Biomedical subjects

P Wilkins Stevens

Publications and source records attributed to P Wilkins Stevens.

5 recordsLinked to original sources

PubMed2003

Improved sensitivity for solid-support invasive cleavage reactions with flow cytometry analysis.

A new configuration of the solid-support invasive cleavage reaction provides a small reaction-volume format for high-sensitivity discrimination of nucleic acid targets with single nucleotide differences. With target concentrations as low as 2 amol/assay, the solid-support invasive cleavage reaction clearly distinguishes single base mutations. Two oligonucleotides tethered to the solid support hybridize to the target nucleic acid, forming a tripartite substrate that can be recognized and cleaved by Cleavase, a structure-specific 5'-nuclease. Each cleavage event yields fluorescence signal on the surface. When microspheres serve as the solid-support surface, analysis by fluorometer imparts real-time information about change in the reaction signal over time. Flow cytometry provides an alternative detection technology that collects endpoint information about the reaction signal on individual microspheres. A reaction volume of 10 microL with as few as 3000 microspheres is sufficient to distinguish single nucleotide differences at target concentrations less than 200 fM. This sensitivity level is within the range required for analysis of SNPs in genomic DNA. In addition, the flow cytometry format has multiplexing potential, making the microsphere-based invasive cleavage assay attractive for high-throughput genomic applications.

Apolipoproteins E↗

PubMed2001

Analysis of single nucleotide polymorphisms with solid phase invasive cleavage reactions.

Using microparticles as the capture surface and fluorescence resonance energy transfer as the detection technology, we have demonstrated the feasibility of performing the invasive cleavage reaction on a solid phase. An effective tool for many genomic applications, the solution phase invasive cleavage assay is a signal amplification method capable of distinguishing nucleic acids that differ by only a single base mutation. The method positions two overlapping oligonucleotides, the probe and upstream oligonucleotides, on the target nucleic acid to create a complex recognized and cleaved by a structure-specific 5'-nuclease. For microarray and other multiplex applications, however, the method must be adapted to a solid phase platform. Effective cleavage of the probe oligonucleotide occurred when either of the two required overlapping oligonucleotides was configured as the particle-bound reagent and also when both oligonucleotides were attached to the solid phase. Positioning probe oligonucleotides away from the particle surface via long tethers improved both the signal and the reaction rates. The particle-based invasive cleavage reaction was capable of distinguishing the ApoE Cys158 and Arg158 alleles at target concentrations as low as 100 amol/assay (0.5 pM).

Alleles↗

PubMed1999

Real-time measurements of DNA hybridization on microparticles with fluorescence resonance energy transfer.

When capture oligonucleotides are tethered on planar surfaces, mass transport limitations influence the kinetics of solid-phase nucleic acid hybridizations. By diffusion theory, however, hybridization of oligonucleotides on microparticles should be reaction-rate limited. In an initial effort to understand the kinetics of microparticle hybridization reactions, we developed a fluorescence resonance energy transfer method for monitoring oligonucleotide hybridization on microparticles. Microparticles were coated with a fluoresceinated oligomer at surface densities of 20, 40, and 80% saturation, hybridized to a complementary oligonucleotide labeled with tetramethylrhodamine, and monitored over time for quenching of the fluorescein signal as hybridization occurred on the particle surface. Association rate constants were compared for microparticle-based hybridization and solution-phase hybridization. Rate constants for hybridizations on the particle surface were about an order of magnitude less than those for hybridization in solution, but decreasing the surface density of the capture oligonucleotide to 20% saturation improved particle hybridization rates. Although a bimolecular reaction model adequately described solution-phase hybridization kinetics, oligonucleotide hybridization on microparticles did not fit this model but exhibited biphasic reaction kinetics. Based on two different lines of reasoning, we argue that microparticle-based oligonucleotide hybridization was indeed reaction-rate limited in our system and not diffusion-rate limited.

Base Sequence↗

PubMed1999

DNA hybridization on microparticles: determining capture-probe density and equilibrium dissociation constants.

Many DNA-probe assays utilize oligonucleotide-coated microparticles for capture of complementary nucleic acids from solution. During development of these assays, as well as in other particle-based nucleic acid applications, it is useful to know both the amount of duplex formation expected under various experimental conditions and the coating density of the capture oligonucleotide on the particle surface. We examined the simplest form of a DNA-probe microparticle assay: hybridization of a particle-bound capture oligonucleotide to its solution-phase complement. Fluorescein-labeled solution-phase oligonucleotide was hybridized to varying amounts of particles, and the amount of labeled oligonucleotide remaining in solution at equilibrium was measured. We present a simple two-state, all-or-none model for bimolecular hybridization of non-self-complementary sequences that can be used to calculate the equilibrium dissociation constant ( Kd ) from hybridization data. With experimental conditions where both the Kd value and the concentration of capture probe in the reaction are small relative to the concentration of labeled complementary oligonucleotide in the reaction, density of the capture probe on the particle's surface can also be determined. Kd values for particle-based hybridization were different from those obtained from solution-phase thermodynamic parameters. At higher temperatures, hybridization on particles was more efficient than hybridization in solution.

DNA↗

PubMed1999

Physicochemical consequences of amino acid variations that contribute to fibril formation by immunoglobulin light chains.

The most common form of systemic amyloidosis originates from antibody light chains. The large number of amino acid variations that distinguish amyloidogenic from nonamyloidogenic light chain proteins has impeded our understanding of the structural basis of light-chain fibril formation. Moreover, even among the subset of human light chains that are amyloidogenic, many primary structure differences are found. We compared the thermodynamic stabilities of two recombinant kappa4 light-chain variable domains (V(L)s) derived from amyloidogenic light chains with a V(L) from a benign light chain. The amyloidogenic V(L)s were significantly less stable than the benign V(L). Furthermore, only the amyloidogenic V(L)s formed fibrils under native conditions in an in vitro fibril formation assay. We used site-directed mutagenesis to examine the consequences of individual amino acid substitutions found in the amyloidogenic V(L)s on stability and fibril formation capability. Both stabilizing and destabilizing mutations were found; however, only destabilizing mutations induced fibril formation in vitro. We found that fibril formation by the benign V(L) could be induced by low concentrations of a denaturant. This indicates that there are no structural or sequence-specific features of the benign V(L) that are incompatible with fibril formation, other than its greater stability. These studies demonstrate that the V(L) beta-domain structure is vulnerable to destabilizing mutations at a number of sites, including complementarity determining regions (CDRs), and that loss of variable domain stability is a major driving force in fibril formation.

Amino Acid Sequence↗