DNA-probes for the highly sensitive identification of single nucleotide polymorphism using single-molecule spectroscopy

This article presents a new, highly sensitive method for the identification of single nucleotide polymorphisms (SNPs) in homogeneous solutions using fluorescently labeled hairpin-structured oligonucleotides (smart probes) and fluorescence single-molecule spectroscopy. While the hairpin probe is closed, fluorescence intensity is quenched due to close contact between the chromophore and several guanosine residues. Upon hybridization to the respective target SNP sequence, contact is lost and the fluorescence intensity increases significantly. High specificity is achieved by blocking sequences containing mismatch with unlabeled oligonucleotides. Time-resolved single-molecule fluorescence spectroscopy enables the detection of individual smart probes passing a small detection volume. This method leads to a subnanomolar sensitivity for this single nucleotide specific DNA assay technique.     

DNA microarrays with unimolecular hairpin double-stranded DNA probes: fabrication and exploration of sequence-specific DNA/protein interactions

We have fabricated double-stranded DNA (dsDNA) microarrays containing unimolecular hairpin dsDNA probes immobilized on glass slides. The unimolecular hairpin dsDNA microarrays were manufactured by four steps: Firstly, synthesizing single-stranded DNA (ssDNA) oligonucleotides with two reverse-complementary sequences at 3' hydroxyl end and an overhang sequence at 5' amino end. Secondly, microspotting ssDNA on glutaraldehyde-derived glass slide to form ssDNA microarrays. Thirdly, annealing two reverse-complementary sequences to form hairpin primer at 3' end of immobilized ssDNA and thus to create partial-dsDNA microarray. Fourthly, enzymatically extending hairpin primer to convert partial-dsDNA microarrays into complete-dsDNA microarray. The excellent efficiency and high accuracy of the enzymatic synthesis were demonstrated by incorporation of fluorescently labeled dUTPs in Klenow extension and digestion of dsDNA microarrays with restriction endonuclease. The accessibility and specificity of the DNA-binding proteins binding to dsDNA microarrays were verified by binding Cy3-labeled NF-kappaB to dsDNA microarrays. The dsDNA microarrays have great potential to provide a high-throughput platform for investigation of sequence-specific DNA/protein interactions involved in gene expression regulation, restriction and so on.     

Solid-phase oligonucleotide synthesis and flow cytometric analysis with microspheres encoded with covalently attached fluorophores

A novel combinatorial approach to synthesize oligonucleotides on fluorescently encoded microspheres based on flow sorting and segmental solid-phase synthesis is described. BODIPY dyes were covalently attached to polystyrene (8.8 microm, 55% DVB) microsphere particles to generate four fluorescently encoded sets. 20-mer oligonucleotide sequences can be synthesized on these microspheres with yields comparable to conventional CPG supports (80% overall yield, average stepwise yield = 99%). The concept of segmental solid-phase synthesis by flow sorting was demonstrated by synthesizing unique 20-mer oligonucleotide sequences on each of four fluorescently encoded microsphere sets by including a flow sorting step (after first eight base additions) and flow cytometric detection of sequences synthesized on each microsphere set by hybridization with fluorescently labeled complementary sequence.     

Hybridization of DNA with oligonucleotides immobilized in a gel: a convenient method for recording single base replacements

In an attempt to develop a reliable system for DNA sequence analysis with multiple hybridization probes, oligonucleotides down to 8 bases long were covalently immobilized in a thin layer of polyacrylamide gel fixed on a glass plate. It was shown possible to detect single base changes in DNA by hybridization of the immobilized oligonucleotides with radioactively and fluorescently labeled DNA fragments. Moreover, it was found that dissociation temperatures of differently GC-rich duplexes could be equalized by appropriate choice of immobilized oligonucleotides concentrations. A model accounting for this phenomenon is presented. In order to make the system more compact, a rectangular matrix of 200 mm dots of immobilized oligonucleotides ("hybridization chip") was designed which offered the sensitivity of 20 attomoles per dot for fluorescent DNA fragment. The applications and perspectives of the approach are discussed.     

Single-base mutational analysis of cancer and genetic diseases using membrane bound modified oligonucleotides.

