Science.gov

Sample records for dna probes

  1. Focus: DNA probes

    SciTech Connect

    Not Available

    1986-11-01

    Progress in the development of DNA probes for the identification and quantitation of specific genetic sequences in biological samples is reviewed. Current research efforts in the development of DNA probes for the diagnosis of a wide variety of bacterial, viral, and other infectious diseases, such as herpes simplex and cytomegalovirus, and inherited genetic diseases such as cystic fibrosis and sickle cell anemia are discussed. Progress in development of DNA probe assays for cancer diagnosis, detection of Salmonella food poisoning, tissue typing (detection of histocompatibility antigens), mutagen screening, and animal diseases, among other applications is included.

  2. Probe and method for DNA detection

    DOEpatents

    Yeh, Hsin-Chih; Werner, James Henry; Sharma, Jaswinder Kumar; Martinez, Jennifer Suzanne

    2013-07-02

    A hybridization probe containing two linear strands of DNA lights up upon hybridization to a target DNA using silver nanoclusters that have been templated onto one of the DNA strands. Hybridization induces proximity between the nanoclusters on one strand and an overhang on the other strand, which results in enhanced fluorescence emission from the nanoclusters.

  3. DAPI: a DNA-specific fluorescent probe.

    PubMed

    Kapuscinski, J

    1995-09-01

    DAPI (4',6-diamidino-2-phenylindole) is a DNA-specific probe which forms a fluorescent complex by attaching in the minor grove of A-T rich sequences of DNA. It also forms nonfluorescent intercalative complexes with double-stranded nucleic acids. The physicochemical properties of the dye and its complexes with nucleic acids and history of the development of this dye as a biological stain are described. The application of DAPI as a DNA-specific probe for flow cytometry, chromosome staining, DNA visualization and quantitation in histochemistry and biochemistry is reviewed. The mechanisms of DAPI-nucleic acid complex formation including minor groove binding, intercalation and condensation are discussed.

  4. DNA probe specific for Legionella pneumophila.

    PubMed Central

    Grimont, P A; Grimont, F; Desplaces, N; Tchen, P

    1985-01-01

    A procedure for preparing a DNA probe to be used in the specific detection of Legionella pneumophila by dot or colony hybridization has been devised. When total DNA from L. pneumophila was used as a radioactive probe, cross-hybridization occurred with DNA from many other species belonging to various families (including Legionellaceae, Enterobacteriaceae, Pseudomonadaceae, and Vibrionaceae). Cross-hybridizing restriction fragments in L. pneumophila ATCC 33152 DNA were identified on Southern blots. When unlabeled DNA from strain ATCC 33152 was cleaved by endonuclease BamHI, the DNA fragments cross-hybridizing with the labeled DNA from all of the other species and genera tested (or with Escherichia coli 16 + 23 S RNA) had a size of 21.4 and 16.2 kilobase pairs (major bands) and 28.0, 12.8, and 10.1 kilobase pairs (minor bands). BamHI restriction fragments of L. pneumophila DNA deprived of the cross-hybridizing fragments were pooled and used as a probe for the detection of L. pneumophila. This probe proved to be specific for L. pneumophila in colony and dot hybridization. It can potentially be used for the detection of L. pneumophila in clinical and water samples. The procedure described can be readily applied to the preparation of probes specific for phylogenetically isolated bacterial species other than L. pneumophila. Images PMID:3980693

  5. Development of DNA probes for Candida albicans

    SciTech Connect

    Cheung, L.L.; Hudson, J.B.

    1988-07-01

    An attempt was made to produce DNA probes that could be used as a rapid and efficient means of detecting candidiasis (invasive Candida infection) in immunocompromised patients. Whole DNA from Candida albicans was digested with restriction endonuclease, and the resulting fragments were randomly cloned into a plasmid vector. Several recombinant plasmids were evaluated for cross-hybridization to various other Candida species, other fungal DNAs, and to nonfungal DNAs. Cross reactions were observed between the probes and different yeasts, but none with unrelated DNAs. Some recombinants were genus-specific, and two of these were applied to the analysis of C. albicans growth curves. It became evident that, although both /sup 32/P- and biotin-labelled probes could be made quite sensitive, a possible limitation in their diagnostic potential was the poor liberation of Candida DNA from cells. Thus, better methods of treatment of clinical specimens will be required before such probes will be useful in routine diagnosis.

  6. SERS gene probe for DNA diagnostics

    NASA Astrophysics Data System (ADS)

    Stokes, David L.; Allain, Leonardo R.; Isola, Narayana R.; Vo-Dinh, Tuan

    2003-07-01

    We describe the development of a surface-enhanced Raman scattering gene (SERGen) probe technology for rapid screening for diseases and pathogens through DNA hybridization assays. The technology combines the use of gene probes labeled with SERS-active markers, and nanostructured metallic platforms for inducing the SERS effect. As a result, SERGen-based methods can offer the spectral selectivity and sensitivity of SERS as well as the molecular specificity of DNA sequence hybridization. Furthermore, these new probe s preclude the use of radioactive labels. As illustrated herein, SERGen probes have been used as primers in polymerase chain reaction (PCR) amplifications of specific DNA sequences, hence further boosting the sensitivity of the technology. We also describe several approaches to developing SERS-active DNA assay platforms, addressing the challenges of making the SERGen technology accessible and practical for clinical settings. The usefulness of the SERGen approach has been demonstrated in the detection of HIV, BRCA1 breast cancer, and BAX genes. There is great potential for the use of numerous SERGen probes for multiplexed detection of multiple biological targets.

  7. Chromosome-specific DNA Repeat Probes

    SciTech Connect

    Baumgartner, Adolf; Weier, Jingly Fung; Weier, Heinz-Ulrich G.

    2006-03-16

    In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with {alpha}-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.

  8. Universal microbial diagnostics using random DNA probes

    PubMed Central

    Aghazadeh, Amirali; Lin, Adam Y.; Sheikh, Mona A.; Chen, Allen L.; Atkins, Lisa M.; Johnson, Coreen L.; Petrosino, Joseph F.; Drezek, Rebekah A.; Baraniuk, Richard G.

    2016-01-01

    Early identification of pathogens is essential for limiting development of therapy-resistant pathogens and mitigating infectious disease outbreaks. Most bacterial detection schemes use target-specific probes to differentiate pathogen species, creating time and cost inefficiencies in identifying newly discovered organisms. We present a novel universal microbial diagnostics (UMD) platform to screen for microbial organisms in an infectious sample, using a small number of random DNA probes that are agnostic to the target DNA sequences. Our platform leverages the theory of sparse signal recovery (compressive sensing) to identify the composition of a microbial sample that potentially contains novel or mutant species. We validated the UMD platform in vitro using five random probes to recover 11 pathogenic bacteria. We further demonstrated in silico that UMD can be generalized to screen for common human pathogens in different taxonomy levels. UMD’s unorthodox sensing approach opens the door to more efficient and universal molecular diagnostics. PMID:27704040

  9. Scanning probe and nanopore DNA sequencing: core techniques and possibilities.

    PubMed

    Lund, John; Parviz, Babak A

    2009-01-01

    We provide an overview of the current state of research towards DNA sequencing using nanopore and scanning probe techniques. Additionally, we provide methods for the creation of two key experimental platforms for studies relating to nanopore and scanning probe DNA studies: a synthetic nanopore apparatus and an atomically flat conductive substrate with stretched DNA molecules.

  10. Monoclonals and DNA probes in diagnostic and preventative medicine

    SciTech Connect

    Gallo, R.C.; Della Povta, G.; Albertini, A.

    1987-01-01

    This book contains 24 selections. Some of the titles are: Use of DNA Probes for Prenatal and Carrier Diagnosis of Hemophilia and Fragile X Mental Retardation; The Application of DNA Probes to Diagnosis and Research of Duchenne Muscular Dystrophy: Clinical Trial, New Probes and Deletion Mapping; Molecular Genetics of the Human Collagens; Molecular Genetics of Human Steroid 21-Hydroxylase Genes; Detection of Hepatitis B Virus DNA and Hepatitis Delta Virus RNA: Implications in Diagnosis and Pathogenesis; and DNA Probes to Evaluate the Possible Association of Papovaviruses with Human Tumors.

  11. Empirical evaluation of oligonucleotide probe selection for DNA microarrays.

    PubMed

    Mulle, Jennifer G; Patel, Viren C; Warren, Stephen T; Hegde, Madhuri R; Cutler, David J; Zwick, Michael E

    2010-03-29

    DNA-based microarrays are increasingly central to biomedical research. Selecting oligonucleotide sequences that will behave consistently across experiments is essential to the design, production and performance of DNA microarrays. Here our aim was to improve on probe design parameters by empirically and systematically evaluating probe performance in a multivariate context. We used experimental data from 19 array CGH hybridizations to assess the probe performance of 385,474 probes tiled in the Duchenne muscular dystrophy (DMD) region of the X chromosome. Our results demonstrate that probe melting temperature, single nucleotide polymorphisms (SNPs), and homocytosine motifs all have a strong effect on probe behavior. These findings, when incorporated into future microarray probe selection algorithms, may improve microarray performance for a wide variety of applications.

  12. Advanced Molecular Probes for Sequence-Specific DNA Recognition

    NASA Astrophysics Data System (ADS)

    Bertucci, Alessandro; Manicardi, Alex; Corradini, Roberto

    DNA detection can be achieved using the Watson-Crick base pairing with oligonucleotides or oligonucleotide analogs, followed by generation of a physical or chemical signal coupled with a transducer device. The nature of the probe is an essential feature which determines the performances of the sensing device. Many synthetic processes are presently available for "molecular engineering" of DNA probes, enabling label-free and PCR-free detection to be performed. Furthermore, many DNA analogs with improved performances are available and are under development; locked nucleic acids (LNA), peptide nucleic acids (PNA) and their analogs, morpholino oligonucleotides (MO) and other modified probes have shown improved properties of affinity and selectivity in target recognition compared to those of simple DNA probes. The performances of these probes in sensing devices, and the requirements for detection of unamplified DNA will be discussed in this chapter. Chemistry and architectures for conjugation of probes to reporter units, surfaces and nanostructures will also be discussed. Examples of probes used in ultrasensitive detection of unamplified DNA are listed.

  13. DNA nanostructure-based imaging probes and drug carriers.

    PubMed

    Zhan, Pengfei; Jiang, Qiao; Wang, Zhen-Gang; Li, Na; Yu, Haiyin; Ding, Baoquan

    2014-09-01

    Self-assembled DNA nanostructures are well-defined nanoscale shapes, with uniform sizes, precise spatial addressability, and excellent biocompatibility. With these features, DNA nanostructures show great potential for biomedical applications; various DNA-based biomedical imaging probes or payload delivery carriers have been developed. In this review, we summarize the recent developments of DNA-based nanostructures as tools for diagnosis and cancer therapy. The biological effects that are brought about by DNA nanostructures are highlighted by in vitro and in vivo imaging, antitumor drug delivery, and immunostimulatory therapy. The challenges and perspectives of DNA nanostructures in the field of nanomedicine are discussed.

  14. Detection of toxoplasma gondii with a DNA molecular beacon probe

    NASA Astrophysics Data System (ADS)

    Xu, Shichao; Yao, Cuicui; Wei, Shuoming; Zhang, Jimei; Sun, Bo; Zheng, Guo; Han, Qing; Hu, Fei; Zhou, Hongming

    2008-12-01

    Toxoplasma gondii is a microscopic parasite that may infect humans, so there is an increasing concern on the early detection of latent Toxoplasma gondii infection in recent years. We currently report a rapid and sensitive method for Toxoplasma gondii based on molecular beacon (MB) probe. The probe based on fluorescence resonance energy transfer (FRET) with a stem-loop DNA oligonucleotide was labeled with CdTe/ZnS quantum dots (energy donor) at 5' end and BHQ-2 (energy acceptor) at 3' end, respectively. The probe was synthesized in PBS buffer at pH 8.2, room temperature for 24 h. Then target DNA was injected under the condition of 37°C, hybridization for 2 h, in Tris-HCl buffer. The data from fluorescence spectrum (FS) showed that ca 65% of emitted fluorescence was quenched, and about 50% recovery of fluorescence intensity was observed after adding target DNA, which indicated that the target DNA was successfully detected by MB probe. The detecting limitation was determined as ca 5 nM. Moreover, specificity of the probe was investigated by adding target DNA with one-base-pair mismatch, the low fluorescence recovery indicated the high specificity. The results showed that the current sensing probe will be a useful and convenient tool in Toxoplasma gondii early detection.

  15. Fluorescent cyanine probe for DNA detection and cellular imaging

    NASA Astrophysics Data System (ADS)

    Zheng, Yong-Chao; Zheng, Mei-Ling; Zhao, Zhen-Sheng; Duan, Xuan-Ming

    2014-03-01

    In our study, two carbazole-based cyanines, 3,6-bis[2-(1-methylpyridinium)vinyl]-9-methyl carbazole diiodide (A) and 6,6'-bis[2-(1-methylpyridinium)vinyl]-bis(9-methyl-carbazol-3yl)methane diiodide (B) were synthesized and employed as light-up probes for DNA and cell imaging. Both of the cyanine probes possess a symmetric structure and bis-cationic center. The obvious induced circular dichroism signals in circular dichroism spectra reveal that the molecules can specifically interact with DNA. Strong fluorescence enhancement is observed when these two cyanines are bound to DNA. These cyanine probes show high binding affinity to oligonucleotides but different binding preferences to various secondary structures. Confocal microscopy images of fixed cell stained by the probes exhibit strong brightness and high contrast in nucleus with a very low cytoplasmic background.

  16. Probing the elastic limit of DNA bending.

    PubMed

    Le, Tung T; Kim, Harold D

    2014-01-01

    Sharp bending of double-stranded DNA (dsDNA) plays an essential role in genome structure and function. However, the elastic limit of dsDNA bending remains controversial. Here, we measured the opening rates of small dsDNA loops with contour lengths ranging between 40 and 200 bp using single-molecule Fluorescence Resonance Energy Transfer. The relationship of loop lifetime to loop size revealed a critical transition in bending stress. Above the critical loop size, the loop lifetime changed with loop size in a manner consistent with elastic bending stress, but below it, became less sensitive to loop size, indicative of softened dsDNA. The critical loop size increased from ∼ 60 bp to ∼ 100 bp with the addition of 5 mM magnesium. We show that our result is in quantitative agreement with the kinkable worm-like chain model, and furthermore, can reproduce previously reported looping probabilities of dsDNA over the range between 50 and 200 bp. Our findings shed new light on the energetics of sharply bent dsDNA.

  17. Utilizing Gold Nanoparticle Probes to Visually Detect DNA Methylation

    NASA Astrophysics Data System (ADS)

    Chen, Kui; Zhang, Mingyi; Chang, Ya-Nan; Xia, Lin; Gu, Weihong; Qin, Yanxia; Li, Juan; Cui, Suxia; Xing, Gengmei

    2016-06-01

    The surface plasmon resonance (SPR) effect endows gold nanoparticles (GNPs) with the ability to visualize biomolecules. In the present study, we designed and constructed a GNP probe to allow the semi-quantitative analysis of methylated tumor suppressor genes in cultured cells. To construct the probe, the GNP surfaces were coated with single-stranded DNA (ssDNA) by forming Au-S bonds. The ssDNA contains a thiolated 5'-end, a regulatory domain of 12 adenine nucleotides, and a functional domain with absolute pairing with methylated p16 sequence (Met- p16). The probe, paired with Met- p16, clearly changed the color of aggregating GNPs probe in 5 mol/L NaCl solution. Utilizing the probe, p16 gene methylation in HCT116 cells was semi-quantified. Further, the methylation of E-cadherin, p15, and p16 gene in Caco2, HepG2, and HCT116 cell lines were detected by the corresponding probes, constructed with three domains. This simple and cost-effective method was useful for the diagnosis of DNA methylation-related diseases.

  18. DNA hybridization probe for clinical diagnosis of Entamoeba histolytica.

    PubMed Central

    Samuelson, J; Acuna-Soto, R; Reed, S; Biagi, F; Wirth, D

    1989-01-01

    As an alternative to microscopic identification of Entamoeba histolytica parasites isolated from stool, a sensitive and species-specific DNA hybridization probe was made for rapid diagnosis of E. histolytica parasites in clinical samples directly applied to nylon membranes. The DNA hybridization probe was made by screening a genomic library of a virulent HM-1:IMSS strain of E. histolytica to detect recombinant plasmids containing highly repeated parasite DNA sequences. Four plasmid clones that reacted across Entamoeba species coded for highly repeated rRNA genes of E. histolytica. Four other plasmid clones were E. histolytica specific in that they bound to four axenized and nine xenic strains of E. histolytica but did not recognize closely related E. histolytica-like Laredo, Entamoeba moshkovskii, or Entamoeba invadens parasites. The diagnostic clones detected as few as eight cultured amoebae and did not distinguish between pathogenic and nonpathogenic zymodemes of E. histolytica. The diagnostic clones were sequenced and contained 145-base-pair sequences which appear to be tandemly repeated in the genome. No stable transcript which is homologous to the diagnostic DNA was detected. In a study of stool samples from Mexico City shown by microscopy to contain E. histolytica, Entamoeba coli, Giardia lamblia, Endolimax nana, Trichuris trichiuria, and Chilomastix mesnili parasites, the DNA hybridization probe demonstrated a sensitivity of 1.0 and a specificity of 0.93. We conclude that the DNA hybridization probe can be used for rapid and accurate diagnosis of E. histolytica parasites. Images PMID:2542361

  19. Colorimetric DNA detection of transgenic plants using gold nanoparticles functionalized with L-shaped DNA probes

    NASA Astrophysics Data System (ADS)

    Nourisaeid, Elham; Mousavi, Amir; Arpanaei, Ayyoob

    2016-01-01

    In this study, a DNA colorimetric detection system based on gold nanoparticles functionalized with L-shaped DNA probes was prepared and evaluated. We investigated the hybridization efficiency of the L-shaped probes and studied the effect of nanoparticle size and the L-shaped DNA probe length on the performance of the as-prepared system. Probes were attached to the surface of gold nanoparticles using an adenine sequence. An optimal sequence of 35S rRNA gene promoter from the cauliflower mosaic virus, which is frequently used in the development of transgenic plants, and the two complementary ends of this gene were employed as model target strands and probe molecules, respectively. The spectrophotometric properties of the as-prepared systems indicated that the large NPs show better changes in the absorption spectrum and consequently present a better performance. The results of this study revealed that the probe/Au-NPs prepared using a vertical spacer containing 5 thymine oligonucleotides exhibited a stronger spectrophotometric response in comparison to that of larger probes. These results in general indicate the suitable performance of the L-shaped DNA probe-functionalized Au-NPs, and in particular emphasize the important role of the gold nanoparticle size and length of the DNA probes in enhancing the performance of such a system.

  20. DNA fingerprints of farm animals generated by microsatellite and minisatellite DNA probes.

    PubMed

    Haberfeld, A; Cahaner, A; Yoffe, O; Plotsky, Y; Hillel, J

    1991-01-01

    A multi-locus DNA probe, R18.1, derived from a bovine genomic library, detected DNA fingerprints of highly polymorphic loci in hybridization to genomic DNA from poultry and sheep, and of moderate polymorphic loci in cattle and human DNA. The average numbers of detected bands in chickens and sheep were 27.8 and 21.4, and the average band sharing levels were 0.25 and 0.33, respectively. In hybridization to cattle and human DNA, the results were less polymorphic; nevertheless, individual identification is feasible using probe R18.1. The results obtained by R18.1 were compared to results obtained by Jeffreys minisatellite probe 33.6 and two microsatellite oligonucleotides, (GT)12 and (GTG)5. The total number of detected loci using probes R18.1 and 33.6 were estimated in chickens through family analysis of broilers and the maximal number of detectable loci was calculated.

  1. DNA-Templated Aptamer Probe for Identification of Target Proteins.

    PubMed

    Bi, Wenjing; Bai, Xue; Gao, Fan; Lu, Congcong; Wang, Ye; Zhai, Guijin; Tian, Shanshan; Fan, Enguo; Zhang, Yukui; Zhang, Kai

    2017-04-04

    Using aptamers as molecular probes for biomarker discovery has attracted a great deal of attention in recent years. However, it is still a big challenge to accurately identify those protein markers that are targeted by aptamers under physiological conditions due to weak and noncovalent aptamer-protein interactions. Herein, we developed an aptamer based dual-probe using DNA-templated chemistry and photo-cross-linking technique for the identification of target proteins that are recognized by aptamers. In this system, the aptamer was modified by a single strand DNA as binding probe (BP), and another complementary DNA with a photoactive group and reporter group was modified as capture probe (CP). BP was first added to recruit the binding protein via aptamer recognition, and subsequently CP was added to let the cross-linker close to the target via DNA self-assembly, and then a covalent bond between CP and its binding protein was achieved via photo-cross-linking reaction. The captured protein can be detected or affinity enrichment using the tag, finally identified by MS. By use of lysozyme as a model substrate, we demonstrated that this multiple functionalized probe can be utilized for a successful labeling and enrichment of target protein even under a complicated and real environment. Thus, a novel method to precisely identify the aptamer-targeted proteins has been developed and it has a potential application for discovery of aptamer-based biomarkers.

  2. Array-based electrical detection of DNA with nanoparticle probes.

    PubMed

    Park, So-Jung; Taton, T Andrew; Mirkin, Chad A

    2002-02-22

    A DNA array detection method is reported in which the binding of oligonucleotides functionalized with gold nanoparticles leads to conductivity changes associated with target-probe binding events. The binding events localize gold nanoparticles in an electrode gap; silver deposition facilitated by these nanoparticles bridges the gap and leads to readily measurable conductivity changes. An unusual salt concentration-dependent hybridization behavior associated with these nanoparticle probes was exploited to achieve selectivity without a thermal-stringency wash. Using this method, we have detected target DNA at concentrations as low as 500 femtomolar with a point mutation selectivity factor of approximately 100,000:1.

  3. Evolving DNA motifs to predict GeneChip probe performance

    PubMed Central

    Langdon, WB; Harrison, AP

    2009-01-01

    Background Affymetrix High Density Oligonuclotide Arrays (HDONA) simultaneously measure expression of thousands of genes using millions of probes. We use correlations between measurements for the same gene across 6685 human tissue samples from NCBI's GEO database to indicated the quality of individual HG-U133A probes. Low correlation indicates a poor probe. Results Regular expressions can be automatically created from a Backus-Naur form (BNF) context-free grammar using strongly typed genetic programming. Conclusion The automatically produced motif is better at predicting poor DNA sequences than an existing human generated RE, suggesting runs of Cytosine and Guanine and mixtures should all be avoided. PMID:19298675

  4. Isolation and characterization of DNA probes for human chromosome 21.

    PubMed

    Watkins, P C

    1990-01-01

    A coordinated effort to map and sequence the human genome has recently become a national priority. Chromosome 21, the smallest human chromosome accounting for less than 2% of the human genome, is an attractive model system for developing and evaluating genome mapping technology. Several strategies are currently being explored including the development of chromosome 21 libraries from somatic cell hybrids as reported here, the cloning of chromosome 21 in yeast artificial chromosomes (McCormick et al., 1989b), and the construction of chromosome 21 libraries using chromosome flow-sorting techniques (Fuscoe et al., 1989). This report describes the approaches used to identify DNA probes that are useful for mapping chromosome 21. Probes were successfully isolated from both phage and cosmid libraries made from two somatic cell hybrids that contain human chromosome 21 as the only human chromosome. The 15 cosmid clones from the WA17 library, reduced to cloned DNA sequences of an average size of 3 kb, total 525 kb of DNA which is approximately 1% of chromosome 21. From these clones, a set of polymorphic DNA markers that span the length of the long arm of chromosome 21 has been generated. All of the probes thus far analyzed from the WA17 libraries have been mapped to chromosome 21 both by physical and genetic mapping methods. It is therefore likely that the WA17 hybrid cell line contains human chromosome 21 as the only human component, in agreement with cytogenetic observation. The 153E7b cosmid libraries will provide an alternative source of cloned chromosome 21 DNA. Library screening techniques can be employed to obtain cloned DNA sequences from the same genetic loci of the two different chromosome 21s. Comparative analysis will allow direct estimation of DNA sequence variation for different regions of chromosome 21. Mapped DNA probes make possible the molecular analysis of chromosome 21 at a level of resolution not achievable by classical cytogenetic techniques (Graw et al

  5. Probe Microscopic Studies of DNA Molecules on Carbon Nanotubes

    PubMed Central

    Umemura, Kazuo; Izumi, Katsuki; Oura, Shusuke

    2016-01-01

    Hybrids of DNA and carbon nanotubes (CNTs) are promising nanobioconjugates for nanobiosensors, carriers for drug delivery, and other biological applications. In this review, nanoscopic characterization of DNA-CNT hybrids, in particular, characterization by scanning probe microscopy (SPM), is summarized. In many studies, topographical imaging by atomic force microscopy has been performed. However, some researchers have demonstrated advanced SPM operations in order to maximize its unique and valuable functions. Such sophisticated approaches are attractive and will have a significant impact on future studies of DNA-CNT hybrids.

