Fluorescent DNA Binding Research Articles

A polymer microfluidic chip for quantitative detection of multiple water- and foodborne pathogens using real-time fluorogenic loop-mediated isothermal amplification

Inexpensive, portable, and easy-to-use devices for rapid detection of microbial pathogens are needed to ensure safety of water and food. In this study, a disposable polymer microfluidic chip for quantitative detection of multiple pathogens using isothermal nucleic acid amplification was developed. The chip contains an array of 15 interconnected reaction wells with dehydrated primers for loop-mediated isothermal amplification (LAMP), and requires only a single pipetting step for dispensing of sample. To improve robustness of loading and amplification, hydrophobic air vents and microvalves were monolithically integrated in the multi-layered structure of the chip using an inexpensive knife plotter. For quantification, LAMP was performed with a highly fluorescent DNA binding dye (SYTO-82) and the reactions monitored in real-time using a low-cost fluorescence imaging system previously developed by our group (Ahmad et al., Biomed. Microdevices 13(5), 929-937). Starting from genomic DNA mixtures, the chip was successfully evaluated for rapid analysis of multiple virulence and marker genes of Salmonella, Campylobacter jejuni, Shigella, and Vibrio cholerae, enabling detection and quantification of 10-100 genomes per μl in less than 20 min. It is anticipated that the microfluidic chip, along with the real-time imaging system, may be a key enabling technology for developing inexpensive and portable systems for on-site screening of multiple pathogens relevant to food and water safety.

Evaluating quantitative methods for measuring plasmid copy numbers in single cells

The life of plasmids is a constant battle against fluctuations: failing to correct copy number fluctuations can increase the plasmid loss rate by many orders of magnitude, as can a failure to more evenly divide the copies between daughters at cell division. Plasmids are therefore long-standing model systems for stochastic processes in cells, much thanks to the efforts of Kurt Nordström to whose memory this issue is dedicated. Here we analyze a range of experimental methods for measuring plasmid copy numbers in single cells, focusing on challenges, trade-offs, and necessary experimental controls. In particular we analyze published and unpublished strategies to infer copy numbers from expression of plasmid-encoded reporters, direct labeling of plasmids with fluorescent probes or DNA binding proteins fused to fluorescent reporters, PCR based methods applied to single cell lysates, and plasmid-specific replication arrest. We conclude that no method currently exists to measure plasmid copy numbers in single cells, and that most methods are overwhelmed by various types of experimental noise. We also discuss how accurate methods can be developed.

Gene-Z: a device for point of care genetic testing using a smartphone

By 2012, point of care (POC) testing will constitute roughly one third of the $59 billion in vitro diagnostics market. The ability to carry out multiplexed genetic testing and wireless connectivity are emerging as key attributes of future POC devices. In this study, an inexpensive, user-friendly and compact device (termed Gene-Z) is presented for rapid quantitative detection of multiple genetic markers with high sensitivity and specificity. Using a disposable valve-less polymer microfluidic chip containing four arrays of 15 reaction wells each with dehydrated primers for isothermal amplification, the Gene-Z enables simultaneous analysis of four samples, each for multiple genetic markers in parallel, requiring only a single pipetting step per sample for dispensing. To drastically reduce the cost and size of the real-time detector necessary for quantification, loop-mediated isothermal amplification (LAMP) was performed with a high concentration of SYTO-81, a non-inhibiting fluorescent DNA binding dye. The Gene-Z is operated using an iPod Touch, which also receives data and carries out automated analysis and reporting via a WiFi interface. This study presents data pertaining to performance of the device including sensitivity and reproducibility using genomic DNA from Escherichia coli and Staphylococcus aureus. Overall, the Gene-Z represents a significant step toward truly inexpensive and compact tools for POC genetic testing.