A convenient format for the detection of PCR amplified sequences is the hybridization of the PCR products to oligonucleotide probes which are immobilized on a solid phase. We describe a new method for site-specific attachment of such probe oligonucleotides to nylon membranes. The method is based on the formation of an amide bond between carboxyl groups present on the membranes and amino-linkers situated on the 5' end of the oligonucleotides. The covalent attachment is via a carbodiimide mediated condensation. The single, 5' end attachment of the oligonucleotides to the membrane surface leaves the probe free to interact with complementary sequences, thus increasing the hybridization efficiency relative to methods where heat or ultraviolet light is used for non-specific fixation. Using biotinylated PCR products in hybridization reactions along with a non-radioactive chemiluminescent detection system, high efficiency hybridization is obtained as well as a very good signal to noise ratio. The method has been applied successfully to the detection of RAS point mutations, cystic fibrosis deletion and point mutations and others. The sensitivity, simplicity and reproducibility of this method make it an ideal tool for the diagnosis of infectious and genetic diseases, as well as analysis of mutations in neoplasias, HLA typing and other areas.     

Microarray fabrication with covalent attachment of DNA using bubble jet technology

We have developed a method for fabricating DNA microarrays that uses a Bubble Jet ink jet device to eject 5'-terminal-thiolated oligonucleotides to a glass surface. The oligonucleotides are covalently attached to the glass surface by heterobifunctional crosslinkers that react with the amino group on the substrate and a thiol group on the oligonucleotide probe. Using this method, we fabricated DNA microarrays that carried 64 groups of 18-mer oligonucleotides encoding all possible three-base mutations in the mutational "hot spot" of the p53 tumor-suppressor gene. These were screened with a fluorescently labeled synthetic 18-mer oligonucleotide derived from the p53 gene, or segments of the p53 gene that had been PCR amplified from genomic DNA of two cell lines of human oral squamous cell carcinoma (SCC). This allowed us to discriminate between matched hybrids and 1 bp-mismatched hybrids.     

Hybridization assay of insect antifreezing protein gene by novel multilayered porous silicon nucleic acid biosensor

A fabrication of a novel simple porous silicon polybasic photonic crystal with symmetrical structure has been reported as a nucleic acid biosensor for detecting antifreeze protein gene in insects (Microdera puntipennis dzhungarica), which would be helpful in the development of some new transgenic plants with tolerance of freezing stress. Compared to various porous silicon-based photonic configurations, porous silicon polytype layered structure is quite easy to prepare and shows more stability; moreover, polybasic photonic crystals with symmetrical structure exhibit interesting optical properties with a sharp resonance in the reflectance spectrum, giving a higher Q factor which causes higher sensitivity for sensing performance. In this experiment, DNA oligonucleotides were immobilized into the porous silicon pores using a standard crosslink chemistry method. The porous silicon polybasic symmetrical structure sensor possesses high specificity in performing controlled experiments with non-complementary DNA. The detection limit was found to be 21.3nM for DNA oligonucleotides. The fabricated multilayered porous silicon-based DNA biosensor has potential commercial applications in clinical chemistry for determination of an antifreeze protein gene or other genes.     

An on-chip thin film photodetector for the quantification of DNA probes and targets in microarrays

A flat microdevice which incorporates a thin-film amorphous silicon (a-Si:H) photodetector with an upper layer of functionalized SiO₂ is used to quantify the density of both immobilized and hybridized DNA oligonucleotides labeled with a fluorophore. The device is based on the photoconductivity of hydrogenated amorphous silicon in a coplanar electrode configuration. Excitation, with near UV/blue light, of a single-stranded DNA molecule tagged with the fluorophore 1-(3-(succinimidyloxycarbonyl)benzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl) pyridinium bromide (PyMPO), results in the emission of visible light. The emitted light is then converted into an electrical signal in the photodetector, thus allowing the optoelectronic detection of the DNA molecules. The detection limit of the present device is of the order of 1 × 10¹² molecules/cm² and is limited by the efficiency of the filtering of the excitation light. A surface density of 33.5 ± 4.0 pmol/cm² was measured for DNA covalently immobilized to the functionalized SiO₂ thin film and a surface density of 3.7 ± 1.5 pmol/cm² was measured for the complementary DNA hybridized to the bound DNA. The detection concept explored can enable on-chip electronic data acquisition, improving both the speed and the reliability of DNA microarrays.     

