Electrostatic-assembly-driven formation of supramolecular rhombus microparticles and their application for fluorescent nucleic acid detection

In this paper, we report on the large-scale formation of supramolecular rhombus microparticles (SRMs) driven by electrostatic assembly, carried out by direct mixing of an aqueous HAuCl(4) solution and an ethanol solution of 4,4'-bipyridine at room temperature. We further demonstrate their use as an effective fluorescent sensing platform for nucleic acid detection with a high selectivity down to single-base mismatch. The general concept used in this approach is based on adsorption of the fluorescently labeled single-stranded DNA (ssDNA) probe by SRM, which is accompanied by substantial fluorescence quenching. In the following assay, specific hybridization with its target to form double-stranded DNA (dsDNA) results in desorption of ssDNA from SRM surface and subsequent fluorescence recovery.     

Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy

Background

Rapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence microscopy and nucleic acid analogues have been proposed so far.

Methods and Results

Here we report a novel enzyme-free approach to efficiently detect cancer mutations. This assay includes gene-specific target enrichment followed by annealing to oligonucleotides containing locked nucleic acids (LNAs) and finally, detection by fluorescence microscopy. The LNA containing probes display high binding affinity and specificity to DNA containing mutations, which allows for the detection of mutation abundance with an intercalating EvaGreen dye. We used a second probe, which increases the overall number of base pairs in order to produce a higher fluorescence signal by incorporating more dye molecules. Indeed we show here that using EvaGreen dye and LNA probes, genomic DNA containing BRAF V600E mutation could be detected by fluorescence microscopy at low femtomolar concentrations. Notably, this was at least 1000-fold above the potential detection limit.

Conclusion

Overall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay, which allows for an accurate estimation of mutant abundance when the detection limit requirement is met. Using fluorescence microscopy, this approach presents the opportunity to detect DNA at single-molecule resolution and directly in the biological sample of choice.     

Simple and sensitive fluorescence sensing of methyl parathion based on the inner filter effect of p-nitrophenol on nitrogen-doped titanium carbide quantum dots

It is of great significance to develop an effective method for methyl parathion (MP) detection. Herein, a novel nitrogen-doped titanium carbide quantum dots (N-Ti₃C₂ QDs) was prepared and used to construct a simple and sensitive fluorescence sensing platform of MP by making use of inner filter effect (IFE). The prepared N-Ti₃C₂ QDs can exhibit strong blue fluorescence at 434 nm. Meanwhile, MP could hydrolyze to produce p-nitrophenol (p-NP) under alkaline conditions, which showed a characteristic ultraviolet-visible (UV-visible) absorption peak at 405 nm, resulting in the fluorescence of N-Ti₃C₂ QDs is effectively quenched by p-NP. In addition, the investigation of time-resolved fluorescence decays indicated that the corresponding quenching mechanism of p-NP on N-Ti₃C₂ QDs is due to the IFE. After optimizing the conditions, the as-developed fluorescence sensing platform displayed wide detection range (0.1–30 μg mL⁻¹) and low detection limit (0.036 μg mL⁻¹) for MP, and it was also successfully applied for MP analysis in real water samples, thus it is expected that this simple, sensitive and enzyme-free sensing platform shows great applications.     

Lateral flow microarrays: a novel platform for rapid nucleic acid detection based on miniaturized lateral flow chromatography

Widely used nucleic acid assays are poorly suited for field deployment where access to laboratory instrumentation is limited or unavailable. The need for field deployable nucleic acid detection demands inexpensive, facile systems without sacrificing information capacity or sensitivity. Here we describe a novel microarray platform capable of rapid, sensitive nucleic acid detection without specialized instrumentation. The approach is based on a miniaturized lateral flow device that makes use of hybridization-mediated target capture. The miniaturization of lateral flow nucleic acid detection provides multiple advantages over traditional lateral flow devices. Ten-microliter sample volumes reduce reagent consumption and yield analyte detection times, excluding sample preparation and amplification, of <120s while providing sub-femtomole sensitivity. Moreover, the use of microarray technology increases the potential information capacity of lateral flow. Coupled with a hybridization-based detection scheme, the lateral flow microarray (LFM) enables sequence-specific detection, opening the door to highly multiplexed implementations for broad-range assays well suited for point-of-care and other field applications. The LFM system is demonstrated using an isothermal amplification strategy for detection of Bacillus anthracis, the etiologic agent of anthrax. RNA from as few as two B. anthracis cells was detected without thermocycling hardware or fluorescence detection systems.     

