Labeled DNA Probes Research Articles

Cytological and molecular characterization of wheat-Hordeum chilense chromosome 7Hch introgression lines

Chromosome 7Hch of Hordeum chilense carries the Phytoene synthase 1 (Psy1) gene encoding the first step in the carotenoid biosynthetic pathway. As such it can be used in the improvement of seed carotenoid content in wheat. Four introgressions of chromosome 7Hch into wheat have been characterized by in situ hybridization of labeled DNA probes and by several sets of DNA markers. Chromosome-specific SSR were used for the identification of wheat chromosomes. Besides 113 conserved orthologous set (COS) markers were tested for homoeologous group 7, of which 97 amplified in H. chilense and 32 were polymorphic between H. chilense and wheat, and 28 expressed sequence tag (EST) barley markers previously allocated to chromosome 7. A total of 60 markers (32 COS and 28 EST) were allocated to chromosome 7Hch with 28 assigned to 7HchS and 22 to 7HchL. A combination of in situ probing and marker genotyping have shown that among the four introgressions there was a substitution line 7Hch (7D), a ditelosomic addition line for the long arm of 7Hch and two homozygous centric translocations 7HchS·2DS and 7HchS·5AL. The Psy1 gene was localized on the short arm of 7Hch. The positions of markers from the international barley consortium map (IBSC2012) were determined and the comparative arm location between H. chilense and H. vulgare is discussed. The genetic stocks characterized here include new wheat-H. chilense recombinations useful for genetic studies and with a potential for breeding.

Transformation of a Ptr ToxB Nonproducing Isolate of Pyrenophora tritici‐repentis with the ToxB Gene Confers Pathogenicity on a Toxin‐Sensitive Wheat Genotype

ABSTRACTThe causal agent of tan spot of wheat, Pyrenophora tritici‐repentis, is a necrotrophic ascomycete that produces multiple host‐selective toxins (HSTs) responsible for the development of destructive foliar lesions. Ptr ToxB, a small proteinaceous HST produced by some races of P. tritici‐repentis, causes extensive chlorosis on toxin‐sensitive wheat genotypes. To investigate the role of Ptr ToxB in the pathogenicity and virulence of P. tritici‐repentis, the open reading frame of the encoding gene, ToxB, was cloned from a wild‐type race 5 isolate of the fungus and transferred into a Ptr ToxB nonproducing isolate of race 2. Ectopic integration of ToxB was confirmed by Southern blotting analysis with a digoxigenin (DIG)‐labeled DNA probe, while the abundance of ToxB transcript in culture was evaluated by quantitative reverse transcriptase polymerase chain reaction (qRT‐PCR). The production and secretion of Ptr ToxB were assessed by Western blotting analysis of the transformed fungal strains. The pathogenicity of the transformants was tested by inoculation on Ptr ToxB‐sensitive and insensitive wheat genotypes. The Ptr ToxB‐producing transformants were able to induce chlorosis in a host‐specific manner only on the toxin‐sensitive host genotype, resulting in significantly higher levels of disease than those caused by the nontransformed isolate. The extent of chlorosis was well correlated with the amount of Ptr ToxB produced. These results demonstrate that the acquisition of Ptr ToxB‐producing ability is a sufficient condition for pathogenicity in P. tritici‐repentis and confirm the role of this toxin in mediating the compatibility between the fungus and different genotypes of its wheat host.

Mechanism of DNA adsorption and desorption on graphene oxide.

