Sensitive DNA Probe Research Articles

Colorimetric detection of SARS‐CoV‐2 variants with paper‐based analytical devices

Colorimetric detection of SARS‐CoV‐2 variants with paper‐based analytical devices

Computer simulation and design of DNA-nanoprobe for fluorescence imaging DNA repair enzyme in living cells

In situ imaging of DNA repair enzymes in living cells gives important insights to diagnosis and explore the formation of various diseases. Fluorescent probes have become a powerful and widely used technique for their high sensitivity and real-time capabilities, but empirical design and optimization of the corresponding probes can be blind and time-consuming. Herein, we report a strategy combining experimental studies with molecular simulation techniques for the rapid and rational design of sensitive fluorescent DNA probes for a representative DNA repair enzyme human apurinic/apyrimidinic endonuclease 1 (APE1). Extended-system Adaptive Biasing Force (eABF) was applied to study the interaction mechanism between DNA probes with respect to the enzyme, based on which a novel sensitive DNA probe was designed efficiently and economically. Product inhibition effect which significantly limited the sensitivity of existing probes was eliminated by decreasing the key interactions between DNA probe products and enzyme. Experimental mechanism studies showed the existence of intramolecular hairpin structure in DNA probes is important for the recognition of APE1 and elimination of product inhibition, which is in consistent with the simulations. The obtained fluorescent DNA nanoprobe (Nanoprobe N) showed a high sensitivity for APE1 with the detection limit as low as 0.5 U/L (∼0.018 pM), and the Nanoprobe N could effectively respond to the variation of APE1 within cells and distinguish cancer cells from normal cells. This work not only demonstrated the effectiveness of molecular simulations in probe design, but also provided a reliable platform for accurate imaging of APE1 and effectors screening at single-cell level.

Ultrafast excited state dynamics and light-switching of [Ru(phen)2(dppz)]2+ in G-quadruplex DNA

The triplet metal to ligand charge transfer (3MLCT) luminescence of ruthenium (II) polypyridyl complexes offers attractive imaging properties, specifically towards the development of sensitive and structure-specific DNA probes. However, rapidly-deactivating dark state formation may compete with 3MLCT luminescence depending on different DNA structures. In this work, by combining femtosecond and nanosecond pump-probe spectroscopy, the 3MLCT relaxation dynamics of [Ru(phen)2(dppz)]2+ (phen = 1,10-phenanthroline, dppz = dipyridophenazine) in two iconic G-quadruplexes has been scrutinized. The binding modes of stacking of dppz ligand on the terminal G-quartet fully and partially are clearly identified based on the biexponential decay dynamics of the 3MLCT luminescence at 620 nm. Interestingly, the inhibited dark state channel in ds-DNA is open in G-quadruplex, featuring an ultrafast picosecond depopulation process from 3MLCT to a dark state. The dark state formation rates are found to be sensitive to the content of water molecules in local G-quadruplex structures, indicating different patterns of bound water. The unique excited state dynamics of [Ru(phen)2(dppz)]2+ in G-quadruplex is deciphered, providing mechanistic basis for the rational design of photoactive ruthenium metal complexes in biological applications.

Sensitive and selective DNA probe based on “turn-on” photoluminescence of C-dots@RGO

In this study, highly hydrophilic and photoluminescent sheets of reduced graphene oxide decorated with carbon dots (C-dots@RGO), methylene blue (MB), and a probe DNA have been used for the detection of DNA. The photoluminescence of C-dots@RGO is quenched by MB, which is restored in the presence of a target DNA. The combination of the C-dots@RGO, MB, and a DNA probe is selective for perfectly matched DNA over mismatched DNA, mainly because relative to single-stranded DNA, double-stranded DNA intercalates more strongly with MB, but interacts more weakly with RGO. In the presence of a target DNA, MB intercalates with the as-formed double-stranded DNA and is released from the surface of C-dots@RGO, leading to "turn-on" photoluminescence. The practicality of this assay has been validated by the determination of tumor suppressor gene BRCA1, with linearity over the concentration range from 25 to 250 nM and a limit of detection (LOD, at a signal-to-noise ratio of 3) of 14.6 nM. The C-dots@RGO probe provides higher specificity towards target DNA than towards common salts, carbohydrates, amino acids, and proteins found in real samples. Having the advantages of simplicity, cost-effectiveness, selectivity, and sensitivity, the DNA-P/C-dots@RGO-MB probe on microwells has been successfully employed for the detection of DNA, suggesting its potential for multiple analyses of DNA targets when various DNA probes are employed.