Poly(m-phenylenediamine) nanospheres and nanorods: selective synthesis and their application for multiplex nucleic acid detection

In this paper, we demonstrate for the first time that poly(m-phenylenediamine) (PMPD) nanospheres and nanorods can be selectively synthesized via chemical oxidation polymerization of m-phenylenediamine (MPD) monomers using ammonium persulfate (APS) as an oxidant at room temperature. It suggests that the pH value plays a critical role in controlling the the morphology of the nanostructures and fast polymerization rate favors the anisotropic growth of PMPD under homogeneous nucleation condition. We further demonstrate that such PMPD nanostructures can be used as an effective fluorescent sensing platform for multiplex nucleic acid detection. A detection limit as low as 50 pM and a high selectivity down to single-base mismatch could be achieved. The fluorescence quenching is attributed to photoinduced electron transfer from nitrogen atom in PMPD to excited fluorophore. Most importantly, the successful use of this sensing platform in human blood serum system is also demonstrated.     

Locked Nucleic Acid Pentamers as Universal PCR Primers for Genomic DNA Amplification

Background

Multiplexing technologies, which allow for simultaneous detection of multiple nucleic acid sequences in a single reaction, can save a lot of time, cost and labor compared to traditional single reaction detection methods. However, the multiplexing method currently used requires precise handiwork and many complicated steps, making a new, simpler technique desirable. Oligonucleotides containing locked nucleic acid residues are an attractive tool because they have strong affinities for their complementary targets, they have been used to avoid dimer formation and mismatch hybridization and to enhance efficient priming. In this study, we aimed to investigate the use of locked nucleic acid pentamers for genomic DNA amplification and multiplex genotyping.

Results

We designed locked nucleic acid pentamers as universal PCR primers for genomic DNA amplification. The locked nucleic acid pentamers were able to prime amplification of the selected sequences within the investigated genomes, and the resulting products were similar in length to those obtained by restriction digest. In Real Time PCR of genomic DNA from three bacterial species, locked nucleic acid pentamers showed high priming efficiencies. Data from bias tests demonstrated that locked nucleic acid pentamers have equal affinities for each of the six genes tested from the Klebsiella pneumoniae genome. Combined with suspension array genotyping, locked nucleic acid pentamer-based PCR amplification was able to identify a total of 15 strains, including 3 species of bacteria, by gene- and species-specific probes. Among the 32 species used in the assay, 28 species and 50 different genes were clearly identified using this method.

Conclusion

As a novel genomic DNA amplification, the use of locked nucleic acid pentamers as universal primer pairs in conjunction with suspension array genotyping, allows for the identification of multiple distinct genes or species with a single amplification procedure. This demonstrates that locked nucleic acid pentamer-based PCR can be utilized extensively in pathogen identification.     

Microwave-accelerated method for ultra-rapid extraction of Neisseria gonorrhoeae DNA for downstream detection

Nucleic acid-based detection of gonorrhea infections typically require a two-step process involving isolation of the nucleic acid, followed by detection of the genomic target often involving polymerase chain reaction (PCR)-based approaches. In an effort to improve on current detection approaches, we have developed a unique two-step microwave-accelerated approach for rapid extraction and detection of Neisseria gonorrhoeae (gonorrhea, GC) DNA. Our approach is based on the use of highly focused microwave radiation to rapidly lyse bacterial cells, release, and subsequently fragment microbial DNA. The DNA target is then detected by a process known as microwave-accelerated metal-enhanced fluorescence (MAMEF), an ultra-sensitive direct DNA detection analytical technique. In the current study, we show that highly focused microwaves at 2.45 GHz, using 12.3-mm gold film equilateral triangles, are able to rapidly lyse both bacteria cells and fragment DNA in a time- and microwave power-dependent manner. Detection of the extracted DNA can be performed by MAMEF, without the need for DNA amplification, in less than 10 min total time or by other PCR-based approaches. Collectively, the use of a microwave-accelerated method for the release and detection of DNA represents a significant step forward toward the development of a point-of-care (POC) platform for detection of gonorrhea infections.     