  6. DNA/DNA in situ hybridization with enzyme linked probes

    SciTech Connect

    Grillo, S.; Mosher, M.; Charles, P.; Henry, S.; Taub, F.

    1987-05-01

    A non-radioactive in situ nucleic acid hybridization method which requires no antibodies, haptens, avidin or biotin intermediateries is presented. Horseradish peroxidase (HRP) labeled nucleic acid probes are hybridized in situ for 2 hours or less, followed by brief washing of hybridized cells and the direct detection of in situ hybrids with diaminobenzidine (DAB). Application of this method to the detection of Human Papilloma Virus (HPV) in human cells is shown.

  7. Repeat sequences from complex ds DNA viruses can be used as minisatellite probes for DNA fingerprinting.

    PubMed

    Crawford, A M; Buchanan, F C; Fraser, K M; Robinson, A J; Hill, D F

    1991-01-01

    In a search for new fingerprinting probes for use with sheep, repeat sequences derived from five poxviruses, an iridovirus and a baculovirus were screened against DNA from sheep pedigrees. Probes constructed from portions of the parapox viruses, orf virus and papular stomatitis virus and the baculovirus from the alfalfa looper, Autographa californica, nuclear polyhedrosis virus all gave fingerprint patterns. Probes from three other poxviruses and an iridovirus did not give useful banding patterns.

  8. Probing Nucleosome Stability with a DNA Origami Nanocaliper.

    PubMed

    Le, Jenny V; Luo, Yi; Darcy, Michael A; Lucas, Christopher R; Goodwin, Michelle F; Poirier, Michael G; Castro, Carlos E

    2016-07-26

    The organization of eukaryotic DNA into nucleosomes and chromatin undergoes dynamic structural changes to regulate genome processing, including transcription and DNA repair. Critical chromatin rearrangements occur over a wide range of distances, including the mesoscopic length scale of tens of nanometers. However, there is a lack of methodologies that probe changes over this mesoscopic length scale within chromatin. We have designed, constructed, and implemented a DNA-based nanocaliper that probes this mesoscopic length scale. We developed an approach of integrating nucleosomes into our nanocaliper at two attachment points with over 50% efficiency. Here, we focused on attaching the two DNA ends of the nucleosome to the ends of the two nanocaliper arms, so the hinge angle is a readout of the nucleosome end-to-end distance. We demonstrate that nucleosomes integrated with 6, 26, and 51 bp linker DNA are partially unwrapped by the nanocaliper by an amount consistent with previously observed structural transitions. In contrast, the nucleosomes integrated with the longer 75 bp linker DNA remain fully wrapped. We found that the nanocaliper angle is a sensitive measure of nucleosome disassembly and can read out transcription factor (TF) binding to its target site within the nucleosome. Interestingly, the nanocaliper not only detects TF binding but also significantly increases the probability of TF occupancy at its site by partially unwrapping the nucleosome. These studies demonstrate the feasibility of using DNA nanotechnology to both detect and manipulate nucleosome structure, which provides a foundation of future mesoscale studies of nucleosome and chromatin structural dynamics.

  9. Probe mapping to facilitate transposon-based DNA sequencing

    SciTech Connect

    Strausbaugh, L.D.; Bourke, M.T.; Sommer, M.T.; Coon, M.E.; Berg, C.M. )

    1990-08-01

    A promising strategy for DNA sequencing exploits transposons to provide mobile sites for the binding of sequencing primers. For such a strategy to be maximally efficient, the location and orientation of the transposon must be readily determined and the insertion sites should be randomly distributed. The authors demonstrate an efficient probe-based method for the localization and orientation of transposon-borne primer sites, which is adaptable to large-scale sequencing strategies. This approach requires no prior restriction enzyme mapping or knowledge of the cloned sequence and eliminates the inefficiency inherent in totally random sequencing methods. To test the efficiency of probe mapping, 49 insertions of the transposon {gamma}{delta} (Tn1000) in a cloned fragment of Drosophila melanogaster DNA were mapped and oriented. In addition, oligonucleotide primers specific for unique subterminal {gamma}{delta} segments were used to prime dideoxynucleotide double-stranded sequencing. These data provided an opportunity to rigorously examine {gamma}{delta} insertion sites. The insertions were quire randomly distributed, even though the target DNA fragment had both A+T-rich and G+C-rich regions; in G+C-rich DNA, the insertions were found in A+T-rich valleys. These data demonstrate that {gamma}{delta} is an excellent choice for supplying mobile primer binding sites to cloned DNA and that transposon-based probe mapping permits the sequences of large cloned segments to be determined without any subcloning.

  10. Identification and epidemiological typing of Naegleria fowleri with DNA probes.

    PubMed Central

    Kilvington, S; Beeching, J

    1995-01-01

    Naegleria fowleri is a small free-living amoeboflagellate found in warm water habitats worldwide. The organism is pathogenic to humans, causing fatal primary amoebic meningoencephalitis. When monitoring the environment for the presence of N. fowleri, it is important to reliably differentiate the organism from other closely related but nonpathogenic species. To this end, we have developed species-specific DNA probes for use in the rapid identification of N. fowleri from the environment. Samples were taken from the thermal springs in Bath, England, and cultured for amoebae. Of 84 isolates of thermophilic Naegleria spp., 10 were identified as N. fowleri by probe hybridization. The identity of these isolates was subsequently confirmed by their specific whole-cell DNA restriction fragment length polymorphisms (RFLPs). One DNA clone was found to contain a repeated element that detected chromosomal RFLPs that were not directly visible on agarose gels. This enabled the further differentiation of strains within geographically defined whole-cell DNA RFLP groups. N. fowleri DNA probes represent a specific and potentially rapid method for the identification of the organism soon after primary isolation from the environment. PMID:7793928

  11. DNA probe and PCR-specific reaction for Lactobacillus plantarum.

    PubMed

    Quere, F; Deschamps, A; Urdaci, M C

    1997-06-01

    A 300 bp DNA fragment of Lactobacillus plantarum isolated by randomly amplified polymorphic DNA (RAPD) analysis was cloned and sequenced. This fragment was tested using a dot-blot DNA hybridization to technique for its ability to identify Lact. plantarum strains. This probe hybridized with all Lact. plantarum strains tested and with some strains of Lact. pentosus, albeit more weakly. Two internal primers of this probe were selected (LbP11 and LbP12) and polymerase chain reaction (PCR) was carried out. All Lact. plantarum strains tested amplified a 250 bp fragment contrary to the other LAB species tested. This specific PCR for Lact. plantarum was also performed from colonies grown on MRS medium with similar results. These methods enabled the rapid and specific detection and identification of Lact. plantarum.

  12. Scanning Probe Microscopy of DNA with a Quartz Tuning Fork

    NASA Astrophysics Data System (ADS)

    King, G. M.; Nunes, G., Jr.

    2001-03-01

    Quartz tuning-forks have recently been put to use as highly sensitive force detectors in atomic force microscopy (AFM).(F.J.Giessibl et al.), Science 289, 422 (2000). In this study we have applied a home-built, tuning-fork based AFM to the investigation of single and double stranded DNA (ssDNA and dsDNA). We operate the microscope in the non-contact mode (typical tip amplitude ~1 nm) with a variety of tips (e.g. Si, Si_3N_4, W). Here we report on recent results showing that the apparent height of plasmid dsDNA on mica substrates depends on both the tip material and imaging frequency shift. This talk will also review our efforts to probe ssDNA with a chemically functionalized tip. Current and future prospects for this dynamic-mode, chemically-sensitive force microscopy technique will be discussed.

  13. A simple and rapid method for the preparation of homologous DNA oligonucleotide hybridization probes from heterologous gene sequences and probes.

    PubMed

    Maxwell, E S; Sarge, K D

    1988-11-30

    We describe a simple and rapid method for the preparation of homologous DNA oligonucleotide probes for hybridization analysis and/or cDNA/genomic library screening. With this method, a synthetic DNA oligonucleotide derived from a known heterologous DNA/RNA/protein sequence is annealed to an RNA preparation containing the gene transcript of interest. Any unpaired 3'-terminal oligonucleotides of the heterologous DNA primer are then removed using the 3' exonuclease activity of the DNA Polymerase I Klenow fragment before primer extension/dideoxynucleotide sequencing of the annealed RNA species with AMV reverse transcriptase. From the determined RNA sequence, a completely homologous DNA oligonucleotide probe is then prepared. This approach has been used to prepare a homologous DNA oligonucleotide probe for the successful library screening of the yeast hybRNA gene starting with a heterologous mouse hybRNA DNA oligonucleotide probe.

  14. Probe DNA-Cisplatin Interaction with Solid-State Nanopores

    NASA Astrophysics Data System (ADS)

    Zhou, Zhi; Hu, Ying; Li, Wei; Xu, Zhi; Wang, Pengye; Bai, Xuedong; Shan, Xinyan; Lu, Xinghua; Nanopore Collaboration

    2014-03-01

    Understanding the mechanism of DNA-cisplatin interaction is essential for clinical application and novel drug design. As an emerging single-molecule technology, solid-state nanopore has been employed in biomolecule detection and probing DNA-molecule interactions. Herein, we reported a real-time monitoring of DNA-cisplatin interaction by employing solid-state SiN nanopores. The DNA-cisplatin interacting process is clearly classified into three stages by measuring the capture rate of DNA-cisplatin adducts. In the first stage, the negative charged DNA molecules were partially discharged due to the bonding of positive charged cisplatin and forming of mono-adducts. In the second stage, forming of DNA-cisplatin di-adducts with the adjacent bases results in DNA bending and softening. The capture rate increases since the softened bi-adducts experience a lower barrier to thread into the nanopores. In the third stage, complex structures, such as micro-loop, are formed and the DNA-cisplatin adducts are aggregated. The capture rate decreases to zero as the aggregated adduct grows to the size of the pore. The characteristic time of this stage was found to be linear with the diameter of the nanopore and this dynamic process can be described with a second-order reaction model. We are grateful to Laboratory of Microfabrication, Dr. Y. Yao, and Prof. R.C. Yu (Institute of Physics, Chinese Academy of Sciences) for technical assistance.

  15. Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

    SciTech Connect

    Greulich-Bode, Karin; Wang, Mei; Rhein, Andreas; Weier, Jingly; Weier, Heinz-Ulli

    2008-12-16

    Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific {approx}6kb plasmid onto an unusually small, {approx}55kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-?B2 locus. The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

  16. Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

    SciTech Connect

    Greulich-Bode, Karin M.; Wang, Mei; Rhein, Andreas P.; Weier, Jingly F.; Weier, Heinz-Ulli G.

    2008-12-04

    Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific {approx}6kb plasmid onto an unusually small, {approx}55kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-{kappa}B2 locus. The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

  17. Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

    PubMed Central

    Greulich-Bode, Karin M; Wang, Mei; Rhein, Andreas P; Weier, Jingly F; Weier, Heinz-Ulli G

    2008-01-01

    Background Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100 kb, careful probe selection and characterization are of paramount importance. Results We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific ~6 kb plasmid onto an unusually small, ~55 kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-κB2 locus. Conclusion The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements. PMID:19108707

  18. DNA Probe Pooling for Rapid Delineation of Chromosomal Breakpoints

    SciTech Connect

    Lu, Chun-Mei; Kwan, Johnson; Baumgartner, Adolf; Weier, Jingly F.; Wang, Mei; Escudero, Tomas; Munne', Santiago; Zitzelsberger, Horst F.; Weier, Heinz-Ulrich

    2009-01-30

    Structural chromosome aberrations are hallmarks of many human genetic diseases. The precise mapping of translocation breakpoints in tumors is important for identification of genes with altered levels of expression, prediction of tumor progression, therapy response, or length of disease-free survival as well as the preparation of probes for detection of tumor cells in peripheral blood. Similarly, in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) for carriers of balanced, reciprocal translocations benefit from accurate breakpoint maps in the preparation of patient-specific DNA probes followed by a selection of normal or balanced oocytes or embryos. We expedited the process of breakpoint mapping and preparation of case-specific probes by utilizing physically mapped bacterial artificial chromosome (BAC) clones. Historically, breakpoint mapping is based on the definition of the smallest interval between proximal and distal probes. Thus, many of the DNA probes prepared for multi-clone and multi-color mapping experiments do not generate additional information. Our pooling protocol described here with examples from thyroid cancer research and PGD accelerates the delineation of translocation breakpoints without sacrificing resolution. The turnaround time from clone selection to mapping results using tumor or IVF patient samples can be as short as three to four days.

  19. Multipyrene Tandem Probes for Point Mutations Detection in DNA

    PubMed Central

    Kholodar, Svetlana A.; Novopashina, Darya S.; Meschaninova, Mariya I.; Venyaminova, Alya G.

    2013-01-01

    Here we report design, synthesis and characterization of highly sensitive, specific and stable in biological systems fluorescent probes for point mutation detection in DNA. The tandems of 3′- and 5′-mono- and bis-pyrene conjugated oligo(2′-O-methylribonucleotides), protected by 3′-“inverted” thymidine, were constructed and their potential as new instruments for genetic diagnostics was studied. Novel probes have been shown to exhibit an ability to form stable duplexes with DNA target due to the stabilizing effect of multiple pyrene units at the junction. The relationship between fluorescent properties of developed probes, the number of pyrene residues at the tandem junction, and the location of point mutation has been studied. On the basis of the data obtained, we have chosen the probes possessing the highest fluorescence intensity along with the best mismatch discrimination and deletion and insertion detection ability. Application of developed probes for detection of polymorphism C677T in MTHFR gene has been demonstrated on model systems. PMID:24455205

  20. Photonic Crystal Biosensor with In-Situ Synthesized DNA Probes for Enhanced Sensitivity

    SciTech Connect

    Hu, Shuren; Zhao, Y.; Retterer, Scott T; Kravchenko, Ivan I; Weiss, Sharon

    2013-01-01

    We report on a nearly 8-fold increase in multi-hole defect photonic crystal biosensor response by incorporating in-situ synthesis of DNA probes, as compared to the conventional functionalization method employing pre-synthesized DNA probe immobilization.

  1. DNA probes and PCR for diagnosis of parasitic infections.

    PubMed Central

    Weiss, J B

    1995-01-01

    DNA probe and PCR-based assays to identify and detect parasites are technically complex; however, they have high sensitivity, directly detect parasites independent of the immunocompetence or previous clinical history of the patient, and can distinguish between organisms that are morphologically similar. Diagnosis of parasites is often based on direct detection by microscopy, which is insensitive and laborious and can lack specificity. Most PCR-based assays were more sensitive than DNA probe assays. The development of PCR-based diagnostic assays requires multiple steps following the initial selection of oligonucleotide primers and reporter probe. Generally, the ability to detect the DNA of one parasite was attained by PCR; however, advances in the preparation of samples for PCR (extraction of DNA while removing PCR inhibitors) will be required to achieve that sensitivity with human specimens. Preliminary PCR systems have been developed for many different parasites, yet few have been evaluated with a large number of clinical specimens and/or under field conditions. Those evaluations are essential for determination of clinical and field utility and performance and of the most appropriate application of the assay. Several situations in which PCR-based diagnosis will result in epidemiologic, medical, or public health advances have been identified. PMID:7704890

  2. Probing the DNA Structural Requirements for Facilitated Diffusion

    PubMed Central

    2015-01-01

    DNA glycosylases perform a genome-wide search to locate damaged nucleotides among a great excess of undamaged nucleotides. Many glycosylases are capable of facilitated diffusion, whereby multiple sites along the DNA are sampled during a single binding encounter. Electrostatic interactions between positively charged amino acids and the negatively charged phosphate backbone are crucial for facilitated diffusion, but the extent to which diffusing proteins rely on the double-helical structure DNA is not known. Kinetic assays were used to probe the DNA searching mechanism of human alkyladenine DNA glycosylase (AAG) and to test the extent to which diffusion requires B-form duplex DNA. Although AAG excises εA lesions from single-stranded DNA, it is not processive on single-stranded DNA because dissociation is faster than N-glycosidic bond cleavage. However, the AAG complex with single-stranded DNA is sufficiently stable to allow for DNA annealing when a complementary strand is added. This observation provides evidence of nonspecific association of AAG with single-stranded DNA. Single-strand gaps, bubbles, and bent structures do not impede the search by AAG. Instead, these flexible or bent structures lead to the capture of a nearby site of damage that is more efficient than that of a continuous B-form duplex. The ability of AAG to negotiate these helix discontinuities is inconsistent with a sliding mode of diffusion but can be readily explained by a hopping mode that involves microscopic dissociation and reassociation. These experiments provide evidence of relatively long-range hops that allow a searching protein to navigate around DNA binding proteins that would serve as obstacles to a sliding protein. PMID:25495964

  3. Chemical Biology Probes from Advanced DNA-encoded Libraries.

    PubMed

    Salamon, Hazem; Klika Škopić, Mateja; Jung, Kathrin; Bugain, Olivia; Brunschweiger, Andreas

    2016-02-19

    The identification of bioactive compounds is a crucial step toward development of probes for chemical biology studies. Screening of DNA-encoded small molecule libraries (DELs) has emerged as a validated technology to interrogate vast chemical space. DELs consist of chimeric molecules composed of a low-molecular weight compound that is conjugated to a DNA identifier tag. They are screened as pooled libraries using selection to identify "hits." Screening of DELs has identified numerous bioactive compounds. Some of these molecules were instrumental in gaining a deeper understanding of biological systems. One of the main challenges in the field is the development of synthesis methodology for DELs.

  4. [Diagnosed tuberculosis using specific DNA probe hybridization methods].

    PubMed

    Furuta, Itaru; Yamazumi, Toshiaki

    2002-11-01

    In Japan, reported cases of tuberculosis had declined nearly every year until 1995. However, in 1997 newly recorded cases began increasing for the first time in more than 38 years. Recent studies using DNA fingerprinting show that person- to person transmission may account for as many as one-third of new cases of tuberculosis in citizen populations. Nucleic acid hybridization methods using specific DNA probes can specifically identify M. tuberculosis and other mycobacterial species. Rapid nucleic acid amplification techniques such as polymerase chain reaction methods allow direct identification of M. tuberculosis in clinical specimens. Is 6110 has been exploited extensively as a clonal marker in molecular epidemiology studies of tuberculosis. The emergence of resistance to antituberculosis drugs is a relevant matter worldwide. A recent genotypic method allows earlier detection of RFP-resistant and INH-resistant stains using probes for mutation in rpoB and in katG.

  5. Toxoplasma gondii DNA detection with a magnetic molecular beacon probe

    NASA Astrophysics Data System (ADS)

    Xu, Shichao; Yao, Cuicui; Wei, Shuoming; Zhang, Jimei; Dai, Zhao; Zheng, Guo; Sun, Bo; Han, Qing; Hu, Fei; Zhou, Hongming

    2008-12-01

    Toxoplasma Gondii infection is widespread in humans worldwide and reported infection rates range from 3%-70%, depending on the populations or geographic areas, and it has been recognized as a potential food safety hazard in our daily life. A magnetic molecular beacon probe (mMBP), based on theory of fluorescence resonance energy transfer (FRET), was currently reported to detect Toxoplasma Gondii DNA. Nano-sized Fe3O4 were primarily prepared by coprecipitation method in aqueous phase with NaOH as precipitator, and was used as magnetic core. The qualified coreshell magnetic quantum dots (mQDs), i.e. CdTe(symbol)Fe3O4, were then achieved by layer-by-layer method when mol ratio of Fe3O4/CdTe is 1/3, pH at 6.0, 30 °C, and reactant solution was refluxed for 30 min, the size of mQDs were determined to be 12-15 nm via transmission electron microscopy (TEM). Over 70% overlap between emission spectrum of mQDs and absorbance spectrum of BHQ-2 was observed, this result suggests the synthesized mQDs and BHQ-2 can be utilized as energy donor and energy acceptor, respectively. The sensing probe was fabricated and a stem-loop Toxoplasma Gondii DNA oligonucleotide was labeled with mQDs at the 5' end and BHQ-2 at 3' end, respectively. Target Toxoplasma gondii DNA was detected under conditions of 37 °C, hybridization for 2h, at pH8.0 in Tris-HCl buffer. About 30% recovery of fluorescence intensity was observed via fluorescence spectrum (FS) after the Toxoplasma gondii DNA was added, which suggested that the Toxoplasma Gondii DNA was successfully detected. Specificity investigation of the mMBP indicated that relative low recovery of fluorescence intensity was obtained when the target DNA with one-base pair mismatch was added, this result indicated the high specificity of the sensing probe. Our research simultaneously indicated that mMBP can be conveniently separated from the unhybridized stem-loop DNA and target DNA, which will be meaningful in DNA sensing and purification process.

  6. Detection of Toxoplasma gondii with a DNA molecular beacon probe

    NASA Astrophysics Data System (ADS)

    Zhou, Cun; Xu, Shichao; Yang, Juan; Zhang, Jimei; Dai, Zhao; Zheng, Guo; Sun, Bo; Sun, Shuqing; Feng, Teilin; Zi, Yan; Liang, Chu; Luo, Hao

    2009-07-01

    Toxoplasma gondii is a kind of microscopic parasite that may infect humans, and there are increasing concerns on the early detection of latent Toxoplasma gondii infection in recent years. This research highlights a new type of molecular beacon (MB) fluorescent probe for Toxoplasma DNA testing. We combined high-efficiency fluorescent inorganic core-shell quantum dots-CdTe/ZnS (as fluorescent energy donor) and BHQ-2 (energy acceptor) to the single-strand DNA of Toxoplasma gondii, and a molecular beacon sensing system based on fluorescence resonance energy transfer (FRET) was achieved. Core-shell quantum dots CdTe/ZnS was firstly prepared in aqueous solution, and the influencing factor of its fluorescent properties, including CdTe/Na2S/Zn(CH3COO)2 (v/v), dependence of reaction time, temperature, and pH, is investigated systematically. The synthesized quantum dots and molecular beacon were characterized by transmission electron microscopy (TEM), ultraviolet-visible spectrophotometer (UV-vis), fluorescent spectrophotometer (FS), respectively. The TEM results showed that CdTe/ZnS core-shell quantum dots is ~11nm in size, and the quantum dots is water-soluble well. The sensing ability of target DNA of assembled MB was investigated, and results showed that the target Toxoplasma gonddi DNA can be successfully detected by measuring the change of fluorescence intensity. The results showed that the current sensing probe will be a useful and convenient tool in Toxoplasma gondii early detection.

  7. Probing Mercury(II)-DNA Interactions by Nanopore Stochastic Sensing

    PubMed Central

    Wang, Guihua; Zhao, Qitao; Kang, Xiaofeng; Guan, Xiyun

    2013-01-01

    In this work, DNA-Hg(II) interactions were investigated by monitoring the translocation of DNA hairpins in a protein ion channel in the absence and presence of metal ions. Our experiments demonstrate that target-specific hairpin structures could be stabilized much more significantly by mercuric ions than by the stem length and the loop size of the hairpin due to the formation of Thymine-Hg(II)-Thymine complexes. In addition, the designed DNA probe allows the development of a highly sensitive nanopore sensor for Hg2+ with a detection limit of 25 nM. Further, the sensor is specific, and other tested metal ions including Pb2+, Cu2+, Cd2+, etc. with concentrations of up to two orders of magnitude greater than that of Hg2+ would not interfere with the mercury detection. PMID:23565989

  8. Epidemiological typing of Moraxella catarrhalis by using DNA probes.

    PubMed Central

    Beaulieu, D; Scriver, S; Bergeron, M G; Low, D E; Parr, T R; Patterson, J E; Matlow, A; Roy, P H

    1993-01-01

    Small-fragment restriction enzyme analysis and DNA-DNA hybridization were used to compare 60 strains of Moraxella catarrhalis isolated from various geographic locations. Restriction enzyme analysis with HaeIII resulted in 46 different patterns, 7 of which were shared by more than one isolate. Hybridizations with two DNA probes resulted in 18 different patterns, 11 of which were shared by more than one isolate. Strains with the same restriction enzyme pattern always had the same hybridization pattern. However, of the 50 strains that shared the 11 hybridization patterns, 39 could be further differentiated by restriction enzyme analysis. We found that hybridization is a method that is specific for the epidemiological typing of M. catarrhalis, but because of limited sensitivity, combination with small-fragment restriction enzyme analysis may be necessary to better determine the relatedness of strains. Images PMID:8096219

  9. Probing Nucleosome Remodeling by Unzipping Single DNA Molecules

    NASA Astrophysics Data System (ADS)

    Wang, Michelle

    2006-03-01

    At the core of eukaryotic chromatin is the nucleosome, which consists of 147 bp of DNA wrapped 1.65 turns around an octamer of histone proteins. Even this lowest level of genomic compaction presents a strong barrier to DNA-binding cellular factors that are required for essential processes such as transcription, DNA replication, recombination and repair. Chromatin remodeling enzymes use the energy of ATP hydrolysis to regulate accessibility of the genetic code by altering chromatin structure. While remodeling enzymes have been the subject of extensive research in recent years, their precise mechanism remains unclear. In order to probe the structure of individual nucleosomes and their remodeling, we assembled a histone octamer onto a DNA segment containing a strong nucleosome positioning sequence. As the DNA double helix was unzipped through the nucleosome using a feedback-enhanced optical trap, the presence of the nucleosome was detected as a series of dramatic increases in the tension in the DNA, followed by sudden tension reductions. Analysis of the unzipping force throughout the disruption accurately revealed the spatial location and fine structure of the nucleosome to near base pair precision. Using this approach, we investigate how remodeling enzymes may alter the location and structure of a nucleosome.

