Cluster-Enhanced Nanopore Sensing of Ovarian Cancer Marker Peptides in Urine.

The development of novel methodologies that can detect biomarkers from cancer or other diseases is both a challenge and a need for clinical applications. This partly motivates efforts related to nanopore-based peptide sensing. Recent work has focused on the use of gold nanoparticles for selective detection of cysteine-containing peptides. Specifically, tiopronin-capped gold nanoparticles, trapped in the cis-side of a wild-type α-hemolysin nanopore, provide a suitable anchor for the attachment of cysteine-containing peptides. It was recently shown that the attachment of these peptides onto a nanoparticle yields unique current signatures that can be used to identify the peptide. In this article, we apply this technique to the detection of ovarian cancer marker peptides ranging in length from 8 to 23 amino acid residues. It is found that sequence variability complicates the detection of low-molecular-weight peptides (<10 amino acid residues), but higher-molecular-weight peptides yield complex, high-frequency current fluctuations. These fluctuations are characterized with chi-squared and autocorrelation analyses that yield significantly improved selectivity when compared to traditional open-pore analysis. We demonstrate that the technique is capable of detecting the only two cysteine-containing peptides from LRG-1, an emerging protein biomarker, that are uniquely present in the urine of ovarian cancer patients. We further demonstrate the detection of one of these LRG-1 peptides spiked into a sample of human female urine.

A combined molecular approach utilizing microbial DNA and microRNAs in a qPCR multiplex for the classification of five forensically relevant body fluids.

Body fluid identification is an essential step in the forensic biology workflow that can assist DNA analysts in determining where to collect DNA evidence. Current presumptive tests lack the specificity that molecular techniques can achieve; therefore, molecular methods, including microRNA (miRNA) and microbial signature characterization, have been extensively researched in the forensic community. Limitations of each method suggest combining molecular markers to increase the discrimination efficiency of multiple body fluids from a single assay. While microbial signatures have been successful in identifying fluids with high bacterial abundances, microRNAs have shown promise in fluids with low microbial abundance (blood and semen). This project synergized the benefits of microRNAs and microbial DNA to identify multiple body fluids using DNA extracts. A reverse transcription (RT)-qPCR duplex targeting miR-891a and let-7g was validated, and miR-891a differential expression was significantly different between blood and semen. The miRNA duplex was incorporated into a previously reported qPCR multiplex targeting 16S rRNA genes of Lactobacillus crispatus, Bacteroides uniformis, and Streptococcus salivarius to presumptively identify vaginal/menstrual secretions, feces, and saliva, respectively. The combined classification regression tree model resulted in the presumptive classification of five body fluids with 94.6% overall accuracy, now including blood and semen identification. These results provide proof of concept that microRNAs and microbial DNA can classify multiple body fluids simultaneously at the quantification step of the current forensic DNA workflow.

A quantifiler™ trio-based HRM screening assay for the accurate prediction of single source versus mixed biological samples.

At present, the forensic DNA workflow is not capable of providing information about the contributor status (single source vs. multiple contributors) of evidentiary samples prior to end-point analysis. This exacerbates the challenges inherent to mixtures and low-template DNA samples. If additional sample information could be provided earlier in the workflow, protocols could be implemented to mitigate these challenges. An integrated Quantiplex®- high resolution melt (HRM) assay was shown to be effective in distinguishing between single source and mixture DNA samples; however, integration of the HRM assay into a more commonly used chemistry would be beneficial to the practitioner community. Thus, the assay was redesigned as an integrated Quantifiler™ Trio-HRM assay, which included the identification of a new DNA-binding dye, an increased reaction volume, and the establishment of new data analysis and standard curve metrics for all targets. This redesigned assay produced quantification values and qualitative values that were comparable to those produced when the same samples were tested using the standard Quantifiler™ Trio chemistry and settings. Further, STR profiles generated with quantification values produced from the integrated Quantifiler™ Trio-HRM assay and standard Quantifiler™ Trio chemistry were complete and fully concordant. Most importantly, the integrated Quantifiler™ Trio-HRM assay was able to accurately predict whether a sample was single source or a mixture 79.2% of the time, demonstrating the potential of this approach. With the incorporation of an expanded training set for prediction modeling, and completion of critical developmental validation studies, this assay could prove useful to the forensic DNA practitioner community.