A label‐free fluorescence method for detection of ureC gene and diagnosis of Helicobacter pylori infection

The feasibility of using a polymerase chain reaction (PCR)‐based label‐free DNA sensor for the detection of Helicobacter pylori is investigated. In particular, H. pylori ureC gene, a specific H. pylori nucleic acid sequence, was selected as the target sequence. In the presence of ureC gene, the target DNA could be amplified to dsDNA with much higher detectable levels. After added the SYBR green I (SGI), the sensing system could show high fluorescence. Thus, the target DNA can be detected by monitoring the change of fluorescence intensity of sensing system. The clinical performance of this method was determined by comparing it with another conventional technique urea breath test (UBT). The result also showed good distinguishing ability between negative and positive patient, which was in good agreement with that obtained by the UBT. It suggests that the label‐free fluorescence‐based method is more suitable for infection confirmation test of H. pylori. This approach offers great potential for simple, sensitive and cost‐effective identification of H. pylori infection.     

Tenascin-R promotes assembly of the extracellular matrix of perineuronal nets via clustering of aggrecan

Perineuronal nets (PNs) in the brains of tenascin-R-deficient (tn-r^−/−) mice develop in temporal concordance with those of wild-type (tn-r^+/+) mice. However, the histological appearance of PNs is abnormal in adult tn-r^−/− mice. Here, we investigated whether similar defects are also seen in dissociated and organotypic cultures from hippocampus and forebrain of tn-r^−/− mice and whether the structure of PNs could be normalized. In tn-r^−/− cultures, accumulations of several extracellular matrix molecules were mostly associated with somata, whereas dendrites were sparsely covered, compared with tn-r^+/+ mice. Experiments to normalize the structure of PNs in tn-r^−/− organotypic slice cultures by depolarization of neurons, or by co-culturing tn-r^+/+ and tn-r^−/− brain slices failed to restore a normal PN phenotype. However, formation of dendritic PNs in cultures was improved by the application of tenascin-R protein and rescued by polyclonal antibodies to aggrecan and a bivalent, but not monovalent form of the lectin Wisteria floribunda agglutinin. These results show that tenascin-R and aggrecan are decisive contributors to formation and stabilization of PNs and that tenascin-R may implement these functions by clustering of aggrecan. Proposed approaches for restoration of normal PN structure are noteworthy in the context of PN abnormalities in neurological disorders, such as epilepsy, schizophrenia and addiction.     

Peroxidative oxidation of leuco-dichlorofluorescein by prostaglandin H synthase in prostaglandin biosynthesis from polyunsaturated fatty acids

Prostaglandin H synthase can oxidize arachidonic acid with leuco-dichlorofluorescein as reducing cosubstrate. Addition of 0.5 mM phenol increases the oxidation of leuco-dichlorofluorescein 5-fold, probably by acting as a cyclic intermediate in the oxidation. Tetramethyl-p-phenylenediamine is also oxidized as cosubstrate. Its oxidation is not influenced by phenol. A stoichiometry of close to one mole of tetramethyl-p-phenylenediamine or leuco-dichlorofluorescein consumed per mole of arachidonic acid was found in the initial phase of the reaction. In the presence of phenol + leuco-dichlorofluorescein, the oxidation rate of arachidonic acid is about 40% lower than with phenol alone as cosubstrate. Since dichlorofluorescein has a molar extinction coefficient of 91 · 10³ at 502 nm, the oxidation of less than 1 μM leuco-dichlorofluorescein can be detected spectrophotometrically. The rate of extinction change with leuco-dichlorofluorescein (at 502 nm) is about 4-fold more rapid than with tetramethyl-p-phenylenediamine (at 611 nm). With this spectrophotometric assay we have confirmed that arachidonic acid, linolenic acid, adrenic acid, γ-linolenic acid, eicosapentaenoic acid, are substrates for prostaglandin H synthase with decreasing reaction rates in the mentioned order. The same order of reaction rates were found when oxygen consumption was measured. The assay also shows that docosahexaenoic acid is substrate for the enzyme. The reaction rate of the enzyme evidently is decreased both by a n − 3 double bond and by deviation from a 20 carbon chain length of the fatty acid substrate.     