  10. Isolation of human minisatellite loci detected by synthetic tandem repeat probes: direct comparison with cloned DNA fingerprinting probes.

    PubMed

    Armour, J A; Vergnaud, G; Crosier, M; Jeffreys, A J

    1992-08-01

    As a direct comparison with cloned 'DNA fingerprinting' probes, we present the results of screening an ordered array Charomid library for hypervariable human loci using synthetic tandem repeat (STR) probes. By recording the coordinates of positive hybridization signals, the subset of clones within the library detected by each STR probe can be defined, and directly compared with the set of clones detected by naturally occurring (cloned) DNA fingerprinting probes. The STR probes vary in the efficiency of detection of polymorphic minisatellite loci; among the more efficient probes, there is a strong overlap with the sets of clones detected by the DNA fingerprinting probes. Four new polymorphic loci were detected by one or more of the STR probes but not by any of the naturally occurring repeats. Sequence comparisons with the probe(s) used to detect the locus suggest that a relatively poor match, for example 10 out of 14 bases in a limited region of each repeat, is sufficient for the positive detection of tandem repeats in a clone in this type of library screening by hybridization. These results not only provide a detailed evaluation of the usefulness of STR probes in the isolation of highly variable loci, but also suggest strategies for the use of these multi-locus probes in screening libraries for clones from hypervariable loci.

  11. Simulation-guided DNA probe design for consistently ultraspecific hybridization

    NASA Astrophysics Data System (ADS)

    Wang, Juexiao Sherry; Zhang, David Yu

    2015-07-01

    Hybridization of complementary sequences is one of the central tenets of nucleic acid chemistry; however, the unintended binding of closely related sequences limits the accuracy of hybridization-based approaches to analysing nucleic acids. Thermodynamics-guided probe design and empirical optimization of the reaction conditions have been used to enable the discrimination of single-nucleotide variants, but typically these approaches provide only an approximately 25-fold difference in binding affinity. Here we show that simulations of the binding kinetics are both necessary and sufficient to design nucleic acid probe systems with consistently high specificity as they enable the discovery of an optimal combination of thermodynamic parameters. Simulation-guided probe systems designed against 44 sequences of different target single-nucleotide variants showed between a 200- and 3,000-fold (median 890) higher binding affinity than their corresponding wild-type sequences. As a demonstration of the usefulness of this simulation-guided design approach, we developed probes that, in combination with PCR amplification, detect low concentrations of variant alleles (1%) in human genomic DNA.

  12. A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III-aided cycling amplification.

    PubMed

    Zeng, Yan; Wan, Yi; Zhang, Dun; Qi, Peng

    2015-01-01

    A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III (Exo-III) aided cycling amplification has been developed. This magneto-DNA duplex probe contains a partly hybrid fluorophore-modified capture probe and a fluorophore-modified signal probe with magnetic microparticle as carrier. In the presence of a perfectly matched target bacterial DNA, blunt 3'-terminus of the capture probe is formed, activating the Exo-III aided cycling amplification. Thus, Exo-III catalyzes the stepwise removal of mononucleotides from this terminus, releasing both fluorophore-modified signal probe, fluorescent dyes of the capture probe and target DNA. The released target DNA then starts a new cycle, while released fluorescent fragments are recovered with magnetic separation for fluorescence signal collection. This system exhibited sensitive detection of bacterial DNA, with a detection limit of 14 pM because of the unique cleavage function of Exo-III, high fluorescence intensity, and separating function of magneto-DNA duplex probes. Besides this sensitivity, this strategy exhibited excellent selectivity with mismatched bacterial DNA targets and other bacterial species targets and good applicability in real seawater samples, hence, this strategy could be potentially used for qualitative and quantitative analysis of bacteria.

  13. DNA hybridization activity of single-stranded DNA-conjugated gold nanoparticles used as probes for DNA detection

    NASA Astrophysics Data System (ADS)

    Kira, Atsushi; Matsuo, Kosuke; Nakajima, Shin-ichiro

    2016-02-01

    Colloidal nanoparticles (NPs) have potential applications in bio-sensing technologies as labels or signal enhancers. In order to meet demands for a development of biomolecular assays by a quantitative understanding of single-molecule, it is necessary to regulate accuracy of the NPs probes modified with biomolecules to optimize the characteristics of NPs. However, to our knowledge, there is little information about the structural effect of conjugated biomolecules to the NPs. In this study, we investigated the contribution of a density of single-stranded DNA (ssDNA) conjugating gold NP to hybridization activity. Hybridization activity decreased in accordance with increases in the density of attached ssDNAs, likely due to electrostatic repulsion generated by negatively charged phosphate groups in the ssDNA backbone. These results highlight the importance of controlling the density of ssDNAs attached to the surface of NPs used as DNA detection probes.

  14. Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ

    DOEpatents

    Gray, Joe W.; Pinkel, Daniel

    1991-01-01

    A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. Probe is constructed by treating DNA with a restriction enzyme and an exonuclease to form template/primers for a DNA polymerase. The digested strand is resynthesized in the presence of labeled nucleoside triphosphate precursor. Labeled single stranded fragments are separated from the resynthesized fragments to form the probe. Target DNA is treated with the same restriction enzyme used to construct the probe, and is treated with an exonuclease before application of the probe. The method significantly increases the efficiency and specificity of hybridization mixtures by increasing effective probe concentration by eliminating self-hybridization between both probe and target DNAs, and by reducing the amount of target DNA available for mismatched hybridizations.

  15. Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ

    DOEpatents

    Gray, J.W.; Pinkel, D.

    1991-07-02

    A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. The probe is constructed by treating DNA with a restriction enzyme and an exonuclease to form template/primers for a DNA polymerase. The digested strand is resynthesized in the presence of labeled nucleoside triphosphate precursor. Labeled single stranded fragments are separated from the resynthesized fragments to form the probe. Target DNA is treated with the same restriction enzyme used to construct the probe, and is treated with an exonuclease before application of the probe. The method significantly increases the efficiency and specificity of hybridization mixtures by increasing effective probe concentration by eliminating self-hybridization between both probe and target DNAs, and by reducing the amount of target DNA available for mismatched hybridizations. No Drawings

  16. DNA probes for papillomavirus strains readied for cervical cancer screening

    SciTech Connect

    Merz, B.

    1988-11-18

    New Papillomavirus tests are ready to come to the aid of the standard Papanicolauo test in screening for cervical cancer. The new tests, which detect the strains of human papillomavirus (HPV) most commonly associated with human cervical cancer, are designed to be used as an adjunct to rather than as a replacement for the Papanicolaou smears. Their developers say that they can be used to indicated a risk of developing cancer in women whose Papanicolaou smears indicate mild cervical dysplasia, and, eventually, to detect papillomavirus infection in normal Papanicolaou smears. The rationale for HPV testing is derived from a growing body of evidence that HPV is a major factor in the etiology of cervical cancer. Three HPV tests were described recently in Chicago at the Third International Conference on Human Papillomavirus and Squamous Cervical Cancer. Each relies on DNA probes to detect the presence of papillomavirus in cervical cells and/or to distinguish the strain of papillomavirus present.

  17. Probing the microenvironments in the grooves of Z-DNA using dan-modified oligonucleotides.

    PubMed

    Kimura, Takumi; Kawai, Kiyohiko; Majima, Tetsuro

    2006-04-14

    The environment-sensitive fluorophore dan (6-dimethylamino-2-acyl-naphthalene)- modified dC or dG bases were introduced into the Z-DNA forming sequence. It was demonstrated that both grooves of Z-DNA are more hydrated than those of B-DNA. Dan will be useful for probing the microenvironments in the grooves among the DNA polymorphs.

  18. Method and apparatus for synthesis of arrays of DNA probes

    DOEpatents

    Cerrina, Francesco; Sussman, Michael R.; Blattner, Frederick R.; Singh-Gasson, Sangeet; Green, Roland

    2002-04-23

    The synthesis of arrays of DNA probes sequences, polypeptides, and the like is carried out using a patterning process on an active surface of a substrate. An image is projected onto the active surface of the substrate utilizing an image former that includes a light source that provides light to a micromirror device comprising an array of electronically addressable micromirrors, each of which can be selectively tilted between one of at least two positions. Projection optics receives the light reflected from the micromirrors along an optical axis and precisely images the micromirrors onto the active surface of the substrate, which may be used to activate the surface of the substrate. The first level of bases may then be applied to the substrate, followed by development steps, and subsequent exposure of the substrate utilizing a different pattern of micromirrors, with further repeats until the elements of a two dimensional array on the substrate surface have an appropriate base bound thereto. The micromirror array can be controlled in conjunction with a DNA synthesizer supplying appropriate reagents to a flow cell containing the active substrate to control the sequencing of images presented by the micromirror array in coordination of the reagents provided to the substrate.

  19. Development of a diagnostic test for Johne's disease using a DNA hybridization probe.

    PubMed Central

    Hurley, S S; Splitter, G A; Welch, R A

    1989-01-01

    A DNA probe, M13 mpHAW71, that detects Mycobacterium paratuberculosis in the fecal material of infected animals was developed for use in the diagnosis of Johne's disease. The probe detected as few as 10(5) M. paratuberculosis when hybridized under stringent conditions to total genomic DNA purified from bovine fecal material. When the probe was used diagnostically, it did not differentiate members of the Mycobacterium avium-M. intracellulare-M. paratuberculosis complex. Compared with culturing, the DNA probe identified 34.4% more mycobacterium-containing fecal samples, and testing took only 72 h to complete. Images PMID:2768445

  20. Surface-Enhanced Raman Scattering Based Nonfluorescent Probe for Multiplex DNA Detection

    PubMed Central

    Sun, Lan; Yu, Chenxu; Irudayaraj, Joseph

    2008-01-01

    To provide rapid and accurate detection of DNA markers in a straightforward, inexpensive and multiplex format, an alternative surface enhanced Raman scattering (SERS) based probe was designed and fabricated to covalently attach both DNA probing sequence and non-fluorescent Raman tags to the surface of gold nanoparticles (DNA-AuP-RTag). The intensity of Raman signal of the probes could be controlled through the surface coverage of the non-fluorescent Raman tags (RTags). Detection sensitivity of these probes could be optimized by fine-tuning the amount of DNA molecules and RTags on the probes. Long-term stability of the DNA-AuP-RTag probes was found to be good (over 3 months). Excellent multiplexing capability of the DNA-AuP-RTag scheme was demonstrated by simultaneous identification of up to eight probes in a mixture. Detection of hybridization of single-stranded DNA (ssDNA) to its complementary targets was successfully accomplished with a long-term goal to use non-fluorescent RTags in a Raman-based DNA microarray platform. PMID:17465531

  1. Detection of foodborne pathogens using DNA probes and a dipstick format.

    PubMed

    Groody, E P

    1996-12-01

    The detection of foodborne microorganisms has traditionally been done using microbiologically based methods. Such "gold standard" methods are generally reliable but have the disadvantages of being labor intensive, subjective, and time consuming. Over the last several years, the development of DNA probe-based methods has simplified the methods used to detect organisms such as Salmonella, Listeria, and E. coli by targeting the unique DNA or RNA sequences of these organisms using DNA probes and nonradioactive detection.

  2. Identification of enterotoxigenic Escherichia coli with synthetic alkaline phosphatase-conjugated oligonucleotide DNA probes.

    PubMed Central

    Seriwatana, J; Echeverria, P; Taylor, D N; Sakuldaipeara, T; Changchawalit, S; Chivoratanond, O

    1987-01-01

    Alkaline phosphatase-conjugated (AP) 26-base oligonucleotide DNA probes were compared with the same probes labeled with gamma-32P for the identification of heat-labile (LT) and heat-stable (ST) enterotoxigenic Escherichia coli (ETEC). The AP oligonucleotide probes were as sensitive as the radiolabeled (RL) probes in detecting LT and STA-2 target cell DNA, but the AP ST probe, which differed from STA-1 by two bases, was less sensitive than the RL probe in detecting STA-1 DNA (6.25 versus 0.78 ng). Of 94 ETEC that were identified with the RL probe, the AP probes detected 93% (28 of 30) of ST, 73% (25 of 34) of LT, and 67% (20 of 30) of LTST ETEC. When colony lysates of these ETEC were examined, the AP probes identified all 94 ETEC. In examinations of stool blots, the RL and AP probes were shown to have sensitivities of 71 and 59%, specificities of 91 and 86%, positive predictive values of 87 and 73%, and negative predictive values of 86 and 74%, respectively. AP oligonucleotide probes to detect ETEC were less sensitive in detecting ETEC by colony or stool blot hybridization than the RL probes but could be used by laboratories without access to radioisotopes to examine colony lysates. Images PMID:3305559

  3. A comparative hybridization analysis of yeast DNA with Paramecium parafusin- and different phosphoglucomutase-specific probes.

    PubMed

    Wyroba, E; Satir, B H

    2000-01-01

    Molecular probes designed for the parafusin (PFUS), the Paramecium exocytic-sensitive phosphoglycoprotein, gave distinct hybridization patterns in Saccharomyces cerevisiae genomic DNA when compared with different phosphoglucomutase specific probes. These include two probes identical to segments of yeast phosphoglucomutase (PGM) genes 1 and 2. Neither of the PGM probes revealed the 7.4 and 5.9 kb fragments in Bgl II-cut yeast DNA digest detected with the 1.6 kb cloned PFUS cDNA and oligonucleotide constructed to the PFUS region (insertion 3--I-3) not found in other species. PCR amplification with PFUS-specific primers generated yeast DNA-species of the predicted molecular size which hybridized to the I-3 probe. A search of the yeast genome database produced an unassigned nucleotide sequence that showed 55% identity to parafusin gene and 37% identity to PGM2 (the major isoform of yeast phosphoglucomutase) within the amplified region.

  4. DNA-probe-target interaction based detection of Brucella melitensis by using surface plasmon resonance.

    PubMed

    Sikarwar, Bhavna; Singh, Virendra V; Sharma, Pushpendra K; Kumar, Ashu; Thavaselvam, Duraipandian; Boopathi, Mannan; Singh, Beer; Jaiswal, Yogesh K

    2017-01-15

    Surface plasmon resonance (SPR) immunosensor using 4-mercaptobenzoic acid (4-MBA) modified gold (4-MBA/Au) SPR chip was developed first time for the detection of Brucella melitensis (B. melitensis) based on the screening of its complementary DNA target by using two different newly designed DNA probes of IS711 gene. Herein, interaction between DNA probes and target molecule are also investigated and result revealed that the interaction is spontaneous. The kinetics and thermodynamic results derived from the experimental data showed that the interaction between complementary DNA targets and probe 1 is more effective than that of probe 2. Equilibrium dissociation constant (KD) and maximum binding capacity of analyte (Bmax) values for the interaction of complementary DNA target with the immobilized DNA probes were calculated by using kinetic evaluation software, and found to be 15.3 pM (KD) and 81.02m° (Bmax) with probe 1 and 54.9pM and 55.29m° (Bmax), respectively. Moreover, real serum samples analysis were also carried out using immobilized probe 1 and probe 2 with SPR which showed the applicability of this methodology and provides an alternative way for the detection of B. melitensis in less than 10min. This remarkable sensing response of present methodology offer a real time and label free detection of biological warfare agent and provide an opportunity to make miniaturized sensor, indicating considerable promise for diverse environmental, bio-defence, clinical diagnostics, food safety, water and security applications.

  5. Non-Covalent Fluorescent Labeling of Hairpin DNA Probe Coupled with Hybridization Chain Reaction for Sensitive DNA Detection.

    PubMed

    Song, Luna; Zhang, Yonghua; Li, Junling; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

    2016-04-01

    An enzyme-free signal amplification-based assay for DNA detection was developed using fluorescent hairpin DNA probes coupled with hybridization chain reaction (HCR). The hairpin DNAs were designed to contain abasic sites in the stem moiety. Non-covalent labeling of the hairpin DNAs was achieved when a fluorescent ligand was bound to the abasic sites through hydrogen bonding with the orphan cytosine present on the complementary strand, accompanied by quench of ligand fluorescence. As a result, the resultant probes, the complex formed between the hairpin DNA and ligand, showed almost no fluorescence. Upon hybridization with target DNA, the probe underwent a dehybridization of the stem moiety containing an abasic site. The release of ligand from the abasic site to the solution resulted in an effective fluorescent enhancement, which can be used as a signal. Compared with a sensing system without HCR, a 20-fold increase in the sensitivity was achieved using the sensing system with HCR. The fluorescent intensity of the sensing system increased with the increase in target DNA concentration from 0.5 nM to 100 nM. A single mismatched target ss-DNA could be effectively discriminated from complementary target DNA. Genotyping of a G/C single-nucleotide polymorphism of polymerase chain reaction (PCR) products was successfully demonstrated with the sensing system. Therefore, integrating HCR strategy with non-covalent labeling of fluorescent hairpin DNA probes provides a sensitive and cost-effective DNA assay.

  6. Evaluation of a prototype DNA probe test for the noncultural diagnosis of gonorrhea.

    PubMed Central

    Granato, P A; Franz, M R

    1989-01-01

    A prototype, nonisotopic, chemiluminescent DNA probe test called the Gen-Probe PACE (Probe Assay-Chemiluminescence Enhanced) system for Neisseria gonorrhoeae (Gen-Probe, San Diego, Calif.) was compared with conventional Martin-Lewis culture medium in JEMBEC plates for the laboratory diagnosis of gonorrhea. This 2-h noncultural assay is based upon the use of an acridinium ester-labeled DNA probe. The rRNA-directed DNA probe hybridizes with the target rRNA, and the hybridized probe is separated from the unhybridized probe through the use of magnetic microparticles. The esterified acridinium is hydrolyzed from the hybridized probe by the addition of an alkaline hydrogen peroxide solution, resulting in the production of visible light which is measured in a luminometer. The amount of light generated is directly proportional to the amount of gonococcal target rRNA present in the sample. A total of 407 clinical specimens (203 urethral and 204 endocervical) were collected from high-risk walk-in patients attending a sexually transmitted disease clinic. Separate patient specimens were collected for culture on Martin-Lewis medium in JEMBEC plates and for DNA probe assay. Statistical analysis of the overall comparative results showed that the DNA probe assay had a sensitivity, specificity, and positive and negative predictive values of 93, 99, 97, and 99%, respectively, in a patient population with a gonococcal disease prevalence of 21%. The results of this comparative study showed that the prototype chemiluminescent DNA probe assay is a rapid and reliable noncultural alternative for the laboratory diagnosis of gonorrhea. PMID:2498388

  7. Probing Minor Groove Hydrogen Bonding Interactions between RB69 DNA Polymerase and DNA

    SciTech Connect

    Xia, Shuangluo; Christian, Thomas D.; Wang, Jimin; Konigsberg, William H.

    2012-09-17

    Minor groove hydrogen bonding (HB) interactions between DNA polymerases (pols) and N3 of purines or O2 of pyrimidines have been proposed to be essential for DNA synthesis from results obtained using various nucleoside analogues lacking the N3 or O2 contacts that interfered with primer extension. Because there has been no direct structural evidence to support this proposal, we decided to evaluate the contribution of minor groove HB interactions with family B pols. We have used RB69 DNA pol and 3-deaza-2'-deoxyadenosine (3DA), an analogue of 2-deoxyadenosine, which has the same HB pattern opposite T but with N3 replaced with a carbon atom. We then determined pre-steady-state kinetic parameters for the insertion of dAMP opposite dT using primer/templates (P/T)-containing 3DA. We also determined three structures of ternary complexes with 3DA at various positions in the duplex DNA substrate. We found that the incorporation efficiency of dAMP opposite dT decreased 10{sup 2}-10{sup 3}-fold even when only one minor groove HB interaction was missing. Our structures show that the HB pattern and base pair geometry of 3DA/dT is exactly the same as those of dA/dT, which makes 3DA an optimal analogue for probing minor groove HB interactions between a DNA polymerase and a nucleobase. In addition, our structures provide a rationale for the observed 10{sup 2}-10{sup 3}-fold decrease in the rate of nucleotide incorporation. The minor groove HB interactions between position n-2 of the primer strand and RB69pol fix the rotomer conformations of the K706 and D621 side chains, as well as the position of metal ion A and its coordinating ligands, so that they are in the optinal orientation for DNA synthesis.

  8. Detecting the effects of toxic agents on spermatogenesis using DNA probes

    SciTech Connect

    Hecht, N.B.

    1987-10-01

    Advances in the molecular biology of spermatogenesis suggest that DNA probes can be used to monitor the effects of toxic agents in male germ cells of mammals. Molecular hybridization analyses with DNA probes can provide a reproducible methodology capable of detecting changes ranging from massive deletions to single base pair substitutions in the genome of exposed individuals. A constantly increasing number of DNA probes that can be used to detect such alterations in human sperm DNA exist for both ubiquitously expressed proteins and for genes solely expressed in the testis. In this chapter, the currently available testicular stage-specific and/or cell type-specific DNA probes and the techniques by which they can be utilized in reproductive toxicology studies are discussed. The advantages, limitations, and future technological advances of this novel biological marker system for the human male reproductive system are also considered.

  9. A Microfluidic Microbeads Fluorescence Assay with Quantum Dots-Bead-DNA Probe.

    PubMed

    Ankireddy, S R; Kim, Jongsung

    2016-03-01

    A microfluidic bead-based nucleic acid sensor for the detection of tumor causing N-Ras genes using quantum dots has been developed. Presently, quantum dots-bead-DNA probe based hybridization detection methods are often called as 'bead based assays' and their success is substantially influenced by the dispensing and manipulation capability of the microfluidic technology. This study reports the detection of N-Ras cancer gene by fluorescence quenching of quantum dots immobilized on the surface of polystyrene beads. A microfluidic chip was constructed in which the quantum dots-bead-DNA probes were packed in the channel. The target DNA flowed across the beads and hybridized with immobilized probe sequences. The target DNA can be detected by the fluorescence quenching of the quantum dots due to their transfer of emission energy to intercalation dye after DNA hybridization. The mutated gene also induces fluorescence quenching but with less degree than the perfectly complementary target DNA.

  10. Effect of salts, solvents and buffer on miRNA detection using DNA silver nanocluster (DNA/AgNCs) probes

    NASA Astrophysics Data System (ADS)

    Shah, Pratik; Cho, Seok Keun; Waaben Thulstrup, Peter; Bhang, Yong-Joo; Ahn, Jong Cheol; Choi, Suk Won; Rørvig-Lund, Andreas; Yang, Seong Wook

    2014-01-01

    MicroRNAs (miRNAs) are small regulatory RNAs (size ˜21 nt to ˜25 nt) which regulate a variety of important cellular events in plants, animals and single cell eukaryotes. Especially because of their use in diagnostics of human diseases, efforts have been directed towards the invention of a rapid, simple and sequence selective detection method for miRNAs. Recently, we reported an innovative method for the determination of miRNA levels using the red fluorescent properties of DNA/silver nanoclusters (DNA/AgNCs). Our method is based on monitoring the emission drop of a DNA/AgNCs probe in the presence of its specific target miRNA. Accordingly, the accuracy and efficiency of the method relies on the sensitivity of hybridization between the probe and target. To gain specific and robust hybridization between probe and target, we investigated a range of diverse salts, organic solvents, and buffer to optimize target sensing conditions. Under the newly adjusted conditions, the target sensitivity and the formation of emissive DNA/AgNCs probes were significantly improved. Also, fortification of the Tris-acetate buffer with inorganic salts or organic solvents improved the sensitivity of the DNA/AgNC probes. On the basis of these optimizations, the versatility of the DNA/AgNCs-based miRNA detection method can be expanded.

  11. Invader probes: Harnessing the energy of intercalation to facilitate recognition of chromosomal DNA for diagnostic applications.

    PubMed

    Guenther, Dale C; Anderson, Grace H; Karmakar, Saswata; Anderson, Brooke A; Didion, Bradley A; Guo, Wei; Verstegen, John P; Hrdlicka, Patrick J

    2015-08-01

    Development of probes capable of recognizing specific regions of chromosomal DNA has been a long-standing goal for chemical biologists. Current strategies such as PNA, triplex-forming oligonucleotides, and polyamides are subject to target choice limitations and/or necessitate non-physiological conditions, leaving a need for alternative approaches. Toward this end, we have recently introduced double-stranded oligonucleotide probes that are energetically activated for DNA recognition through modification with +1 interstrand zippers of intercalator-functionalized nucleotide monomers. Here, probes with different chemistries and architectures - varying in the position, number, and distance between the intercalator zippers - are studied with respect to hybridization energetics and DNA-targeting properties. Experiments with model DNA targets demonstrate that optimized probes enable efficient (C50 < 1 μM), fast (t50 < 3h), kinetically stable (> 24h), and single nucleotide specific recognition of DNA targets at physiologically relevant ionic strengths. Optimized probes were used in non-denaturing fluorescence in situ hybridization experiments for detection of gender-specific mixed-sequence chromosomal DNA target regions. These probes present themselves as a promising strategy for recognition of chromosomal DNA, which will enable development of new tools for applications in molecular biology, genomic engineering and nanotechnology.