SNP genotyping through the melting analysis of unlabelled oligonucleotide applied on dilute PCR amplicon

Adaptation of DNA melting analysis for polymorphic single nucleotides (SNPs) genotyping using an unlabeled oligonucleotide probe for polymorphic DNAs under the presence of fluorescent DNA binding dye necessitates a reaction condition where the probe efficiently associates with a target strand that is PCR amplified. We present experimental evidence that application of an unlabeled probe to a dilute PCR amplicon provides a condition such that the fluorescent signals gained subsequently by probe melting are sufficient to discriminate allelic identities. This approach is best exploited by adapting the multiplexing PCR technique in order to cover multiple SNPs for given samples. 3'-end modification of the probe is unnecessary as the amplicon dilution step provides a way of inactivating the polymerase through divalent cation chelation. With the use of low-cost reagents and ordinary laboratory equipment, this method offers a rapid, simple and cost-efficient way of SNP genotyping.

The Distribution of Cytoplasm and Nuclei within the Extra-radical Mycelia in Glomus intraradices, a Species of Arbuscular Mycorrhizal Fungi

Nuclear distribution within the extra-radical fungal structures and during spore production in the arbuscular mycorrhizae fungus Glomus intraradices was examined using an in vitro monoxenic culture system. A di-compartmental monoxenic culture system was modified using a nitrocellulose membrane and a coverglass slip for detailed observations. Nuclear distribution was observed using the fluorescent DNA binding probes SYBR Green I and DAPI. Both septate and non-septate mycelial regions were observed, but cytoplasmic contents were only found within non-septate mycelia. Nuclear fluorescent staining revealed that the non-septate hyphal region contained nuclei only with cytoplasm, and that nuclear distribution was limited by septa. Swollen hyphal bodies were often associated with septate and empty-looking hyphae. Cytoplasmic contents filled the swollen hyphal body from the non-septate hyphal region following removal of the septa. As a consequence, the swollen body developed into a new spore. These observations provide understanding about the distribution of AM fungal nuclei within extra-radical mycelia and during spore formation. The results suggest a mechanism by which the development of a cytoplasm-containing mycelium is controlled by the formation or removal of septa to efficiently maintain and proliferate essential contents. This mechanism may provide a survival strategy to the fungus.

S146 Microvascular endothelial cell apoptosis and dysregulation of Vascular Endothelial Growth Factor Receptor 2 (VEGF-R2) in response to cigarette smoke. New insights into the pathogenesis of emphysema

Microvascular injury may be a primary mechanism in the pathogenesis of emphysema. Rats treated with a VEGF-R2 antagonist develop emphysema that is caspase-3 dependent. Additionally, patients with emphysema have reduced...

Large T Antigen Promotes JC Virus Replication in G2-arrested Cells by Inducing ATM- and ATR-mediated G2 Checkpoint Signaling

Large T antigen (TAg) of the human polyomavirus JC virus (JCV) possesses DNA binding and helicase activities, which, together with various cellular proteins, are required for replication of the viral genome. We now show that JCV-infected cells expressing TAg accumulate in the G(2) phase of the cell cycle as a result of the activation of ATM- and ATR-mediated G(2) checkpoint pathways. Transient transfection of cells with a TAg expression vector also induced G(2) checkpoint signaling and G(2) arrest. Analysis of TAg mutants with different subnuclear localizations suggested that the association of TAg with cellular DNA contributes to the induction of G(2) arrest. Abrogation of G(2) arrest by inhibition of ATM and ATR, Chk1, and Wee1 suppressed JCV genome replication. In addition, abrogation of the G(2)-M transition by Cdc2 depletion disabled Wee1 depletion-induced suppression of JCV genome replication, suggesting that JCV replication is facilitated by G(2) arrest resulting from G(2) checkpoint signaling. Moreover, inhibition of ATM and ATR by caffeine suppressed JCV production. The observation that oligodendrocytes productively infected with JCV in vivo also undergo G(2) arrest suggests that G(2) checkpoint inhibitors such as caffeine are potential therapeutic agents for JCV infection.

