Saliva identification in forensic samples by automated microextraction and intact mass analysis of statherin.

The enzyme α-amylase has long been a commonly targeted protein in serological tests for saliva. While being especially abundant in saliva, α-amylase is detectable in vaginal secretions, sweat, fecal matter, breast milk and other matrices. As a result, assays for α-amylase only provide a presumptive indication of saliva.

High-throughput seminal fluid identification by automated immunoaffinity mass spectrometry.

The identification of semen during a criminal investigation may be a critical component in the prosecution of a sexual assault. Commonly employed enzymatic and affinity-based methods for detection lack specificity, are time-consuming, and only provide a presumptive indication that semen is present where microscopic visualization is unable to meet the throughput demands. Contrary to traditional approaches, protein mass spectrometry provides true confirmatory results, but multiday sample preparation and nanoflow sample separation requirements have limited the practical applicability of these approaches.

Developmental validation of a multiplex proteomic assay for the identification of forensically relevant biological fluids.

The aim of this study was to validate a multiplex proteomic assay for the identification of high-specificity protein biomarkers by multiple reaction monitoring mass spectrometry on a triple quadrupole mass spectrometer for the accurate, reliable, and confirmatory identification of bodily fluids commonly encountered in a forensic context. This includes the identification of peripheral blood, semen, saliva, urine, and vaginal/menstrual fluid. The assay is able to efficiently identify pure or mixed stains through the identification of target peptide fragments originating from tissue-specific proteins including: uromodulin from urine; prostatic acid phosphatase, prostate specific antigen and semenogelin-II for semen; statherin, submaxillary gland androgen-regulated protein 3B and amylase for saliva; cornulin, martrigel-induced gene C4 protein, suprabasin and neutrophil gelatinase-associated lipocalin for vaginal/menstrual fluid; and alpha-1 antitrypsin, hemopexin, and hemoglobin subunit beta for peripheral blood.

Direct seminal fluid identification by protease-free high-resolution mass spectrometry.

Serological screening of sexual assault evidence has traditionally focused on enzyme activity and immunochromatographic assays that provide only a presumptive indication of seminal fluid and have limited sensitivity relative to DNA testing. Seminal fluid detection based on protein mass spectrometry represents a "Next Gen" serological technology that overcomes the specificity and sensitivity limitations of traditional serological screening but requires time-consuming sample preparation protocols. This paper describes a novel "peptidomics" approach to seminal fluid detection that eliminates the need for lengthy trypsin digestion.

Effects of organic acids and common household products on the occurrence of false positive test results using immunochromatographic assays.

Forensic serological analyses often rely on lateral flow immunochromatographic assays to detect proteins that are characteristic of forensically relevant body fluids. In this study, we demonstrate that a positive result, however, is not limited to target protein binding. Citric and lactic acids at various pH levels were tested using 9 different commercial immunochromatographic assays.

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.

Discovery of highly specific protein markers for the identification of biological stains.

DNA profiling has transformed the field of forensic biology by making it possible to individualize biological stains. The identification of the stain itself, however, continues to present forensic serologists with significant challenges. Current antibody- and enzyme activity-based assays yield only presumptive results as detection in nontarget body fluids or cross-reactivity with nonhuman sources have both been well documented.

Evolution of gnathostome prodynorphin and proenkephalin: characterization of a shark proenkephalin and prodynorphin cDNAs.

Analyses of prodynorphin and proenkephalin cDNAs cloned from the central nervous system of the shark, Heterodontus portusjacksoni, provided additional evidence that these two opioid precursor-coding genes were most likely directly derived from a common ancestral gene. The two cDNAs could be aligned by inserting only seven gaps. The prodynorphin cDNA encodes five opioid sequences which could be aligned to opioid positions B through F in the proenkephalin cDNA.

Comparative analysis of the HV1 and HV2 regions of human mitochondrial DNA by denaturing high-performance liquid chromatography.

Denaturing high-performance liquid chromatography (DHPLC) was evaluated as a sequencing-independent means of detecting the presence of sequence differences in pair-wise mixtures of nonconcordant amplicons of human mitochondrial DNA (mtDNA). A total of 920 pair-wise combinations of HV1 and HV2 mtDNA amplicons from 95 individuals were assayed by DHPLC for sequence concordance/nonconcordance. For the 72 combinations of amplicons from different individuals who shared identical DNA sequences, DHPLC assays consistently indicated sequence concordance between the samples.

Validation of reduced-scale reactions for the Quantifiler Human DNA kit.

Accurate quantification of DNA samples is an important step in obtaining accurate and reproducible short tandem repeat (STR) profiles. Quantitative real-time-PCR has improved the speed and accuracy of DNA quantification over earlier methods, albeit at significantly greater cost per reaction. Here, the performance of reduced volume (10 microL) DNA quantification assays using the Quantifiler Human DNA Quantification Kit was evaluated using commercial standards and single source biological stains (e.