  12. Invader probes: Harnessing the energy of intercalation to facilitate recognition of chromosomal DNA for diagnostic applications†

    PubMed Central

    Guenther, Dale C.; Anderson, Grace H.; Karmakar, Saswata; Anderson, Brooke A.; Didion, Bradley A.; Guo, Wei; Verstegen, John P.; Hrdlicka, Patrick J.

    2015-01-01

    Development of probes capable of recognizing specific regions of chromosomal DNA has been a long-standing goal for chemical biologists. Current strategies such as PNA, triplex-forming oligonucleotides, and polyamides are subject to target choice limitations and/or necessitate non-physiological conditions, leaving a need for alternative approaches. Toward this end, we have recently introduced double-stranded oligonucleotide probes that are energetically activated for DNA recognition through modification with +1 interstrand zippers of intercalator-functionalized nucleotide monomers. Here, probes with different chemistries and architectures – varying in the position, number, and distance between the intercalator zippers – are studied with respect to hybridization energetics and DNA-targeting properties. Experiments with model DNA targets demonstrate that optimized probes enable efficient (C50 < 1 μM), fast (t50 < 3h), kinetically stable (> 24h), and single nucleotide specific recognition of DNA targets at physiologically relevant ionic strengths. Optimized probes were used in non-denaturing fluorescence in situ hybridization experiments for detection of gender-specific mixed-sequence chromosomal DNA target regions. These probes present themselves as a promising strategy for recognition of chromosomal DNA, which will enable development of new tools for applications in molecular biology, genomic engineering and nanotechnology. PMID:26240741

  13. Conditionally fluorescent molecular probes for detecting single base changes in double-stranded DNA.

    PubMed

    Chen, Sherry Xi; Zhang, David Yu; Seelig, Georg

    2013-09-01

    Small variations in nucleic acid sequences can have far-reaching phenotypic consequences. Reliably distinguishing closely related sequences is therefore important for research and clinical applications. Here, we demonstrate that conditionally fluorescent DNA probes are capable of distinguishing variations of a single base in a stretch of target DNA. These probes use a novel programmable mechanism in which each single nucleotide polymorphism generates two thermodynamically destabilizing mismatch bubbles rather than the single mismatch formed during typical hybridization-based assays. Up to a 12,000-fold excess of a target that contains a single nucleotide polymorphism is required to generate the same fluorescence as one equivalent of the intended target, and detection works reliably over a wide range of conditions. Using these probes we detected point mutations in a 198 base-pair subsequence of the Escherichia coli rpoB gene. That our probes are constructed from multiple oligonucleotides circumvents synthesis limitations and enables long continuous DNA sequences to be probed.

  14. Study on the SPR responses of various DNA probe concentrations by parallel scan spectral SPR imaging

    NASA Astrophysics Data System (ADS)

    Ma, Suihua; Liu, Le; Lu, Weiping; Zhang, Yaou; He, Yonghong; Guo, Jihua

    2008-12-01

    SPR sensors have become a high sensitive and label free method for characterizing and quantifying chemical and biochemical interactions. However, the relations between the SPR refractive index response and the property (such as concentrations) of biochemical probes are still lacking. In this paper, an experimental study on the SPR responses of varies concentrations of Legionella pneumophila mip DNA probes is presented. We developed a novel two-dimensional SPR sensing technique-parallel scan spectral SPR imaging-to detect an array of mip gene probes. This technique offers quantitative refractive index information with a high sensing throughput. By detecting mip DNA probes with different concentrations, we obtained the relations between the SPR refractive index response and the concentrations of mip DNA probes. These results are valuable for design and developing SPR based mip gene biochips.

  15. Conditionally fluorescent molecular probes for detecting single base changes in double-stranded DNA

    NASA Astrophysics Data System (ADS)

    Chen, Sherry Xi; Zhang, David Yu; Seelig, Georg

    2013-09-01

    Small variations in nucleic acid sequences can have far-reaching phenotypic consequences. Reliably distinguishing closely related sequences is therefore important for research and clinical applications. Here, we demonstrate that conditionally fluorescent DNA probes are capable of distinguishing variations of a single base in a stretch of target DNA. These probes use a novel programmable mechanism in which each single nucleotide polymorphism generates two thermodynamically destabilizing mismatch bubbles rather than the single mismatch formed during typical hybridization-based assays. Up to a 12,000-fold excess of a target that contains a single nucleotide polymorphism is required to generate the same fluorescence as one equivalent of the intended target, and detection works reliably over a wide range of conditions. Using these probes we detected point mutations in a 198 base-pair subsequence of the Escherichia coli rpoB gene. That our probes are constructed from multiple oligonucleotides circumvents synthesis limitations and enables long continuous DNA sequences to be probed.

  16. DNA probes for identification of enteroinvasive Escherichia coli.

    PubMed Central

    Gomes, T A; Toledo, M R; Trabulsi, L R; Wood, P K; Morris, J G

    1987-01-01

    Eighty-one Escherichia coli strains belonging to all known invasive O serogroups were tested with two distinct invasiveness probes (pMR17 and pSF55). All 54 Sereny test-positive strains and 5 strains that lost Sereny positivity during storage hybridized with both probes. Probe-positive strains carried a 120- to 140-megadalton plasmid, did not produce lysine decarboxylase, and, with the exception of certain serotypes, were nonmotile. Motile strains of serotype O144:H25 were for the first time characterized as invasive by hybridization with the probes. PMID:3312292

  17. Energy-transfer-based wavelength-shifting DNA probes with "clickable" cyanine dyes.

    PubMed

    Holzhauser, Carolin; Rubner, Moritz M; Wagenknecht, Hans-Achim

    2013-05-01

    The insertion of cyanine dye azides as energy donor dyes via postsynthetic "click"-type cycloaddition chemistry with e.g. a new thiazole orange azide combined with thiazole red yields dual emitting DNA probes with good fluorescence readout properties.

  18. Computer programs used to aid in the selection of DNA hybridization probes.

    PubMed Central

    Raupach, R E

    1984-01-01

    This paper describes a package of three programs which used together aid in selecting the best possible sequence to be used as a DNA hybridization probe. This system searches an amino acid sequence for four adjacent amino acids with the fewest possible corresponding mRNA sequences, calculates their probability of occurrence, and locates the positions of wobbles and mismatches between the DNA hybridization probe and the possible mRNA sequences. PMID:6546442

  19. Neutral red as a specific light-up fluorescent probe for i-motif DNA.

    PubMed

    Xu, Lijun; Wang, Jine; Sun, Na; Liu, Min; Cao, Yi; Wang, Zhili; Pei, Renjun

    2016-12-06

    We report a specific light-up fluorescent probe for i-motif DNA for the first time. Compared with the previously reported probes, neutral red could selectively interact with an i-motif and show a significant increase in its fluorescence. This feature makes it advantageous for designing label-free fluorescent sensing systems.

  20. Polyamide fluorescent probes for visualization of repeated DNA sequences in living cells.

    PubMed

    Nozeret, Karine; Loll, François; Escudé, Christophe; Boutorine, Alexandre S

    2015-03-02

    DNA imaging in living cells usually requires transgenic approaches that modify the genome. Synthetic pyrrole-imidazole polyamides that bind specifically to the minor groove of double-stranded DNA (dsDNA) represent an attractive approach for in-cell imaging that does not necessitate changes to the genome. Nine hairpin polyamides that target mouse major satellite DNA were synthesized. Their interactions with synthetic target dsDNA fragments were studied by thermal denaturation, gel-shift electrophoresis, circular dichroism, and fluorescence spectroscopy. The polyamides had different affinities for the target DNA, and fluorescent labeling of the polyamides affected their affinity for their targets. We validated the specificity of the probes in fixed cells and provide evidence that two of the probes detect target sequences in mouse living cell lines. This study demonstrates for the first time that synthetic compounds can be used for the visualization of the nuclear substructures formed by repeated DNA sequences in living cells.

  1. Employing double-stranded DNA probes on colloidal substrates for competitive hybridization events

    NASA Astrophysics Data System (ADS)

    Baker, Bryan Alexander

    DNA has found application beyond its biological function in the cell in a variety of materials assembly systems as well as nucleic acid-based detection devices. In the current research, double-stranded DNA probes are applied in both a colloidal particle assembly and fluorescent assay approach utilizing competitive hybridization interactions. The responsiveness of the double-stranded probes (dsProbes) was tuned by sequence design and tested against a variety of nucleic acid targets. Chapter 1 provides a review of the particle substrate used in the current research, colloidal particles, as well as examines previous applications of DNA in assembly and nucleic acid detection formats. Chapter 2 discusses the formation of fluorescent satellites, or similarly termed fluorescent micelles, via DNA hybridization. The effects of DNA duplex sequence, temperature at which assembly occurs, and oligonucleotide density are variables considered with preferential assembly observed for low oligonucleotide density particles. Chapter 3 demonstrates the controlled disassembly of these satellite structures via competitive hybridization with a soluble target strand. Chapter 4 examines DNA duplexes as fluorescent dsProbes and characterizes the kinetics of competitive hybridization between immobilized dsProbes and solution targets of interest. The sequence-based affinities of dsProbes as well as location of an embedded target sequence are both variables explored in this study. Based on the sequence design of the dsProbes, a range of kinetics responses are observed. Chapter 5 also examines the kinetics of competitive hybridization with dsProbes but with a focus on the specificity of competitive target by including mismatches within a short 15 base competitive target. Chapter 6 examines the effects of dsProbe orientation relative to the particle surface as well as substrate particle size. The kinetics of displacement of DNA targets with those of RNA targets of analogous sequence are also

  2. [Cu(phen)2](2+) acts as electrochemical indicator and anchor to immobilize probe DNA in electrochemical DNA biosensor.

    PubMed

    Yang, Linlin; Li, Xiaoyu; Li, Xi; Yan, Songling; Ren, Yinna; Wang, Mengmeng; Liu, Peng; Dong, Yulin; Zhang, Chaocan

    2016-01-01

    We demonstrate a novel protocol for sensitive in situ label-free electrochemical detection of DNA hybridization based on copper complex ([Cu(phen)2](2+), where phen = 1,10-phenanthroline) and graphene (GR) modified glassy carbon electrode. Here, [Cu(phen)2](2+) acted advantageously as both the electrochemical indicator and the anchor for probe DNA immobilization via intercalative interactions between the partial double helix structure of probe DNA and the vertical aromatic groups of phen. GR provided large density of docking site for probe DNA immobilization and increased the electrical conductivity ability of the electrode. The modification procedure was monitored by electrochemical impedance spectroscopy (EIS). Square-wave voltammetry (SWV) was used to explore the hybridization events. Under the optimal conditions, the designed electrochemical DNA biosensor could effectively distinguish different mismatch degrees of complementary DNA from one-base mismatch to noncomplementary, indicating that the biosensor had high selectivity. It also exhibited a reasonable linear relationship. The oxidation peak currents of [Cu(phen)2](2+) were linear with the logarithm of the concentrations of complementary target DNA ranging from 1 × 10(-12) to 1 × 10(-6) M with a detection limit of 1.99 × 10(-13) M (signal/noise = 3). Moreover, the stability of the electrochemical DNA biosensor was also studied.

  3. 16S rRNA gene probe quantitates residual host cell DNA in pharmaceutical-grade plasmid DNA.

    PubMed

    Wang, Kai-Yu; Guo, Ying-Jun; Sun, Shu-Han; Shi, Ke; Zhang, Shu; Wang, Kai-Hui; Yi-Zhang; Chen, Zu-Huan

    2006-03-24

    The development and widespread use of DNA-based vaccination against infectious pathogens have been a great triumph of medical science. Quality control of DNA vaccines as biopharmaceutical productions is a problem to solve. Residual genomic DNA of engineering bacteria has been identified as a potential risk factor, so whose level must be controlled under the regulatory standards. We report a dot-blot hybridization method to detect residual host cell DNA in purified DNA vaccines. The assay utilizes PCR amplified and digoxigenin-labeled Escherichia coli 16S rRNA gene as probe. The sensitivity of the dot-blot hybridization assay with E. coli 16S rRNA gene probe was evaluated in comparison with single copy UidR gene probe. The optimized dot-blot hybridization assay had both low background and a suitable sensitivity, detecting 10 pg of residual E. coli DNA. The method is suitable in the routine use of measuring the levels of residual E. coli DNA in the pharmaceutical-grade DNA vaccine.

  4. Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes.

    PubMed

    García-Cañas, Virginia; Mondello, Monica; Cifuentes, Alejandro

    2011-05-01

    New DNA amplification methods are continuously developed for sensitive detection and quantification of specific DNA target sequences for, e.g. clinical, environmental or food applications. These new applications often require the use of long DNA oligonucleotides as probes for target sequences hybridization. Depending on the molecular technique, the length of DNA probes ranges from 40 to 450 nucleotides, solid-phase chemical synthesis being the strategy generally used for their production. However, the fidelity of chemical synthesis of DNA decreases for larger DNA probes. Defects in the oligonucleotide sequence result in the loss of hybridization efficiency, affecting the sensitivity and selectivity of the amplification method. In this work, an enzymatic procedure has been developed as an alternative to solid-phase chemical synthesis for the production of long oligonucleotides. The enzymatic procedure for probe production was based on ligation of short DNA sequences. Long DNA probes were obtained from smaller oligonucleotides together with a short sequence that acts as bridge stabilizing the molecular complex for DNA ligation. The ligation reactions were monitored by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) using a bare fused-silica capillary. The capillary gel electrophoresis-LIF method demonstrated to be very useful and informative for the characterization of the ligation reaction, providing important information about the nature of some impurities, as well as for the fine optimization of the ligation conditions (i.e. ligation cycles, oligonucleotide and enzyme concentration). As a result, the yield and quality of the ligation product were highly improved. The in-lab prepared DNA probes were used in a novel multiplex ligation-dependent genome amplification (MLGA) method for the detection of genetically modified maize in samples. The great possibilities of the whole approach were demonstrated by the specific and sensitive

  5. Magnetic microparticle-based multiplexed DNA detection with biobarcoded quantum dot probes.

    PubMed

    Xiang, Dong-shan; Zeng, Guo-ping; He, Zhi-ke

    2011-07-15

    We have developed a new analytical method to detect multiple DNA simultaneously based on the biobarcoded CdSe/ZnS quantum dot (QD) and magnetic microparticle (MMP). It was demonstrated by using oligonucleotide sequences of 64 bases associated with human papillomavirus 16 and 18 L1 genes (HPV-16 and HPV-18) as model systems. This analytical system involves three types of probes, a MMP probe and two streptavidin-modified QD probes. The MMPs are functionalized with HPV-16 and HPV-18 captures DNA to form MMP probes. The QDs are conjugated with HPV-16 or HPV-18 probe DNA along with FAM- or Rox-labeled random DNA to form HPV-16 and HPV-18 QD probes, respectively. A one-step hybridization reaction was performed by mixing the MMP probes, HPV-16 and HPV-18 target DNA (T-16 and T-18), HPV-16 and HPV-18 QD probes. Afterwards, the hybrid-conjugated microparticles were separated by a magnet and heated to remove the MMPs. Finally, the detections of T-16 and T-18 were done by measuring fluorescence signals of FAM and Rox, respectively. Under the optimum conditions, the fluorescence intensity exhibited a good linear dependence on target DNA concentration in the range from 8 × 10⁻¹¹ to 8 × 10⁻⁹ M. The detection limit of T-16 is up to 7 × 10⁻¹¹ M (3σ), and that of T-18 is 6 × 10⁻¹¹ M. Compared with other biobarcode assay methods, the proposed method that QDs were used as the solid support has some advantages including shorter preparation time of QD probes, faster binding kinetics and shorter analytical time. Besides, it is simple and accurate.

  6. Commercial DNA Probes for Mycobacteria Incorrectly Identify a Number of Less Frequently Encountered Species▿ †

    PubMed Central

    Tortoli, Enrico; Pecorari, Monica; Fabio, Giuliana; Messinò, Massimino; Fabio, Anna

    2010-01-01

    Although commercially available DNA probes for identification of mycobacteria have been investigated with large numbers of strains, nothing is known about the ability of these probes to identify less frequently encountered species. We analyzed, with INNO LiPA MYCOBACTERIA (Innogenetics) and with GenoType Mycobacterium (Hein), 317 strains, belonging to 136 species, 61 of which had never been assayed before. INNO LiPA misidentified 20 taxa, the majority of which cross-reacted with the probes specific for Mycobacterium fortuitum and the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum group. GenoType misidentified 28 taxa, most of which cross-reacted with M. intracellulare and M. fortuitum probes; furthermore, eight species were not recognized as members of the genus Mycobacterium. Among 54 strains investigated with AccuProbe (Gen-Probe), cross-reactions were detected for nine species, with the probes aiming at the M. avium complex being most involved in cross-reactions. PMID:19906898

  7. Ultrasensitive flow injection chemiluminescence detection of DNA hybridization using signal DNA probe modified with Au and CuS nanoparticles.

    PubMed

    Zhang, Shusheng; Zhong, Hua; Ding, Caifeng

    2008-10-01

    A novel and sensitive flow injection chemiluminescence assay for sequence-specific DNA detection based on signal amplification with nanoparticles (NPs) is reported in the present work. The "sandwich-type" DNA biosensor was fabricated with the thiol-functionalized capture DNA first immobilized on an Au electrode and hybridized with one end of target DNA, the other end of which was recognized with a signal DNA probe labeled with CuS NPs and Au NPs on the 3'- and 5'-terminus, respectively. The hybridization events were monitored by the CL intensity of luminol-H2O2-Cu(2+) after the cupric ions were dissolved from the hybrids. We demonstrated that the incorporation of Au NPs in this sensor design significantly enhanced the sensitivity and the selectivity because a single Au NP can be loaded with hundreds of signal DNA probe strands, which were modified with CuS NPs. The ratios of Au NPs, signal DNA probes, and CuS NPs modified on the gold electrode were approximately 1/101/103. A preconcentration process of cupric ions performed by anodic stripping voltammetry technology further increased the sensor performance. As a result of these two combined effects, this DNA sensor could detect as low as femtomolar target DNA and exhibited excellent selectivity against two-base mismatched DNA. Under the optimum conditions, the CL intensity was increased with the increase of the concentration of target DNA in the range of 2.0 x 10(-14)-2.0 x 10(-12) M. A detection limit of 4.8 x 10(-15) M target DNA was achieved.

  8. Directly incorporating fluorochromes into DNA probes by PCR increases the efficience of fluorescence in situ hybridization

    SciTech Connect

    Dittmer, Joy

    1996-05-01

    The object of this study was to produce a directly labeled whole chromosome probe in a Degenerative Oligonucleotide Primed-Polymerase Chain Reaction (DOP-PCR) that will identify chromosome breaks, deletions, inversions and translocations caused by radiation damage. In this study we amplified flow sorted chromosome 19 using DOP-PCR. The product was then subjected to a secondary DOP PCR amplification, After the secondary amplification the DOP-PCR product was directly labeled in a tertiary PCR reaction with rhodamine conjugated with dUTP (FluoroRed) to produce a DNA fluorescent probe. The probe was then hybridized to human metaphase lymphocytes on slides, washed and counterstained with 4{prime},6-diamino-2-phenylindole (DAPI). The signal of the FluoroRed probe was then compared to a signal of a probe labeled with biotin and stained with avidin fluorescein isothio cynate (FITC) and anti-avidin FITC. The results show that the probe labeled with FluoroRed gave signals as bright as the probe with biotin labeling. The FluoroRed probe had less noise than the biotin labeled probe. Therefore, a directly labeled probe has been successfully produced in a DOP-PCR reaction. In future a probe labeled with FluoroRed will be produced instead of a probe labeled with biotin to increase efficiency.

  9. Ribosomal DNA spacer probes for yeast identification: studies in the genus Metschnikowia.

    PubMed

    Henriques, M; Sá-Nogueira, I; Giménez-Jurado, G; van Uden, N

    1991-02-01

    To test whether DNA probes derived from ribosomal DNA spacer sequences are suitable for rapid and species-specific yeast identification, a pilot study was undertaken. A 7.7 kb entire ribosomal DNA unit of the type strain of Metschnikowia reukaufii was isolated, cloned and mapped. A 0.65 kb BamHI-HpaI fragment containing non-transcribed spacer sequences was amplified and selected for testing as a 32P hybridization probe with total DNA from the type strains of M. reukaufii, M. pulcherrima, M. lunata, M. bicuspidata, M. australis, M. zobellii, M. krissii, five other strains identified as M. reukaufii and strains of Schizosaccharomyces pombe, Hansenula canadensis, Saccharomyces cerevisiae and Yarrowia lipolytica. The probe hybridized exclusively with DNA from the type strain and four other strains of M. reukaufii. DNA from one strain labelled M. reukaufii did not hybridize with the probe. Subsequent % G + C comparison and DNA-DNA reassociation with the type strain revealed that the non-hybridizing strain does not belong to the species M. reukaufii.

  10. Construction of specific DNA probe for the detection of Salmonella in food.

    PubMed

    Pilantanapak, A; Jayanetra, P; Panbangred, W; Klungthong, C; Bangtrakulnonth, A

    1997-03-01

    The Salmonella specific DNA fragment from genomic DNA of S. typhimurium ATCC 23566 was cloned in E. coli and successfully used as a digoxigenin labeled probe for detecting the presence of Salmonella serotypes in both artificially contaminated food and natural contaminated food samples.

  11. Hairpin DNA probe based surface plasmon resonance biosensor used for the activity assay of E. coli DNA ligase.

    PubMed

    Luan, Qingfen; Xue, Ying; Yao, Xin; Lu, Wu

    2010-02-01

    Using hairpin DNA probe self-structure change during DNA ligation process, a sensitive, label-free and simple method of E. coli DNA ligase assay via a home-built high-resolution surface plasmon resonance (SPR) instrument was developed. The DNA ligation process was monitored in real-time and the effects of single-base mutation on the DNA ligation process were investigated. Then an assay of E. coli DNA ligase was completed with a lower detection limit (0.6 nM), wider concentration range and better reproducibility. Moreover, the influence of Quinacrine on the activity of E. coli DNA ligase was also studied, which demonstrated that our method was useful for drug screening.

  12. Data Mining Empowers the Generation of a Novel Class of Chromosome-specific DNA Probes

    SciTech Connect

    Zeng, Hui; Weier, Heinz-Ulrich G.; Kwan, Johnson; Wang, Mei; O'Brien, Benjamin

    2011-03-08

    Probes that allow accurate delineation of chromosome-specific DNA sequences in interphase or metaphase cell nuclei have become important clinical tools that deliver life-saving information about the gender or chromosomal make-up of a product of conception or the probability of an embryo to implant, as well as the definition of tumor-specific genetic signatures. Often such highly specific DNA probes are proprietary in nature and have been the result of extensive probe selection and optimization procedures. We describe a novel approach that eliminates costly and time consuming probe selection and testing by applying data mining and common bioinformatics tools. Similar to a rational drug design process in which drug-protein interactions are modeled in the computer, the rational probe design described here uses a set of criteria and publicly available bioinformatics software to select the desired probe molecules from libraries comprised of hundreds of thousands of probe molecules. Examples describe the selection of DNA probes for the human X and Y chromosomes, both with unprecedented performance, but in a similar fashion, this approach can be applied to other chromosomes or species.

  13. A DNA probe based on phosphorescent resonance energy transfer for detection of transgenic 35S promoter DNA.

    PubMed

    Lv, Jinzhi; Miao, Yanming; Yang, Jiajia; Qin, Jin; Li, Dongxia; Yan, Guiqin

    2017-05-15

    A QDs-DNA nano-probe was made by combining Mn-doped ZnS room-temperature phosphorescence (RTP) quantum dots (QDs) and DNA. Then an RTP sensor for quantitative detection of genetically-modified mark sequence cauliflower mosaic virus 35S promoter (Ca MV 35S) DNA was built on basis of phosphorescent resonance energy transfer (PRET). The underlying principles were that a QDs-DNA water-soluble nano-probe was built by connecting single-strand DNA to the surfaces of QDs via a ligand exchange method. This probe had good RTP performance and could well identify Ca MV 35S. Thereby, the simple, rapid and efficient detection of genetically-modified organisms was realized. With the increase of target DNA sequence, the phosphorescent intensity of QDs was gradually reduced due to the energy transfer between QDs and the organic quencher BHQ2. This sensor had a detection limit of 4.03nM and a detection range of 12-300nM. Moreover, this sensor had high selectivity. This sensor could effectively detect the target DNA compared with mismatched and random sequences. Thus, this method is very promising for biological analysis.