Graphene oxide (GO) adsorbing a fluorophore-labeled single-stranded (ss) DNA serves as a sensor system because subsequent desorption of the adsorbed probe DNA from GO in the presence of complementary target DNA enhances the fluorescence. In this study, we investigated the interaction of single- and double-stranded (ds) DNAs with GO by using a fluorescently labeled DNA probe. Although GO is known to preferentially interact with ssDNA, we found that dsDNA can also be adsorbed on GO, albeit with lower affinity. Furthermore, the status of ssDNA or dsDNA previously adsorbed on the GO surface was investigated by adding complementary or noncomplementary DNA (cDNA or non-cDNA) to the adsorption complex. We observed that hybridization occurred between the cDNA and the probe DNA on the GO surface. On the basis of the kinetics driven by the incoming additional DNA, we propose a mechanism for the desorption of the preadsorbed probe DNA from the GO surface: the desorption of the GO-adsorbed DNA was facilitated following its hybridization with cDNA on the GO surface; when the GO surface was almost saturated with the adsorbed DNA, nonspecific desorption dominated the process through a simple displacement of the GO-adsorbed DNA molecules by the incoming DNA molecules because of the law of mass action. Our results can be applied to design appropriate DNA probes and to choose proper GO concentrations for experimental setups to improve specific signaling in many biosensor systems based on the GO platform.

Aneugenic effects of epirubicin in somatic and germinal cells of male mice.

The ability of the antineoplastic agent epirubicin to induce aneuploidy and meiotic delay in the somatic and germinal cells of male mice was investigated by fluorescence in situ hybridization assay using labeled DNA probes and BrdU-incorporation assay. Mitomycin C and colchicine were used as positive controls for clastogen and aneugen, respectively, and these compounds produced the expected responses. The fluorescence in situ hybridization assay with a centromeric DNA probe for erythrocyte micronuclei showed that epirubicin is not only clastogenic but also aneugenic in somatic cells in vivo. By using the BrdU-incorporation assay, it could be shown that the meiotic delay caused by epirubicin in germ cells was approximately 48 h. Disomic and diploid sperm were shown in epididymal sperm hybridized with DNA probes specific for chromosomes 8, X and Y after epirubicin treatment. The observation that XX- and YY-sperm significantly prevailed over XY-sperm indicates missegregation during the second meiotic division. The results also suggest that earlier prophase stages contribute less to epirubicin-induced aneuploidy. Both the clastogenic and aneugenic potential of epirubicin can give rise to the development of secondary tumors and abnormal reproductive outcomes in cured cancer patients and medical personnel exposed to epirubicin.

Addressing unmet clinical needs: FISHing for bile duct cancer.

Addressing unmet clinical needs: FISHing for bile duct cancer.

Highly sensitive photoelectrochemical assay for DNA methyltransferase activity and inhibitor screening by exciton energy transfer coupled with enzyme cleavage biosensing strategy

Highly sensitive DNA methyltransferase (MTase) activity and inhibitor screening photoelectrochemical (PEC) assay was developed based on the exciton energy transfer (EET) effect coupled with site-specific cleavage of restriction endonuclease (HpaII). The assay was designed by integrating the Au nanoparticles (NPs) labeled probe DNA (pDNA-Au) with CdSe quantum dots (QDs). The strong EET effect between Au NPs and CdSe QDs resulted in the dramatic decrease of photocurrent signal. The pDNA carried a sensing region for specifically recognizing target DNA (tDNA) and hybridizing with it to form a DNA duplex. With the site-specific cleavage of HpaII, the DNA duplex could be cleaved and Au NPs would be released, which broke the EET and resulted in the restoration of photocurrent signal. However, when the DNA duplex was methylated by M.SssI MTase, this cleavage of HpaII was blocked, and therefore the unbroken EET effect kept the lower photocurrent signal. That was, the restored photocurrent was inversely proportional to the MTase activity. Based on this strategy, the PEC assay could determine as low as ~0.0042U/mL of M.SssI MTase with a linear range from 0.01 to 150U/mL. In addition, the assay could be used for the screening of the inhibitors of MTase. This PEC assay provides a promising platform for monitoring the activity and inhibition of DNA MTase, and thus shows a great potential in cancer diagnostics and anti-cancer drugs discovery.

A label-free bioluminescent sensor for real-time monitoring polynucleotide kinase activity.