A tetraphenylethene-based zinc complex as a sensitive DNA probe by coordination interaction.

We developed a new DNA probe by utilizing the coordination interaction of Zn(2+) with DNA and the consequent emission. Because the coordination interactions do not depend on the length of the DNA, the new probe exhibited much higher sensitivity for the detection of short ssDNA than the corresponding probe based on electrostatic interactions.

The analytical application and spectral investigation of DNA–CPB–emodin and sensitive determination of DNA by resonance Rayleigh light scattering technique

A new sensitive DNA probe containing cetylpyridinium bromide (CPB) and emodin (an effective component of Chinese herbal medicine) was developed using the resonance Rayleigh light scattering (RLS) technique. A novel assay was first developed to detect DNA at nanogram level based on the ternary system of DNA–CPB–emodin. The RLS signal of DNA was enhanced remarkably in the presence of emodin–CPB, and the enhanced RLS intensity at 340.0nm was in direct proportion to DNA concentration in the range of 0.01–2.72μgmL−1 with a good linear relationship. The detection limit was 1.5ngmL−1. Three synthetic DNA samples were measured obtaining satisfactory results, the recovery was 97.6–107.3%.

Relationship between p53 overexpression, human papillomavirus infection, and lifestyle in Indian patients with head and neck cancers

Exposure to pollutants in the environment, tobacco and alcohol consumption, poor oral hygiene and opportunistic viral infections are important aetiological factors in head and neck cancers. In this study, we evaluate the complex interrelationships between these factors and molecular events such as p53 overexpression in causation of head and neck cancers. Tissue samples from 110 patients with histopathologically confirmed carcinoma of head and neck were analyzed from our tissue biorepository with patient consent. Data pertaining to their dietary habits, tobacco and alcohol consumption were abstracted. P53 overexpression was analysed by immunohistochemistry and HPV (high-risk genotype) were studied by Chromogenic in situ Hybridization using an ultra sensitive DNA probe. Chi-square analysis was done to determine relationships between proportions of dependent and independent variables. Bivariate relationships were determined between these variables using Spearman's rank correlation. Linear regression analysis was used to determine the best predictor variable influencing p53 expression. Tobacco consumption especially smoking cigarettes and all forms of tobacco consumption put together and HPV infection significantly influenced p53 overexpression. Forty-five percent of the studied cohort was positive for HPV. Regression analysis showed interaction between tobacco and HPV infection to be a primary predictor (β = 0.31, p = 0.02) for p53 expression. Tobacco in any form: chewing, smoking and snuffing, along with HPV infection is significantly associated with p53 overexpression. There is a high prevalence of HPV infection (45%) in Indian patients suggesting its possible role in the aetiology of head and neck cancer.

Methylene blue as a DNA probe for a comparative study of Cd 2+, Pb 2+ and Cr 3+ ions binding to calf thymus DNA

