ABSTRACT
We have demonstrated a proof of concept with affinity-based purification of sperm cells from mock forensic samples using SOMAmer™ reagents, DNA-based affinity reagents developed by SomaLogic, Inc. SOMAmer reagents were selected in vitro using whole-cell SELEX to bind specifically with intact, detergent-treated sperm cells. Successful separation of sperm from epithelial cells and their debris was demonstrated using buccal swabs with added semen. Primarily male DNA profiles were generated from sperm cells eluted from the types of cotton swabs typically used for rape kit evidence collection. The quality of sperm DNA isolated from samples purified using SOMAmers is comparable to existing commercially available differential extraction-based methods at higher sperm concentrations. This purification method is simple, offers relatively rapid (<2hr) sperm purification, and can potentially be automated using robotic workstations. This work serves as proof of concept that demonstrates the first use of SOMAmer reagents as affinity ligands to bind sperm cells. With further development, this technique can potentially be used for high-throughput sexual assault forensic casework.
Subject(s)
DNA/isolation & purification , Spermatozoa/cytology , Cell Separation , DNA Fingerprinting , Epithelial Cells/cytology , Female , Forensic Genetics , Humans , Indicators and Reagents , Male , Specimen Handling/instrumentationABSTRACT
We present an electrokinetic technique to increase the reaction rate and sensitivity of bead-based assays. We use isotachophoresis (ITP) to preconcentrate and co-focus target molecules and beads into a single ITP zone. The process achieves rapid mixing, stirring, and strongly increases the binding reaction rate. We demonstrate our assay with quantitative detection of 24â nt single-stranded DNA over a dynamic range of three orders of magnitude and multiplexed detection of ten target species per sample. We show that ITP can achieve approximately the same sensitivity as a well-stirred standard reaction in 60-fold reduced reaction time (20â min versus 20â h). Alternately, compared to standard reaction times of 30â min, we show that 20â min ITP hybridization can achieve 5.3-fold higher sensitivity.
Subject(s)
Isotachophoresis , Limit of DetectionABSTRACT
Recently, we reported a SOMAmer-based, highly multiplexed assay for the purpose of biomarker identification. To enable seamless transition from highly multiplexed biomarker discovery assays to a format suitable and convenient for diagnostic and life-science applications, we developed a streamlined, plate-based version of the assay. The plate-based version of the assay is robust, sensitive (sub-picomolar), rapid, can be highly multiplexed (upwards of 60 analytes), and fully automated. We demonstrate that quantification by microarray-based hybridization, Luminex bead-based methods, and qPCR are each compatible with our platform, further expanding the breadth of proteomic applications for a wide user community.
Subject(s)
Biological Assay/methods , Biomarkers/analysis , Diagnostic Techniques and Procedures , Oligonucleotides/metabolism , Proteomics/methods , Automation , Case-Control Studies , Humans , Limit of Detection , Nucleic Acids/metabolism , Oligonucleotides/chemistry , Reproducibility of Results , TitrimetryABSTRACT
We have expressed and purified 13 proteins predicted to be required for B. subtilis DNA replication. When combined with a circular DNA template with a 5' unpaired flap, these proteins reconstitute replication of both the leading and lagging strands at the physiological rate. Consistent with the in vivo requirement for two DNA polymerase III replicases for B. subtilis chromosomal replication, both PolC and DnaE are required for reconstitution of the replication fork in vitro. Leading strand synthesis requires PolC plus ten proteins; lagging strand synthesis additionally requires primase and DnaE. DnaE does not serve as the lagging strand replicase, like DNA polymerase delta in eukaryotes, but instead functions like eukaryotic DNA polymerase alpha, adding a stretch of deoxynucleotides to the RNA primer before handoff to PolC. Primase equilibrates with the fork prior to synthesis of each Okazaki fragment, and its concentration controls the frequency of initiation and Okazaki fragment size.
Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/physiology , DNA Replication/physiology , DNA-Directed DNA Polymerase/physiology , Models, Genetic , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA/biosynthesis , DNA Polymerase III/genetics , DNA Polymerase III/metabolism , DNA Polymerase III/physiology , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolismABSTRACT
DNA replication is highly regulated in most organisms. Although much research has focused on mechanisms that regulate initiation of replication, mechanisms that regulate elongation of replication are less well understood. We characterized a mechanism that regulates replication elongation in the bacterium Bacillus subtilis. Replication elongation was inhibited within minutes after amino acid starvation, regardless of where the replication forks were located on the chromosome. We found that small nucleotides ppGpp and pppGpp, which are induced upon starvation, appeared to inhibit replication directly by inhibiting primase, an essential component of the replication machinery. The replication forks arrested with (p)ppGpp did not recruit the recombination protein RecA, indicating that the forks are not disrupted. (p)ppGpp appear to be part of a surveillance mechanism that links nutrient availability to replication by rapidly inhibiting replication in starved cells, thereby preventing replication-fork disruption. This control may be important for cells to maintain genomic integrity.