Polynucleotide kinase (PNK) plays a crucial role in maintaining the genomic stability of cells and is becoming a potential target in the radio-therapeutic treatment of cancers. The fluorescent method is usually used to measure the PNK activity, but it is impossible to obtain the real-time monitoring without the employment of the labeled DNA probes. Here, we report a label-free bioluminescent sensor for PNK activity assay through real-time monitoring of the phosphorylation-dependent DNA ligation reaction. In this bioluminescent sensor, two hairpin DNA probes with 5'-protruding terminal are designed as the phosphate acceptor, and the widely used phosphate donor of ATP is substituted by dCTP. In the absence of PNK, the ligation reaction cannot be triggered due to the lack of 5'-phosphoryl groups in the probes, and the background signal is negligible. With the addition of PNK, the phosphorylation-ligation reaction of the probes is initiated with the release of AMP, and the subsequent conversion of AMP to ATP leads to the generation of distinct bioluminescence signal. The PNK activity assay can be performed in real time by continuously monitoring the bioluminescence signal. This bioluminescent sensor is much simpler, label-free, cost-effective, and free from the autofluorescence interference of biological matrix, and can be further used for quantitative, kinetic, and inhibition assay.

The Microscopic Examination of Phytophthora cinnamomi in Plant Tissues Using Fluorescent In Situ Hybridization

The microscopic examination of Phytophthora cinnamomi in plant tissues is often difficult as structures such as hyphae, chlamydospores and oospores are frequently indistinguishable from those of other fungi and oomycetes, with histological stains not enabling species differentiation. This lack of staining specificity makes the localization of P. cinnamomi hyphae and reproductive structures within plant tissues difficult, especially in woody tissues. This study demonstrates that with the use of a species-specific fluorescently labelled DNA probe, P. cinnamomi can be specifically detected and visualized directly using fluorescent in situ hybridization (FISH) without damage to plant or pathogen cell integrity or the need for subculturing. This approach provides a new application for FISH with potential use in the detailed study of plant–pathogen interactions in plants.

Expression of the glycine transporter type 1 (GlyT‐1A) is upregulated by ATF‐4 following physiological stress in human intestinal epithelial cells (1109.14)

Downstream of activating transcription factor 4 (ATF‐4) in Caco‐2 cells in response to stress. In the present study we show that increased expression of the GlyT‐1a gene following stress is dependent on elements contained within the 5’untranslated region (UTR). Caco‐2 cells transfected with plasmid constructs containing sequences representative of the GlyT‐1a proximal promoter and 5’UTR upstream of a beta‐galactosidase reporter showed increased reporter activity following treatment with thapsigargin (Tg), tunicamycin (Tu), amino acid (AA) starvation, tertbutylhydroquinone(tBHQ) or Diethyl maleate (DEM). Short labeled DNA probes containing a potential amino acid response element (AARE) located in exon 1 of the GLYT‐1a gene demonstrated binding by ATF‐4 in gel shift and super shift assays. qPCR assays performed on short genomic DNA fragments isolated from Caco‐2 cells by chromatin immunoprecipitation (ChIP) with antibodies against ATF4 demonstrated 9, 5 and 2‐fold enrichment of the ATF‐4/AARE interaction at the 5’‐UTR region of GlyT1a following Tu, AA starvation and DEM treatment respectively. Our data suggests that response elements within the 5’UTR including exon 1 and the first 85 bases of exon 2 are required for transcriptional induction of GlyT1a. We propose the direct interaction of ATF‐4 at the identified AARE drives transcriptional up‐regulation of GlyT‐1a following nutrient, oxidative and endoplasmic reticulum stress.Grant Funding Source: University of Newcastle

Quantitative Determination of Sequence-specific Nucleic Acid Based on Fluorescent Labeled DNA Probe and Dye SYBR Green I

Quantitative Determination of Sequence-specific Nucleic Acid Based on Fluorescent Labeled DNA Probe and Dye SYBR Green I

