Single-molecule fluorescence techniques are used to detect a specific
nucleic acid sequence in a mixture of unrelated sequences. Co-hybridization of a pair oligonucleotide hybridization probes, each
labeled with a spectrally-distinct fluorophore and complementary to a
specific sub-sequence of the target nucleic acid, forms a fluorescent
adduct containing both fluorophores. The presence of the specific
sequence is signaled by the simultaneous detection of both fluorophore labels on a single target fragment. We demonstrate quantitative detection of target nucleic acid sequences at fragment concentrations as low as 100 fM with a simple instrument that uses low-power, continuous-wave laser excitation. Furthermore, we show that a cross-correlation analysis of the arrival times of individual
single-molecule fluorescence photon bursts detected in spectrally
separate channels permits quantitative detection of the dual-color
labeled species at concentrations approximately 1000x lower
than can be quantitatively detected using the photon cross-correlation between the two detection channels. We also demonstrate that a pair of quencher-labeled oligonucleotides each complementary to the fluorescent hybridization probes can be used to reduce unbound probe fluorescence, substantially improving the sensitivity of the assay. We use this approach to detect β-actin messenger RNA (mRNA) transcripts.
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.
Our current experiments further the development of a laser- based technique capable of sequencing an individual strand of DNA. We report the detection and identification of fluorescently labeled nucleotides enzymatically cleaved from DNA strands suspended in flow. We used fluorescence lifetime, fluorescence intensity, or a correlated measure of the intensity and lifetime to identify each individual tagged base traversing the detection region with high accuracy. DNA strands containing a single tetramethylrhodamine labeled uracil and/or a single Rhodamine 6G labeled cytosine were attached to polystyrene microspheres. An optical trap was used to capture and hold a single DNA-laden microsphere nominally 20 microns upstream of the detection region of an ultra- sensitive flow cytometer. The addition of an exonuclease cleaved bases from the 3' end of the fluorescently labeled strand. The cleaved, labeled nucleotides were carried by the flow downstream and detected and identified one-at-a-time with high efficiency by laser-induced fluorescence.
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.
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