-
Methods in Molecular Biology (Clifton,... 2022High-throughput DNA fluorescence in situ hybridization (hiFISH) combines multicolor combinatorial DNA FISH staining with automated image acquisition and analysis to...
High-throughput DNA fluorescence in situ hybridization (hiFISH) combines multicolor combinatorial DNA FISH staining with automated image acquisition and analysis to visualize and localize tens to hundreds of genomic loci in up to millions of cells. hiFISH can be used to measure physical distances between pairs of genomic loci, radial distances from genomic loci to the nuclear edge or center, and distances between genomic loci and nuclear structures defined by protein or RNA markers. The resulting large datasets of 3D spatial distances can be used to study cellular heterogeneity in genome architecture and the molecular mechanisms underlying this phenomenon in a variety of cellular systems. In this chapter we provide detailed protocols for hiFISH to measure distances between genomic loci, including all steps involved in DNA FISH probe design and preparation, cell culture, DNA FISH staining in 384-well imaging plates, automated image acquisition and analysis, and, finally, statistical analysis.
Topics: Cell Nucleus; DNA; DNA Probes; Genome; In Situ Hybridization, Fluorescence
PubMed: 35867253
DOI: 10.1007/978-1-0716-2497-5_12 -
The FEBS Journal Nov 2007Optical-fiber bundles have been employed as a versatile substrate for the fabrication of high-density microwell arrays. In this minireview, we discuss the application of... (Review)
Review
Optical-fiber bundles have been employed as a versatile substrate for the fabrication of high-density microwell arrays. In this minireview, we discuss the application of optical-fiber-bundle arrays for a variety of biological problems. For genomics studies and microbial pathogen detection, individual beads have been functionalized with DNA probes and then loaded into the microwells. In addition, beads differentially responsive to vapors have been employed in an artificial olfaction system. Microwell arrays have also been loaded with living cells to monitor their individual response to biologically active compounds over long periods. Finally, the microwells have been sealed to enclose single enzyme molecules that can be used to measure individual molecule catalytic activity.
Topics: Biosensing Techniques; DNA Probes; Fiber Optic Technology; Microarray Analysis; Molecular Probe Techniques; Optical Fibers
PubMed: 17937772
DOI: 10.1111/j.1742-4658.2007.06078.x -
Angewandte Chemie (International Ed. in... Sep 2021Threading intercalators bind DNA with high affinities. Here, we describe single-molecule studies on a cell-permeant luminescent dinuclear ruthenium(II) complex that has...
Threading intercalators bind DNA with high affinities. Here, we describe single-molecule studies on a cell-permeant luminescent dinuclear ruthenium(II) complex that has been previously shown to thread only into short, unstable duplex structures. Using optical tweezers and confocal microscopy, we show that this complex threads and locks into force-extended duplex DNA in a two-step mechanism. Detailed kinetic studies reveal that an individual stereoisomer of the complex exhibits the highest binding affinity reported for such a mono-intercalator. This stereoisomer better preserves the biophysical properties of DNA than the widely used SYTOX Orange. Interestingly, threading into torsionally constrained DNA decreases dramatically, but is rescued on negatively supercoiled DNA. Given the "light-switch" properties of this complex on binding DNA, it can be readily used as a long-lived luminescent label for duplex or negatively supercoiled DNA through a unique "load-and-lock" protocol.
Topics: Coordination Complexes; DNA; DNA Probes; Molecular Structure; Ruthenium
PubMed: 34378843
DOI: 10.1002/anie.202108077 -
Proceedings of the National Academy of... Aug 2023Electronic detection of DNA oligomers offers the promise of rapid, miniaturized DNA analysis across various biotechnological applications. However, known all-electrical...
Electronic detection of DNA oligomers offers the promise of rapid, miniaturized DNA analysis across various biotechnological applications. However, known all-electrical methods, which solely rely on measuring electrical signals in transducers during probe-target DNA hybridization, are prone to nonspecific electrostatic and electrochemical interactions, subsequently limiting their specificity and detection limit. Here, we demonstrate a nanomechanoelectrical approach that delivers ultra-robust specificity and a 100-fold improvement in detection limit. We drive nanostructural DNA strands tethered to a graphene transistor to oscillate in an alternating electric field and show that the transistor-current spectra are characteristic and indicative of DNA hybridization. We find that the inherent difference in pliability between unpaired and paired DNA strands leads to the spectral characteristics with minimal influence from nonspecific electrostatic and electrochemical interactions, resulting in high selectivity and sensitivity. Our results highlight the potential of high-performance DNA analysis based on miniaturized all-electronic settings.
