Verification of protein biomarker specificity for the identification of biological stains by quadrupole time‐of‐flight mass spectrometry

Advances in proteomics technology over the past decade offer forensic serologists a greatly improved opportunity to accurately characterize the tissue source from which a DNA profile has been developed. Such information can provide critical context to evidence and can help to prioritize downstream DNA analyses. Previous proteome studies compiled panels of “candidate biomarkers” specific to each of five body fluids (i.e ., peripheral blood, vaginal/menstrual fluid, seminal fluid, urine, and saliva). Here, a multiplex quadrupole time‐of‐flight mass spectrometry assay has been developed in order to verify the tissue/body fluid specificity the 23 protein biomarkers that comprise these panels and the consistency with which they can be detected across a sample population of 50 humans. Single‐source samples of these human body fluids were accurately identified by the detection of one or more high‐specificity biomarkers. Recovery of body fluid samples from a variety of substrates did not impede accurate characterization and, of the potential inhibitors assayed, only chewing tobacco juice appeared to preclude the identification of a target body fluid. Using a series of 2‐component mixtures of human body fluids, the multiplex assay accurately identified both components in a single‐pass. Only in the case of saliva and peripheral blood did matrix effects appear to impede the detection of salivary proteins.     

High-resolution melt analysis for the detection of skin/sweat via DNA methylation

The determination of tissue type is important when reconstructing a crime scene as skin cells may indicate innocent contact, whereas other types of cells, such as blood and semen, may indicate foul play. Up to now, there has been no specific DNA methylation-based marker to distinguish skin cell DNA from other body fluids. The goal of this study is to develop a DNA methylation-based assay to detect and identify skin cells collected at forensic crime scenes for use in DNA typing. For this reason, we have utilized a DNA methylation chip array-based genome-wide association study to identify skin-specific DNA methylation markers. DNA obtained from skin along with other body fluids, such as semen, saliva, blood, and vaginal epithelia, were tested using five genes that were identified as sites for potential new epigenetic skin markers. Samples were collected, bisulfite converted, and subjected to real-time polymerase chain reaction (PCR) with high-resolution melt analysis. In our studies, when using WDR11, PON2, and NHSL1 assays with bisulfite-modified PCR, skin/sweat amplicons melted at lower temperatures compared to blood, saliva, semen, and vaginal epithelia. One-way analysis of variance demonstrates that these three skin/sweat markers are significantly different when compared with other body fluids (p < 0.05). These results demonstrate that high-resolution melt analysis is a promising technology to detect and identify skin/sweat DNA from other body fluids.     

A new set of DIP‐SNP markers for detection of unbalanced and degraded DNA mixtures

Unbalanced and degraded mixtures (UDM) are frequently encountered during forensic DNA analysis. For example, forensic DNA units regularly encounter DNA mixture signal where the DNA signal from the alleged offender is masked or swamped by high quantities of DNA from the victim. Our previous data presented a new kind of DNA markers that composed of a deletion/insertion polymorphism (DIP) and a SNP and we termed this new kind of microhaplotypes DIP‐SNP (combination of DIP and SNP). Since such markers could be designed short enough for degraded DNA amplification, we hypothesized that DIP‐SNP markers are applicable for typing of UDM. In this study, we developed a new set of DIP‐SNPs with short amplicons which were complement to our prior developed system. The multiplex PCR and SNaPshot assay were established for 20 DIP‐SNPs in a Chinese Han population. The DIP‐SNPs were capable of detecting the minor contributor's allele in home‐made DNA mixture with sensitivities from 1:100 to 1:1000 with a total of 1 –10 ng input DNA. Moreover, this system successfully typed the degraded DNA whether it came from the single source or mixture samples. In Chinese population, the system showed an average informative value of 0.293 and combined informative value of 0.998363862. Our results demonstrated that DIP‐SNPs may serve as a valuable tool in detection of UDM in forensic medicine.     

