We have developed a SNP scoring platform, yielding high throughput, inexpensive assays. The basic platform uses fluorescently labeled DNA fragments bound to microspheres, which are analyzed using flow cytometry. SNP scoring is performed using minisequencing primers and fluorescently labeled dideoxynucleotides. Furthermore, multiplexed microspheres make it possible to score hundreds of SNPs simultaneously. Multiplexing, coupled with high throughput rates allow inexpensive scoring of several million SNPs/day. GAMMArrays use universal tags that consist of computer designed, unique DNA tails. These are incorporated into each primer, and the reverse-component is attached to a discrete population of microspheres in a multiplexed set. This enables simultaneous minisequencing of many SNPs in solution, followed by capture onto the appropriate microsphere for multiplexed analysis by flow cytometry. We present results from multiplexed SNP analyses of bacterial pathogens, and human mtDNA variation. Analytes are performed on PCR amplicons, each containing numerous SNPs scored simultaneously. In addition, these assays easily integrate into conventional liquid handling automation, and require no unique instrumentation for setup and analysis. Very high signal-to-noise ratios, ease of setup, flexibility in format and scale, and low cost make these assays extremely versatile and valuable tools for a wide variety of SNP scoring applications.
Flow cytometry is uniquely capable of making sensitive and quantitative multiparameter fluorescence measurements with discrimination of free from particle-bound fluorophore. Recent advances in mixing and sample delivery have extended these capabilities into the sub-second time domain. Access to these time scales has enabled us to use flow cytometry to measure molecular interactions. Using the general approach of immobilizing one molecule on a microsphere and fluorescently labeling another, we have been able to make real-time measurements of ligand-receptor and enzyme-substrate interactions involving proteins, nucleic acids, carbohydrates, and lipids. We are developing schemes for immobilizing active biological molecules in defined and homogeneous orientations relative to the surface. We are also developing approaches for homogeneous fluorescent labeling of active biomolecules and calibration schemes for quantitative measurements by flow cytometry. We will present several examples of applications of this new technology, including DNA- and protein-protein interactions, nucleic acid hybridization, and interactions on artificial membrane surfaces. These approaches should have wide applications for mechanistic analysis, diagnostics, and drug development.
Functional analysis of the human genome, including the quantification of differential gene expression and the identification of polymorphic sites and disease genes, is an important element of the Human Genome Project. Current methods of analysis are mainly gel-based assays that are not well- suited to rapid genome-scale analyses. To analyze DNA sequence on a large scale, robust and high throughput assays are needed. We are developing a suite of microsphere-based approaches employing fluorescence detection to screen and analyze genomic sequence. Our approaches include competitive DNA hybridization to measure DNA or RNA targets in unknown samples, and oligo ligation or extension assays to analyze single-nucleotide polymorphisms. Apart from the advantages of sensitivity, simplicity, and low sample consumption, these flow cytometric approaches have the potential for high throughput multiplexed analysis using multicolored microspheres and automated sample handling.
Flow cytometry discriminates particle associated fluorescence from the fluorescence of the surrounding medium. It permits assemblies of macromolecular complexes on beads or cells to be detected in real-time with precision and specificity. We have investigated two types of robust sample handling systems which provide sub-second resolution and high throughput: (1) mixers which use stepper-motor driven syringes to initiate chemical reactions in msec time frames; and (2) flow injection controllers with valves and automated syringes used in chemical process control. In the former system, we used fast valves to overcome the disparity between mixing 100 (mu) ls of sample in 100 msecs and delivering sample to a flow cytometer at 1 (mu) l/sec. Particles were detected within 100 msec after mixing, but unstable flow was created which lasted for 1 sec after injection of the sample into the flow cytometer. We used optical criteria to discriminate particles which were out of alignment due to the unstable flow. Complex sample handling protocols involving multiple mixing steps and sample dilution have also been achieved. With the latter system we were able to automate sample handling and delivery with intervals of a few seconds. We used a fluidic approach to defeat the instability caused by sample introduction. By controlling both sheath and sample with individual syringes, the period of instability was reduced to approximately 200 msecs. Automated sample handling and sub-second resolution should permit broad analytical and diagnostic applications of flow cytometry.
We describe the application of single molecule detection (SMD) technologies for the analysis of natural and synthetic transport systems. The need for advanced analytical procedures of these complex and important systems is presented with the specific enhancements afforded by SMD with flowing sample streams. In contrast to bulk measurements which yield only average values, measurement of individual species allows creation of population histograms form heterogeneous samples. The data are acquired in minutes and the analysis requires relatively small sample quantities. Preliminary data are presented from the analysis of low density lipoprotein, and multilamellar and unilamellar vesicles.
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