A fluorescence assay for the trace detection of protamine and heparin

Here we designed a fluorescence sensing strategy for the determination of protamine, heparin and trypsin. A fluorescein isothiocyanate (FITC)-labeled DNA probe could wrap around protamine molecules tightly via electrostatic interactions accompanied by the fluorescence quenching of the DNA probe. In the presence of heparin, protamine preferred to bind to heparin instead of DNA due to the stronger affinity of heparin to protamine, and the fluorescence could be restored. Based on the fact that the protamine which is rich in arginine could be hydrolyzed by the addition of trypsin, the fluorescence intensity of the DNA/protamine complex could also be linked to the trypsin concentration. By measuring the fluorescence intensity changes, the concentrations of protamine, heparin and trypsin were determined. Under the optimized conditions, the linear response range was obtained from 2.5 to 17.5 ng mL−1, 0.156–1.875 ng mL−1 and 62.5 to 10 000 ng mL−1 with the low detection limit of 2.2 ng mL−1, 0.078 ng mL−1 and 30.2 ng mL−1 for protamine, heparin and trypsin, respectively.

Electrochemical biosensor for DNA damage detection based on exonuclease III digestions

Detection of DNA damage is significant to evaluate genotoxicity of new chemicals in the early stages of its development. In this work, we described an electrochemical biosensor for simple, rapid and sensitive detection of specific sequences of DNA. Ferrocene (Fc)-labeled probe DNA first hybridized with capture DNA 1 to form double strands in the hybridization solution. In the presence of target DNA, the Fc-labeled probe DNA will be released by the digestions of exonuclease III and further hybridize with the capture DNA 2 immobilized on a gold electrode, producing strong electrochemical signals. On the other hand, Exonuclease III (Exo III) leaded to recycling and reuse of the target DNA, which also resulted in signal amplification. The present method can discriminate single base mismatched DNA from complementary DNA and has been successfully applied to detect DNA damage induced by chemical reagents and UV radiation.

Ultrasensitive Detection of Telomerase Activity at the Single-Cell Level

Human telomerase is a ribonucleoprotein complex which adds the repeats of TTAGGG on the telomeric ends of chromosomal DNA. Even though the vast majority of human cancers express telomerase activity, most human somatic cells lack the telomerase activity; consequently, telomerase has been regarded as both a biomarker for early cancer diagnosis and a therapeutic target. Here we develop a simple, rapid and highly sensitive method for the detection of telomerase activity at the single-cell level using telomeres-induced two-stage isothermal amplification-mediated chemiluminescence assay. In the presence of telomerase, the telomere repeats of (TTAGGG)n are added to the 3' end of telomerase substrate primer, which can be converted to the template of strand displacement amplification (SDA) for the generation of short oligonucleotides, catalytic DNAzymes, and the telomere repeats of (TTAGGG)n. The short oligonucleotide might function as a new trigger to bind the free telomerase substrate primer and consequently initiate an isothermally exponential amplification reaction (EXPAR), generating a large number of catalytic DNAzymes. Both the DNAzymes and the G-rich telomeric repeat units can interact with hemin to form the catalytic hemin-G-quadruplex nanostructures, which can catalyze the generation of amplified chemiluminescence signals in the presence of luminol and H2O2. While in the absence of telomerase, the two-stage isothermal amplification cannot be initiated, and no chemiluminescence signal is observed. Owing to the high amplification efficiency of two-stage isothermal amplification as well as the high sensitivity and wide dynamic range of the chemiluminescence assay, the proposed method can sensitively measure the synthetic telomerase product TPC8 with a detection limit of as low as 0.1 aM and a large dynamic range of 10 orders of magnitude from 0.1 aM to 1 nM and can even detect the telomerase activity from a single HeLa cancer cell without the need for any labeled DNA probes. The proposed method can be further used to screen the anticancer drugs and might provide a promising approach for the discovery of new anticancer drugs.

Chemiluminescence resonance energy transfer biosensing platform for site-specific determination of DNA methylation and assay of DNA methyltransferase activity using exonuclease III-assisted target recycling amplification