Design and optimization of a 16S microbial qPCR multiplex for the presumptive identification of feces, saliva, vaginal and menstrual secretions.

Molecular methods for body fluid identification have been extensively researched in the forensic community over the last decade, mostly focusing on RNA-based methods. Microbial DNA analysis has long been used for forensic applications, such as postmortem interval estimations, but only recently has it been applied to body fluid identification. High-throughput sequencing of the 16S ribosomal RNA gene by previous research groups revealed that microbial signatures and abundances vary across human body fluids at the genus and/or species taxonomic level. Since quantitative PCR is still the current technique used in forensic DNA analysis, the purpose of this study was to design a qPCR multiplex targeting the 16S gene of Bacteroides uniformis, Streptococcus salivarius, and Lactobacillus crispatus that can distinguish between feces, saliva, and vaginal/menstrual secretions, respectively. Primers and probes were designed at the species level because these bacteria are highly abundant within their respective fluid. The validated 16S triplex was evaluated in DNA extracts from thirty donors of each body fluid. A classification regression tree model resulted in 96.5% classification accuracy of the population data, which demonstrates the ability of this 16S triplex to presumptively identify these fluids with high confidence at the quantification step of the forensic workflow using minimal input volume of DNA extracted from evidentiary samples.

Comparison of DNA typing success in compromised blood and touch samples based on sampling swab composition.

Sample collection at the crime scene can introduce variations in DNA recovery based upon the substrate from which a sample is collected, the material of the collection device used, or the storage conditions after collection. There are many factors during this process that can degrade the sample during drying and storage, and before DNA extraction can be performed. The purpose of this study was to evaluate and compare the performance of standard cotton swab collection with the Bode BioSafe® swab, which includes both a desiccant at the swab head and proprietary compounds to prevent degradation of the sample during sample collection and preservation. Blood and touch DNA samples were collected from porous and nonporous substrates and stored at elevated temperatures to simulate accelerated time. DNA quantification and STR profile data were used to assess the performance of the swabs. BioSafe® swab collection resulted in similar DNA yields from blood samples and significantly higher DNA yields from touch samples when compared to collection with cotton swabs. BioSafe® swabs also resulted in higher DNA integrity during long-term storage, increased STR profile success and improved retention of low-level contributor alleles.

The effects of dithiothreitol (DTT) on fluorescent qPCR dyes.

DNA extractions of semen samples commonly utilize dithiothreitol (DTT) to reduce and disrupt disulfide bonds. Although traditional extraction techniques remove DTT before downstream analyses, the forensic DNA community has recently explored Y-screening, direct amplification, and direct cell lysis assays that omit purification but employ reducing agents to lyse spermatozoa. This study examined the impact of residual DTT on downstream processes involving fluorescent dyes. Quantification using Investigator® Quantiplex HYres revealed a significant increase in the male DNA yield (p = 0.00056) and a >150,000,000-fold increase in the male:human DNA ratio when DTT remained in extracts versus when it was filtered out using a traditional purification method. When DTT was present with Quantifiler™ Trio, the true mean DNA yield for the large autosomal target significantly increased (p = 0.038) and the average reported DNA yields increased 1.1-fold, >9.5-fold, and 1.3-fold for the small autosomal, large autosomal, and male targets, respectively. DTT-spiked DNA standards from both kits were impacted similarly to samples with residual DTT, demonstrating that observed effects were related to DTT and not the extraction method. This study corroborates other reports that DTT adversely affects multiple dyes (e.g., Cy5, Quasar 670, SYBR Green I, TMR, and Mustang Purple® ). Overall, DTT causes inaccurate quantities and, consequently, inaccurate calculated male:female ratios when used in conjunction with these kits. Thus, implementation of newer direct-to-PCR assays incorporating DTT should either be avoided or used only with carefully evaluated, compatible dyes.

microRNA Detection in Blood, Urine, Semen, and Saliva Stains After Compromising Treatments.