Topics: DNA; Nucleic Acid Hybridization; DNA Probes; Graphite; Hybridization, Genetic; Biosensing Techniques
PubMed: 37549255
DOI: 10.1073/pnas.2306130120 -
Methods (San Diego, Calif.) Apr 2016In situ hybridization is the technique by which specific RNA or DNA molecules are detected in cytological preparations. Basically it involves formation of a hybrid... (Review)
Review
In situ hybridization is the technique by which specific RNA or DNA molecules are detected in cytological preparations. Basically it involves formation of a hybrid molecule between an endogenous single-stranded RNA or DNA in the cell and a complementary single-stranded RNA or DNA probe. In its original form the probe was labeled with (3)H and the hybrid was detected by autoradiography. The first successful experiments in 1968 involved detection of the highly amplified ribosomal DNA in oocytes of the frog Xenopus, followed soon after by the reiterated "satellite DNA" in mouse and Drosophila chromosomes. Fluorescent probes were developed about ten years later.
Topics: Animals; Autoradiography; DNA; DNA Probes; Drosophila melanogaster; Fluorescent Dyes; History, 20th Century; History, 21st Century; In Situ Hybridization; Larva; Mice; Oocytes; Polytene Chromosomes; RNA; Salivary Glands; Tritium; Xenopus laevis
PubMed: 26655524
DOI: 10.1016/j.ymeth.2015.11.026 -
RNA (New York, N.Y.) Dec 2018Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive P-labeled DNA or RNA oligonucleotide probes....
Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive P-labeled DNA or RNA oligonucleotide probes. Alternatively, nonradioactive northern blot relies on chemiluminescent reactions triggered by horseradish peroxidase (HRP) conjugated probes. The use of regulated radioactive material and the complexity of chemiluminescent reactions and detection have hampered the adoption of northern blot techniques by the wider biomedical research community. Here, we describe a sensitive and straightforward nonradioactive northern blot method, which utilizes near-infrared (IR) fluorescent dye-labeled probes (irNorthern). We found that irNorthern has a detection limit of ∼0.05 femtomoles (fmol), which is slightly less sensitive than P-Northern. However, we found that the IR dye-labeled probe maintains the sensitivity after multiple usages as well as long-term storage. We also present alternative irNorthern methods using a biotinylated DNA probe, a DNA probe labeled by terminal transferase, or an RNA probe labeled during in vitro transcription. Furthermore, utilization of different IR dyes allows multiplex detection of different RNA species. Therefore, irNorthern represents a more convenient and versatile tool for RNA detection compared to traditional northern blot analysis.
Topics: Blotting, Northern; DNA Probes; Fluorescent Dyes; Nucleic Acid Hybridization; RNA; RNA Probes
PubMed: 30201850
DOI: 10.1261/rna.068213.118 -
Journal of Visualized Experiments : JoVE Feb 2022Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic marks in a...
Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic marks in a single cell and requiring data from other cells to distinguish signal from experimental background noise in a single cell. This paper describes a method to reuse the same single cell for iterative epigenomic analyses. In this experimental method, cellular proteins are first anchored to a polyacrylamide polymer instead of crosslinking them to protein and DNA, alleviating structural bias. This critical step allows repeated experiments with the same single cell. Next, a random primer with a scaffold sequence for proximity ligation is annealed to the genomic DNA, and the genomic sequence is added to the primer by extension using a DNA polymerase. Subsequently, an antibody against an epigenetic marker and control IgG, each labeled with different DNA probes, are bound to the respective targets in the same single cell. Proximity ligation is induced between the random primer and the antibody by adding a connector DNA with complementary sequences to the scaffold sequence of the random primer and the antibody-DNA probe. This approach integrates antibody information and nearby genome sequences in a single DNA product of proximity ligation. By enabling repeated experiments with the same single cell, this method allows an increase in data density from a rare cell and statistical analysis using only IgG and antibody data from the same cell. The reusable single cells prepared by this method can be stored for at least a few months and reused later to broaden epigenetic characterization and increase data density. This method provides flexibility to researchers and their projects.
Topics: DNA; DNA Probes; DNA-Directed DNA Polymerase; Epigenome; Epigenomics
PubMed: 35225292
DOI: 10.3791/63456 -
Biosensors & Bioelectronics Mar 2021An electrical immuno-sandwich assay utilizing an electrokinetic-based streaming current method for signal transduction is proposed. The method records the changes in...
