Parallel production of oligonucleotide arrays using membranes and reagent jet printing.

Here, we present a low-cost method to produce compact arrays using microporous materials and reagent jetting. Oligonucleotides are immobilized on membrane sheets as a series of lines. The membrane sheet is then rolled and bound, and the spiral structure is cut like a "jelly roll" to produce identical arrays. The spiral arrays behave much like larger formats using membranes, and hybridization detection can be accomplished using standard signal-generation mechanisms. The method is particularly useful for producing identical arrays from pre-synthesized oligonucleotides.

The utility of optical waveguide DNA array hybridization and melting for rapid resolution of mismatches, and for detection of minor mutant components in the presence of a majority of wild type sequence: Statistical model and supporting data.

We have derived a statistical model to determine binding and dissociation kinetics of oligonucleotide-antibody-microparticle complexes to oligonucleotides on a surface illuminated by the evanescent wave. The model supports the extremely rapid binding kinetics previously observed and predicts very rapid thermal dissociation kinetics. Experimental results confirmed the model and showed that the most rapid heating available achieved meltoff in 8 s with discrimination of single base differences. Similarly, a model of particulate labels with multiple points of interaction predicted that the system would be very sensitive to a minority of matched interactions in the presence of a majority of mismatched interactions. Conversely, the system would be relatively insensitive to the presence of a higher proportion of matched species. These predictions are born out by experiment. A 1:10 mixture of matched:mismatched gave a melting curve which was approximately the sum of melting curves of the two species alone, and a 50:50 mixture gave a melting curve almost indistinguishable from the highest melting component. Thus, the system will be amenable to detection of infrequent mutations in the presence of an excess of the wild type allele provided the mutant sequence is present on the chip. Further, it lends itself to high throughput automation due to the rapidity with which the thermal melt can be achieved.

Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides.

The challenge of the Human Genome Project is to increase the rate of DNA sequence acquisition by two orders of magnitude to complete sequencing of the human genome by the year 2000. The present work describes a rapid detection method using a two-dimensional optical wave guide that allows measurement of real-time binding or melting of a light-scattering label on a DNA array. A particulate label on the target DNA acts as a light-scattering source when illuminated by the evanescent wave of the wave guide and only the label bound to the surface generates a signal. Imaging/visual examination of the scattered light permits interrogation of the entire array simultaneously. Hybridization specificity is equivalent to that obtained with a conventional system using autoradiography. Wave guide melting curves are consistent with those obtained in the liquid phase and single-base discrimination is facile. Dilution experiments showed an apparent lower limit of detection at 0.4 nM oligonucleotide. This performance is comparable to the best currently known fluorescence-based systems. In addition, wave guide detection allows manipulation of hybridization stringency during detection and thereby reduces DNA chip complexity. It is anticipated that this methodology will provide a powerful tool for diagnostic applications that require rapid cost-effective detection of variations from known sequences.

Relation of personality characteristics and color preferences.

150 students responded to the Edwards Personal Preference Schedule and ranked color plates from the Luscher Color Test. Regression analysis yielded no relationship of predictive significance between the two sets of variables. An ancillary analysis using sets of random numbers in place of the color preference data yielded results basically similar to those produced in the earlier analysis. Males and females rank ordered the colors identically except for yellow, which was significantly more preferred by males than females.

Some correlates of trust.

Trust has been variously defined by behavioral scientists and not very thoroughly investigated. In this study trust was defined as an expectancy held by an individual that the behavior of another person or a group would be altruistic and personally beneficial. An attempt was made, using this conceptual definition, to identify some personality and behavioral correlates of trust. Seven interpersonal relations groups with approximately 10 male and female undergraduates per group were studied with use of the Janis and Field self-esteem inventory, Schutz's FIRO-B scale, and the Rotter internal-external scale. It was discovered that a trusted person is one who is highly influential, has an internal locus of control, a low need to control others, high self-esteem, and is open to being influenced by others.

Isoelectric focusing of a dimerizing solute in rapid chemical equilibrium: comparison of simulation procedures.

The approach to isoelectric focusing equilibrium of a rapidly dimerizing solute was simulated by two different computing procedures: a stationary-grid model developed by Cann and Stimpson and a distorted-grid technique derived from the method of Cox. The results given by the two models were virtually identical at all times during the approach to equilibrium. Of the two procedures, the distorted-grid method has an advantage in computing time, while the stationary-grid model is applicable to a broader range of transport experiments. The effect on the focusing experiment of varying the electric field was examined by distorted-grid simulations. When the field was increased, the equilibrium distribution sharpened somewhat and the peak of the concentration profile shifted toward the isoelectric position of the dimer. The rate of approach to equilibrium was approximately proportional to the field strength.

Isoelectric focusing of interacting systems. I. Carrier ampholyte-induced macromolecular isomerization.

A phenomenological theory of isoelectric focusing is formulated for rapidly reversible, ampholyte-induced macromolecular isomerization. The calculations reveal that such interactions can give well resolved, bimodal transient and equilibrium isoelectric focusing patterns in which the two peaks correspond to different chemical equilibrium compositions and not to separated isomers. The kinetics of approach to the equilibrium pattern are characteristically biphasic: During the first phase, which is controlled by the rate of migration of the isomers in the electric field, two peaks are positioned in the region between the isoelectric points of the two isomers; one of the peaks then grows slowly at the expense of the other with a diffusion-dominated rate. The kinetics are dependent upon the initial distribution of macromolecule in the isoelectric focusing column, and in certain cases only a single peak is apparent during the first phase. These findings have practical implications for unambiguous interpretation of isoelectric focusing patterns, furnish explanations for hitherto puzzling experimental observations, and provide theoretical insights required for application of isoelectric focusing to the detection and characterization of macromolecular interactions in general.

Isoelectric focusing of interacting systems. II. pH-dependent conformational transitions.

Transient and equilibrium isoelectric focusing patterns have been computed for pH-dependent conformational transitions in the limits of complete cooperativity and instantaneous chemical equilibration. Transitions induced by the binding of a relatively large number of hydrogen ions by the macromolecule give well resolved bimodal equilibrium patterns, provided that the resulting conformer has the lower isoelectric point. The corresponding transient patterns may be either bimodal or virtually unimodal for practical times of operation depending upon the point of insertion of the sample into the pH gradient and the stoichiometry of the interaction. A macromolecule undergoing sequential transitions can give multimodal isoelectric focussing patterns.