Highly sensitive detection of hybridization of oligonucleotides to specific sequences of nucleic acids by application of fluorescence resonance energy transfer

We show a new application of fluorescence resonance energy transfer (FRET) in two stages to detect specific sequences of nucleic acids. In the first stage, two fluorescently tagged oligonucleotides hybridize with a complementary target molecule to produce FRET. The sequences of the oligonucleotides and spectral properties of fluorophores are chosen to provide a basis for an efficient energy transfer. In the next step, the specificity of hybridization is tested by competition of labeled probes with an excess of unlabeled oligonucleotides of the same sequence. The resulting emission spectra, one obtained in the excess of unlabeled donor probe and the other produced in the excess of unlabeled acceptor probe, are compared with the spectrum from the first stage to look for differences in the emission pattern of the fluorescent labels. We show that it is possible to detect the existence of specific hybrids composed of the two probes and complementary target molecule even in very unfavorable conditions, such as the presence of unhybridized probes in the final reaction mixture, secondary nonacceptor quenching of donor probe fluorescence, and strong background emission of acceptor produced by its direct excitation with a donor excitation light.     

Analysis and purification of synthetic oligonucleotides by reversed-phase high-performance liquid chromatography with photodiode array and mass spectrometry detection.

Native and modified synthetic oligonucleotides were purified by reversed-phase HPLC using volatile ion-pairing mobile phases. Purification of 10-90 nmol of oligonucleotides in a single injection was demonstrated using a 4.6 x 75-mm HPLC column packed with porous 2.5 microm C18 sorbent. Separation of target products from N-1 failure fragments was achieved for oligonucleotides in the 4- to 60-mer size range. We employed a combination of absorbance and mass spectrometry detection to identify by-products of oligonucleotide synthesis. This method was also employed for analysis and purification of fluorescently labeled oligonucleotides.     

Oligonucleotide microchips as genosensors for determinative and environmental studies in microbiology.

The utility of parallel hybridization of environmental nucleic acids to many oligonucleotides immobilized in a matrix of polyacrylamide gel pads on a glass slide (oligonucleotide microchip) was evaluated. Oligonucleotides complementary to small-subunit rRNA sequences of selected microbial groups, encompassing key genera of nitrifying bacteria, were shown to selectively retain labeled target nucleic acid derived from either DNA or RNA forms of the target sequences. The utility of varying the probe concentration to normalize hybridization signals and the use of multicolor detection for simultaneous quantitation of multiple probe-target populations were demonstrated.     

Single nucleotide polymorphic discrimination by an electronic dot blot assay on semiconductor microchips

We have developed a rapid assay for single nucleotide polymorphism (SNP) detection that utilizes electronic circuitry on silicon microchips. The method was validated by the accurate discrimination of blinded DNA samples for the complex quadra-allelic SNP of mannose binding protein. The microchip directed the transport, concentration, and attachment of amplified patient DNA to selected electrodes (test sites) creating an array of DNA samples. Through control of the electric field, the microchip enabled accurate genetic identification of these samples using fluorescently labeled DNA reporter probes. The accuracy of this approach was established by internal controls of dual labeled reporters and by using mismatched sequences in addition to the wild-type and variant reporter sequences to validate the SNP-genotype. The ability to customize this assay for multiple genes has advantages over other existing approaches.     

Ultrahigh-resolution optical trap with single-fluorophore sensitivity

We present a single-molecule instrument that combines a time-shared ultrahigh-resolution dual optical trap interlaced with a confocal fluorescence microscope. In a demonstration experiment, we observed individual single fluorophore-labeled DNA oligonucleotides to bind and unbind complementary DNA suspended between two trapped beads. Simultaneous with the single-fluorophore detection, we clearly observed coincident angstrom-scale changes in tether extension. Fluorescence readout allowed us to determine the duplex melting rate as a function of force. The new instrument will enable the simultaneous measurement of angstrom-scale mechanical motion of individual DNA-binding proteins (for example, single-base-pair stepping of DNA translocases) along with the detection of properties of fluorescently labeled protein (for example, internal configuration).     