Specific Interaction between Tomato HsfA1 and HsfA2 Creates Hetero-oligomeric Superactivator Complexes for Synergistic Activation of Heat Stress Gene Expression

In plants, a family of more than 20 heat stress transcription factors (Hsf) controls the expression of heat stress (hs) genes. There is increasing evidence for the functional diversification between individual members of the Hsf family fulfilling distinct roles in response to various environmental stress conditions and developmental signals. In response to hs, accumulation of both heat stress proteins (Hsp) and Hsfs is induced. In tomato, the physical interaction between the constitutively expressed HsfA1 and the hs-inducible HsfA2 results in synergistic transcriptional activation (superactivation) of hs gene expression. Here, we show that the interaction is strikingly specific and not observed with other class A Hsfs. Hetero-oligomerization of the two-component Hsfs is preferred to homo-oligomerization, and each Hsf in the HsfA1/HsfA2 hetero-oligomeric complex has its characteristic contribution to its function as superactivator. Distinct regions of the oligomerization domain are responsible for specific homo- and hetero-oligomeric interactions leading to the formation of hexameric complexes. The results are summarized in a model of assembly and function of HsfA1/A2 superactivator complexes in hs gene regulation.

A comparison of fluorescent DNA binding dyes for flow cytometric analysis of sporulating Saccharomyces cerevisiae

Flow cytometry is important tool for investigating DNA replication in sporulating Saccharomyces cerevisiae. However, flow cytometry data from maturing spores is often difficult to interpret due to extensive broadening of the fluorescence peaks. This problem is markedly improved by treatment of the spores with potassium hydroxide prior to staining.

Fluorescent Single-Stranded DNA Binding Protein as a Probe for Sensitive, Real-Time Assays of Helicase Activity

The formation and maintenance of single-stranded DNA (ssDNA) are essential parts of many processes involving DNA. For example, strand separation of double-stranded DNA (dsDNA) is catalyzed by helicases, and this exposure of the bases on the DNA allows further processing, such as replication, recombination, or repair. Assays of helicase activity and probes for their mechanism are essential for understanding related biological processes. Here we describe the development and use of a fluorescent probe to measure ssDNA formation specifically and in real time, with high sensitivity and time resolution. The reagentless biosensor is based on the ssDNA binding protein (SSB) from Escherichia coli, labeled at a specific site with a coumarin fluorophore. Its use in the study of DNA manipulations involving ssDNA intermediates is demonstrated in assays for DNA unwinding, catalyzed by DNA helicases.

DNA and BSA binding studies of novel tetracationic phthalocyanines

The novel tetracationic metallophthalocyanines having diethylmethyl aminoethylsulfanyl groups on the periphery have been synthesized and characterized. The compounds tend to form aggregates in aqueous solutions. The aggregation of ZnPc 4+ or CoPc 4+, but not that of CuPc 4+, was hindered by the addition of surfactants (Triton X-100 and Tween 20). Using absorption and fluorescence spectroscopic techniques, DNA and BSA binding properties of tetracationic ZnPc in the presence of the surfactants were also investigated.

Fluorometric Assay for Testing Rifampin Susceptibility of Mycobacterium tuberculosis Complex

The emergence and transmission of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) have raised concern about diagnostic delay associated with culture-based drug susceptibility testing methods. The association between rifampin resistance and MDR-TB or XDR-TB makes it an important genetic marker for genotypic drug susceptibility testing. In this article, we describe the analysis of the physical properties of the rifampin resistance-determining region (RRDR) in the rpoB gene by high-resolution thermal melt analysis as a method for detecting rifampin resistance in Mycobacterium tuberculosis complex. The RRDR from the M. tuberculosis complex was amplified by PCR from DNA templates extracted from sputum cultures of M. tuberculosis or the laboratory strain (H37Rv) in the presence of a fluorescent DNA binding dye. Subsequent mixing of the amplification products from the respective sputum cultures and the laboratory strain and thermocycling allowed the formation of DNA duplexes. The thermal denaturation properties of these DNA duplexes were determined by measuring the derivative of the intensity of fluorescence at different temperatures. Analysis of DNA extracted from 153 sputum cultures showed a sensitivity of 98% and a specificity of 100% for the detection of rifampin resistance compared to the "gold standard" culture-based phenotyping method. No statistical difference was detected in the performance of the method when applied to crude DNA from 134 boiled cultures. This method, named "FAST-Rif" ("fluorometric assay for susceptibility testing of rifampin"), allowed the rapid, reliable, and easy detection of genotypic rifampin resistance as a marker for MDR-TB and XDR-TB.