Rectangular coordination polymer nanoplates: large-scale, rapid synthesis and their application as a fluorescent sensing platform for DNA detection

In this paper, we report on the large-scale, rapid synthesis of uniform rectangular coordination polymer nanoplates (RCPNs) assembled from Cu(II) and 4,4'-bipyridine for the first time. We further demonstrate that such RCPNs can be used as a very effective fluorescent sensing platform for multiple DNA detection with a detection limit as low as 30 pM and a high selectivity down to single-base mismatch. The DNA detection is accomplished by the following two steps: (1) RCPN binds dye-labeled single-stranded DNA (ssDNA) probe, which brings dye and RCPN into close proximity, leading to fluorescence quenching; (2) Specific hybridization of the probe with its target generates a double-stranded DNA (dsDNA) which detaches from RCPN, leading to fluorescence recovery. It suggests that this sensing system can well discriminate complementary and mismatched DNA sequences. The exact mechanism of fluorescence quenching involved is elucidated experimentally and its use in a human blood serum system is also demonstrated successfully.     

Comparison of DNA and RNA quantification methods suitable for parameter estimation in metabolic modeling of microorganisms

Recent developments in cellular and molecular biology require the accurate quantification of DNA and RNA in large numbers of samples at a sensitivity that enables determination on small quantities. In this study, five current methods for nucleic acid quantification were compared: (i) UV absorbance spectroscopy at 260 nm, (ii) colorimetric reaction with orcinol reagent, (iii) colorimetric reaction based on diphenylamine, (iv) fluorescence detection with Hoechst 33258 reagent, and (v) fluorescence detection with thiazole orange reagent. Genomic DNA of three different microbial species (with widely different G+C content) was used, as were two different types of yeast RNA and a mixture of equal quantities of DNA and RNA. We can conclude that for nucleic acid quantification, a standard curve with DNA of the microbial strain under study is the best reference. Fluorescence detection with Hoechst 33258 reagent is a sensitive and precise method for DNA quantification if the G+C content is less than 50%. In addition, this method allows quantification of very low levels of DNA (nanogram scale). Moreover, the samples can be crude cell extracts. Also, UV absorbance at 260 nm and fluorescence detection with thiazole orange reagent are sensitive methods for nucleic acid detection, but only if purified nucleic acids need to be measured.     

Determination of nucleic acid by [tetra‐(3‐methoxy‐4‐hydroxyphenyl)]–Tb3+ porphyrin as the fluorescence spectral probe in bis(2‐ethylhexyl)sulfosuccinate sodium salt micelle system

A new system for the determination of nucleic acid by rare earth metallic porphyrin of [tetra‐(3‐methoxy‐4‐hydroxyphenyl)]–Tb³⁺ [T(3‐MO‐4HP)–Tb³⁺] porphyrin as fluorescence spectral probe has been developed in this paper. Nucleic acid can enhance the fluorescence intensity of the T(3‐MO‐4HP)–Tb³⁺ porphyrin in the presence of bis(2‐ethylhexyl)sulfosuccinate sodium salt(AOT) micelle. In pH 8.00 Tris–HCl buffer solution, under optimum conditions, the enhanced fluorescence intensity is in proportion to the concentration of nucleic acids in the range of 0.05–3.00 µg mL^?1 for calf thymus DNA (ct DNA) and 0.03–4.80 µg mL^?1 for fish sperm DNA(fs DNA). Their detection limits are 0.03 and 0.01 µg mL^?1, respectively. In addition, the binding interaction mechanism between T(3‐MO‐4HP)–Tb³⁺ porphyrin and ct DNA is also investigated by resonance scattering and fluorescence spectra. The maximum binding number is calculated by molar ratio method. The new system can be used for the determination of nucleic acid in pig liver, yielding satisfactory results. Copyright © 2009 John Wiley & Sons, Ltd.     

Clinical validation of integrated nucleic acid and protein detection on an electrochemical biosensor array for urinary tract infection diagnosis

Background

Urinary tract infection (UTI) is a common infection that poses a substantial healthcare burden, yet its definitive diagnosis can be challenging. There is a need for a rapid, sensitive and reliable analytical method that could allow early detection of UTI and reduce unnecessary antibiotics. Pathogen identification along with quantitative detection of lactoferrin, a measure of pyuria, may provide useful information towards the overall diagnosis of UTI. Here, we report an integrated biosensor platform capable of simultaneous pathogen identification and detection of urinary biomarker that could aid the effectiveness of the treatment and clinical management.