  14. Probing the binding mode of psoralen to calf thymus DNA.

    PubMed

    Zhou, Xiaoyue; Zhang, Guowen; Wang, Langhong

    2014-06-01

    The binding properties between psoralen (PSO) and calf thymus DNA (ctDNA) were predicted by molecular docking, and then determined with the use of UV-vis absorption, fluorescence, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy, coupled with DNA melting and viscosity measurements. The data matrix obtained from UV-vis spectra was resolved by multivariate curve resolution-alternating least squares (MCR-ALS) approach. The pure spectra and the equilibrium concentration profiles for PSO, ctDNA and PSO-ctDNA complex extracted from the highly overlapping composite response were obtained simultaneously to evaluate the PSO-ctDNA interaction. The intercalation mode of PSO binding to ctDNA was supported by the results from the melting studies, viscosity measurements, iodide quenching and fluorescence polarization experiments, competitive binding investigations and CD analysis. The molecular docking prediction showed that the specific binding most likely occurred between PSO and adenine bases of ctDNA. FT-IR spectra studies further confirmed that PSO preferentially bound to adenine bases, and this binding decreased right-handed helicity of ctDNA and enhanced the degree of base stacking with the preservation of native B-conformation. The calculated thermodynamic parameters indicated that hydrogen bonds and van der Waals forces played a major role in the binding process.

  15. Preparation and chromatographic use of 5'-fluorescent-labelled DNA probes.

    PubMed

    Tous, G; Fausnaugh, J; Vieira, P; Stein, S

    1988-07-01

    A convenient procedure for synthesizing and purifying fluorescently-labelled short DNA probes is reported. DNA probes were chemically synthesized on an automated instrument using the "Aminolink" reagent in the final cycle to attach a primary amino group at the 5'-terminus in the final step. The synthetic oligonucleotides were purified by polyacrylamide urea gel electrophoresis, followed by reversed-phase high-performance liquid chromatography (HPLC). The oligomers were then allowed to react with a fluorescent compound, and the products were separated by HPLC with consecutive detection by UV absorption and fluorescence. Gel permeation chromatography demonstrated that the fluorescent probes were able to form stable hybrids with complementary oligodeoxynucleotides. Furthermore, essentially 100% of the purified fluorescent probe was capable of hybridizing to its complementary strand. Special precautions in handling the fluorescent probes, such as stability, were investigated.

  16. Whole chromosomal DNA probes for rapid identification of Mycobacterium tuberculosis and Mycobacterium avium complex.

    PubMed Central

    Roberts, M C; McMillan, C; Coyle, M B

    1987-01-01

    Whole chromosomal DNA probes were used to identify clinical isolates of Mycobacterium tuberculosis, Mycobacterium avium complex, and Mycobacterium gordonae. The probe for M. tuberculosis was prepared from Mycobacterium bovis BCG, which has been shown to be closely related to M. tuberculosis. A probe for the M. avium complex was prepared from three strains representing each of the three DNA homology groups in the M. avium complex. The probes were used in dot blot assays to identify clinical isolates of mycobacteria. The dot blot test correctly identified 57 of the 61 (93%) cultures grown on solid media, and 100% of antibiotic-treated broth-grown cells were correctly identified. Identification by dot blot required a maximum of 48 h. When the probes were tested against 63 positive BACTEC (Johnston Laboratories, Inc., Towson, Md.) cultures of clinical specimens, 59% were correctly identified. However, of the 14 BACTEC cultures that had been treated with antibiotics before being lysed, 13 (93%) were correctly identified. PMID:3112180

  17. Probing DNA by 2-OG-dependent dioxygenase

    PubMed Central

    Tsai, Chi-Lin; Tainer, John A.

    2014-01-01

    TET-mediated 5-methyl cytosine (5mC) oxidation acts in epigenetic regulation, stem cell development, and cancer. Hu et al. now determine the crystal structure of the TET2 catalytic domain bound to DNA, shedding light on 5mC-DNA substrate recognition and the catalytic mechanism of 5mC oxidation. PMID:24360270

  18. Detection of infectious laryngotracheitis virus infected cells with cloned DNA probes.

    PubMed Central

    Nagy, E

    1992-01-01

    A genomic library of infectious laryngotracheitis virus (ILTV) DNA BamH1 fragments was prepared and two cloned fragments were evaluated for their potential as probes for the detection of ILTV infected cells. The virus was purified by a modified sucrose density gradient procedure for the isolation of pure ILTV DNA. A genomic library was constructed using BamH1-digested ILTV DNA and pGEM7 as a vector. A 1.1 kb cloned BamH1 fragment of ILTV DNA was tested in a slot or dot blot assay for the detection of ILTV infected cells. The limit of detection for this probe was at least 0.12 ng of pure ILTV DNA. The probe was able to identify both chicken embryo liver (CELi) cells and choriallantoic membranes infected with ILTV. Chicken embryo liver cells infected with several field isolates and a vaccine strain of ILTV were positive by dot blot analysis using this probe. Some qualitative differences in the degree of hybridization to cells infected by different ILTV isolates were observed. Uninfected cells and cells infected with fowlpox virus, turkey herpesvirus, Marek's disease virus or Newcastle disease virus were negative by the same assay. Compared with the 1.1 kb fragment, a larger 6 kb cloned BamH1 fragment of ILTV DNA showed a stronger hybridization signal to DNA from ILTV infected cells. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:1316798

  19. Label-free DNA hybridization detection by various spectroscopy methods using triphenylmethane dyes as a probe

    NASA Astrophysics Data System (ADS)

    Tu, Jiaojiao; Cai, Changqun; Ma, Ying; Luo, Lin; Weng, Chao; Chen, Xiaoming

    2012-12-01

    A new assay is developed for direct detection of DNA hybridization using triphenylmethane dye as a probe. It is based on various spectroscopic methods including resonance light scattering (RLS), circular dichroism (CD), ultraviolet spectra and fluorescence spectra, as well as atomic force microscopy (AFM), six triphenylmethane dyes interact with double strand DNA (dsDNA) and single strand DNA (ssDNA) were investigated, respectively. The interaction results in amplified resonance light scattering signals and enables the detection of hybridization without the need for labeling DNA. Mechanism investigations have shown that groove binding occurs between dsDNA and these triphenylmethane dyes, which depends on G-C sequences of dsDNA and the molecular volumes of triphenylmethane dyes. Our present approaches display the advantages of simple and fast, accurate and reliable, and the artificial samples were determined with satisfactory results.

  20. Isolation by genomic subtraction of DNA probes specific for Erwinia carotovora subsp. atroseptica.

    PubMed Central

    Darrasse, A; Kotoujansky, A; Bertheau, Y

    1994-01-01

    Erwinia carotovora subsp. atroseptica is a pathogen of potatoes in Europe because of its ability to induce blackleg symptoms early in the growing season. However, E. carotovora subsp. carotovora is not able to produce such severe symptoms under the same conditions. On the basis of the technique described by Straus and Ausubel (Proc. Natl. Acad. Sci. USA 87:1889-1893, 1990), we isolated DNA sequences of E. carotovora subsp. atroseptica 86.20 that were absent from the genomic DNA of E. carotovora subsp. carotovora CH26. Six DNA fragments ranging from ca. 180 to 400 bp were isolated, cloned, and sequenced. Each fragment was further hybridized with 130 microorganisms including 87 E. carotovora strains. One probe was specific for typical E. carotovora subsp. atroseptica strains, two probes hybridized with all E. carotovora subsp. atroseptica strains and with a few E. carotovora subsp. carotovora strains, and two probes recognized only a subset of E. carotovora subsp. atroseptica strains. The last probe was absent from the genomic DNA of E. carotovora subsp. carotovora CH26 but was present in the genomes of many strains, including those of other species and genera. This probe is homologous to the putP gene of Escherichia coli, which encodes a proline carrier. Further use of the probes is discussed. Images PMID:8117082

  1. New hairpin-structured DNA probes: alternatives to classical molecular beacons

    NASA Astrophysics Data System (ADS)

    Friedrich, Achim; Habl, Gregor; Sauer, Markus; Wolfrum, Jürgen; Hoheisel, Jörg; Marmé, Nicole; Knemeyer, Jens-Peter

    2007-02-01

    In this article we report on two different classes of self-quenching hairpin-structured DNA probes that can be used as alternatives to Molecular Beacons. Compared to other hairpin-structured DNA probes, the so-called smart probes are labeled with only one extrinsic dye. The fluorescence of this dye is efficiently quenched by intrinsic guanine bases via a photo-induced electron transfer reaction in the closed hairpin. After hybridization to a target DNA, the distance between dye and the guanines is enlarged and the fluorescence is restored. The working mechanism of the second class of hairpin DNA probes is similar, but the probe oligonucleotide is labeled at both ends with an identical chromophore and thus the fluorescence of the closed hairpin is reduced due to formation of non-fluorescent dye dimers. Both types of probes are appropriate for the identification of single nucleotide polymorphisms and in combination with confocal single-molecule spectroscopy sensitivities in the picomolar range can be achieved.

  2. Self-quenching DNA probes based on aggregation of fluorescent dyes

    NASA Astrophysics Data System (ADS)

    Schafer, Gabriela; Muller, Matthias; Hafner, Bernhard; Habl, Gregor; Nolte, Oliver; Marme, Nicole; Knemeyer, Jens-Peter

    2005-04-01

    Here we present a novel class of self-quenching, double-labeled DNA probes based on the formation of non fluorescent H-type dye dimers. We therefore investigated the aggregation behavior of the red-absorbing oxazine derivative MR121 and found a dimerization constant of about 3000 M-1. This dye was successfully used to develop hairpin-structured as well as linear self-quenching DNA probes that report the presence of the target DNA by an increase of the fluorescence intensity by a factor of 3 to 12. Generally fluorescence quenching of the hairpin-structure probes is more efficient compared to the linear probes, whereas the kinetic of the fluorescence increase is significantly slower. The new probes were used for the identification of different mycobacteria and their antibiotic resistant species. As a test system a probe for the identification of a DNA sequence specific for the Mycobacterium xenopi was synthesized differing from the sequence of the Mycobacterium fortuitum by 6 nucleotides. Furthermore we developed a method for the discrimination between the sequences of the wild type and an antibiotic resistant species of Mycobacterium tuberculosis. Both sequences differ by just 2 nucleotides and were detected specifically by the use of competing olignonucleotides.

  3. Use of DNA Probes for Diagnosis of Infectious Diseases.

    DTIC Science & Technology

    1987-01-01

    cloned D sequences and mre recently clinical and enviromental sales. The fundamental ongets behind nuc -leic acid inr** m~licatigos. Nucleic acid probe...Professor of Pldical icrobiology and Virology In the School of Public Health and is Director of the Naval Biosciences Laboratory. The research reported here was supported by the Office of Naval Research. - ~-w~ -

  4. A method for recovering strand-specific probes from nick-translated DNA fragments.

    PubMed

    Dutton, F L; Chovnick, A

    1984-07-01

    A method of preparing strand-specific probes for DNA X DNA or DNA X RNA hybridizations is described. Double-stranded DNA fragments are first isolated from any recombinant DNA clone containing the desired sequence, and then labeled in vitro by nick-translation (T. Maniatis, A. Jeffrey, and D. G. Kleid (1975) Proc. Natl. Acad. Sci. USA 72, 1184-1188; P. W. J. Rigby, M. Dieckmann, C. Rhodes, and P. Berg (1977) J. Mol. Biol. 113, 237-251). Sequences homologous to the desired strand are captured by annealing the denatured nick-translate to viral strands of an appropriate M13 clone, and recovered by elution of the resulting hybrids from a column of agarose A50M (Bio-Rad). By this method, separate probes with specificity to either strand, as well as the double-stranded probe, may conveniently be prepared from a single nick-translation reaction. Probes may be obtained which are homologous either to the full length of the cloned region or to selected portions thereof by selecting appropriate M13 clones for annealing. The probe is recovered as a population of fragments several hundred bases or less in length, which have been found ideal for saturating liquid hybridizations, and should be similarly well suited for in situ hybridizations to cytological preparations.

  5. Effect of different concentration of HPV DNA probe immobilization for cervical cancer detection based IDE biosensor

    NASA Astrophysics Data System (ADS)

    Roshila, M. L.; Hashim, U.; Azizah, N.; Nadzirah, Sh.; Arshad, M. K. Md; Ruslinda, A. R.; Gopinath, Subash C. B.

    2017-03-01

    This paper principally delineates to the detection process of Human Papillomavirus (HPV) DNA test. HPV is an extremely common virus infection that infected to human by the progressions cell in the cervix cell. The types of HPV that give a most exceedingly awful infected with cervical cancer is 16 and 18 other than 31 and 45. The HPV DNA probe is immobilized with a different concentration to stabilize the sensitivity. A technique of rapid and sensitive for the HPV identification was proposed by coordinating basic DNA extraction with a quality of DNA. The extraction of the quality of DNA will make a proficiency of the discovery procedure. It will rely on the sequence of the capture probes and the way to support their attached. The fabrication, surface modification, immobilization and hybridization procedures are described by current-voltage (I-V) estimation by utilizing KEITHLEY 6487. This procedure will play out a decent affectability and selectivity of HPV discovery.

  6. Effect of structure on sensing performance of a target induced signaling probe shifting DNA-based (TISPS-DNA) sensor.

    PubMed

    Yu, Xiang; Yu, Zhigang; Li, Fengqin; Xu, Yanmei; He, Xunjun; Xu, Lan; Shi, Wenbing; Zhang, Guiling; Yan, Hong

    2017-05-15

    A type of "signal on" displacement-based sensors named target induced signaling probe shifting DNA-based (TISPS-DNA) sensor were developed for a designated DNA detection. The signaling mechanism of the signaling probe (SP) shifting different from the classical conformation/flexibility change mode endows the sensor with high sensitivity. Through using thiolated or no thiolated capturing probe (CP), two 3-probe sensing structures, sensor-1 and sensor-2, were designed and constructed. The systematical comparing research results show that both sensors exhibit some similarities or big differences in sensing performance. On the one hand, the similarity in structures determines the similarity in some aspects of signaling mechanism, background signal, signal changing form, anti-fouling ability and versatility; on the other hand, the slight difference in structures also results in two opposite hybridization modes of gradual increasing resistance and gradual decreasing resistance which can affect the hybridization efficiency between the assistant probe (AP) and the SP, further producing some big differences in sensing performance, for example, apparently different signal enhancement (SE) change, point mutation discrimination ability and response speed. Under the optimized fabrication and detection conditions, both sensors feature high sensitivity for target DNAs with the detection limits of ∼10 fM for sensor-1 and ∼7 fM for sensor-2, respectively. Among many acquired sensing virtues, the sensor-1 shows a peculiar specificity adjustability which is also a highlight in this work.

  7. Probing the Conformational Distributions of Sub-Persistence Length DNA

    SciTech Connect

    Mastroianni, Alexander; Sivak, David; Geissler, Phillip; Alivisatos, Paul

    2009-06-08

    We have measured the bending elasticity of short double-stranded DNA (dsDNA) chains through small-angle X-ray scattering from solutions of dsDNA-linked dimers of gold nanoparticles. This method, which does not require exertion of external forces or binding to a substrate, reports on the equilibrium distribution of bending fluctuations, not just an average value (as in ensemble FRET) or an extreme value (as in cyclization), and in principle provides a more robust data set for assessing the suitability of theoretical models. Our experimental results for dsDNA comprising 42-94 basepairs (bp) are consistent with a simple worm-like chain model of dsDNA elasticity, whose behavior we have determined from Monte Carlo simulations that explicitly represent nanoparticles and their alkane tethers. A persistence length of 50 nm (150 bp) gave a favorable comparison, consistent with the results of single-molecule force-extension experiments on much longer dsDNA chains, but in contrast to recent suggestions of enhanced flexibility at these length scales.

  8. Determination for Enterobacter cloacae based on a europium ternary complex labeled DNA probe

    NASA Astrophysics Data System (ADS)

    He, Hui; Niu, Cheng-Gang; Zeng, Guang-Ming; Ruan, Min; Qin, Pin-Zhu; Liu, Jing

    2011-11-01

    The fast detection and accurate diagnosis of the prevalent pathogenic bacteria is very important for the treatment of disease. Nowadays, fluorescence techniques are important tools for diagnosis. A two-probe tandem DNA hybridization assay was designed for the detection of Enterobacter cloacae based on time-resolved fluorescence. In this work, the authors synthesized a novel europium ternary complex Eu(TTA) 3(5-NH 2-phen) with intense luminescence, high fluorescence quantum yield and long lifetime before. We developed a method based on this europium complex for the specific detection of original extracted DNA from E. cloacae. In the hybridization assay format, the reporter probe was labeled with Eu(TTA) 3(5-NH 2-phen) on the 5'-terminus, and the capture probe capture probe was covalent immobilized on the surface of the glutaraldehyde treated glass slides. The original extracted DNA of samples was directly used without any DNA purification and amplification. The detection was conducted by monitoring the fluorescence intensity from the glass surface after DNA hybridization. The detection limit of the DNA was 5 × 10 -10 mol L -1. The results of the present work proved that this new approach was easy to operate with high sensitivity and specificity. It could be conducted as a powerful tool for the detection of pathogen microorganisms in the environment.

  9. Immobilization of human papillomavirus DNA probe for surface plasmon resonance imaging

    NASA Astrophysics Data System (ADS)

    Chong, Xinyuan; Ji, Yanhong; Ma, Suihua; Liu, Le; Liu, Zhiyi; Li, Yao; He, Yonghong; Guo, Jihua

    2009-08-01

    Human papillomavirus (HPV) is a kind of double-stranded DNA virus whose subspecies have diversity. Near 40 kinds of subspecies can invade reproductive organ and cause some high risk disease, such as cervical carcinoma. In order to detect the type of the subspecies of the HPV DNA, we used the parallel scan spectral surface plasmon resonance (SPR) imaging technique, which is a novel type of two- dimensional bio-sensing method based on surface plasmon resonance and is proposed in our previous work, to study the immobilization of the HPV DNA probes on the gold film. In the experiment, four kinds of the subspecies of the HPV DNA (HPV16, HPV18, HPV31, HPV58) probes are fixed on one gold film, and incubate in the constant temperature condition to get a HPV DNA probe microarray. We use the parallel scan spectral SPR imaging system to detect the reflective indices of the HPV DNA subspecies probes. The benefits of this new approach are high sensitive, label-free, strong specificity and high through-put.

  10. Real-time observation of DNA repair: 2-aminopurine as a molecular probe

    NASA Astrophysics Data System (ADS)

    Krishnan, Rajagopal; Butcher, Christina E.; Oh, Dennis H.

    2008-02-01

    Triplex forming oligos (TFOs) that target psoralen photoadducts to specific DNA sequences have generated interest as a potential agent in gene therapy. TFOs also offer an opportunity to study the mechanism of DNA repair in detail. In an effort to understand the mechanism of DNA repair at a specific DNA sequence in real-time, we have designed a plasmid containing a psoralen reaction site adjacent to a TFO binding site corresponding to a sequence within the human interstitial collagenase gene. Two 2-aminopurine residues incorporated into the purine-rich strand of the TFO binding site and located within six nucleotides of the psoralen reaction site serve as molecular probes for excision repair events involving the psoralen photoadducts on that DNA strand. In duplex DNA, the 2-aminopurine fluorescence is quenched. However, upon thermal or formamide-induced denaturation of duplex DNA to single stranded DNA, the 2-aminopurine fluorescence increases by eight fold. These results suggest that monitoring 2-aminopurine fluorescence from plasmids damaged by psoralen TFOs may be a method for measuring excision of single-stranded damaged DNA from the plasmid in cells. A fluorescence-based molecular probe to the plasmid may significantly simplify the real-time observation of DNA repair in both populations of cells as well as single cells.

  11. Mechanisms of small molecule–DNA interactions probed by single-molecule force spectroscopy

    PubMed Central

    Almaqwashi, Ali A.; Paramanathan, Thayaparan; Rouzina, Ioulia; Williams, Mark C.

    2016-01-01

    There is a wide range of applications for non-covalent DNA binding ligands, and optimization of such interactions requires detailed understanding of the binding mechanisms. One important class of these ligands is that of intercalators, which bind DNA by inserting aromatic moieties between adjacent DNA base pairs. Characterizing the dynamic and equilibrium aspects of DNA-intercalator complex assembly may allow optimization of DNA binding for specific functions. Single-molecule force spectroscopy studies have recently revealed new details about the molecular mechanisms governing DNA intercalation. These studies can provide the binding kinetics and affinity as well as determining the magnitude of the double helix structural deformations during the dynamic assembly of DNA–ligand complexes. These results may in turn guide the rational design of intercalators synthesized for DNA-targeted drugs, optical probes, or integrated biological self-assembly processes. Herein, we survey the progress in experimental methods as well as the corresponding analysis framework for understanding single molecule DNA binding mechanisms. We discuss briefly minor and major groove binding ligands, and then focus on intercalators, which have been probed extensively with these methods. Conventional mono-intercalators and bis-intercalators are discussed, followed by unconventional DNA intercalation. We then consider the prospects for using these methods in optimizing conventional and unconventional DNA-intercalating small molecules. PMID:27085806

  12. Identification of Streptococcus pneumoniae with a DNA probe.

    PubMed Central

    Denys, G A; Carey, R B

    1992-01-01

    The Accuprobe Streptococcus pneumoniae Culture Identification Test (Gen-Probe, Inc.) was evaluated with 172 isolates of S. pneumoniae and 204 nonpneumococcal isolates. The sensitivity and specificity of the Accuprobe test were 100%. Optimum results were obtained when four or more discrete colonies were selected for testing. The Accuprobe test was determined to be an accurate and rapid method for identification of S. pneumoniae. PMID:1400974

  13. Controlling microarray DNA hybridization efficiency by probe-surface distance and external surface electrostatics

    NASA Astrophysics Data System (ADS)

    Qamhieh, K.; Pettitt, B. Montgomery

    2015-03-01

    DNA microarrays are analytical devices designed to determine the composition of multicomponent solutions of nucleic acids, DNA or RNA. These devices are promising technology for diverse applications, including sensing, diagnostics, and drug/gene delivery. Here, we modify a hybridization adsorption isotherm to study the effects of probe-surface distance and the external electrostatic fields, on the oligonucleotide hybridization in microarray and how these effects are varies depending on surface probe density and target concentration. This study helps in our understanding on-surface hybridization mechanisms, and from it we can observe a significant effect of the probe-surface distance, and the external electrostatic fields, on the hybridization yield. In addition we present a simple new criteria to control the oligonucleotide hybridization efficiency by providing a chart illustrating the effects of all factors on the DNA-hybridization efficiency.

  14. Coaxial atomic force microscope probes for dielectrophoresis of DNA under different buffer conditions

    NASA Astrophysics Data System (ADS)

    Tao, Yinglei; Kumar Wickramasinghe, H.

    2017-02-01

    We demonstrate a coaxial AFM nanoprobe device for dielectrophoretic (DEP) trapping of DNA molecules in Tris-EDTA (TE) and phosphate-buffered saline (PBS) buffers. The DEP properties of 20 nm polystyrene beads were studied with coaxial probes in media with different conductivities. Due to the special geometry of our DEP probe device, sufficiently high electric fields were generated at the probe end to focus DNA molecules with positive DEP. DEP trapping for both polystyrene beads and DNA molecules was quantitatively analyzed over the frequency range from 100 kHz to 50 MHz and compared with the Clausius-Mossotti theory. Finally, we discussed the negative effect of medium salinity during DEP trapping.

  15. Synthetic exfoliative toxin A and B DNA probes for detection of toxigenic Staphylococcus aureus strains.

    PubMed Central

    Rifai, S; Barbancon, V; Prevost, G; Piemont, Y

    1989-01-01

    Two methods for the detection of exfoliative toxin (ET) from Staphylococcus aureus were compared: (i) a phenotypic assay, electrosyneresis, and (ii) a genotypic assay, staphylococcal DNA hybridization with oligodeoxynucleotide probes. The probes were chosen from the previously determined sequences of serotype A and B of ET, one probe for serotype A and another for serotype B. Strains exhibiting ET production in electrosyneresis always possessed the ET gene(s). Conversely, some strains not exhibiting ET production in electrosyneresis harbored the ET gene(s). The latter strains produced levels of ET. ET-negative phage group 2 strains of S. aureus as well as tested coagulase-negative staphylococci did not possess the ET gene(s). The sensitivity of the DNA hybridization technique was 10(6) bacteria or 100 ng of genomic DNA. Images PMID:2715322

  16. A new strategy for site-specific alkylation of DNA using oligonucleotides containing an abasic site and alkylating probes.

    PubMed

    Sato, Norihiro; Tsuji, Genichiro; Sasaki, Yoshihiro; Usami, Akira; Moki, Takuma; Onizuka, Kazumitsu; Yamada, Ken; Nagatsugi, Fumi

    2015-10-14

    Selective chemical reactions with DNA, such as its labelling, are very useful in many applications. In this paper, we discuss a new strategy for the selective alkylation of DNA using an oligonucleotide containing an abasic site and alkylating probes. We designed three probes consisting of 2-AVP as a reactive moiety and three kinds of binding moiety with high affinity to duplex DNA. Among these probes, Hoechst-AVP probe exhibited high selectivity and efficient reactivity to thymine bases at the site opposite an abasic site in DNA. Our method is potentially useful for inducing site-directed reactions aimed at inhibiting polymerase reactions.