Expression profile analysis and stability evaluation of 18 small RNAs in the Chinese Han population

In recent years, differentially expressed small RNAs have been widely used to identify the compositions of forensically relevant biological samples, and a vast number of such RNA candidates have been proposed. Nevertheless, when assessing the expression levels of target small RNAs using relative quantitative analysis methods, credible internal controls are usually required for reliable data normalization. Therefore, the identification of optimal reference genes is an important task. In this study, the expression profile of 18 small RNA reference genes was characterized in the Chinese Han population using TaqMan real-time quantitative PCR. Systematic evaluations of these candidate genes were performed based on their expression levels and stability in several common types of body fluids (i.e., venous blood, menstrual blood, saliva, semen, and vaginal secretions). Analysis results from the ΔCq method, BestKeeper, NormFinder, and geNorm were integrated by RefFinder for ranking and comparing the candidates in each type of body fluid. Among all the candidates, miR-191 was identified as the most suitable reference gene because it had a favorable ranking value in all tested samples. In addition, miR-423, miR-93, miR-484, and let-7i were also shown to be applicable reference genes. Overall, this study provides detailed assessment results of these candidate genes in different body fluids; thus, it could be used as a guide for the selection of reference genes according to their performance in the sample of choice.     

The effect of infertile semen on the mRNA-based body fluid identification

In the past decade, mRNA markers have been well demonstrated as promising molecular markers in forensic body fluid identification (BFI), and successfully used in wide applications. Several studies have assessed the performance of semen-specific mRNA markers in distinguishing semen from other common body fluids at the crime scene. Infertility has been reported as a global health problem that is affecting approximately 15% of couples worldwide. Therefore, it is important for forensic researchers to consider the impact of infertility on semen identification. This study aimed to explore the effect of semen from infertile men (hereinafter “infertile semen”) on BFI and to identify semen-specific mRNAs that can efficiently and accurately distinguish normal and infertile semen samples from other body fluids. Results showed that the selected five mRNAs (KLK3, TGM4, SEMG1, PRM1, and PRM2) performed a significantly high semen specificity in normal semen. Moreover, KLK3 was slightly influenced by infertile semen samples with over 98% positive results in all semen samples. The accuracy to predict normal semen reached up to 96.6% using the discrimination function Y1 with KLK3 and PRM1. However, when the infertile semen samples were included in discrimination function (function Y2 with KLK3), the accuracy rate of semen identification (including the normal and infertile semen) was down to 89.5%. Besides, the sensitivity of multiplex assay could reach down to 50pg. Our results suggest that it is important to consider the presence of infertile semen when using mRNAs to identify semen samples, which would have a far-reaching impact in forensic identification.     

Isoelectric focusing of group specific component in semen and vaginal stains on ultrathin immobilized pH gradient gels

Subtyping of the group specific component in secretions of semen and vaginal fluids is impossible with conventional detection systems. By means of a highly sensitive alkaline-phosphatase secondary antibody system the group specific component can reliably be detected in semen stains. Results with vaginal swabs were inconsistent and in saliva stains Gc activity could not be detected.     

Extended specificity studies of mRNA assays used to infer human organ tissues and body fluids

Messenger RNA (mRNA) profiling is a technique increasingly applied for the forensic identification of body fluids and skin. More recently, an mRNA‐based organ typing assay was developed which allows for the inference of brain, lung, liver, skeletal muscle, heart, kidney, and skin tissue. When applying this organ typing system in forensic casework for the presence of animal, rather than human, tissue is an alternative scenario to be proposed, for instance that bullets carry cell material from a hunting event. Even though mRNA profiling systems are commonly in silico designed to be primate specific, physical testing against other animal species is generally limited. In this study, human specificity of the organ tissue inferring system was assessed against organ tissue RNAs of various animals. Results confirm human specificity of the system, especially when utilizing interpretation rules considering multiple markers per cell type. Besides, we cross‐tested our organ and body fluid mRNA assays against the target types covered by the other assay. Marker expression in the nontarget organ tissues and body fluids was observed to a limited extent, which emphasizes the importance of involving the case‐specific context of the forensic samples in deciding which mRNA profiling assay to use and when for interpreting results.     

Subtyping of group specific component (GC) in human semen, blood and vaginal fluid by isoelectric focusing in immobilized pH gradients

The group specific component (GC) is stable and well suited for forensic casework. Isoelectric focusing of common GC variants from semen, seminal fluid, vaginal fluid and semen stains, on Immobiline DryPlates, pH 4.5-5.4, is of practical value in criminal investigations of sexual deliquencies. GC is present in normospermia and azoospermia seminal fluids and found in about 20% of the vaginal secretions. The GC patterns observed were similar and in accordance with the bands of the individual GC type in plasma/serum.     