Thiol-responsive pro-fluorophore labeling: Synthesis of a pro-fluorescent labeled oligonucleotide for monitoring cellular uptake

Pro-fluorescent labeled oligonucleotides are potential alternative tools to classical fluorescently labeled oligonucleotides for monitoring cellular uptake. Here, we report the design and synthesis of a thiol-responsive pro-fluorophore labeled oligonucleotide, and its fluorescence responsivity to glutathione in the test tube and live cells.     

DNA sequencing by denaturation: principle and thermodynamic simulations

We describe a new DNA sequencing method called sequencing by denaturation (SBD). A Sanger dideoxy sequencing reaction is performed on the templates on a solid surface to generate a ladder of DNA fragments randomly terminated by fluorescently labeled dideoxyribonucleotides. The labeled DNA fragments are sequentially denatured from the templates and the process is monitored by measuring the change in fluorescence intensities from the surface. By analyzing the denaturation profiles, the base sequence of the template can be determined. Using thermodynamic principles, we simulated the denaturation profiles of a series of oligonucleotides ranging from 12 to 32 bases and developed a base-calling algorithm to decode the sequences. These simulations demonstrate that DNA molecules up to 20 bases can be sequenced by SBD. Experimental measurements of the melting profiles of DNA fragments in solution confirm that DNA sequences can be determined by SBD. The potential limitations and advantages of SBD are discussed. With SBD, millions of sequencing reactions can be performed on a small area on a surface in parallel with a very small amount of sequencing reagents. Therefore, DNA sequencing by SBD could potentially result in a significant increase in speed and reduction in cost in large-scale genome resequencing.     

Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy

We demonstrate the specific identification of single nucleotide polymorphism (SNP) responsible for rifampicin resistance of Mycobacterium tuberculosis applying fluorescently labeled DNA-hairpin structures (smart probes) in combination with single-molecule fluorescence spectroscopy. Smart probes are singly labeled hairpin-shaped oligonucleotides bearing a fluorescent dye at the 5′ end that is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs, reflected in a strong increase in fluorescence intensity. An excess of unlabeled (‘cold’) oligonucleotides was used to prevent the formation of secondary structures in the target sequence and thus facilitates hybridization of smart probes. Applying standard ensemble fluorescence spectroscopy we demonstrate the identification of SNPs in PCR amplicons of mycobacterial rpoB gene fragments with a detection sensitivity of 10⁻⁸ M. To increase the detection sensitivity, confocal fluorescence microscopy was used to observe fluorescence bursts of individual smart probes freely diffusing through the detection volume. By measuring burst size, burst duration and fluorescence lifetime for each fluorescence burst the discrimination accuracy between closed and open (hybridized) smart probes could be substantially increased. The developed technique enables the identification of SNPs in 10⁻¹¹ M solutions of PCR amplicons from M.tuberculosis in only 100 s.     

In vitro protein microarrays for detecting protein-protein interactions: application of a new method for fluorescence labeling of proteins

Protein microarrays or proteome chips are potentially powerful tools for comprehensive analysis of protein-protein interactions. In interaction analysis, a set of immobilized proteins is arrayed on slides and each slide is probed with a set of fluorescently labeled proteins. Here we have developed and tested an in vitro protein microarray, in which both arraying and probing proteins were prepared by cell-free translation. The in vitro synthesis of fluorescently labeled proteins was accomplished by a new method: a fluorophore-puromycin conjugate was incorporated into a protein at the C-terminus on the ribosome. The resulting fluorescently labeled proteins were confirmed to be useful for probing protein-protein interactions on protein microarrays in model experiments. Since the in vitro protein microarrays can easily be extended to a high-throughput format and also combined with in vitro display technologies such as the streptavidin-biotin linkage in emulsions method (Doi and Yanagawa, FEBS Lett. 1999, 457, 227-230), our method should be useful for large-scale analysis of protein-protein interactions.