  17. Probing the linearity and nonlinearity in DNA sequences

    NASA Astrophysics Data System (ADS)

    Tsonis, Anastasios A.; Heller, Fred L.; Tsonis, Panagiotis A.

    2002-09-01

    In this paper, we apply the principles of information theory that relate to the definition of nonlinear predictability, which is a measure that describes both the linear and nonlinear components of a system. By comparing this measure to a measure of linear predictability, one can assess whether a given system has a strong linear or a strong nonlinear component. This provides insights as to whether the system should be modeled by a nonlinear or a linear model. We apply these ideas to DNA sequences. Our results, which extend previous results on this issue indicate that all DNA sequences (coding and noncoding) exhibit strong nonlinear structure. At the same time the results provide insights to understand DNA structure and possible clues about evolutionary mechanisms.

  18. Probing DNA with micro- and nanocapillaries and optical tweezers

    NASA Astrophysics Data System (ADS)

    Steinbock, L. J.; Otto, O.; Skarstam, D. R.; Jahn, S.; Chimerel, C.; Gornall, J. L.; Keyser, U. F.

    2010-11-01

    We combine for the first time optical tweezer experiments with the resistive pulse technique based on capillaries. Quartz glass capillaries are pulled into a conical shape with tip diameters as small as 27 nm. Here, we discuss the translocation of λ-phage DNA which is driven by an electrophoretic force through the nanocapillary. The resulting change in ionic current indicates the folding state of single λ-phage DNA molecules. Our flow cell design allows for the straightforward incorporation of optical tweezers. We show that a DNA molecule attached to an optically trapped colloid is pulled into a capillary by electrophoretic forces. The detected electrophoretic force is in good agreement with measurements in solid-state nanopores.

  19. Clinical application of novel sample processing technology for the identification of salmonellae by using DNA probes.

    PubMed

    Scholl, D R; Kaufmann, C; Jollick, J D; York, C K; Goodrum, G R; Charache, P

    1990-02-01

    Two hundred and fifty clinical fecal specimens collected over a 7-month period were analyzed for the presence of salmonellae by a rapid DNA hybridization procedure. Hybridizations were performed by using a novel specimen processing protocol called wicking and a previously unreported 1,600-base-pair probe cloned from Salmonella enteritidis DNA. The probe was shown to be reactive with all 70 Salmonella serotypes tested and not reactive with 101 stock strains of other enteric bacteria. Southern analysis of 30 Salmonella isolates representing 22 serotypes suggested that the probe sequence was highly conserved, appearing as a 1,600-base-pair band in a BglII digest of isolate DNA in 29 of 30 isolates and as a 2,300-base-pair fragment in 1 of the isolates. The probe correctly identified all salmonellae (nine isolates) among 47 H2S-producing colonies tested from among 250 clinical specimens cultured on xylose-lysine-desoxycholate medium. Salmonellae grown on xylose-lysine-desoxycholate medium gave consistently higher hybridization values than did those grown on either MacConkey or Hektoen enteric agar. In addition, of eight gram-negative broth enrichments in which salmonellae were identified by conventional means, seven were probe positive. The use of this nucleic acid probe and hybridization technique provides a simple and rapid identification of Salmonella species.

  20. Development of a specific biotinylated DNA probe for the detection of Renibacterium salmoninarum.

    PubMed Central

    Hariharan, H; Qian, B; Despres, B; Kibenge, F S; Heaney, S B; Rainnie, D J

    1995-01-01

    A specific DNA probe for the identification of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), was developed from one of 3 clones pRS47, pRS49, and pRS26 of 5.1 kb, 5.3 kb, and 11.3 kb, respectively. The biotinylated pRS47/BamHI insert probe was tested on 3 dilutions of DNA extracted from 3 strains of R. salmoninarum and from 1 strain each of Arthrobacter protophormiae, Aeromonas salmonicida, Corynebacterium aquaticum, Carnobacterium piscicola, Listonella anguillarum, Micrococcus luteus, Pseudomonas fluorescens, Vibrio ordalii, and Yersinia ruckeri. In a dot blot assay, this probe hybridized only with the DNA from the R. salmoninarum strains. When used on kidney samples from fish challenged with R. salmoninarum, the dot blot hybridization assay with the probe was found to be as sensitive as culture. In a fluorescent antibody test, samples that were negative in culture and dot blot hybridization showed no more than one fluorescing cell in 50 microscopic fields examined. This DNA probe, therefore, has the potential for use in the diagnosis of BKD of fish. Images Fig. 2. Fig. 3. PMID:8548693

  1. Using Amino-Labeled Nucleotide Probes for Simultaneous Single Molecule RNA-DNA FISH

    PubMed Central

    Wu, Jun; Shao, Fangwei; Zhang, Li-Feng

    2014-01-01

    Using amino-labeled oligonucleotide probes, we established a simple, robust and low-noise method for simultaneous detection of RNA and DNA by fluorescence in situ hybridization, a highly useful tool to study the large pool of long non-coding RNAs being identified in the current research. With probes either chemically or biologically synthesized, we demonstrate that the method can be applied to study a wide range of RNA and DNA targets at the single-cell and single-molecule level in cellular contexts. PMID:25226542

  2. The effects of multiple probes on the hybridization of target DNA on surfaces.

    PubMed

    Welling, Ryan C; Knotts, Thomas A

    2015-01-07

    DNA microarrays have disruptive potential in many fields including genetics and medicine, but the technology has yet to find widespread clinical use due to poor reliability. Microarrays work on the principle of hybridization and can only be as dependable as this process is reliable. As such, a significant amount of theoretical research has been done to understand hybridization on surfaces on the molecular level. Previous simulations of a target strand with a single, surface-tethered probe molecule have yielded valuable insights, but such is an ideal system and little is known about the effects of multiple probes-a situation that more closely approximates the real system. This work uses molecular simulation to determine the specific differences in duplex stability between one, three, six, and nine tethered probes on a surface. The results show that it is more difficult for a single target to hybridize to a probe as the number of probes on the surface increases due to crowding effects; however, once hybridized, the duplex is more stable than when fewer probes are present. The data also indicate that hybridization of a target to a probe on the face of a group of probes is more stable than hybridization to probes at the edge or center locations. Taken as a whole, the results offer new insights into the cause of the poor reproducibility exhibited by microarrays.

  3. Study of concentration of HPV DNA probe immobilization for cervical cancer detection based IDE biosensor

    NASA Astrophysics Data System (ADS)

    Roshila, M. L.; Hashim, U.; Azizah, N.

    2016-07-01

    This paper mainly illustrates regarding the detection process of Human Papillomavirus (HPV) DNA probe. HPV is the most common virus that infected to human by a sexually transmitted virus. The most common high-risk HPV are 16 and 18. Interdigitated electrode (IDE) device used as based of Titanium Dioxide (TiO2) acts as inorganic surface, where by using APTES as a linker between inorganic surface and organic surface. A strategy of rapid and sensitive for the HPV detection was proposed by integrating simple DNA extraction with a gene of DNA. The extraction of the gene of DNA will make an efficiency of the detection process. It will depend on the sequence of the capture probes and the way to support their attached. The fabrication, surface modification, immobilization and hybridization processes are characterized by current voltage (I-V) measurement by using KEITHLEY 6487. This strategy will perform a good sensitivity of HPV detection.

  4. New naphthalimide modified polyethylenimine nanoparticles as fluorescent probe for DNA detection

    NASA Astrophysics Data System (ADS)

    Liang, Shucai; Yu, Hui; Xiang, Jin; Yang, Wang; Chen, Xiaohui; Liu, Yanbin; Gao, Chen; Yan, Guoping

    2012-11-01

    A new naphthalimide modified polyethylenimine (PEI) nanoparticles (called NPEI-NPs) was synthesized and applied as fluorescent probe for rapid, selective and sensitive fluorometric detection of trace DNA. The synthesis involved the covalent modification of PEI with 4-butylamino-N-carboxymethyl-1,8-naphthalimide(BACMN) for getting amphiphilic polymer. Then the amphiphilic polymer was self-assembled in water to give the NPEI-NPs. NPEI-NPs was soluble in water and emitted fluorescence at 545 nm with exciting at 460 nm. The fluorescence spectra resulting from the interaction between NPEI-NPs and DNA indicated that the fluorescence of NPEI-NPs increased in the present of DNA. Therefore, a fluorescence enhancement method was developed for the determination of trace fish sperm DNA (fsDNA) and calf thymus DNA (ctDNA). Under the optimal conditions, the calibration curves were linear over the concentration ranges of 0.05-2.8 μg/mL for fsDNA and 0.08-3.0 μg/mL for ctDNA. The detection limits for fsDNA and ctDNA were 1.6 and 2.0 ng/mL, respectively. The proposed method has been employed to quantify DNA in synthetic samples with the satisfactory results.

  5. Platinated DNA oligonucleotides: new probes forming ultrastable conjugates with graphene oxide

    NASA Astrophysics Data System (ADS)

    Wang, Feng; Liu, Juewen

    2014-05-01

    Metal containing polymers have expanded the property of polymers by involving covalently associated metal complexes. DNA is a special block copolymer. While metal ions are known to influence DNA, little is explored on its polymer property when strong metal complexes are associated. In this work, we study cisplatin modified DNA as a new polymer and probe. Out of the complexes formed between cisplatin-A15, HAuCl4-A15, Hg2+-T15 and Ag+-C15, only the cisplatin adduct is stable under the denaturing gel electrophoresis condition. Each Pt-nucleobase bond gives a positive charge and thus makes DNA a zwitterionic polymer. This allows ultrafast adsorption of DNA by graphene oxide (GO) and the adsorbed complex is highly stable. Non-specific DNA, protein, surfactants and thiolated compounds cannot displace platinated DNA from GO, while non-modified DNA is easily displaced in most cases. The stable GO/DNA conjugate is further tested for surface hybridization. This is the first demonstration of using metallated DNA as a polymeric material for interfacing with nanoscale materials.Metal containing polymers have expanded the property of polymers by involving covalently associated metal complexes. DNA is a special block copolymer. While metal ions are known to influence DNA, little is explored on its polymer property when strong metal complexes are associated. In this work, we study cisplatin modified DNA as a new polymer and probe. Out of the complexes formed between cisplatin-A15, HAuCl4-A15, Hg2+-T15 and Ag+-C15, only the cisplatin adduct is stable under the denaturing gel electrophoresis condition. Each Pt-nucleobase bond gives a positive charge and thus makes DNA a zwitterionic polymer. This allows ultrafast adsorption of DNA by graphene oxide (GO) and the adsorbed complex is highly stable. Non-specific DNA, protein, surfactants and thiolated compounds cannot displace platinated DNA from GO, while non-modified DNA is easily displaced in most cases. The stable GO/DNA conjugate

  6. DNA-Dye-Conjugates: Conformations and Spectra of Fluorescence Probes

    PubMed Central

    Beierlein, Frank R.; Paradas Palomo, Miguel; Sharapa, Dmitry I.; Zozulia, Oleksii; Mokhir, Andriy; Clark, Timothy

    2016-01-01

    Extensive molecular-dynamics (MD) simulations have been used to investigate DNA-dye and DNA-photosensitizer conjugates, which act as reactants in templated reactions leading to the generation of fluorescent products in the presence of specific desoxyribonucleic acid sequences (targets). Such reactions are potentially suitable for detecting target nucleic acids in live cells by fluorescence microscopy or flow cytometry. The simulations show how the attached dyes/photosensitizers influence DNA structure and reveal the relative orientations of the chromophores with respect to each other. Our results will help to optimize the reactants for the templated reactions, especially length and structure of the spacers used to link reporter dyes or photosensitizers to the oligonucleotides responsible for target recognition. Furthermore, we demonstrate that the structural ensembles obtained from the simulations can be used to calculate steady-state UV-vis absorption and emission spectra. We also show how important quantities describing the quenching of the reporter dye via fluorescence resonance energy transfer (FRET) can be calculated from the simulation data, and we compare these for different relative chromophore geometries. PMID:27467071

  7. Effects of DNA probe and target flexibility on the performance of a "signal-on" electrochemical DNA sensor.

    PubMed

    Wu, Yao; Lai, Rebecca Y

    2014-09-02

    We report the effect of the length and identity of a nontarget binding spacer in both the probe and target sequences on the overall performance of a folding-based electrochemical DNA sensor. Six near-identical DNA probes were used in this study; the main differences between these probes are the length (6, 10, or 14 bases) and identity (thymine (T) or adenine (A)) of the spacer connecting the two target binding domains. Despite the differences, the signaling mechanism of these sensors remains essentially the same. The methylene blue (MB)-modified probe assumes a linear unstructured conformation in the absence of the target; upon hybridization to the target, the probe adopts a "close" conformation, resulting in an increase in the MB current. Among the six sensors, the T14 and A14 sensors showed the largest signal increase upon target hybridization, highlighting the significance of probe flexibility on sensor performance. In addition to the target without a midsequence spacer, 12 other targets, each with a different oligo-T or oligo-A spacer, were used to elucidate the effect of target flexibility on the sensors' signaling capacity. For all six sensors, hybridization to targets with a 2- or 3-base spacer resulted in the largest signal increase. Higher signal enhancement was also observed with targets with an oligo-A spacer. For this sensor design, addition of a long nontarget binding spacer to the probe sequence is advantageous, as it provides flexibility for optimal target capture. The length of the spacer in the target sequence, however, should be adequately long to enable efficient hybridization yet does not introduce undesirable electrostatic and crowding effects.

  8. Efficient in situ detection of mRNAs using the Chlorella virus DNA ligase for padlock probe ligation.

    PubMed

    Schneider, Nils; Meier, Matthias

    2017-02-01

    Padlock probes are single-stranded DNA molecules that are circularized upon hybridization to their target sequence by a DNA ligase. In the following, the circulated padlock probes are amplified and detected with fluorescently labeled probes complementary to the amplification product. The hallmark of padlock probe assays is a high detection specificity gained by the ligation reaction. Concomitantly, the ligation reaction is the largest drawback for a quantitative in situ detection of mRNAs due to the low affinities of common DNA or RNA ligases to RNA-DNA duplex strands. Therefore, current protocols require that mRNAs be reverse transcribed to DNA before detection with padlock probes. Recently, it was found that the DNA ligase from Paramecium bursaria Chlorella virus 1 (PBCV-1) is able to efficiently ligate RNA-splinted DNA. Hence, we designed a padlock probe assay for direct in situ detection of mRNAs using the PBCV-1 DNA ligase. Experimental single-cell data were used to optimize and characterize the efficiency of mRNA detection with padlock probes. Our results demonstrate that the PBCV-1 DNA ligase overcomes the efficiency limitation of current protocols for direct in situ mRNA detection, making the PBCV-1 DNA ligase an attractive tool to simplify in situ ligation sequencing applications.

  9. Molecular cloning of verrucosidin-producing Penicillium polonicum genes by differential screening to obtain a DNA probe.

    PubMed

    Aranda, E; Rodríguez, M; Benito, M J; Asensio, M A; Córdoba, J J

    2002-06-05

    A differential molecular screening procedure was developed to obtain DNA clones enriched for verrucosidin-related genes that could be used as DNA probes to detect verrucosidin-producing Penicillium polonicum. Permissive and nonpermissive conditions for verrucosidin production were selected to obtain differentiated poly (A)+ RNA for the cloning strategy. P. polonicum yielded the highest amount of verrucosidin when cultured in malt extract broth at 25 degrees C without shaking. These conditions were selected as verrucosidin permissive conditions. When shaking was applied to the verrucosidin permissive conditions, verrucosidin was not detected. Approximately 5000 transformants were obtained for the library of DNA fragments from verrucosidin-producing P. polonicum and hybridized with cDNA probes obtained from poly (A)+ RNA of permissive and nonpermissive conditions. A total of 120 clones hybridized only with the permissive cDNA probes. From these, eight representative DNA inserts selected on the basis of size and labelled with fluorescein-dUTP were assayed as DNA probes in the second differential screening by Northern hybridization. Probe SVr1 gave a strong hybridization signal selectively with poly (A)+ RNAs from high verrucosidin production. When this probe was assayed by dot blot hybridization with DNA of different moulds species, hybridization was detected only with DNA from the verrucosidin-producing strain. The strategy used in this work has proved to be useful to detect unknown genes related to mycotoxins. In addition, the DNA probe obtained should be considered for the detection of verrucosidin-producing moulds.

  10. General method for cloning amplified DNA by differential screening with genomic probes.

    PubMed Central

    Brison, O; Ardeshir, F; Stark, G R

    1982-01-01

    Mutant Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, have amplified the gene coding for the multifunctional protein (CAD) that includes this activity. The average amount of DNA amplified is approximately 500 kilobases per gene copy, about 20 times the length of the CAD gene itself. A differential screening method which uses genomic DNAs as probes was developed to isolate recombinant phage containing fragments of amplified DNA. One probe was prepared by reassociating fragments of total genomic DNA from 165-28, a mutant cell line with 190 times the wild-type complement of CAD genes, until all of the sequences repeated about 200 times were annealed and then isolating the double-stranded DNA with hydroxyapatite.This DNA was highly enriched in sequences from the entire amplified region, whereas the same sequences were very rare in DNA prepared similarly from wild-type cells. After both DNAs were labeled by nick translation, highly repeated sequences were removed by hybridization to immobilized total genomic DNA from wild-type cells. A library of cloned DNA fragments from mutant 165-28 was screened with both probes, and nine independent fragments containing about 165 kilobases of amplified DNA, including the CAD gene, have been isolated so far. These cloned DNAs can be used to study the structure of the amplified region, to evaluate the nature of the amplification event, and to investigate gene expression from the amplified DNA. For example, one amplified fragment included a gene coding for a 3.8-kilobase, cytoplasmic, polyadenylated RNA which was overproduced greatly in cells resistant to N-(phosphonacetyl)-L-aspartate. The method for cloning amplified DNA is general and can be used to evaluate the possible involvement of gene amplification in phenomena such as drug resistance, transformation, or differentiation. DNA fragments corresponding to any region amplified about 10-fold or

  11. The effects of multiple probes on the hybridization of target DNA on surfaces

    NASA Astrophysics Data System (ADS)

    Welling, Ryan C.; Knotts, Thomas A.

    2015-01-01

    DNA microarrays have disruptive potential in many fields including genetics and medicine, but the technology has yet to find widespread clinical use due to poor reliability. Microarrays work on the principle of hybridization and can only be as dependable as this process is reliable. As such, a significant amount of theoretical research has been done to understand hybridization on surfaces on the molecular level. Previous simulations of a target strand with a single, surface-tethered probe molecule have yielded valuable insights, but such is an ideal system and little is known about the effects of multiple probes—a situation that more closely approximates the real system. This work uses molecular simulation to determine the specific differences in duplex stability between one, three, six, and nine tethered probes on a surface. The results show that it is more difficult for a single target to hybridize to a probe as the number of probes on the surface increases due to crowding effects; however, once hybridized, the duplex is more stable than when fewer probes are present. The data also indicate that hybridization of a target to a probe on the face of a group of probes is more stable than hybridization to probes at the edge or center locations. Taken as a whole, the results offer new insights into the cause of the poor reproducibility exhibited by microarrays.

  12. Probing Evolutionary Patterns in Neotropical Birds through DNA Barcodes

    PubMed Central

    Kerr, Kevin C. R.; Lijtmaer, Darío A.; Barreira, Ana S.; Hebert, Paul D. N.; Tubaro, Pablo L.

    2009-01-01

    Background The Neotropical avifauna is more diverse than that of any other biogeographic region, but our understanding of patterns of regional divergence is limited. Critical examination of this issue is currently constrained by the limited genetic information available. This study begins to address this gap by assembling a library of mitochondrial COI sequences, or DNA barcodes, for Argentinian birds and comparing their patterns of genetic diversity to those of North American birds. Methodology and Principal Findings Five hundred Argentinian species were examined, making this the first major examination of DNA barcodes for South American birds. Our results indicate that most southern Neotropical bird species show deep sequence divergence from their nearest-neighbour, corroborating that the high diversity of this fauna is not based on an elevated incidence of young species radiations. Although species ages appear similar in temperate North and South American avifaunas, patterns of regional divergence are more complex in the Neotropics, suggesting that the high diversity of the Neotropical avifauna has been fueled by greater opportunities for regional divergence. Deep genetic splits were observed in at least 21 species, though distribution patterns of these lineages were variable. The lack of shared polymorphisms in species, even in species with less than 0.5M years of reproductive isolation, further suggests that selective sweeps could regularly excise ancestral mitochondrial polymorphisms. Conclusions These findings confirm the efficacy of species delimitation in birds via DNA barcodes, even when tested on a global scale. Further, they demonstrate how large libraries of a standardized gene region provide insight into evolutionary processes. PMID:19194495

  13. Quantum interference in DNA bases probed by graphene nanoribbons

    NASA Astrophysics Data System (ADS)

    Jeong, Heejeong; Seul Kim, Han; Lee, Sung-Hoon; Lee, Dongho; Hoon Kim, Yong; Huh, Nam

    2013-07-01

    Based on first-principles nonequilibrium Green's function calculations, we demonstrate quantum interference (QI) effects on the tunneling conductance of deoxyribonucleic acid bases placed between zigzag graphene nanoribbon electrodes. With the analogy of QI in hydrocarbon ring structures, we hypothesize that QI can be well preserved in the π-π coupling between the carbon-based electrode and a single DNA base. We demonstrate indications of QI, such as destructively interfered anti-resonance or Fano-resonance, that affect the variation of tunneling conductance depending on the orientation of a base. We find that guanine, with a 10-fold higher transverse conductance, can be singled out from the other bases.

  14. DNA-based digital tension probes reveal integrin forces during early cell adhesion

    PubMed Central

    Zhang, Yun; Ge, Chenghao; Zhu, Cheng; Salaita, Khalid

    2014-01-01

    Mechanical stimuli profoundly alter cell fate, yet the mechanisms underlying mechanotransduction remain obscure due to a lack of methods for molecular force imaging. Here, to address this need, we develop a new class of molecular tension probes that function as a switch to generate a 20–30-fold increase in fluorescence upon experiencing a threshold piconewton force. The probes employ immobilized DNA-hairpins with tunable force response thresholds, ligands, and fluorescence reporters. Quantitative imaging reveals that integrin tension is highly dynamic and increases with an increasing integrin density during adhesion formation. Mixtures of fluorophore-encoded probes show integrin mechanical preference for cyclized-RGD over linear-RGD peptides. Multiplexed probes with variable guanine-cytosine content within their hairpins reveal integrin preference for the more stable probes at the leading tip of growing adhesions near the cell edge. DNA-based tension probes are among the most sensitive optical force reporters to date, overcoming the force and spatial-resolution limitations of traction force microscopy. PMID:25342432

  15. Combination probes with intercalating anchors and proximal fluorophores for DNA and RNA detection

    PubMed Central

    Qiu, Jieqiong; Wilson, Adam; El-Sagheer, Afaf H.; Brown, Tom

    2016-01-01

    A new class of modified oligonucleotides (combination probes) has been designed and synthesised for use in genetic analysis and RNA detection. Their chemical structure combines an intercalating anchor with a reporter fluorophore on the same thymine nucleobase. The intercalator (thiazole orange or benzothiazole orange) provides an anchor, which upon hybridisation of the probe to its target becomes fluorescent and simultaneously stabilizes the duplex. The anchor is able to communicate via FRET to a proximal reporter dye (e.g. ROX, HEX, ATTO647N, FAM) whose fluorescence signal can be monitored on a range of analytical devices. Direct excitation of the reporter dye provides an alternative signalling mechanism. In both signalling modes, fluorescence in the unhybridised probe is switched off by collisional quenching between adjacent intercalator and reporter dyes. Single nucleotide polymorphisms in DNA and RNA targets are identified by differences in the duplex melting temperature, and the use of short hybridization probes, made possible by the stabilisation provided by the intercalator, enhances mismatch discrimination. Unlike other fluorogenic probe systems, placing the fluorophore and quencher on the same nucleobase facilitates the design of short probes containing multiple modifications. The ability to detect both DNA and RNA sequences suggests applications in cellular imaging and diagnostics. PMID:27369379

  16. DNA-Based MRI Probes for Specific Detection of Chronic Exposure to Amphetamine in Living Brains

    PubMed Central

    Liu, Christina H.; Ren, Jia Q.; Yang, Jinsheng; Liu, Charng-ming; Mandeville, Joseph B.; Rosen, Bruce R.; Bhide, Pradeep G.; Yanagawa, Yuchio; Liu, Philip K.