Hyperspectral Image Analysis of Live Cells in Various Cell Cycle Stages

In this study we have explored the use of hyperspectral imaging (HSI) to determine the cell-cycle status of live cells in culture. Live cancer cell lines in culture were either synchronized by release from nocodazole or arrested in various cell-cycle phases with serum starvation (G1), aphidicolin (S), or nocodazole (G2/M). The live cells were then stained with the fluorescent DNA binding dyes Heochst 33342 or Dyecycle orange along with propidium iodide or Mitotracker green. Microscopic HSI data was then collected using the PARISS HSI system. Classified spectra were incorporated into spectral libraries; and all spectra acquired from each sample were correlated with library spectra to a user-determined confidence threshold, generating a unique spectral signature for each sample. Examination of these spectral signatures revealed that all cell cycle phases could be objectively differentiated. Ongoing studies employing other viable cell fluorescent dyes, and dyes in combination may provide more robust spectral signatures defining the status and condition of living cells.

Study of Bacteriophage T4-encoded Dam DNA (Adenine-N6)-methyltransferase Binding with Substrates by Rapid Laser UV Cross-linking

DNA methyltransferases of the Dam family (including bacteriophage T4-encoded Dam DNA (adenine-N(6))-methyltransferase (T4Dam)) catalyze methyl group transfer from S-adenosyl-L-methionine (AdoMet), producing S-adenosyl-L-homocysteine (AdoHcy) and methylated adenine residues in palindromic GATC sequences. In this study, we describe the application of direct (i.e. no exogenous cross-linking reagents) laser UV cross-linking as a universal non-perturbing approach for studying the characteristics of T4Dam binding with substrates in the equilibrium and transient modes of interaction. UV irradiation of the enzyme.substrate complexes using an Nd(3+):yttrium aluminum garnet laser at 266 nm resulted in up to 3 and >15% yields of direct T4Dam cross-linking to DNA and AdoMet, respectively. Consequently, we were able to measure equilibrium constants and dissociation rates for enzyme.substrate complexes. In particular, we demonstrate that both reaction substrates, specific DNA and AdoMet (or product AdoHcy), stabilized the ternary complex. The improved substrate affinity for the enzyme in the ternary complex significantly reduced dissociation rates (up to 2 orders of magnitude). Several of the parameters obtained (such as dissociation rate constants for the binary T4Dam.AdoMet complex and for enzyme complexes with a nonfluorescent hemimethylated DNA duplex) were previously inaccessible by other means. However, where possible, the results of laser UV cross-linking were compared with those of fluorescence analysis. Our study suggests that rapid laser UV cross-linking efficiently complements standard DNA methyltransferase-related tools and is a method of choice to probe enzyme-substrate interactions in cases in which data cannot be acquired by other means.

Validation of a high-throughput in vitro alkaline elution/rat hepatocyte assay for DNA damage

In vitro alkaline elution is a sensitive and specific short term assay which measures DNA strand breakage in a mammalian test system (primary rat hepatocytes). This lab has previously demonstrated the performance of the assay with known genotoxic and non-genotoxic compounds. The methodology employed has relatively low sample throughput and is labor-intensive, requiring a great deal of manual processing of samples in a format that is not amenable to automation. Here, we present an automated version of the assay. This high-throughput alkaline elution assay (HT-AE) was made possible through 3 key developments: (1) DNA quantitation using PicoGreen ® and OliGreen ® fluorescent DNA binding dyes; (2) design and implementation of a custom automation system; and (3) reducing the assay to a 96-well plate format. The assay can now be run with 5–50 mg of test compound. HT-AE was validated in a similar manner as the original assay, including assessment of non-genotoxic and non-carcinogenic compounds and evaluation of cytotoxicity to avoid confounding effects of toxicity-associated DNA degradation. The validation test results from compounds of known genotoxic potential were used to set appropriate criteria to classify alkaline elution results for genotoxicity.