An electrical immuno-sandwich assay utilizing an electrokinetic-based streaming current method for signal transduction is proposed. The method records the changes in streaming current, first when a target molecule binds to the capture probes immobilized on the inner surface of a silica micro-capillary, and then when the detection probes interact with the bound target molecules on the surface. The difference in signals in these two steps constitute the response of the assay, which offers better target selectivity and a linear concentration dependent response for a target concentration within the range 0.2-100 nM. The proof of concept is demonstrated by detecting different concentrations of Immunoglobulin G (IgG) in both phosphate buffered saline (PBS) and spiked in E. coli cell lysate. A superior target specificity for the sandwich assay compared to the corresponding direct assay is demonstrated along with a limit of detection of 90 pM in PBS. The prospect of improving the detection sensitivity was theoretically analysed, which indicated that the charge contrast between the target and the detection probe plays a crucial role in determining the signal. This aspect was then experimentally validated by modulating the zeta potential of the detection probe by conjugating negatively charged DNA oligonucleotides. The length of the conjugated DNA was varied from 5 to 30 nucleotides, altering the zeta potential of the detection probe from -9.3 ± 0.8 mV to -20.1 ± 0.9 mV. The measurements showed a clear and consistent enhancement of detection signal as a function of DNA lengths. The results presented here conclusively demonstrate the role of electric charge in detection sensitivity as well as the prospect for further improvement. The study therefore is a step forward in developing highly selective and sensitive electrokinetic assays for possible application in clinical investigations.
Topics: Biosensing Techniques; DNA; DNA Probes; Escherichia coli; Sensitivity and Specificity
PubMed: 33421763
DOI: 10.1016/j.bios.2020.112917 -
Nucleic Acids Research Sep 2002Molecular beacons (MBs) are hairpin-like fluorescent DNA probes that have single-mismatch detection capability. Although they are extremely useful for many...
Molecular beacons (MBs) are hairpin-like fluorescent DNA probes that have single-mismatch detection capability. Although they are extremely useful for many solution-based nucleic acid detections, MBs are expensive probes for applications that require the use of a large number of different DNA probes due to the high cost and tedious procedures associated with probe synthesis and purification. In addition, since both ends of MB probes are covalently modified with chromophores, they do not offer the flexibility for fluorophore change and the capability for surface immobilization through free DNA ends. In this report, we describe an alternative form of MB, denoted tripartite molecular beacon (TMB), that may help overcome these problems. A TMB uses an unmodified oligodeoxyribonucleotide that forms a MB-like structure with two universal single-stranded arms to bring on a universal pair of oligodeoxyribonucleotides modified separately with a fluorophore and a quencher. We found that TMBs are as effective as standard MBs in signaling the presence of matching nucleic acid targets and in precisely discriminating targets that differ by a single nucleotide. TMBs have the necessary flexibility that may make MBs more affordable for various nucleic acid detection applications.
Topics: Base Sequence; DNA; DNA Probes; Fluorescent Dyes; Nucleic Acid Conformation; Nucleic Acid Hybridization; Oligonucleotides
PubMed: 12235396
DOI: 10.1093/nar/gnf093 -
Bioconjugate Chemistry Sep 2022Fluorescent DNA probes were prepared in a modular approach using the "click" post-synthetic modification strategy. The new glycol-based module and DNA building block...
Fluorescent DNA probes were prepared in a modular approach using the "click" post-synthetic modification strategy. The new glycol-based module and DNA building block place just two carbons between the phosphodiester bridges and anchor the dye by an additional alkyne group. This creates a stereocenter in the middle of this artificial nucleoside substitute. Both enantiomers and a variety of photostable cyanine-styryl dyes as well as thiazole orange derivatives were screened as "clicked" conjugates in different surrounding DNA sequences. The combination of the ()-configured DNA anchor and the cyanylated cyanine-styryl dye shows the highest fluorescence light-up effect of 9.2 and a brightness of approximately 11,000 M cm. This hybridization sensitivity and fluorescence readout were further developed utilizing electron transfer and energy transfer processes. The combination of the hybridization-sensitive DNA building block with the nucleotide of 5-nitroindole as an electron acceptor and a quencher increases the light-up effect to 20 with the DNA target and to 15 with the RNA target. The fluorescence readout could significantly be enhanced to values between 50 and 360 by the use of energy transfer to a second DNA probe with commercially available dyes, like Cy3.5, Cy5, and Atto590, as energy acceptors at the 5'-end. The latter binary probes shift the fluorescent readout from the range of 500-550 nm to the range of 610-670 nm. The optical properties make these fluorescent DNA probes potentially useful for RNA imaging. Due to the strong light-up effect, they will not require washing procedures and will thus be suitable for live-cell imaging.
Topics: Alkynes; DNA; DNA Probes; Fluorescent Dyes; Glycols; Nucleosides; Nucleotides; RNA
PubMed: 35995426
DOI: 10.1021/acs.bioconjchem.2c00241