    2009-01-01

    We designed phosphorothioate-modified DNA probes linked to superparamagnetic iron oxide nanoparticles (SPION) for in vivo magnetic resonance imaging (MRI) of fosB and ΔfosB mRNA after amphetamine (AMPH) exposure in mice. Specificity of both the fosB and ΔfosB probes was verified by in vitro reverse transcriptase-PCR amplification to a single fragment of total cDNA obtained from acutely AMPH-exposed mouse brains. We confirmed time-dependent uptake and retention profiles of both probes in neurons of GAD67-green fluorescent protein knock-in mice. MRI signal of SPION-labeled fosB probe delivered via intracerebroventricular route was elevated in both acutely and chronically AMPH-exposed mice; the signal was suppressed by dopaminergic receptor antagonist pretreatment. SPION-labeled ΔfosB probe signal elevation occurred only in chronically AMPH-exposed mice. The in vivo target specificity of these probes permits reliable MRI visualization of AMPH-induced differential elevations of fosB and ΔfosB mRNA in living brains. PMID:19710318

  17. Probing DNA helicase kinetics with temperature-controlled magnetic tweezers.

    PubMed

    Gollnick, Benjamin; Carrasco, Carolina; Zuttion, Francesca; Gilhooly, Neville S; Dillingham, Mark S; Moreno-Herrero, Fernando

    2015-03-18

    Motor protein functions like adenosine triphosphate (ATP) hydrolysis or translocation along molecular substrates take place at nanometric scales and consequently depend on the amount of available thermal energy. The associated rates can hence be investigated by actively varying the temperature conditions. In this article, a thermally controlled magnetic tweezers (MT) system for single-molecule experiments at up to 40 °C is presented. Its compact thermostat module yields a precision of 0.1 °C and can in principle be tailored to any other surface-coupled microscopy technique, such as tethered particle motion (TPM), nanopore-based sensing of biomolecules, or super-resolution fluorescence imaging. The instrument is used to examine the temperature dependence of translocation along double-stranded (ds)DNA by individual copies of the protein complex AddAB, a helicase-nuclease motor involved in dsDNA break repair. Despite moderately lower mean velocities measured at sub-saturating ATP concentrations, almost identical estimates of the enzymatic reaction barrier (around 21-24 k(B)T) are obtained by comparing results from MT and stopped-flow bulk assays. Single-molecule rates approach ensemble values at optimized chemical energy conditions near the motor, which can withstand opposing loads of up to 14 piconewtons (pN). Having proven its reliability, the temperature-controlled MT described herein will eventually represent a routinely applied method within the toolbox for nano-biotechnology.

  18. Probing DNA Helicase Kinetics with Temperature‐Controlled Magnetic Tweezers

    PubMed Central

    Gollnick, Benjamin; Carrasco, Carolina; Zuttion, Francesca; Gilhooly, Neville S.; Dillingham, Mark S.

    2015-01-01

    Motor protein functions like adenosine triphosphate (ATP) hydrolysis or translocation along molecular substrates take place at nanometric scales and consequently depend on the amount of available thermal energy. The associated rates can hence be investigated by actively varying the temperature conditions. In this article, a thermally controlled magnetic tweezers (MT) system for single‐molecule experiments at up to 40 °C is presented. Its compact thermostat module yields a precision of 0.1 °C and can in principle be tailored to any other surface‐coupled microscopy technique, such as tethered particle motion (TPM), nanopore‐based sensing of biomolecules, or super‐resolution fluorescence imaging. The instrument is used to examine the temperature dependence of translocation along double‐stranded (ds)DNA by individual copies of the protein complex AddAB, a helicase‐nuclease motor involved in dsDNA break repair. Despite moderately lower mean velocities measured at sub‐saturating ATP concentrations, almost identical estimates of the enzymatic reaction barrier (around 21–24 k B T) are obtained by comparing results from MT and stopped‐flow bulk assays. Single‐molecule rates approach ensemble values at optimized chemical energy conditions near the motor, which can withstand opposing loads of up to 14 piconewtons (pN). Having proven its reliability, the temperature‐controlled MT described herein will eventually represent a routinely applied method within the toolbox for nano‐biotechnology. PMID:25400244

  19. Development of a biotinylated DNA probe for detection of infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Deering, R.E.; Arakawa, C.K.; Oshima, K.H.; O'Hara, P.J.; Landolt, M.L.; Winton, J.R.

    1991-01-01

    A nonrad~oact~ve DNA probe assay was developed to detect and ~dent~fy infect~ous hernatopoiet~c necrosls virus (IHNV) uslng a dot blot format The probe a synthet~c DNA oligonucleot~de labeled enzymatlcally w~th biotln hybnd~zed spec~f~cally w~th nucleocaps~d mRNA extracted from Infected cells early In the vlrus repl~cation cycle A rap~d guan~dln~um th~ocyanate based RNA extraction method uslng RNAzol B and rn~crocentrifuge tubes eff~c~ently pioduced h~gh qual~ty RNA from 3 commonly used f~sh cell llnes, CHSE-214, CHH-1, and EPC The probe reacted with 6 d~verse ~solates of IHNV, but d~d not react \

  20. Bulged Invader probes: activated duplexes for mixed-sequence dsDNA recognition with improved thermodynamic and kinetic profiles.

    PubMed

    Guenther, Dale C; Karmakar, Saswata; Hrdlicka, Patrick J

    2015-10-18

    Double-stranded oligonucleotides with +1 interstrand zipper arrangements of intercalator-functionalized nucleotides are energetically activated for recognition of mixed-sequence double-stranded DNA. Incorporation of nonyl (C9) bulges at specific positions of these probes, results in more highly affine (>5-fold), faster (>4-fold) and more persistent dsDNA recognition relative to conventional Invader probes.

  1. Cyanines as new fluorescent probes for DNA detection and two-photon excited bioimaging.

    PubMed

    Feng, Xin Jiang; Wu, Po Lam; Bolze, Frédéric; Leung, Heidi W C; Li, King Fai; Mak, Nai Ki; Kwong, Daniel W J; Nicoud, Jean-François; Cheah, Kok Wai; Wong, Man Shing

    2010-05-21

    A series of cyanine fluorophores based on fused aromatics as an electron donor for DNA sensing and two-photon bioimaging were synthesized, among which the carbazole-based biscyanine exhibits high sensitivity and efficiency as a fluorescent light-up probe for dsDNA, which shows selective binding toward the AT-rich regions. The synergetic effect of the bischromophoric skeleton gives a several-fold enhancement in a two-photon absorption cross-section as well as a 25- to 100-fold enhancement in two-photon excited fluorescence upon dsDNA binding.

  2. DNase-activatable fluorescence probes visualizing the degradation of exogenous DNA in living cells

    NASA Astrophysics Data System (ADS)

    Gong, Ping; Shi, Bihua; Zhang, Pengfei; Hu, Dehong; Zheng, Mingbin; Zheng, Cuifang; Gao, Duyang; Cai, Lintao

    2012-03-01

    This work presents a method to visualize the degradation of exogenous DNA in living cells using a novel type of activatable fluorescence imaging probe. Deoxyribonuclease (DNase)-activatable fluorescence probes (DFProbes) are composed of double strands deoxyribonucleic acid (dsDNA) which is labeled with fluorophore (ROX or Cy3) and quencher on the end of one of its strands, and stained with SYBR Green I. In the absence of DNase, DFProbes produce the green fluorescence signal of SYBR Green I. In the presence of DNase, SYBR Green I is removed from the DFProbes and the labeled fluorophore is separated from the quencher owing to the degradation of DFProbes by DNase, resulting in the decrease of the green fluorescence signal and the occurrence of a red fluorescence signal due to fluorescence resonance energy transfer (FRET). DNase in biological samples was detected using DFProbes and the fluorescence imaging in living cells was performed using DFprobe-modified Au nanoparticles. The results show that DFProbes have good responses to DNase, and can clearly visualize the degradation of exogenous DNA in cells in real time. The well-designed probes might be useful in tracing the dynamic changes of exogenous DNA and nanocarriers in vitro and in vivo.This work presents a method to visualize the degradation of exogenous DNA in living cells using a novel type of activatable fluorescence imaging probe. Deoxyribonuclease (DNase)-activatable fluorescence probes (DFProbes) are composed of double strands deoxyribonucleic acid (dsDNA) which is labeled with fluorophore (ROX or Cy3) and quencher on the end of one of its strands, and stained with SYBR Green I. In the absence of DNase, DFProbes produce the green fluorescence signal of SYBR Green I. In the presence of DNase, SYBR Green I is removed from the DFProbes and the labeled fluorophore is separated from the quencher owing to the degradation of DFProbes by DNase, resulting in the decrease of the green fluorescence signal and the

  3. A universal design for a DNA probe providing ratiometric fluorescence detection by generation of silver nanoclusters

    NASA Astrophysics Data System (ADS)

    Del Bonis-O'Donnell, Jackson Travis; Vong, Daniel; Pennathur, Sumita; Fygenson, Deborah Kuchnir

    2016-07-01

    DNA-stabilized silver nanoclusters (AgNCs), the fluorescence emission of which can rival that of typical organic fluorophores, have made possible a new class of label-free molecular beacons for the detection of single-stranded DNA. Like fluorophore-quencher molecular beacons (FQ-MBs) AgNC-based molecular beacons (AgNC-MBs) are based on a single-stranded DNA that undergoes a conformational change upon binding a target sequence. The new conformation exposes a stretch of single-stranded DNA capable of hosting a fluorescent AgNC upon reduction in the presence of Ag+ ions. The utility of AgNC-MBs has been limited, however, because changing the target binding sequence unpredictably alters cluster fluorescence. Here we show that the original AgNC-MB design depends on bases in the target-binding (loop) domain to stabilize its AgNC. We then rationally alter the design to overcome this limitation. By separating and lengthening the AgNC-stabilizing domain, we create an AgNC-hairpin probe with consistent performance for arbitrary target sequence. This new design supports ratiometric fluorescence measurements of DNA target concentration, thereby providing a more sensitive, responsive and stable signal compared to turn-on AgNC probes. Using the new design, we demonstrate AgNC-MBs with nanomolar sensitivity and singe-nucleotide specificity, expanding the breadth of applicability of these cost-effective probes for biomolecular detection.DNA-stabilized silver nanoclusters (AgNCs), the fluorescence emission of which can rival that of typical organic fluorophores, have made possible a new class of label-free molecular beacons for the detection of single-stranded DNA. Like fluorophore-quencher molecular beacons (FQ-MBs) AgNC-based molecular beacons (AgNC-MBs) are based on a single-stranded DNA that undergoes a conformational change upon binding a target sequence. The new conformation exposes a stretch of single-stranded DNA capable of hosting a fluorescent AgNC upon reduction in the

  4. Probing Y-shaped DNA structure with time-resolved FRET

    NASA Astrophysics Data System (ADS)

    Chatterjee, Subhasish; Lee, Jong Bum; Valappil, Nikesh V.; Luo, Dan; Menon, Vinod M.

    2012-02-01

    Self-assembly based on nucleic acid systems has become highly attractive for bottom-up fabrication of programmable matter due to the highly selective molecular recognition property of biomolecules. In this context, Y-shaped DNA (Y-DNA) provides an effective building block for forming unique self-assembled large-scale architectures. The dimension and growth of the nano- and microstructures depend significantly on the configurational stability of Y-DNA as a building block. Here we present structural studies of Y-DNA systems using a time-resolved FRET (Förster resonance energy transfer) technique. A fluorophore (Alexa 488) and an acceptor (DABCYL) were placed at two different ends of Y-DNA, and the lifetime of the fluorophore was measured to probe the relative distance between the donor and acceptor. Our results confirmed different distances between the arms of the Y-DNA and highlighted the overall structural integrity of the Y-DNA system as a leading building block for molecular self-assembly. Temperature dependent lifetime measurements indicated configurational changes in the overall Y-DNA nanoarchitecture above 40 °C.Self-assembly based on nucleic acid systems has become highly attractive for bottom-up fabrication of programmable matter due to the highly selective molecular recognition property of biomolecules. In this context, Y-shaped DNA (Y-DNA) provides an effective building block for forming unique self-assembled large-scale architectures. The dimension and growth of the nano- and microstructures depend significantly on the configurational stability of Y-DNA as a building block. Here we present structural studies of Y-DNA systems using a time-resolved FRET (Förster resonance energy transfer) technique. A fluorophore (Alexa 488) and an acceptor (DABCYL) were placed at two different ends of Y-DNA, and the lifetime of the fluorophore was measured to probe the relative distance between the donor and acceptor. Our results confirmed different distances between

  5. Derivation of DNA probes for enumeration of a specific strain of Lactobacillus acidophilus in piglet digestive tract samples.

    PubMed Central

    Rodtong, S; Dobbinson, S; Thode-Andersen, S; McConnell, M A; Tannock, G W

    1993-01-01

    Four DNA probes were derived that hybridized specifically to DNA from Lactobacillus acidophilus O. The probes were constructed by randomly cloning lactobacillus DNA in plasmid vector pBR322. Two of the probes (pSR1 and pSR2) were composed of vector and plasmid DNA inserts (3.6 and 1.6 kb, respectively); the others (pSR3 and pSR4) were composed of vector and chromosomally derived inserts (6.9 and 1.4 kb, respectively). The probes were used to enumerate, by colony hybridization, strain O in digestive tract samples collected from piglets inoculated 24 hours previously with a culture of the strain. The probes did not hybridize to DNA from lactobacilli inhabiting the digestive tract of uninoculated piglets. Strain O made up about 10% of the total lactobacillus population of the pars esophagea and about 20% of the population in other digestive tract samples. Images PMID:8285690

  6. Electrochemical impedance probing of DNA hybridisation on oligonucleotide-functionalised polypyrrole.

    PubMed

    Tlili, Chaker; Korri-Youssoufi, Hafsa; Ponsonnet, Laurence; Martelet, Claude; Jaffrezic-Renault, Nicole J

    2005-11-15

    We report a new approach for detecting DNA hybridisation using non faradaic electrochemical impedance spectroscopy. The technique was applied to a system of DNA probes bearing amine groups that are immobilized by covalent grafting on a supporting polypyrrole matrix functionalised with activated ester groups. The kinetics of the attachment of the ss-DNA probe was monitored using the temporal evolution of the open circuit potential (OCP). This measurement allows the determination of the time necessary for the chemical reaction of ss-DNA probe into the polypyrrole backbone. The hybridisation reactions with the DNA complementary target and non complementary target were investigated by non faradaic electrochemical impedance spectroscopy. Results show a significant modification in the Nyquist plot upon addition of the complementary target whereas, in presence of the non complementary target, the Nyquist plot is not modified. The spectra, in the form of Nyquist plot, were analysed with the Randles circuit. The transfer charge resistance R(2) shows a linear variation versus the complementary target concentration. Sensitivity and detection limit (0.2nM) were determined and detection limit was lower of one order of magnitude than that obtained with the same system and measuring variation of the oxidation current at constant potential.

  7. DNA Probes As Potential Tools for the Detection of Marteilia refringens.

    PubMed

    Le Roux F; Audemard; Barnaud; Berthe

    1999-11-01

    Since its first description, the paramyxean parasite Marteilia refringens has been recognized as a significant pathogen of bivalve mollusks. The existence of a complex life cycle was postulated by many authors. Here we report the development of DNA-based detection assays as powerful tools to elucidate the Marteilia refringens life cycle. After alignment of the Marteilia refringens ribosomal DNA small subunit sequence with those of various eukaryotic organisms, polymerase chain reaction primers were designed. Specific primers were used to amplify DNA extracted from purified Marteilia refringens and infected hosts. The specificity of amplified fragments was confirmed by Southern blotting with an oligoprobe. For in situ hybridization, four probes were tested for specific detection of 18S rRNA isolated from Marteilia refringens and other eukaryotic cells by Northern blotting. The most specific probe, Smart 2, was successfully used to detect Marteilia refringens by in situ hybridization in infected oysters and mussels.

  8. Dinuclear Ruthenium(II) Complexes as Two-Photon, Time-Resolved Emission Microscopy Probes for Cellular DNA**

    PubMed Central

    Baggaley, Elizabeth; Gill, Martin R; Green, Nicola H; Turton, David; Sazanovich, Igor V; Botchway, Stanley W; Smythe, Carl; Haycock, John W; Weinstein, Julia A; Thomas, Jim A

    2014-01-01

    The first transition-metal complex-based two-photon absorbing luminescence lifetime probes for cellular DNA are presented. This allows cell imaging of DNA free from endogenous fluorophores and potentially facilitates deep tissue imaging. In this initial study, ruthenium(II) luminophores are used as phosphorescent lifetime imaging microscopy (PLIM) probes for nuclear DNA in both live and fixed cells. The DNA-bound probes display characteristic emission lifetimes of more than 160 ns, while shorter-lived cytoplasmic emission is also observed. These timescales are orders of magnitude longer than conventional FLIM, leading to previously unattainable levels of sensitivity, and autofluorescence-free imaging. PMID:24458590

  9. Design and evaluation of Bacteroides DNA probes for the specific detection of human fecal pollution

    SciTech Connect

    Kreader, C.A.

    1995-04-01

    Because Bacteroides spp. are obligate anaerobes that dominate the human fecal flora, and because some species may live only in the human intestine, these bacteria might be useful to distinguish human from nonhuman sources of fecal pollution. To test this hypothesis, PCR primers specific for 16S rRNA gene sequences of Bacteroides distasonis, B. thetaiotaomicron, and B. vulgatus were designed. Hybridization with species-specific internal probes was used to detect the intended PCR products. Extracts from 66 known Bacteroides strains, representing 10 related species, were used to confirm the specificity of these PCR-hybridization assays. To test for specificity in feces, procedures were developed to prepare DNA of sufficient purity for PCR. Extracts of feces from 9 humans and 70 nonhumans (cats, dogs, cattle, hogs, horses, sheep, goats, and chickens) were each analyzed with and without an internal positive control to verify that PCR amplification was not inhibited by substances in the extract. In addition, serial dilutions from each extract that tested positive were assayed to estimate the relative abundance of target Bacteroides spp. in the sample. Depending on the primer-probe set used, either 78 or 67% of the human fecal extracts tested had high levels of target DNA. On the other hand, only 7 to 11% of the nonhuman extracts tested had similarly high levels of target DNA. An additional 12 to 20% of the nonhuman extracts had levels of target DNA that were 100- to 1,000-fold lower than those found in humans. Although the B. vulgatus probes detected high levels of their target DNA in most of the house pets, similarly high levels of target DNA were found only in a few individuals from other groups of nonhumans. Therefore, the results indicate that these probes can distinguish human from non human feces in many cases. 50 refs., 5 figs., 2 tabs.

  10. Differential diagnosis of Taenia saginata and Taenia solium infections: from DNA probes to polymerase chain reaction.

    PubMed

    González, Luis Miguel; Montero, Estrella; Sciutto, Edda; Harrison, Leslie J S; Parkhouse, R Michael E; Garate, Teresa

    2002-04-01

    The objective of this work was the rapid and easy differential diagnosis of Taenia saginata and T. solium. First, a T. saginata size-selected genomic deoxyribonucleic acid (gDNA) library was constructed in the vector lambda gt10 using the 2-4 kb fraction from the parasite DNA digested with EcoR1, under 'star' conditions. After differential screening of the library and hybridization analysis with DNA from T. saginata, T. solium, T. taeniaeformis, T. crassiceps, and Echinococcus granulosus (bovine, porcine, and human), 2 recombinant phages were selected. They were designated HDP1 and HDP2. HDP1 reacted specifically with T. saginata DNA, and HDP2 recognized DNA from both T. saginata and T. solium. The 2 DNA probes were then sequenced and further characterized. HDP1 was a repetitive sequence with a 53 bp monomeric unit repeated 24 times in direct tandem along the 1272 bp fragment, while the 3954 bp HDP2 was not a repetitive sequence. Using the sequencing data, oligonucleotides were designed and used in a polymerase chain reaction (PCR). The 2 selected oligonucleotides from probe HDP1 (PTs4F1 and PTs4R1) specifically amplified gDNA from T. saginata, but not T. solium or other related cestodes, with a sensitivity of < 10 pg of T. saginata gDNA, about the quantity of DNA in one taeniid egg. The 3 oligonucleotides selected from the HDP2 sequence (PTs7S35F1, PTs7S35F2, and PTs7S35R1) allowed the differential amplification of gDNA from T. saginata, T. solium and E. granulosus in a multiplex PCR, again with a sensitivity of < 10 pg. These diagnostic tools have immediate application in the differential diagnosis of T. solium and T. saginata in humans and in the diagnosis of dubious cysts in the slaughterhouse. We also hope to apply them to epidemiological surveys of, for example, soil and water in endemic areas.

  11. Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures

    SciTech Connect

    Solomon, M.J.; Varshavsky, A.

    1985-10-01

    Formaldehyde (HCHO) produces DNA-protein crosslinks both in vitro and in vivo. Simian virus 40 (SV40) chromosomes that have been fixed by prolonged incubation with HCHO either in vitro or in vivo (within SV40-infected cells) can be converted to nearly protein-free DNA by limit-digestion with Pronase in the presence of NaDodSO/sub 4/. The remaining Pronase-resistant DNA-peptide adducts retard the DNA upon gel electrophoresis, allowing resolution of free and crosslink-containing DNA. Though efficiently crosslinking histones to DNA within nucleosomes both in vitro and in vivo, HCHO does not crosslink either purified lac repressor to lac operator-containing DNA or an (A + T)-DNA-binding protein (..cap alpha..-protein) to its cognate DNA in vitro. Furthermore, a protein that does not bind to DNA, such as serum albumin, is not crosslinked to DNA by HCHO even at extremely high protein concentrations. These properties of HCHO as a DNA-protein crosslinker are used to probe the distribution of nucleosomes in vivo. It is shown that there are no HCHO-crosslinkable DNA-protein contacts in a subset of SV40 chromosomes in vivo within a 325-base-pair stretch that spans the exposed (nuclease-hypersensitive) region of the SV40 chromosomes. This replication origin-proximal region has been found previously to lack nucleosomes in a subset of isolated SV40 chromosomes. Other applications of the HCHO technique are discussed, including the possibility of obtaining base-resolution in vivo nucleosome footprints.

  12. Selectivity of Enzymatic Conversion of Oligonucleotide Probes during Nucleotide Polymorphism Analysis of DNA

    PubMed Central

    Vinogradova, O.A.

    2010-01-01

    The analysis of DNA nucleotide polymorphisms is one of the main goals of DNA diagnostics. DNA–dependent enzymes (DNA polymerases and DNA ligases) are widely used to enhance the sensitivity and reliability of systems intended for the detection of point mutations in genetic material. In this article, we have summarized the data on the selectiveness of DNA–dependent enzymes and on the structural factors in enzymes and DNA which influence the effectiveness of mismatch discrimination during enzymatic conversion of oligonucleotide probes on a DNA template. The data presented characterize the sensitivity of a series of DNA–dependent enzymes that are widely used in the detection of noncomplementary base pairs in nucleic acid substrate complexes. We have analyzed the spatial properties of the enzyme–substrate complexes. These properties are vital for the enzymatic reaction and the recognition of perfect DNA–substrates. We also discuss relevant approaches to increasing the selectivity of enzyme–dependent reactions. These approaches involve the use of modified oligonucleotide probes which “disturb” the native structure of the DNA–substrate complexes. PMID:22649627

  13. Spin-labeled psoralen probes for the study of DNA dynamics

    SciTech Connect

    Spielmann, H.P.; Chi, D.Y.; Hunt, N.G.

    1995-11-14

    Six nitroxide spin-labeled psoralen derivatives have been synthesized and evaluated as probes for structural and dynamic studies. Sequence specific photoaddition of these derivatives to DNA oligonucleotides resulted in site-specifically cross-linked and spin-labeled oligomers. Comparison of the general line shape features of the observed electron paramagnetic resonance (EPR) spectra of several duplexes ranging in size from 8 to 46 base pairs with simulated EPR spectra indicate that the nitroxide spin-labeled probe reports the global tumbling motion of the oligomers. While there is no apparent large amplitude motion of the psoralen other than the overall tumbling of DNA on the time scales investigated, there are no indications of bending and other residual motions. The (A)BC excinuclease DNA repair system detects structural or dynamic features of the DNA that distinguish between damaged and undamaged DNA and are independent of the intrinsic structure of the lesion. NMR studies have shown that psoralen-cross-linked DNA has altered backbone dynamics and conformational populations in the immediate vicinity of the adduct. We suggested that the signal for recognition of a lesion to be repaired is in the sugar-phosphate backbone and not in the damaged base(s). 71 refs., 11 figs., 1 tab.

  14. [Fluorescence in situ hybridization with DNA probes derived from individual chromosomes and chromosome regions].

    PubMed

    Bogomolov, A G; Karamysheva, T V; Rubtsov, N B

    2014-01-01

    A significant part of the eukaryotic genomes consists of repetitive DNA, which can form large clusters or distributed along euchromatic chromosome regions. Repeats located in chromosomal regions make a problem in analysis and identification of the chromosomal material with fluorescence in situ hybridization (FISH). In most cases, the identification of chromosome regions using FISH requires detection of the signal produced with unique sequences. The feasibility, advantages and disadvantages of traditional methods of suppression of repetitive DNA hybridization, methods of repeats-free probe construction and methods of chromosome-specific DNA sequences visualization using image processing of multicolor FISH results are considered in the paper. The efficiency of different techniques for DNA probe generation, different FISH protocols, and image processing of obtained microscopic images depends on the genomic size and structure of analyzing species. This problem was discussed and different approaches were considered for the analysis of the species with very large genome, rare species and species which specimens are too small in size to obtain the amount of genomic and Cot-1 DNA required for suppression of repetitive DNA hybridization.