Methylene blue (MB) was developed as a sensitive DNA probe for a comparative study of Cd 2+, Pb 2+ and Cr 3+ ions binding with calf thymus DNA (ctDNA). The fluorescence intensity of the MB–ctDNA system increased dramatically when heavy metal ions (Cd 2+, Pb 2+ and Cr 3+ ions) were added, which indicated that some of the bound MB molecules were released from the ctDNA base pairs. To compare the binding affinity of these three different heavy metal ions with ctDNA, the relationships between the fluorescence intensity of the MB–ctDNA–M (Metal ions) system and the concentration ratio of [M]/[DNA(p)] were investigated. The results showed that the order of the binding affinity of heavy metal ions with ctDNA had the following sequence: Cr 3+> Cd 2+>Pb 2+. This order was further proved by the effects of heavy metal ions on the number of MB bound to ctDNA, the measurements of binding constants of these heavy metal ions to ctDNA, and the effects of heavy metal ions on the absorption of the MB–ctDNA system. In addition, the interaction mechanisms of Cd 2+, Pb 2+ and Cr 3+ ions with ctDNA were also discussed in detail. These results indicated that their interaction mechanisms are related to the concentration ratios of heavy metal ions to DNA.

Sensitive DNA probe for photochemical ligation prepared in click chemistry

We report a new photosensitive probe, that takes advantage of the electronic structural changes associated with the triazole ring formed in the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition. We succeeded in performing photoligation on the second time scale. Huisgen cycloaddition are employed to various labeling. As an example, we report a procedure for preparing the DNA probe for quick SNP typing.

Click Chemistry as an Efficient Method for Preparing a Sensitive DNA Probe for Photochemical Ligation

Click Chemistry as an Efficient Method for Preparing a Sensitive DNA Probe for Photochemical Ligation

Detection of translocation rob(1;29) in bull sperm using a specific DNA probe

The Robertsonian translocation rob(1;29), connected with reduced fertility, is widespread in different cattle breeds all over the world. After laser microdissection, DOP-PCR, cloning and sequencing, a highly sensitive translocation-specific DNA probe, suitable for detection of rob(1;29) in cattle metaphase and interphase cells, including spermatozoa was designed. Sperm samples of five heterozygous translocation carriers were analyzed using this probe and a control probe for chromosome 6. One thousand decondensed spermatozoa from each bull were scored. Signals of the translocation-specific probe were detected in 48.8, 50.9, 50.1, 51.8, and 54.8% of spermatozoa, respectively. In contrast, semen samples from five chromosomally normal bulls showed only signals of the control probe for chromosome 6. Semen from a chimeric (XX/XY) bull, showing 57.5% of 59,XX,rob(1;29) and 42.5% of 60,XY cells in cultured peripheral lymphocytes, was also examined using this probe. No sperm head with signal of the translocation-specific probe was observed among 1,000 spermatozoa analyzed in this bull, demonstrating that female cells do not pass through the process of spermatogenesis.

Exciton interaction in molecular beacons: a sensitive sensor for short range modifications of the nucleic acid structure.

Molecular beacons are hairpin-shaped, single-stranded oligonucleotides constituting sensitive fluorescent DNA probes widely used to report the presence of specific nucleic acids. In its closed form the stem of the hairpin holds the fluorophore covalently attached to one end, close to the quencher, which is covalently attached to the other end. Here we report that in the closed form the fluorophore and the quencher form a ground state intramolecular heterodimer whose spectral properties can be described by exciton theory. Formation of the heterodimers was found to be poorly sensitive to the stem sequence, the respective positions of the dyes and the nature of the nucleic acid (DNA or RNA). The heterodimer allows strong coupling between the transition dipoles of the two chromophores, leading to dramatic changes in the absorption spectrum that are not compatible with a Förster-type fluorescence resonance energy transfer (FRET) mechanism. The excitonic heterodimer and its associated absorption spectrum are extremely sensitive to the orientation of and distance between the dyes. Accordingly, the application of molecular beacons can be extended to monitoring short range modifications of the stem structure. Moreover, the excitonic interaction was also found to operate for doubly end-labeled duplexes.

Molecular beacons: novel fluorescent probes.

These sensitive fluorescent DNA probes can be used for real-time biomolecular recognition of unseparated target species.

Detection of Fastidious Bacteria Causing Citrus Greening Disease by Nonradioactive DNA Probes.