Detection and Identification of Cytochrome P-450 2C9 Alleles *1, *2, and *3 by High-Resolution Melting Curve Analysis of PCR Amplicons

High-resolution melting curve analysis using a fluorescent DNA binding dye can detect sequence variations in a closed-tube system without labeled primers or probes. We developed and verified a melting analysis assay for common single nucleotide polymorphisms of cytochrome P-450 (CYP) 2C9 that affect warfarin metabolism. We used this method to genotype 84 patients receiving warfarin. For wild-type, *1/*1, 50% fluorescence corresponded to a mean+/-SD of 87.17+/-0.05 degrees C, whereas *2/*2 was 0.4 degrees C lower. The *1/*2 melting curve was easily distinguished from *1/*1 and *2/*2 based on transition temperature and shape. Exon 7 showed a more complex melting curve; however, genotypes *1/*1, *1/*3, and *3/*3 were easily distinguishable. Melting curves were highly reproducible (SD of temperature for multiple fluorescence values 0.04 degrees C-0.11 degrees C; mean, 0.06 degrees C). Heterozygotes (*1/*2 or *1/*3) required significantly lower mean maintenance warfarin doses compared with wild-type (30.67 and 29.56 vs 42.81 mg/wk; P<.05). High-resolution melting analysis provides a simple and accurate method for genotyping of CYP2C9.

17. Visualization of the Intranuclear rAAV Replication Centers and Their Co-Localization with Double-Stranded DNA Break Repair Proteins

We have developed a method for the visualization of the sites of conversion of the ssDNA AAV genome to dsDNA inside the cell nucleus. This method is based on the utilization of a recombinant AAVlacO.14 vector, containing 112 LacR binding sites (lacO) cloned between the viral ITRs, which is used for the infection of cells that constitutively express a LacR-GFP fusion protein. The rationale of this approach is that the fluorescent sequence-specific DNA binding protein only binds its target site when this is present in a dsDNA form; this permits visualization and monitoring of ss to ds AAV genome conversion over time.

Determination of cell concentration in a plant cell suspension using a fluorescence microplate reader

Microscopic counting of plant cells is a very tedious and time-consuming process and is therefore seldom used to evaluate plant cell number on a routine basis. This study describes a fast and simple method to evaluate cell concentration in a plant cell suspension using a fluorescence microplate reader. Eschscholtzia californica cells were fixed in a mix of methanol and acetic acid (3:1) and stained with a fluorescent DNA binding dye (Hoechst 33258). Readings were done in a fluorescence microplate reader at 360/465 nm. Specific binding of the dye to double-stranded DNA was significantly favored over unspecific binding when 1.0 M Tris buffer at pH 7.5 containing 1.0 M NaCl and 75 microg ml(-1) of Hoechst 33258 was used. Fluorescence readings must be done between 4 min and 12 min following the addition of the staining solution to the sample. The microplate counting method provides a convenient, rapid and sensitive procedure for determining the cell concentration in plant cell suspensions. The assay has a linear detection range from 0.2 x 10(6) cells to 10.0 x 10(6) cells per milliliter (actual concentration in the tested cell suspension). The time needed to perform the microplate counting was 10% of that needed for the microscopic enumeration. However, this microplate counting method can only be used on genetically stable cell lines and on asynchronous cell suspensions.

Cell Classification in Human Peripheral Blood Using the Amnis ImageStream® System.