连接反应介导的等位基因特异性扩增-微流控芯片电泳法同时检测多个SNP位点

以CYP2D6基因中的6个SNP位点为测定对象, 开展多个SNP位点同时测定的方法学研究.     

Application of NMR metabolomics to search for human disease biomarkers

Since antiquity, humans have used body fluids like saliva, urine and sweat for the diagnosis of diseases. The amount, color and smell of body fluids are still used in many traditional medical practices to evaluate an illness and make a diagnosis. The development and application of analytical methods for the detailed analysis of body fluids has led to the discovery of numerous disease biomarkers. Recently, mass spectrometry (MS), nuclear magnetic resonance spectroscopy (NMR), and multivariate statistical techniques have been incorporated into a multidisciplinary approach to profile changes in small molecules associated with the onset and progression of human diseases. The goal of these efforts is to identify metabolites that are uniquely correlated with a specific human disease in order to accurately diagnose and treat the malady. In this review we will discuss recent developments in sample preparation, experimental techniques, the identification and quantification of metabolites, and the chemometric tools used to search for biomarkers of human diseases using NMR.     

High speed single nucleotide polymorphism typing of a hereditary haemochromatosis mutation with capillary array electrophoresis microplates

A single nucleotide polymorphism (SNP) typing assay is developed and evaluated on a microfabricated capillary array electrophoresis system. Using fluorescently labeled allele-specific primers, the S65C (193A-->T) substitution associated with hereditary haemochromatosis in the HFE gene is genotyped. The covalently labeled polymerase chain reaction (PCR) products are separated on a microfabricated radial capillary array electrophoresis microplate using nondenaturing gel media in under two minutes. Detection is accomplished with a laser-excited rotary confocal scanner. The Rox-labeled A-allele specific amplicon (211 bp) is differentiated from the R110-labeled T-allele specific amplicon (201 bp) by both size and color. This study demonstrates the feasibility of using allele-specific PCR with covalently labeled primers for high speed fluorescent SNP typing on microfabricated radial capillary array electrophoresis microplates.     

SNPlexing the human Y-chromosome: a single-assay system for major haplogroup screening

SNPs are one of the main sources of DNA variation among humans. Their unique properties make them useful polymorphic markers for a wide range of fields, such as medicine, forensics, and population genetics. Although several high-throughput techniques have been (and are being) developed for the vast typing of SNPs in the medical context, population genetic studies involve the typing of few and select SNPs for targeted research. This results in SNPs having to be typed in multiple reactions, consuming large amounts of time and of DNA. In order to improve the current situation in the area of human Y-chromosome diversity studies, we decided to employ a system based on a multiplex oligo ligation assay/PCR (OLA/PCR) followed by CE to create a Y multiplex capable of distinguishing, in a single reaction, all the major haplogroups and as many subhaplogroups on the Y-chromosome phylogeny as possible. Our efforts resulted in the creation of a robust and accurate 35plex (35 SNPs in a single reaction) that when tested on 165 human DNA samples from different geographic areas, proved capable of assigning samples to their corresponding haplogroup.     

Detection of bacteria from biological mixtures using immunomagnetic separation combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A rapid method for identifying specific bacteria from complex biological mixtures using immunomagnetic separation coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been developed. The technique employs commercially available magnetic beads coated with polycolonal antibodies raised against specific bacteria and whole cell analysis by MALDI-MS. A suspension of a bacterial mixture is mixed with the immunomagnetic beads specific for the target microorganism. After a short incubation period (20 mins) the bacteria captured by the beads are washed, resuspended in deionized H(2)O and directly applied onto a MALDI probe. Liquid suspensions containing bacterial mixtures can be screened within 1 h total analysis time. Positive tests result in the production of a fingerprint mass spectrum primarily consisting of protein biomarkers characteristic of the targeted microorganism. Using this procedure, Salmonella choleraesuis was isolated and detected from standard bacterial mixtures and spiked samples of river water, human urine, and chicken blood.     