Evaluation of microRNA (miRNA) expression as a potential method for forensic body fluid identification has been the subject of investigation over the past several years. Because of their size and encapsulation within proteins and lipids, miRNAs are inherently less susceptible to degradation than other RNAs. In this work, blood, urine, semen, and saliva were exposed to environmental and chemical conditions mimicking sample compromise at the crime scene. For many treated samples, including 100% of blood samples, miRNAs remained detectable, comparable to the untreated control. Sample degradation varied by body fluid and treatment, with blood remarkably resistant, while semen and saliva are more susceptible to environmental insult. Body fluid identification using relative miRNA expression of blood and semen of the exposed samples was 100% and 94%, respectively. Given the overall robust results herein, the case is strengthened for the use of miRNAs as a molecular method for body fluid identification.

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.

Optical tweezers as an effective tool for spermatozoa isolation from mixed forensic samples.

A single focus optical tweezer is formed when a laser beam is launched through a high numerical aperture immersion objective. This objective focuses the beam down to a diffraction-limited spot, which creates an optical trap where cells suspended in aqueous solutions can be held fixed. Spermatozoa, an often probative cell type in forensic investigations, can be captured inside this optical trap and dragged one by one across millimeter-length distances in order to create a cluster of cells which can be subsequently drawn up into a capillary for collection. Sperm cells are then ejected onto a sterile cover slip, counted, and transferred to a tube for DNA analysis workflow. The objective of this research was to optimize sperm cell collection for maximum DNA yield, and to determine the number of trapped sperm cells necessary to produce a full STR profile. A varying number of sperm cells from both a single-source semen sample and a mock sexual assault sample were isolated utilizing optical tweezers and processed using conventional STR analysis methods. Results demonstrated that approximately 50 trapped spermatozoa were required to obtain a consistently full DNA profile. A complete, single-source DNA profile was also achieved by isolating sperm cells via optical trapping from a mixture of sperm and vaginal epithelial cells. Based on these results, optical tweezers are a viable option for forensic applications such as separation of mixed populations of cells in forensic evidence.

An Optimized Centrifugal Method for Separation of Semen from Superabsorbent Polymers for Forensic Analysis.

Connection of a perpetrator to a sexual assault is best performed through the confirmed presence of semen, thereby proving sexual contact. Evidentiary items can include sanitary napkins or diapers containing superabsorbent polymers (SAPs), complicating spermatozoa visualization and DNA analysis. In this report, we evaluated the impact of SAPS on the current forensic DNA workflow, developing an efficient centrifugal protocol for separating spermatozoa from SAP material. The optimized filtration method was compared to common practices of excising the top layer only, resulting in significantly higher sperm yields when a core sample of the substrate was taken. Direct isolation of the SAP-containing materials without filtering resulted in 20% sample failure; additionally, SAP material was observed in the final eluted DNA samples, causing physical interference. Thus, use of the described centrifugal-filtering method is a simple preliminary step that improves spermatozoa visualization and enables more consistent DNA yields, while also avoiding SAP interference.

Infrared Laser Heating Applied to Nanopore Sensing for DNA Duplex Analysis.