Amnis Corporation's ImageStream® system combines the sample handling and quantitative power of flow cytometry with high-resolution brightfield, darkfield, and fluorescence cellular imagery. The system simultaneously generates up to six images of each cell in flow and can acquire data sets consisting of tens of thousands of cells in just a few minutes, while offering fluorescence sensitivity equal to or better than flow cytometry. The image data are analyzed using Amnis' IDEAS® software, which automatically calculates over 200 morphometric and photometric features and associated statistics for each cell, identifying unique cell groups based not only on their fluorescence intensity signature but also on their morphological characteristics. The software offers the ability to view the imagery associated with any cell in a scatter plot, perform “virtual cell sorts” of user-specified sub-populations, and generate custom features of biological significance (e.g. N/C ratio). The ImageStream platform's ability to quantitate morphologic and immunofluorescent differences between very large numbers of cells in suspension make it particularly well suited for hematology.In the present study, human peripheral blood mononuclear cells were stained with a fluorescent DNA binding dye to reveal nuclear morphology, as well as fluorescently labeled mAb to various CD markers. Five images of each cell were acquired: brightfield (transmitted light), darkfield (laser side scatter), and three fluorescent colors for nuclear imagery and quantitation of the CD marker abundance. The object of the study was to identify morphometric parameters in the brightfield, darkfield, and nuclear imagery that would prove useful in hematologic cell type classification. The mAb to CD antigens provided a positive control for use in the evaluation of the of the various morphometric parameters. Parameters with discriminating power included cellular size and texture, darkfield intensity and granularity, and nuclear fluorescence intensity, texture, and shape. Cell types that could be automatically discriminated using these parameters in lieu of immunofluorescent markers included neutrophils, eosinophils, monocytes, and lymphocytes (including putative activated lymphocytes).In addition to forming the basis for an advanced ImageStream hematology platform, it is envisioned that the automated morphometric classification of blood cells will act as the foundation for a wide range of image-based cellular assays performed in peripheral blood (e.g. NF-kB translocation, apoptosis, mAb compartmentalization), allowing the differential quantitation of assay results in various cell types for the purposes of basic research, drug discovery, and clinical diagnostics.

Microtubule Stress Modifies Intra-Nuclear Location of Msh2 in Mouse Embryonic Fibroblasts

The maintenance of genomic stability in mitotic and meiotic cycles through mismatch repair (MMR) demands the co-ordination of MMR functions with multiple processes including cell cycle traverse, linked changes in microtubule dynamics, protein translocation at chromatin sites and checkpoint activation. We have studied changes in the intracellular location of the MMR protein Msh2 in response to mitosis, microtubule disruption by colcemid and DNA damage induction by cis-platin in mouse embryonic fibroblasts (MEFs). Image analysis indicated that MEFs have a normally high nuclear retention of Msh2 during interphase with a precipitous dispersal of protein from chromatin sites into the cytoplasm at mitosis. Dispersal was also observed in cisplatin- and colcemid-treated interphase MEFs without any change in the overall Msh2 levels throughout the cell cycle. There was no evidence of co-localisation of the punctate cytoplasmic Msh2 foci with any microtubule structures and knockout of Msh2 altered neither the extent of microtubule disruption nor the functional activation of the spindle assembly checkpoint by colcemid. Critically, extra-nuclear relocation of protein did not alter the ability to mount an Msh2-dependent G2 checkpoint delay in response to cisplatin-induced DNA damage. Depletion of the nuclear pool of Msh2 protein in cells undergoing dispersal was found to involve a rapid relocation of protein from AT-rich chromatin sites as defined by co-association studies exploiting a newly-characterised base-pair preference of the fluorescent DNA binding probe DRAQ5. The study reveals the unexpected mobility of MMR protein pools during the MEF cell cycle and in response to different stress-inducing agents. The results link for the first time microtubule-integrity with intra-nuclear Msh2 protein dynamics. The high nuclear retention of Msh2 in interphase MEFs is in contrast to human tumour cells while the observations on protein dispersal suggest that only low levels of nuclear-located Msh2 are needed for G2 checkpoint activation by DNA damage.