Methodology/Principal Findings

The integrated pathogen 16S rRNA and host lactoferrin detection using the biosensor array was performed on 113 clinical urine samples collected from patients at risk for complicated UTI. For pathogen detection, the biosensor used sandwich hybridization of capture and detector oligonucleotides to the target analyte, bacterial 16S rRNA. For detection of the protein biomarker, the biosensor used an analogous electrochemical sandwich assay based on capture and detector antibodies. For this assay, a set of oligonucleotide probes optimized for hybridization at 37°C to facilitate integration with the immunoassay was developed. This probe set targeted common uropathogens including E. coli, P. mirabilis, P. aeruginosa and Enterococcus spp. as well as less common uropathogens including Serratia, Providencia, Morganella and Staphylococcus spp. The biosensor assay for pathogen detection had a specificity of 97% and a sensitivity of 89%. A significant correlation was found between LTF concentration measured by the biosensor and WBC and leukocyte esterase (p<0.001 for both).

Conclusion/Significance

We successfully demonstrate simultaneous detection of nucleic acid and host immune marker on a single biosensor array in clinical samples. This platform can be used for multiplexed detection of nucleic acid and protein as the next generation of urinary tract infection diagnostics.     

Simple System for Isothermal DNA Amplification Coupled to Lateral Flow Detection

Infectious disease diagnosis in point-of-care settings can be greatly improved through integrated, automated nucleic acid testing devices. We have developed an early prototype for a low-cost system which executes isothermal DNA amplification coupled to nucleic acid lateral flow (NALF) detection in a mesofluidic cartridge attached to a portable instrument. Fluid handling inside the cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard scalable manufacturing techniques such as injection molding. Nucleic acid amplification occurs in a two-layer pouch that enables efficient heat transfer. We have demonstrated as proof of principle the amplification and detection of Mycobacterium tuberculosis (M.tb) genomic DNA in the cartridge, using either Loop Mediated Amplification (LAMP) or the Exponential Amplification Reaction (EXPAR), both coupled to NALF detection. We envision that a refined version of this cartridge, including upstream sample preparation coupled to amplification and detection, will enable fully-automated sample-in to answer-out infectious disease diagnosis in primary care settings of low-resource countries with high disease burden.     

A nucleotide-independent nitroxide probe reports on site-specific stereomeric environment in DNA

In this report, stereospecific structural and dynamic features in DNA are studied using the site-directed spin labeling technique. A stable nitroxide radical, 1-oxyl-4-bromo-2,2,5,5-tetramethylpyrroline (R5a), was attached postsynthetically to phosphorothioates that were chemically introduced, one at a time, at five sites of a DNA duplex. The two phosphorothioate diastereomers (R_p or S_p) were separated, and nitroxide rotational motions were monitored using electron paramagnetic resonance spectroscopy. The resulting spectra vary according to diastereomer identity and location of the labeling site, with R_p-R5a spectra effectively reporting on local DNA structural features and S_p-R5a spectra sensing variations in local DNA motions. This establishes R_p- and S_p-R5a as unique probes for investigating nucleic acids in a site- and stereospecific manner, which may aid studies of stereospecific DNA/protein interactions. In addition, weighted averages of individual R_p and S_p spectra match those of R5a attached to mixed diastereomers. This suggests that R5a linked to mixed diastereomers reports on the composite behaviors of R_p- and S_p-R5a and is useful in initial probing of the DNA local environment. This work advances understanding of R5a/DNA coupling, and is a key step forward in developing a nucleotide-independent spectroscopic probe for studying nucleic acids.     