Analysis and interpretation of mixture DNA using AS‐PCR of mtDNA

Semi‐nested PCR with allele‐specific (AS) primers and sequencing of mitochondrial DNA (mtDNA) were performed to analyze and interpret DNA mixtures, especially when biological materials were degraded or contained a limited amount of DNA. SNP‐STR markers were available to identify the minor DNA component using AS‐PCR; moreover, SNPs in mtDNA could be used when the degraded or limited amounts of DNA mixtures were not successful with SNP‐STR markers. Five pairs of allele‐specific primers were designed based on three SNPs (G15043A, T16362C, and T16519C). The sequence of mtDNA control region of minor components was obtained using AS‐PCR and sequencing. Sequences of the amplification fragments were aligned and compared with the sequences of known suspects or databases. When this assay was used with the T16362C and T16519C SNPs, we found it to be highly sensitive for detecting small amounts of DNA (～30 pg) and analyzing DNA mixtures of two contributors, even at an approximately 1‰ ratio of minor and major components. An exception was tests based on the SNP G15043A, which required approximately 300 pg of a 1% DNA mixture. In simulated three contributor DNA mixtures (at rate of 1:1:1), control region fragments from each contributor were detected and interpreted. AS‐PCR combined with semi‐nested PCR was successfully used to identify the mtDNA control region of each contributor, providing biological evidence for excluding suspects in forensic cases, especially when biological materials were degraded or had a limited amount of DNA.     

A data-driven,high-throughput methodology to determine tissue-specific differentially methylated regions able to discriminate body fluids

Tissue-specific differentially methylated regions (tDMRs) are regions of the genome with methylation patterns that modulate gene expression in those tissue types. The detection of tDMRs in forensic evidence can permit the identification of body fluids at trace levels. In this report, we have performed a bioinformatic analysis of an existing array dataset to determine if new tDMRs could be identified for use in body fluid identification from forensic evidence. Once these sites were identified, primers were designed and bisulfite modification was performed. The relative methylation level for each body fluid at a given locus was then determined using qPCR with high-resolution melt analysis (HRM). After screening 127 tDMR's in multiple body fluids, we were able to identify four new markers able to discriminate blood (2 markers), vaginal epithelia (1 marker) and buccal cells (1 marker). One marker for each target body fluid was also tested with pyrosequencing showing results consistent with those obtained by HRM. This work successfully demonstrates the ability of in silico analysis to develop a novel set of tDMRs capable of being differentiated by real time PCR/HRM. The method can rapidly determine the body fluids left at crime scenes, assisting the triers of fact in forensic casework.     

A strategy to identify specific biomarkers related to the effects of a PCDD/F mixture on the immune system of marine mammals

The cell lines chosen have demonstrated a positive response in terms of cell proliferation and associated modifications in proteins content, evaluated through DNA and proteins synthesis, at environmentally relevant dose of dioxins, brought by a typical environmental PCDD/F mixture. The response is time and species dependent. After completion of the identification of proteins affected by the intoxication, we will identify a set of specific proteins whose expression is correlated to the dioxin dose and submit the cell culture to the treatment with a single chemical as well as with mixtures. We hope that this will allow us to construct and validate a set of protein biomarkers of exposure to pollutants that will show a predictive aspect for unknown chemicals. The quantitative analysis of the set of biomarkers can then be a more specific bioassay and an alternative to physico-chemical or other already established bioanalytical methods for screening purposes.     

A SNaPshot assay for genotyping 44 individual identification single nucleotide polymorphisms

Single nucleotide polymorphisms (SNPs), which have relatively low mutation rates and can be genotyped after PCR with shorter amplicons compared with short tandem repeats (STRs), are being considered as potentially useful markers in forensic DNA analysis. Those SNPs with high heterozygosity and low Fst (F-statistics) in human populations are described as individual identification SNPs, which perform the same function as STRs used in forensic routine work. In the present study, we developed a multiplex typing method for analyzing 44 selected individual identification SNPs simultaneously by using multiplex PCR reaction in association with fluorescent labeled single base extension (SBE) technique. PCR primers were designed and the lengths of the amplicons ranged from 69 to 125?bp. The population genetics data of 79 unrelated Chinese individuals for the 44 SNP loci were investigated and a series of experiments were performed to validate the characteristic of the SNP multiplex typing assay, such as sensitivity, species specificity and the performance in paternity testing and analysis of highly degraded samples. The results showed that the 44-SNPs multiplex typing assay could be applied in forensic routine work and provide supplementary data when STRs analysis was partial or failed.     