Temperature studies coupled with resistive-pulse nanopore sensing enable the quantification of a variety of important thermodynamic properties at the single-molecule limit. Previous demonstrations of nanopore sensing with temperature control have utilized bulk chamber heating methodologies. This approach makes it difficult to rapidly change temperatures and enable optical access for other analytical techniques (i.e., single-molecule fluorescence). To address these issues, researchers have explored laser-based methodologies through either direct infrared (IR) absorption or plasmonic assisted heating. In this paper, we demonstrate the use of IR-based direct absorption heating with the DNA sensing capabilities of a biological nanopore. The IR heating enables rapid changes of the temperature in and around an α-hemolysin pore, and we use this to explore melting properties for short (≤50 bp) double-stranded DNA homopolymers. We also demonstrate that the IR heating enables one to measure the percentage of different-sized DNA molecules in a binary mixture.

Dual Action of miR-125b As a Tumor Suppressor and OncomiR-22 Promotes Prostate Cancer Tumorigenesis.

MicroRNAs (miRs) are a novel class of small RNA molecules, the dysregulation of which can contribute to cancer. A combinatorial approach was used to identify miRs that promote prostate cancer progression in a unique set of prostate cancer cell lines, which originate from the parental p69 cell line and extend to a highly tumorigenic/metastatic M12 subline. Together, these cell lines are thought to mimic prostate cancer progression in vivo. Previous network analysis and miR arrays suggested that the loss of hsa-miR-125b together with the overexpression of hsa-miR-22 could contribute to prostate tumorigenesis. The dysregulation of these two miRs was confirmed in human prostate tumor samples as compared to adjacent benign glandular epithelium collected through laser capture microdissection from radical prostatectomies. In fact, alterations in hsa-miR-125b expression appeared to be an early event in tumorigenesis. Reverse phase microarray proteomic analysis revealed ErbB2/3 and downstream members of the PI3K/AKT and MAPK/ERK pathways as well as PTEN to be protein targets differentially expressed in the M12 tumor cell compared to its parental p69 cell. Relevant luciferase+3'-UTR expression studies confirmed a direct interaction between hsa-miR-125b and ErbB2 and between hsa-miR-22 and PTEN. Restoration of hsa-miR-125b or inhibition of hsa-miR-22 expression via an antagomiR resulted in an alteration of M12 tumor cell behavior in vitro. Thus, the dual action of hsa-miR-125b as a tumor suppressor and hsa-miR-22 as an oncomiR contributed to prostate tumorigenesis by modulations in PI3K/AKT and MAPK/ERK signaling pathways, key pathways known to influence prostate cancer progression.

Separation of uncompromised whole blood mixtures for single source STR profiling using fluorescently-labeled human leukocyte antigen (HLA) probes and fluorescence activated cell sorting (FACS).

Analysis of biological mixtures is a significant problem for forensic laboratories, particularly when the mixture contains only one cell type. Contributions from multiple individuals to biologic evidence can complicate DNA profile interpretation and often lead to a reduction in the probative value of DNA evidence or worse, its total loss. To address this, we have utilized an analytical technique that exploits the intrinsic immunological variation among individuals to physically separate cells from different sources in a mixture prior to DNA profiling. Specifically, we applied a fluorescently labeled antibody probe to selectively bind to one contributor in a mixture through allele-specific interactions with human leukocyte antigen (HLA) proteins that are expressed on the surfaces of most nucleated cells. Once the contributor's cells were bound to the probe, they were isolated from the mixture using fluorescence activated cell sorting (FACS)-a high throughput technique for separating cell populations based on their optical properties-and then subjected to STR analysis. We tested this approach on two-person and four-person whole blood mixtures where one contributor possessed an HLA allele (A*02) that was not shared by other contributors to the mixture. Results showed that hybridization of the mixture with a fluorescently-labeled antibody probe complimentary to the A*02 allele's protein product created a cell population with a distinct optical profile that could be easily differentiated from other cells in the mixture. After sorting the cells with FACS, genetic analysis showed that the STR profile of this cell population was consistent with that of the contributor who possessed the A*02 allele. Minor peaks from the A*02 negative contributor(s) were observed but could be easily distinguished from the profile generated from A*02 positive cells. Overall, this indicates that HLA antibody probes coupled to FACS may be an effective approach for generating STR profiles of individual contributors from forensic mixtures.