ABSTRACT
DNA mixtures from 3 or more contributors have proven difficult to analyze using the current state-of-the-art method of short-tandem repeat (STR) amplification followed by capillary electrophoresis (CE). Here we analyze samples from both laboratory-defined mixtures and complex multi-contributor touch samples using a single nucleotide polymorphism (SNP) panel comprised of 2311 low-minor-allele-frequency loci, combined with massively parallel sequencing (MPS). This approach demonstrates that as many as 10 people can be identified in touch samples using a threshold of -Log P(RMNE) of 6, and a detection rate of 18-94 % across 10 different materials using a threshold of -Log P(RMNE) of 2. Thirty-two false positives were observed in 100 touch samples.
Subject(s)
DNA/genetics , Forensic Genetics/methods , Gene Frequency , High-Throughput Nucleotide Sequencing , Polymorphism, Single Nucleotide , Alleles , DNA Fingerprinting , Humans , TouchABSTRACT
High-throughput sequencing (HTS) of large panels of single nucleotide polymorphisms (SNPs) provides an alternative or complimentary approach to short tandem repeats (STRs) panels for the analysis of complex DNA mixture forensic samples. For STRs, methods to estimate individual contribution concentrations compare capillary electrophoresis peak heights, peak areas, or HTS allele read counts within a mixture. This article introduces three approaches (mean, median, and slope methods) for estimating individual DNA contributions to forensic mixtures for HTS/massively parallel sequencing (MPS) SNP panels. For SNPs, the major:minor allele ratios or counts, unique to each contributor, were compared to estimate contributor proportion within the mixture using the mean, median, and slope intercept for these alleles. The estimates for these three methods were typically within 5% of planned experimental contributions for defined mixtures.
Subject(s)
DNA/genetics , High-Throughput Nucleotide Sequencing , Polymorphism, Single Nucleotide , Alleles , DNA Fingerprinting , Forensic Genetics/methods , Humans , Linear ModelsABSTRACT
We report the use of genetically engineered cells in a pathogen identification sensor. This sensor uses B lymphocytes that have been engineered to emit light within seconds of exposure to specific bacteria and viruses. We demonstrated rapid screening of relevant samples and identification of a variety of pathogens at very low levels. Because of its speed, sensitivity, and specificity, this pathogen identification technology could prove useful for medical diagnostics, biowarfare defense, food- and water-quality monitoring, and other applications.
Subject(s)
B-Lymphocytes , Bacteria/isolation & purification , Bacteriological Techniques , Biosensing Techniques , Viruses/isolation & purification , Aequorin/biosynthesis , Antibodies, Bacterial/immunology , Antibodies, Viral/immunology , B-Lymphocytes/immunology , Bacillus anthracis/immunology , Bacillus anthracis/isolation & purification , Bacteria/immunology , Cell Line , Colony Count, Microbial , Encephalitis Virus, Venezuelan Equine/immunology , Encephalitis Virus, Venezuelan Equine/isolation & purification , Escherichia coli O157/immunology , Escherichia coli O157/isolation & purification , Foot-and-Mouth Disease Virus/immunology , Foot-and-Mouth Disease Virus/isolation & purification , Immunoglobulin Variable Region/immunology , Light , Receptors, Antigen, B-Cell/immunology , Sensitivity and Specificity , Time Factors , Transfection , Viruses/immunology , Yersinia pestis/immunology , Yersinia pestis/isolation & purificationABSTRACT
The mammalian zona pellucida (ZP) consists of three glycoproteins (ZP1, ZP2 and ZP3), which are variably conserved among species at the genomic and amino acid levels. In order to evaluate the expression of ZP during ovarian development, a population of antibodies was selected that recognize species conserved antigenic domains of the three ZP proteins. Domain specific antibodies were selected from sera of rabbits immunized with all three native pig ZP proteins by elution of antibodies bound to each of the three human ZP recombinant proteins expressed from cDNAs, using the baculovirus expression system in insect cells. Immunoblot analysis was used to characterize the specificity of the antibodies and immunohistochemistry was used to evaluate the stage specific expression of ZP proteins during ovarian follicular development of the mouse, baboon and human. This study demonstrates that the conserved domains of all three ZP proteins are localized in the oocyte extracellular ZP matrix as well as in a subset of granulosa cells. However, this expression does vary among species with respect to the stage and cell type during early stages of ovarian follicular development. These antibodies should serve as excellent markers for evaluating early stages of human ovarian follicular development and in the development of contraceptive agents.
Subject(s)
Antigens/analysis , Egg Proteins/immunology , Membrane Glycoproteins/immunology , Ovary/chemistry , Receptors, Cell Surface , Animals , Antibody Specificity , Antigens/immunology , Baculoviridae/genetics , DNA, Complementary/genetics , Egg Proteins/genetics , Electrophoresis, Polyacrylamide Gel , Extracellular Matrix/chemistry , Female , Granulosa Cells/chemistry , Humans , Immunoblotting , Immunohistochemistry , Membrane Glycoproteins/genetics , Ovarian Follicle/physiology , Recombinant Proteins/immunology , Species Specificity , Spodoptera/metabolism , Swine , Transfection , Zona Pellucida/chemistry , Zona Pellucida/ultrastructure , Zona Pellucida GlycoproteinsABSTRACT
Rran-dependent nuclear transport requires a nuclear pool of RanGTP both for the assembly of export complexes and the disassembly of import complexes. Accordingly, in order for these processes to proceed, Ran-dependent nuclear import and export assays in vitro require the addition of GTP to produce RanGTP. Notably, no ATP requirement can be detected for these transport processes in vitro. But in vivo, when cells are depleted of ATP by the addition of sodium azide and 2-deoxyglucose to block ATP production by oxidative phosphorylation and glycolysis, respectively, Ran-dependent nuclear import and export are rapidly inhibited. This raised the question of whether there is an ATP requirement for these nuclear transport pathways in an intact cell that has remained undetected in vitro. Here we report that the free (but not total) GTP concentration rapidly drops to an undetectable level upon ATP depletion as does the availability of RanGTP. Our conclusion is that the inhibition of Ran-dependent nuclear transport observed upon ATP depletion in vivo results from a shortage of RanGTP rather than the inhibition of some ATP-dependent process.