Electrostatic analysis of the hepatitis C virus NS3 helicase reveals both active and allosteric site locations

Multi-conformation continuum electrostatics (MCCE) was used to analyze various structures of the NS3 RNA helicase from the hepatitis C virus in order to determine the ionization state of amino acid side chains and their pK_as. In MCCE analyses of HCV helicase structures that lacked ligands, several active site residues were identified to have perturbed pK_as in both the nucleic acid binding site and in the distant ATP-binding site, which regulates helicase movement. In all HCV helicase structures, Glu493 was unusually basic and His369 was abnormally acidic. Both these residues are part of the HCV helicase nucleic acid binding site, and their roles were analyzed by examining the pH profiles of site-directed mutants. Data support the accuracy of MCCE predicted pK_a values, and reveal that Glu493 is critical for low pH enzyme activation. Several key residues, which were previously shown to be involved in helicase-catalyzed ATP hydrolysis, were also identified to have perturbed pK_as including Lys210 in the Walker-A motif and the DExD/H-box motif residues Asp290 and His293. When DNA was present in the structure, the calculated pK_as shifted for both Lys210 and Asp290, demonstrating how DNA binding might lead to electrostatic changes that stimulate ATP hydrolysis.     

Note from the Biological Stain Commission: Certification of Wright Stain Solution

DNA fluorochrome staining with Hoechst 33258 bisbenzimide is commonly used for detection of mycoplasma contamination in cell cultures. Photobleaching of Hoechst 33258 is pronounced under the conditions of intense illumination, high magnification and resolution required for detection of mycoplasmas. To reduce photobleaching we investigated the effects of some antioxidant molecules, p-phenylenediamine (PPD), n-propyl gallate (NPG) and 1,4-diazabicyclo(2,2,2)octane (DABCO), which are known to reduce the fading rate of fluorescein. Mycoplasma-contaminated cell monolayers were stained with Hoechst 33258 and mounted in glycerol containing different amounts of antioxidant additives. The cells were examined in an epifluorescence microscope, and the emitted light intensity was recorded. Results showed that PPD and, to a lower degree, NPG, retarded the photobleaching of Hoechst 33258-stained cells, whereas DABCO was not effective. However, fluorescence half-life was increased about three-fold by NPG and almost 20-fold by PPD. The rate of fluorescence fading of Hoechst 33258 can therefore be retarded by PPD, with obvious advantages for reading and photographic recording of results.     

Genotoxicity testing of five compounds in three Drosophila short-term somatic assays

To provide further background data for the somatic mutation and/or recombination tests in Drosophila melanogaster, we have evaluated the responses in 3 assyas (zeste-white, white-ivory and wing spot) of 5 chemicals classified by the U.S. National Toxicology Program (NTP) as genotoxic non-carcinogens (or ambiguous). The selected compounds were 2-chloromethylpyridine, 1-nitronaphthalene, 4-nitro-o-phenylenediamine, 3-nitropropionic acid and p-phenylenediamine. Our results show that all the compounds tested produce significant increases in the frequency of mutant clones, in at least one of the assays, p-phenylenediamine being the compound which present a clearer mutagenic activity, and the wing spot test, the assay the detects more genotoxic compounds (4/5).     

Detection of DNA Damage in Prokaryotes by Terminal Deoxyribonucleotide Transferase-Mediated dUTP Nick End Labeling

Numerous agents can damage the DNA of prokaryotes in the environment (e.g., reactive oxygen species, irradiation, and secondary metabolites such as antibiotics, enzymes, starvation, etc.). The large number of potential DNA-damaging agents, as well as their diverse modes of action, precludes a simple test of DNA damage based on detection of nucleic acid breakdown products. In this study, free 3′-OH DNA ends, produced by either direct damage or excision DNA repair, were used to assess DNA damage. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) is a procedure in which 3′-OH DNA ends are enzymatically labeled with dUTP-fluorescein isothiocyanate using TdT. Cells labeled by this method can be detected using fluorescence microscopy or flow cytometry. TUNEL was used to measure hydrogen peroxide-induced DNA damage in the archaeon Haloferax volcanii and the bacterium Escherichia coli. DNA repair systems were implicated in the hydrogen peroxide-dependent generation of 3′-OH DNA ends by the finding that the protein synthesis inhibitors chloramphenicol and diphtheria toxin blocked TUNEL labeling of E. coli and H. volcanii, respectively. DNA damage induced by UV light and bacteriophage infection was also measured using TUNEL. This methodology should be useful in applications where DNA damage and repair are of interest, including mutant screening and monitoring of DNA damage in the environment.