Site-specific determination of DNA methylation and assay of MTase activity can be used for determining specific cancer types, providing insights into the mechanism of gene repression, and developing novel drugs to treat methylation-related diseases. Herein, we develop a simple and highly sensitive chemiluminescence (CL) biosensing platform for site-specific determination of DNA methylation using Exonuclease III (Exo III)-assisted target recycling signal amplification. After bisulfite treatment of mixture of methylated DNA and unmethylated DNA, methylated DNA can hybridize with fluorescein (FAM)-labeled probe DNA to form double-stranded DNA (dsDNA), removing the FAM-labeled probe DNA from the surface of grapheme oxide, and the chemiluminescence resonance energy transfer (CRET) sensing signal can be observed and then amplified using Exo III-based recycling strategy.The biosensing platform exhibits excellent high sensitivity, and it can ever distinguish as low as 0.002% methylation level from the mixture, which is superior to most currently reported methods used for DNA methylation assay. In addition, the proposed method can also be used to sensitively assay MTase activity with determination limit of 0.007U/mL. This CL biosensing offers the advantages of being facile, sensitive, rapid and cost-effective. These features make the system promising for future use for early cancer diagnosis and discover of new anticancer drugs.

Dendrimer-encapsulated copper as a novel oligonucleotides label for sensitive electrochemical stripping detection of DNA hybridization

This paper describes the synthesis and characterization of a novel electrochemical label for sensitive electrochemical stripping detection of DNA hybridization based on dendrimer-encapsulated copper. The generation 4.5 (G 4.5) carboxyl-terminated poly(amidoamine) dendrimer with a trimesyl core was used as a template for synthesis of Cu2+/dendrimer nanocomposites (Cu-DNCs). Ratios of Cu2+/dendrimer were optimized in order to obtain stable nanocomposites with maximal copper loading in the interior of a polymeric shell. Cu-DNCs labeled DNA probe was employed for determining a target ssDNA immobilized on multi-walled carbon nanotubes-modified glassy carbon electrode (GCE) based on a specific hybridization reaction. The hybridization events were monitored by electrochemical detection of Cu anchored on the hybrids after the release in a diluted nitric acid by anodic stripping differential pulse voltammetry (ASDPV). The results showed that only a complementary sequence could form a dsDNA with the Cu-DNCs DNA probe and give an obvious electrochemical signal. The non-complementary sequence exhibited negligible signal change compared with the blank measurement (means: the electrode containing no target DNA incubating in hybridization buffer solution containing Cu-DNCs DNA probe for a certain time). The use of Cu encapsulated-dendrimer as tags and ASDPV for the detection of the released Cu ions could enhance the hybridization signal, and result in the increase of the sensitivity for the target DNA. Under the conditions employed here, the detection limit for measuring the full complementary sequence is down to pM level.

Comparison of polyurethane and epoxy resist master mold for nanoscale soft lithography

Soft lithography at the nanoscale requires a nanostructured silicon master mold generated by ebeam lithography and reactive ion etching. Such a fabrication is both expensive and time consuming for an expected industrial use of polydimethylsiloxane (PDMS) stamps in soft lithography. Our work focuses on a easy - low cost - technology to duplicate silicon master molds with nanoscale structures. Hence, master silicon molds patterned with nanometer scale lines have been replicated into epoxy resist and polyurethane by a process that implicated the following steps. First, the PDMS stamp made from a silicon master mold was used as template to copy this original mold into epoxy resist or polyurethane layer by nanoimprint. After characterization by Atomic Force Microscopy (AFM), these replicated molds were used to generate a second generation of PDMS stamps, which were ultimately employed to transfer labeled DNA probes on epoxysilane slides by soft lithography. Fluorescent microscopy reveals that molecular nanoscale patterns produced by these second generation PDMS stamps were exquisitely identical to those from the original PDMS stamp. As a conclusion, the epoxy resist or polyurethane are very attractive and cost-effective substrate to reproduce at large scale PDMS stamps initially made on a nanostructured silicon master mold.