The greening fastidious bacteria (GFB), also tentatively named Liberobacter, cause citrus greening disease. They unevenly inhabit the sieve tubes of host plants in low concentration. A highly sensitive and specific DNA probe, developed with DNA cloning methods has been used to detect GFB in infected citrus hosts. One of the clones containing a 0.24-kb GFB-specific DNA fragment was labeled with biotinylated nucleotides by a PCR-labeling technique. A dot hybridization assay with the biotin-labeled DNA probe has been successfully used for detecting GFB in various citrus hosts including mandarins, tangors, sweet oranges and pummelos. This probe could specifically react with all GFB strains from several Asian countries, but not with those from South Africa. The probe developed has specificity and sensitivity sufficient enough to detect minute levels of GFB infection and, therefore, can be used in quarantine of the Asian greening disease.

Quantification of Gaeumannomyces graminis var. tritici in infected roots and soil using slot-blot hybridization

A slot-blot hybridization assay for quantifying Gaeumannomyces graminis var. tritici, in diseased roots and in soil, using a specific and sensitive DNA probe (pG158), has been developed. The pG158 probe hybridized strongly to pathogenic isolates of G. graminis var. tritici, moderately to G. graminis var. avenae and did not hybridize to non-pathogenic G. graminis var. graminis and to a wide range of other soil fungi. The pG158 contains a dispersed highly repeated sequence which provides the high level of sensitivity needed for a quantitative diagnostic test. The amount of Gaeumannomyces graminis var. tritici in soil can be estimated directly from the test soil without growing the susceptible plants or culturing the fungus. pG158 detects approximately 1·0 ng and 0·3 ng of G. graminis var. tritici DNA in 1 μg total DNA extracted from diseased roots and from highly infested soil organic matter, respectively. Both the high copy clone, pG158 and a single copy clone, pG217, clearly distinguished the three varieties of G. graminis and can be used for accurate intra-specific classifications of G. graminis isolates. The pG158 probe provides a tool for field researchers to determine levels of pathogenic isolates of G. graminis in soils and to study the ecology of this fungus.

A repetitive DNA probe for the sensitive detection of Fasciola hepatica infected snails

Epizootiologic studies on F. hepatica frequently use microscopic techniques for the detection of infected snails, however, the poor efficiency, sensitivity, and specificity associated with these techniques limit their usefulness. A DNA-based test for the identification of snails infected with larval stages of F. hepatica would solve these problems and enable a level of detection accuracy previosly unavailable. We have cloned and sequenced a 124 bp fragment of repetitive DNA from F. hepatica which hybridizes specifically with DNA of F. hepatica but not with DNA of its snail intermediate hosts Fossaria cubensis and Pseudosuccinea columella, or with DNA of Fascioloides magna and Paramphistomum liorchis, ruminant trematodes which share the same intermediate host and same enzootic range as F. hepatica. Using this 124 bp fragment as a probe, infection in saails was detected immediately following miracidial penetration, thus a sensitivity equivalent to the minimum biologic unit of the parasite was achieved. This 124 bp repeated sequence belongs to a large family of 124 bp repeats that share a high level of sequence identity and constitute spproximately 15% of the F. hepatica genome. We also report here the development of a quick and inexpensive DNA extraction protocol for use in field-collected snails. Thus, we have developed both a highly sensitive and specific DNA probe and a means to use the probe in a large epizootiologic study of F. hepatica where thousands of fieldcollected snails need to be assayed for infection.

