal swabs, gags, and cigarettes. Furthermore, DNA and DNA banding patterns were obtained from actual forensic evidentiary samples containing mixed saliva/semen stains. The DNA banding patterns obtained from saliva or saliva-stained material were indistinguishable from the patterns obtained from blood or hair from the same individual. Intact DNA was readily isolated and DNA banding patterns were obtained from saliva stored at -20 degrees C and dried saliva stains stored under varying conditions. We conclude that saliva and saliva-stained material can be good sources of DNA for analysis and for DNA typing in certain forensic settings.     

Advanced statistical analysis of Raman spectroscopic data for the identification of body fluid traces: Semen and blood mixtures

Conventional confirmatory biochemical tests used in the forensic analysis of body fluid traces found at a crime scene are destructive and not universal. Recently, we reported on the application of near-infrared (NIR) Raman microspectroscopy for non-destructive confirmatory identification of pure blood, saliva, semen, vaginal fluid and sweat. Here we expand the method to include dry mixtures of semen and blood. A classification algorithm was developed for differentiating pure body fluids and their mixtures. The classification methodology is based on an effective combination of Support Vector Machine (SVM) regression (data selection) and SVM Discriminant Analysis of preprocessed experimental Raman spectra collected using an automatic mapping of the sample. This extensive cross-validation of the obtained results demonstrated that the detection limit of the minor contributor is as low as a few percent. The developed methodology can be further expanded to any binary mixture of complex solutions, including but not limited to mixtures of other body fluids.     

混合斑的DNA分型解析

综述了常染色体STR、Amelogenin、Y染色体STR、线粒体DNA和单核苷酸多态性等DNA检测方法在解释混合斑检验结果应用中的进展。对混合斑的统计学方法也作了总结。     

Detection and quantification of the age-related point mutation A189G in the human mitochondrial DNA

Mutation analysis in the mitochondrial DNA (mtDNA) control region is widely used in population genetic studies as well as in forensic medicine. Among the difficulties linked to the mtDNA analysis, one can find the detection of heteroplasmy, which can be inherited or somatic. Recently, age-related point mutation A189G was described in mtDNA and shown to accumulate with age in muscles. We carried out the detection of this 189 heteroplasmic point mutation using three technologies: automated DNA sequencing, Southern blot hybridization using a digoxigenin-labeled oligonucleotide probe, and peptide nucleic acid (PNA)/real-time PCR combined method on different biological samples. Our results give additional information on the increase in mutation frequency with age in muscle tissue and revealed that the PNA/real-time PCR is a largely more sensitive method than DNA sequencing for heteroplasmy detection. These investigations could be of interest in the detection and interpretation of mtDNA heteroplasmy in anthropological and forensic studies.     

Alpha-amylase kinetic test in bodily single and mixed stains

Recently, in Italy, a murder and a putative sexual violence was accomplished on a child. A bodily fluids mixture on the child's underwear between the victim (female) and the suspect (male) was ascertained by short tandem repeat (STR) DNA typing and, due to the absence of seminal fluid, saliva from the suspect and urine from the child was hypothesized. In order to investigate the possibility of specifically and rapidly detecting saliva stains both alone and mixed with other bodily fluids, we used a quantitative spectrophotometric technique, named Amylase test, for the detection of alpha-amylases. We determined alpha-amylase activity and reaction kinetic curves in several samples collected from the child's underwear. In order to confirm our intuition, we first tested saliva, perspiration, and urine, singularly and in mixtures; second, several forensic stains including saliva, perspiration, urine stains, saliva/perspiration, and saliva/urine mixture stains were tested. Evaluating alpha-amylase activity values and time-course curves' behavior of alpha-amylase reactions we were able to recognize successfully, in all cases, the presence of saliva and to distinguish it specifically from other bodily fluids containing alpha-amylase. A further confirmation of our result was provided by STR DNA typing on several areas of the underwear: a clear correlation between alpha-amylases activity and male DNA was detected on all the samples evaluated.