Resolving mtDNA mixtures by denaturing high-performance liquid chromatography and linkage phase determination.

Mitochondrial DNA (mtDNA) sequencing can provide crucial information to forensic investigators when the quantity and quality of DNA would otherwise be limiting. The difficulty of analyzing mtDNA mixtures, however, has been a significant obstacle to its broader use in forensics. Denaturing high-performance liquid chromatography (DHPLC) in combination with direct sequencing makes it possible to determine the linkage phase of individual amplicons in a mixture.

Seasonal changes in CRF-I and urotensin I transcript levels in masu salmon: correlation with cortisol secretion during spawning.

Pacific salmon employ a semelparous reproductive strategy where sexual maturation is followed by rapid senescence and death. Cortisol overproduction has been implicated as the central physiologic event responsible for the post-spawning demise of these fish. Cortisol homeostasis is regulated through the action of hormones of the hypothalamus-pituitary-interrenal (HPI) axis.

Separating human DNA mixtures using denaturing high-performance liquid chromatography.

DNA mixtures represent challenging samples that are rarely amenable to direct DNA sequence analysis and many of the strategies available to separate mixtures are both labor and time intensive. Denaturing high-performance liquid chromatography is an accurate and rapid approach for the detection and scoring of mutations. It can also be used to separate DNA mixtures.

Characterizing a proopiomelanocortin cDNA cloned from the brain of the Bichir, Polypterus senegalus: evaluating phylogenetic relationships among ray-finned fish.

There is general agreement that the polypteriform fishes, like Polypterus senegalus, constitute a unique lineage in the evolution of the vertebrates. However, the precise position of these fishes had been a point of controversy since the time of Darwin and Huxley. There is now consensus that the polypteriform fishes are members of superorder Actinopterygii.

Clinical applications of denaturing high-performance liquid chromatography-based genotyping.

Aim: To develop and evaluate heteroduplex forming templates (HFTs) as a common set of molecular standards for genotyping by denaturing high-performance liquid chromatography (DHPLC) using hypervariable regions of human mitochondrial DNA (mtDNA) as a model system.

Methods: Hypervariable regions 1 and 2 from the mtDNA D-loop of 22 maternally related and unrelated human volunteers were amplified by polymerase chain reaction (PCR) and individually mixed with each of three HFTs. Following denaturation and reannealing of the mixture, the resulting hetero- and homoduplicies were separated by DHPLC using temperature-modulated heteroduplex analysis.

Presence of the delta-MSH sequence in a proopiomelanocortin cDNA cloned from the pituitary of the galeoid shark, Heterodontus portusjacksoni.

Since a fourth MSH sequence, delta-MSH, has been detected in the proopiomelanocortin (POMC) gene of a dogfish and a stingray, members of superorder Squalea (class Chondrichthyes), it is possible that this novel MSH sequence might be a feature common to the POMC genes of all modern sharks and rays. As an initial step towards addressing this question, a full-length POMC cDNA was cloned and sequenced from the pituitary of the Port Jackson shark, Heterodontus portusjacksoni. The Port Jackson shark represents one of the oldest lineages in superorder Galea, and this superorder together with superorder Squalea form infraclass Neoselachii (the extant sharks and rays).

Evaluating the radiation of the POMC gene in teleosts: characterization of American eel POMC.

A distinctive feature of the pituitary hormone precursor, proopiomelanocortin (POMC), is the presence of multiple melanocortin core sequences (HFRW), and one copy of the opioid, beta-endorphin. In the older lineages of ray-finned fish (i.e.

Forensic utility of mitochondrial DNA analysis based on denaturing high-performance liquid chromatography.

Aim: To determine the forensic utility for pairwise DNA comparisons and DNA mixture resolution with denaturing high-performance liquid chromatography (DHPLC) of human mitochondrial DNA (mtDNA).

Methods: MtDNA hypervariable regions (HV) 1 and 2 from the mtDNA D-loop were amplified by the polymerase chain reaction and mixed between known and unknown sample sources. The DNA mixtures were denatured and reannealed, and the resultant homo- and heteroduplices were evaluated by temperature-modulated heteroduplex analysis by the DHPLC method.

Analyzing the evolution of the opioid/orphanin gene family.

Advances in molecular biology have made it possible to rapidly obtain the amino acid sequence of neuropeptide precursors-either by cloning and sequencing the cDNA that encodes the precursor, or by reconstructing the arrangement of exons and introns in a neuropeptide-coding gene through genomic approaches. The databases generated from these molecular approaches have been used to design probes to identify the cells that express the gene, or to ascertain the rate of expression of the gene, and even to predict the post-translational modifications that can generate functional neuropeptides from a biologically inert precursor. Although the power of these approaches is substantial, it is appreciated that a gene sequence or an mRNA sequence reflects the potential products that may be assembled in a secretory cell.