Nucleic acid probes as a diagnostic method for tickborne hemoparasites of veterinary importance

An increased number of articles on the use of nucleic acid-based hybridization techniques for diagnostic purposes have been recently published. This article reviews nucleic acid-based hybridization as an assay to detect hemoparasite infections of economic relevance in veterinary medicine. By using recombinant DNA techniques, selected clones containing inserts of Anaplasma, Babesia, Cowdria or Theileria genomic DNA sequences have been obtained, and they are now available to be utilized as specific, highly sensitive DNA or RNA probes to detect the presence of the hemoparasite DNA in an infected animal. Either in an isotopic or non-isoropic detection system, probes have allowed scientists to test for—originally in samples collected from experimentally infected animals and later in samples collected in the field—the presence of hemoparasites during the prepatent, patent, convalescent, and chronic periods of the infection in the host. Nucleic acid probes have given researchers the opportunity to carry out genomic analysis of parasite DNA to differentiate hemoparasite species and to identify genetically distinct populations among and within isolates, strains and clonal populations. Prevalence of parasite infection in the tick vector can now be accomplished more specifically with the nucleic acid probes. Lately, with the advent of the polymerase chain reaction technique, small numbers of hemoparasites can be positively identified in the vertebrate host and tick vector. These techniques can be used to assess the veterinary epidemiological situation in a particular geographical region for the planning of control measures.

A sensitive DNA probe for the detection of Polymyxa betae in sugar beet roots

A Polymyxa betae genomic DNA fragment of 1·9 kb has been cloned from infected sugar beet roots and shown to contain DNA sequences that are repeated on at least four different chromosomes. A probe generated from this fragment detected four isolates of P. betae in infected roots and did not cross hybridize with DNA from healthy sugar beet, contaminating microorganisms commonly found within the sugar beet rhizosphere, or isolates of the closely related Polymyxa graminis and Olpidium brassicae. This probe provides a molecular detection method for P. betae which is quicker and likely to be more sensitive than conventional microscopic examination.

A highly specific and sensitive DNA probe derived from chromosomal DNA of Helicobacter pylori is useful for typing H. pylori isolates

HindIII-digested DNA fragments derived from an EcoRI-digested 6.5-kb fragment of chromosomal DNA prepared from Helicobacter pylori ATCC 43629 (type strain) were cloned into the pUC19 vector. A 0.86-kb insert was identified as a potential chromosomal DNA probe. The specificity of the probe was evaluated by testing 166 non-H. pylori bacterial strains representing 38 genera and 91 species which included aerobic, anaerobic, and microaerophilic flora of the upper and lower gastrointestinal tracts. None of the 166 non-H. pylori strains hybridized with this probe (100% specificity), and the sensitivity of this probe was also 100% when H. pylori isolates from 72 patients with gastritis and with the homologous ATCC type strain were tested by dot blot hybridization. The capability of this probe for differentiating between strains of H. pylori was evaluated by Southern blot hybridization of HaeIII-digested chromosomal DNA from 68 clinical isolates and the homologous ATCC type strain of H. pylori. Fifty-one unique hybridization patterns were seen among the 69 strains tested, demonstrating considerable genotypic variation among H. pylori clinical isolates. We propose that this probe would be of significant value for conducting epidemiologic studies.

Development of a DNA probe for identification of bovine herpesvirus 4

Summary A sensitive and specific dna probe for detection and identification of bovine herpesvirus 4 (bhv-4) was developed. Cloned fragments from a library of HindIII fragments of the bhv-4 (DN-599) genome were labeled with 32P or digoxigenin and were tested for sentitivity and specificity in detecting viral dna by dot-blot hybridization. Two probes were identified that detected 10 pg of purified viral dna, and detected viral dna in 0.001 μg of total dna extracted from bhv-4-infected cells. Both probes labeled with 32P and 1 labeled with digoxigenin detected viral dna in samples prepared from cells infected with 2 prototype strains (DN-599 and Movar 33/63) and 4 field isolates of bhv-4. The dna probes did not hybridize to total dna prepared from uninfected bovine cells or from cells infected with bhv-1, bhv-2, alcelaphine herpesvirus 1, pseudorabies virus, or equine herpesvirus 1. One probe, labeled with digoxigenin, was tested further by dot-blot hybridization with infected cell lysates that were simply treated with sodium dodecyl sulfate and proteinase K prior to application to the membrane, avoiding extensive dna purification procedures. This simplified procedure also resulted in specific detection of field isolates of bhv-4 and prototype strains of bhv-4.