A rapid and sensitive non‐radioactive method applicable for genome‐wide analysis of Saccharomyces cerevisiae genes involved in small RNA biology

Conventional isolation and detection methods for small RNAs from yeast cells have been designed for a limited number of samples. In order to be able to conduct a genome-wide assessment of how each gene product impacts upon small RNAs, we developed a rapid method for analysing small RNAs from Saccharomyces cerevisiae wild-type (wt) and mutants cells in the deletion and temperature-sensitive (ts) collections. Our method implements three optimized techniques: a procedure for growing small yeast cultures in 96-deepwell plates, a fast procedure for small RNA isolation from the plates, and a sensitive non-radioactive northern method for RNA detection. The RNA isolation procedure requires only 4 h for processing 96 samples, is highly reproducible and yields RNA of good quality and quantity. The non-radioactive northern method employs digoxigenin (DIG)-labelled DNA probes and chemiluminescence. It detects femtomole levels of small RNAs within 1 min exposure time. We minimized the processing time for large-scale analysis and optimized the stripping and reprobing procedures for analyses of multiple RNAs from a single membrane. The method described is rapid, sensitive, safe and cost-effective for genome-wide screens of novel genes involved in the biogenesis, subcellular trafficking and stability of small RNAs. Moreover, it will be useful to educational laboratory class venues and to research institutions with limited access to radioisotopes or robots.

DETECTION OF SAG1 AND BAG1 Toxoplasma gondii DNA PROBES LABELLED WITH DIGOXIGENIN-11-dUTP

The objective of this research was to detect a minimum concentration of the probes that could be used for dot blot hybridization analysis. The method required labeled DNA probes. In this study a non-radioactive label of Digoxigenin-11-dUTP was used for labeling the Sag1 and the Bag1 of Toxoplasma gondii DNA probe. Labeling method for the probes was done according to the random primed labeling technique. The result showed that 0.67 pg/µl Sag1 probe and 0.58 pg/µl Bag1 probe could be detected by anti-Dig-antibody. It could be concluded that 0.67 pg/µl Sag1 probe and 0.58 pg/µl Bag1 probe could be used to diagnose toxoplasmosis by dot blot hybridization method.

Real-Time Detection of Transcription Factors Using Target-Converted Helicase-Dependent Amplification Assay with Zero-Background Signal

Highly sensitive detection of transcription factors is essential to the evaluation of cellular development and the disease state. However, so far most detection methods are usually laborious and time-consuming with a poor sensitivity. Here, we demonstrate a simple and ultrasensitive approach for transcription factor detection based on the target-converted helicase-dependent amplification assay. We employ a hairpin probe bearing the transcription factor binding site to convert the protein signal to the DNA signal, which can be further amplified by helicase-dependent amplification. With the digestion of excess probes by the exonucleases and the subsequent one primer-triggered high fidelity amplification, zero-background signal can be achieved in the absence of a transcription factor. This method exhibits excellent specificity and high sensitivity with the detection limit of 9.3× 10(-13) M and the detection range over 4 orders of magnitude, which is superior to most currently used approaches for transcription factor detection. Moreover, the proposed method has significant advantages of simple, rapid, and low cost without the need of any labeled DNA probes and might be extended to selectively detect various DNA-binding proteins by simply changing the binding-site sequences of hairpin probes.

Fabrication of CdTe@SiO2 nanoprobes for sensitive electrogenerated chemiluminescence detection of DNA damage

Detection of DNA damage is significant for the evaluation of genotoxicity of new chemicals in the early stages of its development. An electrogenerated chemiluminescence (ECL) biosensor was fabricated to detect specific sequences of DNA by using CdTe@SiO2 as nanoprobes for signal amplification. This DNA biosensor was constructed by self-assembly of an aminated capture DNA on the glass carbon electrode. DNA detection was realized by outputting a remarkable ECL signal of the CdTe@SiO2 labeled probe DNA. When the target DNA was introduced into the system, it was complementary to the probe DNA at the one-half-segment and complementary to the capture DNA at the other half-segment, resulting in the formation of a stable duplex complex. As a result, the CdTe@SiO2 labeled probe was proximate to the electrode surface and the ECL was observed. This DNA biosensor was proved to have a low detection limit (0.03 nM) and a wide dynamic range (from 0.1 nM to 2 μM). Most importantly, the sensing system could differentiate the single base mismatched DNA from the complementary DNA. It was successfully applied to study the damage to DNA caused by several genotoxicity chemicals, which was rapid, simple, reliable and sensitive compared to the classical biological methods.