Genetic and expression analysis of all non‐synonymous single nucleotide polymorphisms in the human deoxyribonuclease I‐like 1 and 2 genes

Members of the human DNase I family, DNase I‐like 1 and 2 (DNases 1L1 and 1L2), with physiological role(s) other than those of DNase I, possess three and one non‐synonymous SNPs in the genes, respectively. However, only limited population data are available, and the effect of these SNPs on the catalytic activity of the enzyme remains unknown. Genotyping of all the non‐synonymous SNPs was performed in three ethnic groups including six different populations using the PCR‐RFLP method newly developed. Asian and African groups including Japanese, Koreans, Ghanaians and Ovambos were typed as a single genotype at each SNP, but polymorphism at only SNP V122I in DNase 1L1 was found in Caucasian groups including Germans and Turks; thus a Caucasian‐specific allele was identified. The DNase 1L1 and 1L2 genes show relatively low genetic diversity with regard to these non‐synonymous SNPs. The level of activity derived from the V122I, Q170H and D227A substituted DNase 1L1 corresponding to SNPs was similar to that of the wild‐type, whereas replacement of the Asp residue at position 197 in the DNase 1L2 protein with Ala, corresponding to SNP D197A, reduced its activity greatly. Thus, SNP V122I in DNase 1L1 exhibiting polymorphism exerts no effect on the catalytic activity, and furthermore SNP D197A in DNase 1L2, affecting its catalytic activity, shows no polymorphism. These findings permit us to postulate that the non‐synonymous SNPs identified in the DNase 1L1 and 1L2 genes may exert no influence on the activity levels of DNases 1L1 and 1L2 in human populations.     

Separation mixed semen of two individuals using magnetic beads coupled ABH blood group antibody

In sexual assault cases, one of the most common samples collected is a mixed semen stain, which is often found on the vagina, female underwear, or bed sheets. However, it is usually difficult to identify the perpetrator based on this sample alone. One technique that has been developed to address this issue is magnetic bead-based separation. This method involves using modified magnetic microspheres to capture and enrich specific target cells, in this case, sperm cells. In this study, we utilized magnetic beads coupled with ABH blood group antibody to isolate sperm cells from an individual of a single ABO blood type. Subsequently, polymerase chain reaction amplification and capillary electrophoresis were employed to perform the genotyping the short tandem repeat (STR) loci. This approach allows for the identification of different individuals in a mixed seminal stain sample from two individuals, by first separating sperm cells based on ABH antigen differences and subsequently utilizing autosomal STR typing on the enriched single blood group cells.     

1H NMR metabolite fingerprinting as a new tool for body fluid identification in forensic science

In this feasibility study, we propose, for the first time, ¹H NMR spectroscopy coupled with mathematical strategies as a valid tool for body fluid (BF) trace identification in forensic science. In order to assess the ability of this approach to identify traces composed either by a single or by two different BFs, samples of blood, urine, saliva, and semen were collected from different donors, and binary mixtures were prepared. ¹H NMR analyses were carried out for all samples. Spectral data of the whole set were firstly submitted to unsupervised principal component analysis (PCA); it showed that samples of the same BF cluster well on the basis of their characterizing molecular components and that mixtures exhibit intermediate characteristics among BF typologies. Furthermore, samples were divided into a training set and a test set. An average NMR spectral profile for each typology of BF was obtained from the training set and validated as representative of each BF class. Finally, a fitting procedure, based on a system of linear equations with the four obtained average spectral profiles, was applied to the test set and the mixture samples; it showed that BFs can be unambiguously identified, even as components of a mixture. The successful use of this mathematical procedure has the advantage, in forensics, of overcoming bias due to the analyst's personal judgment. We therefore propose this combined approach as a valid, fast, and non‐destructive tool for addressing the challenges in the identification of composite traces in forensics. Copyright © 2013 John Wiley & Sons, Ltd.