  15. Single and multiple molecular beacon probes for DNA hybridization studies on a silica glass surface

    NASA Astrophysics Data System (ADS)

    Fang, Xiaohong; Liu, Xiaojing; Tan, Weihong

    1999-05-01

    Surface immobilizable molecular beacons have been developed for DNA hybridization studies on a silica glass plate. Molecular beacons are a new class of oligonucleotide probes that have a loop-and-stem structure with a fluorophore and a quencher attached to the two ends of the stem. They only emit intense fluorescence when hybridize to their target molecules. This provides an excellent selectivity for the detection of DNA molecules. We have designed biotinylated molecular beacons which can be immobilized onto a solid surface. The molecular beacon is synthesized using DABCYL as the quencher and an optical stable dye, tetramethylrhodamine, as the fluorophore. Mass spectrometry is used to confirm the synthesized molecular beacon. The molecular beacons have been immobilized onto a silica surface through biotin-avidin binding. The surface immobilized molecular beacons have been used for the detection of target DNA with subnanomolar analytical sensitivity. have also immobilized two different molecular beacons on a silica surface in spatially resolved microscopic regions. The hybridization study of these two different molecular beacon probes has shown excellent selectivity for their target sequences. The newly designed molecular beacons are intended for DNA molecular interaction studies at an interface and for the development of ultrasensitive DNA sensors for a variety of applications including disease diagnosis, disease mechanism studies, new drug development, and in the investigation of molecular interactions between DNA molecules and other interesting biomolecules.

  16. Nanopore-based DNA-probe sequence-evolution method unveiling characteristics of protein-DNA binding phenomena in a nanoscale confined space.

    PubMed

    Liu, Nannan; Yang, Zekun; Lou, Xiaoding; Wei, Benmei; Zhang, Juntao; Gao, Pengcheng; Hou, Ruizuo; Xia, Fan

    2015-04-07

    Almost all of the important functions of DNA are realized by proteins which interact with specific DNA, which actually happens in a limited space. However, most of the studies about the protein-DNA binding are in an unconfined space. Here, we propose a new method, nanopore-based DNA-probe sequence-evolution (NDPSE), which includes up to 6 different DNA-probe systems successively designed in a nanoscale confined space which unveil the more realistic characteristics of protein-DNA binding phenomena. There are several features; for example, first, the edge-hindrance and core-hindrance contribute differently for the binding events, and second, there is an equilibrium between protein-DNA binding and DNA-DNA hybridization.

  17. Novel molecular beacon DNA probes for protein-nucleic acid interaction studies

    NASA Astrophysics Data System (ADS)

    Li, Jianwei J.; Perlette, John; Fang, Xiaohong; Kelley, Shannon; Tan, Weihong

    2000-03-01

    We report a novel approach to study protein-nucleic acid interactions by using molecular beacons (MBs). Molecular beacons are hairpin-shaped DNA oligonucleotide probes labeled with a fluorophore and a quencher, and can report the presence of target DNA/RNA sequences. MBs can also report the existence of single-stranded DNA binding proteins (SSB) through non-sequence specific binding. The interaction between SSB and MB has resulted in significant fluorescence restoration of the MB. The fluorescence enhancement brought by SSB and by complementary DNA is very comparable. The molar ratio of the binding between SSB and the molecular beacon is 1:1 with a binding constant of 2 X 107 M-1. Using the MB-SSB binding, we are able to determine SSB at 2 X 10-10 M with a conventional spectrometer. We have also applied MB DNA probes for the analysis of an enzyme lactic dehydrogenase (LDH), and for the investigation of its binding properties with ssDNA. The biding process between MB and different isoenzymes of LDH has been studied. We also show that there are significant differences in MB binding affinity to different proteins, which will enable selective binding studies of a variety of proteins. This new approach is potentially useful for protein-DNA/RNA interaction studies that require high sensitivity, speed and convenience. The results also open the possibility of using easily obtainable, custom designed, modified DNA molecules for studies of drug interactions and targeting. Our results demonstrate that MB can be effectively used for sensitive protein quantitation and for efficient protein-DNA interaction studies. MB has the signal transduction mechanism built within the molecule, and can thus be used for quick protein assay development and for real-time measurements.

  18. DNA probe culture confirmation assay for identification of thermophilic Campylobacter species.

    PubMed Central

    Tenover, F C; Carlson, L; Barbagallo, S; Nachamkin, I

    1990-01-01

    We studied the ability of a new DNA probe-based assay system to correctly identify isolates of the thermophilic campylobacters Campylobacter jejuni, C. coli, and C. laridis grown in vitro. We examined 424 organisms, including 214 Campylobacter isolates and 210 other aerobic and anaerobic isolates. The probe assay, which uses a new homogeneous system in which all reactions take place within a single tube, demonstrated 100% accuracy, producing neither false-positive nor false-negative results. The assay does not, however, distinguish among C. jejuni, C. coli, and C. laridis. PMID:2380357

  19. The detection of HBV DNA with gold-coated iron oxide nanoparticle gene probes

    NASA Astrophysics Data System (ADS)

    Xi, Dong; Luo, XiaoPing; Lu, QiangHua; Yao, KaiLun; Liu, ZuLi; Ning, Qin

    2008-03-01

    Gold-coated iron oxide nanoparticle Hepatitis B virus (HBV) DNA probes were prepared, and their application for HBV DNA measurement was studied. Gold-coated iron oxide nanoparticles were prepared by the citrate reduction of tetra-chloroauric acid in the presence of iron oxide nanoparticles which were added as seeds. With a fluorescence-based method, the maximal surface coverage of hexaethiol 30-mer oligonucleotides and the maximal percentage of hybridization strands on gold-coated iron oxide nanoparticles were (120 ± 8) oligonucleotides per nanoparticle, and (14 ± 2%), respectively, which were comparable with those of (132 ± 10) and (22 ± 3%) in Au nanoparticle groups. Large network aggregates were formed when gold-coated iron oxide nanoparticle HBV DNA gene probe was applied to detect HBV DNA molecules as evidenced by transmission electron microscopy and the high specificity was verified by blot hybridization. Our results further suggested that detecting DNA with iron oxide nanoparticles and magnetic separator was feasible and might be an alternative effective method.

  20. Recognition of DNA abasic site nanocavity by fluorophore-switched probe: Suitable for all sequence environments

    NASA Astrophysics Data System (ADS)

    Wang, Ying; Hu, Yuehua; Wu, Tao; Zhang, Lihua; Liu, Hua; Zhou, Xiaoshun; Shao, Yong

    2016-01-01

    Removal of a damaged base in DNA produces an abasic site (AP site) nanocavity. If left un-repaired in vivo by the specific enzyme, this nanocavity will result in nucleotide mutation in the following DNA replication. Therefore, selective recognition of AP site nanocavity by small molecules is important for identification of such DNA damage and development of genetic drugs. In this work, we investigate the fluorescence behavior of isoquinoline alkaloids including palmatine (PAL), berberine (BER), epiberberine (EPI), jatrorrhizine (JAT), coptisine (COP), coralyne (COR), worenine (WOR), berberrubine (BEU), sanguinarine (SAN), chelerythrine (CHE), and nitidine (NIT) upon binding with the AP nanocavity. PAL is screened out as the most efficient fluorophore-switched probe to recognize the AP nanocavity over the fully matched DNA. Its fluorescence enhancement occurs for all of the AP nanocavity sequence environments, which has not been achieved by the previously used probes. The bridged π conjugation effect should partially contribute to the AP nanocavity-specific fluorescence, as opposed to the solvent effect. Due to the strong binding with the AP nanocavity, PAL will find wide applications in the DNA damage recognition and sensor development.

  1. Synthesis of RNA probes by the direct in vitro transcription of PCR-generated DNA templates.

    PubMed

    Urrutia, R; McNiven, M A; Kachar, B

    1993-05-01

    We describe a novel method for the generation of RNA probes based on the direct in vitro transcription of DNA templates amplified by polymerase chain reaction (PCR) using primers with sequence hybrids between the target gene and those of the T7 and T3 RNA polymerases promoters. This method circumvents the need for cloning and allows rapid generation of strand-specific RNA molecules that can be used for the identification of genes in hybridization experiments. We have successfully applied this method to the identification of DNA sequences by Southern blot analysis and library screening.

  2. Molecular hybridization with DNA-probes as a laboratory diagnostic test for influenza viruses.

    PubMed

    Pljusnin, A Z; Rozhkova, S A; Nolandt, O V; Bryantseva, E A; Kuznetsov, O K; Noskov, F S

    1987-01-01

    The possibilities of using DNA-copies of different influenza A virus genes cloned with recombinant bacterial plasmids for the detection of virus-specific RNA by molecular dot-hybridization were analyzed. High specificity of RNA identification has been demonstrated and it has been shown expedient to use DNA-probes with high-conservative virus genes (polymerase, nucleoprotein, or matrix) for the detection of influenza A virus subtypes (H1N1, H2N2, H3N2) and probes with corresponding hemagglutinin genes for the differentiation of the subtypes H3N2 and H1N1. The results of nasopharyngeal specimens testing proved the effectiveness of molecular dot-hybridization in epidemiological studies of influenza outbreaks, especially of mixed etiology.

  3. Probe classification of on-off type DNA microarray images with a nonlinear matching measure

    NASA Astrophysics Data System (ADS)

    Ryu, Munho; Kim, Jong Dae; Min, Byoung Goo; Kim, Jongwon; Kim, Y. Y.

    2006-01-01

    We propose a nonlinear matching measure, called counting measure, as a signal detection measure that is defined as the number of on pixels in the spot area. It is applied to classify probes for an on-off type DNA microarray, where each probe spot is classified as hybridized or not. The counting measure also incorporates the maximum response search method, where the expected signal is obtained by taking the maximum among the measured responses of the various positions and sizes of the spot template. The counting measure was compared to existing signal detection measures such as the normalized covariance and the median for 2390 patient samples tested on the human papillomavirus (HPV) DNA chip. The counting measure performed the best regardless of whether or not the maximum response search method was used. The experimental results showed that the counting measure combined with the positional search was the most preferable.

  4. A probe-based mapping strategy for DNA sequencing with mobile primers

    SciTech Connect

    Strausbaugh, L.D.; Berg, C.M.

    1991-01-01

    Research on DNA sequencing continued. The specific areas of research targeted for the period of this Progress Report included three general phases: (1) optimization of probe-mapping by both the development of new transposons and the design of stream-lined methods for mapping; (2) application of transposon-based methods to larger plasmids and cosmids; and (3) initiation of PCR-based applications of transposons.

  5. A probe-based mapping strategy for DNA sequencing with mobile primers. Progress report

    SciTech Connect

    Strausbaugh, L.D.; Berg, C.M.

    1991-12-31

    Research on DNA sequencing continued. The specific areas of research targeted for the period of this Progress Report included three general phases: (1) optimization of probe-mapping by both the development of new transposons and the design of stream-lined methods for mapping; (2) application of transposon-based methods to larger plasmids and cosmids; and (3) initiation of PCR-based applications of transposons.

  6. Characterization of an In Vivo Z-DNA Detection Probe Based on a Cell Nucleus Accumulating Intrabody.

    PubMed

    Gulis, Galina; Silva, Izabel Cristina Rodrigues; Sousa, Herdson Renney; Sousa, Isabel Garcia; Bezerra, Maryani Andressa Gomes; Quilici, Luana Salgado; Maranhao, Andrea Queiroz; Brigido, Marcelo Macedo

    2016-09-01

    Left-handed Z-DNA is a physiologically unstable DNA conformation, and its existence in vivo can be attributed to localized torsional distress. Despite evidence for the existence of Z-DNA in vivo, its precise role in the control of gene expression is not fully understood. Here, an in vivo probe based on an anti-Z-DNA intrabody is proposed for native Z-DNA detection. The probe was used for chromatin immunoprecipitation of potential Z-DNA-forming sequences in the human genome. One of the isolated putative Z-DNA-forming sequences was cloned upstream of a reporter gene expression cassette under control of the CMV promoter. The reporter gene encoded an antibody fragment fused to GFP. Transient co-transfection of this vector along with the Z-probe coding vector improved reporter gene expression. This improvement was demonstrated by measuring reporter gene mRNA and protein levels and the amount of fluorescence in co-transfected CHO-K1 cells. These results suggest that the presence of the anti-Z-DNA intrabody can interfere with a Z-DNA-containing reporter gene expression. Therefore, this in vivo probe for the detection of Z-DNA could be used for global correlation of Z-DNA-forming sequences and gene expression regulation.

  7. Regulation of DNA Metabolism by DNA-Binding Proteins Probed by Single Molecule Spectroscopy

    DTIC Science & Technology

    2006-12-05

    denaturation The Watson - Crick double- helix is the thermodynamically stable configuration of a DNA molecule under physiological conditions (normal salt and...room/body temperature). This stability is effected (a) by Watson - Crick H-bonding, that is essential for the specificity of base pairing, i.e., for...guarantees the high level of fidelity during replication and transcription. (b) The second contribution to DNA helix stability comes from base-stacking

  8. An AFLP-based procedure for the efficient mapping of mutations and DNA probes in barley.

    PubMed Central

    Castiglioni, P; Pozzi, C; Heun, M; Terzi, V; Müller, K J; Rohde, W; Salamini, F

    1998-01-01

    A strategy based upon AFLP markers for high-efficiency mapping of morphological mutations and DNA probes to linkage groups in barley is presented. First, 511 AFLP markers were placed on the linkage map derived from the cross Proctor x Nudinka. Second, loci controlling phenotypic traits were assigned to linkage groups by AFLP analysis, using F2 populations consisting of 30-50 mutant plants derived from crosses of the type "mutant x Proctor" and "mutant x Nudinka." To map DNA probes, 67 different wild-type barley lines were selected to generate F2 populations by crossing with Proctor and Nudinka. F2 plants that were polymorphic for a given RFLP fragment were classified into genotypic classes. Linkage of the RFLP polymorphism to 1 of the 511 AFLP loci was indicated by cosegregation. The use of the strategy is exemplified by the mapping of the mutation branched-5 to chromosome 2 and of the DNA probes Bkn2 and BM-7 to chromosomes 5 and 1, respectively. Map expansion and marker order in map regions with dense clustering of markers represented a particular problem. A discussion considering the effect of noncanonical recombinant products on these two parameters is provided. PMID:9691056

  9. Brain-specific expression of MAP2 detected using a cloned cDNA probe

    PubMed Central

    1986-01-01

    We describe the isolation of a set of overlapping cDNAs encoding mouse microtubule associated protein 2 (MAP2), using an anti-MAP antiserum to screen a mouse brain cDNA expression library cloned in bacteriophage lambda gt11. The authenticity of these clones was established by the following criteria: (a) three non-identical clones each expressing a MAP2 immunoreactive fusion protein were independently isolated from the expression library; each of these clones cross-hybridized at the nucleic acid level; (b) anti-MAP antiserum was affinity purified using nitrocellulose-bound fusion protein; these antibodies detected only MAP2 in an immunoblot experiment of whole brain microtubule protein; (c) a series of cDNA "walking" experiments was done so as to obtain a non-overlapping cloned fragment corresponding to a different part of the same mRNA molecule. Upon subcloning this non-overlapping fragment into plasmid expression vectors, a fusion protein was synthesized that was immunoreactive with an anti-MAP2 specific antiserum. Thus, a single contiguous cloned mRNA molecule encodes at least two MAP2-specific epitopes; (d) the cloned cDNA probes detect an mRNA species in mouse brain that is of a size (approximately 9 kb) consistent with the coding capacity required by a 250,000-D protein. The MAP2-specific cloned cDNA probes were used in RNA blot transfer experiments to assay for the presence of MAP2 mRNA in a variety of mouse tissues. Though brain contained abundant quantities of MAP2 mRNA, no corresponding sequences were detectable in RNA prepared from liver, kidney, spleen, stomach, or thymus. We conclude that the expression of MAP2 is brain-specific. Use of the MAP2 specific cDNA probes in genomic Southern blot transfer experiments showed the presence of a single gene encoding MAP2 in mouse. The microheterogeneity of MAP2 is therefore ascribable either to alternative splicing within a single gene, or to posttranslational modification(s), or both. Under conditions of low

  10. BaitFisher: A Software Package for Multispecies Target DNA Enrichment Probe Design.

    PubMed

    Mayer, Christoph; Sann, Manuela; Donath, Alexander; Meixner, Martin; Podsiadlowski, Lars; Peters, Ralph S; Petersen, Malte; Meusemann, Karen; Liere, Karsten; Wägele, Johann-Wolfgang; Misof, Bernhard; Bleidorn, Christoph; Ohl, Michael; Niehuis, Oliver

    2016-07-01

    Target DNA enrichment combined with high-throughput sequencing technologies is a powerful approach to probing a large number of loci in genomes of interest. However, software algorithms that explicitly consider nucleotide sequence information of target loci in multiple reference species for optimizing design of target enrichment baits to be applicable across a wide range of species have not been developed. Here we present an algorithm that infers target DNA enrichment baits from multiple nucleotide sequence alignments. By applying clustering methods and the combinatorial 1-center sequence optimization to bait design, we are able to minimize the total number of baits required to efficiently probe target loci in multiple species. Consequently, more loci can be probed across species with a given number of baits. Using transcript sequences of 24 apoid wasps (Hymenoptera: Crabronidae, Sphecidae) from the 1KITE project and the gene models of Nasonia vitripennis, we inferred 57,650, 120-bp-long baits for capturing 378 coding sequence sections of 282 genes in apoid wasps. Illumina reduced-representation library sequencing confirmed successful enrichment of the target DNA when applying these baits to DNA of various apoid wasps. The designed baits furthermore enriched a major fraction of the target DNA in distantly related Hymenoptera, such as Formicidae and Chalcidoidea, highlighting the baits' broad taxonomic applicability. The availability of baits with broad taxonomic applicability is of major interest in numerous disciplines, ranging from phylogenetics to biodiversity monitoring. We implemented our new approach in a software package, called BaitFisher, which is open source and freely available at https://github.com/cmayer/BaitFisher-package.git.

  11. Accuracy of the Clinical Diagnosis of Vaginitis Compared to a DNA Probe Laboratory Standard

    PubMed Central

    Lowe, Nancy K.; Neal, Jeremy L.; Ryan-Wenger, Nancy A.

    2009-01-01

    Objective To estimate the accuracy of the clinical diagnosis of the three most common causes of acute vulvovaginal symptoms (bacterial vaginosis, candidiasis vaginitis, and trichomoniasis vaginalis) using a traditional, standardized clinical diagnostic protocol compared to a DNA probe laboratory standard. Methods This prospective clinical comparative study had a sample of 535 active duty United States military women presenting with vulovaginal symptoms. Clinical diagnoses were made by research staff using a standardized protocol of history, physical examination including pelvic examination, determination of vaginal pH, vaginal fluid amines test, and wet-prep microscopy. Vaginal fluid samples were obtained for DNA analysis. The research clinicians were blinded to the DNA results. Results The participants described a presenting symptom of abnormal discharge (50%), itching/irritation (33%), malodor (10%), burning (4%), or others such as vulvar pain and vaginal discomfort. According to laboratory standard, there were 225 cases (42%) of bacterial vaginosis 76 cases (14%) of candidiasis vaginitis, 8 cases (1.5%) of trichomoniasis vaginalis, 87 cases of mixed infections (16%), and 139 negative cases (26%). For each single infection, the clinical diagnosis had a sensitivity and specificity of 80.8% and 70.0% for bacterial vaginosis; 83.8% and 84.8% for candidiasis vaginitis; and 84.6% and 99.6% for trichomoniasis vaginalis when compared to the DNA probe standard. Conclusion Compared to a DNA probe standard, clinical diagnosis is 81-85% sensitive and 70- 99% specific for bacterial vaginosis, candida vaginitis, and trichomoniasis. Even under research conditions that provided clinicians with sufficient time and materials to conduct a thorough and standardized clinical evaluation, the diagnosis and therefore, subsequent treatment of these common vaginal problems remains difficult. PMID:19104364

  12. Chromosomal DNA probes for the identification of asaccharolytic anaerobic pigmented bacterial rods from the oral cavity of cats.

    PubMed

    Love, D N; Bailey, G D; Bastin, D

    1992-06-01

    A dot-blot hybridisation assay using isolated high molecular weight DNA as whole chromosomal probes of the cat pigmented asaccharolytic Bacteroides/Porphyromonas species was used against both purified high molecular weight DNA and DNA released on membranes from whole cells for the identification of B. salivosus and for its differentiation from the other anaerobic species isolated from normal and diseased mouths of cats and horses. 32P-labelled probes were compared with digoxigenin (DIG)-labelled probes (Boehringer-Mannheim). The whole chromosomal probes were specific--differentiating B. salivosus from a variety of species (including members of the genera Bacteroides, Fusobacterium, Eubacterium, and Prevotella) found in normal and abnormal mouths of cats and horses. Likewise, asaccharolytic black pigmented Group 2 strains were distinguishable from all strains tested. However, cat strains of P. gingivalis which show 68-76% DNA-DNA homology with human strain P. gingivalis ATCC 33277T, were not distinguishable from each other using either 32P-labelled or DIG-labelled probes. The minimum amount of pure Bacteroides DNA which could be detected by the 32P-labelled probe was 100-300 pg, while the amount of pure DNA detected by the DIG system was 1-3 mg after room temperature colour development for 1 h and 100-300 pg after 6 h colour development.

  13. Viral Contribution to Dissolved DNA in the Marine Environment as Determined by Differential Centrifugation and Kingdom Probing

    PubMed Central

    Jiang, S. C.; Paul, J. H.

    1995-01-01

    Dissolved or filterable (<0.2-(mu)m-pore-size filter) DNA is a ubiquitous component of the dissolved organic matter in the surface waters of this planet. In an effort to understand the composition and possible sources, we subjected dissolved DNA concentrated by vortex flow filtration from offshore and coastal environments to differential centrifugation and probing with 16S rRNA-targeted kingdom oligonucleotide probes. Initial studies with calf thymus soluble DNA and T2 phage particles indicated that high-speed ultracentrifugation (201,000 x g for 90 min), a method to separate viral particles from soluble DNA used by other investigators, resulted in pelleting of nearly all the DNA and virus particles. Lower-speed centrifugation (11,200 to 25,800 x g for 90 min) resulted in >99% of the virus particles being collected in the pellet and (equiv)65% of the calf thymus DNA remaining in the supernatant. Employing this approach, we estimate that approximately 50% of the filterable DNA from marine environments is truly soluble or free DNA and that the other half is composed of bound forms (viral particles and, potentially, colloids). Of the bound form, 17 to 30% could be accounted for by viral particles, by calculating the amount of viral DNA on the basis of viral abundance, leaving a portion of the bound form uncharacterized. Kingdom probing with universal, eubacterial, and eucaryotic probes indicated that dissolved DNA hybridized with all of these probes, while purified standard viral DNAs did not, or hybridized only slightly with the universal probe (tailed oligonucleotide only). Collectively, these data indicate that DNA in viral particles is a small component of the dissolved DNA, the majority being of eubacterial and eucaryotic origin. PMID:16534913

  14. Development of a simple and rapid assay for methylase activity based on DNA hairpin probe and Sybr Green I

    NASA Astrophysics Data System (ADS)

    Long, Yi; Zhou, Xiaoming

    2012-03-01

    Methylase is vital for a large number of biological reactions. Here we developed a new method for DNA methylase activity analysis. In this paper, a DNA hairpin probe with a sequence of 5'-GATC-3' in the stem region was designed. The 5'-GATC-3' sequence was targeted by Dam MTase and was methylated. Subsequently, restriction enzyme Dpnl recognized the site and cut it. Then the haipin probe was transformed into three single stranded DNA. This enzymatic process can be monitored by the change of SYBR green I fluorescence. The current label free assay is an useful tool for DNA methylase activity analysis due to its simplicity, speedability, and low cost.

  15. Development of a simple and rapid assay for methylase activity based on DNA hairpin probe and Sybr Green I

    NASA Astrophysics Data System (ADS)

    Long, Yi; Zhou, Xiaoming

    2011-11-01

    Methylase is vital for a large number of biological reactions. Here we developed a new method for DNA methylase activity analysis. In this paper, a DNA hairpin probe with a sequence of 5'-GATC-3' in the stem region was designed. The 5'-GATC-3' sequence was targeted by Dam MTase and was methylated. Subsequently, restriction enzyme Dpnl recognized the site and cut it. Then the haipin probe was transformed into three single stranded DNA. This enzymatic process can be monitored by the change of SYBR green I fluorescence. The current label free assay is an useful tool for DNA methylase activity analysis due to its simplicity, speedability, and low cost.

  16. A novel DNA tetrahedron-hairpin probe for in situ"off-on" fluorescence imaging of intracellular telomerase activity.

    PubMed

    Feng, Qiu-Mei; Zhu, Meng-Jiao; Zhang, Ting-Ting; Xu, Jing-Juan; Chen, Hong-Yuan

    2016-04-21

    A novel three-dimensionally structured DNA probe is reported to realize in situ"off-on" imaging of intracellular telomerase activity. The probe consists of a DNA tetrahedron and a hairpin DNA on one of the vertices of the DNA tetrahedron. It is composed of four modified DNA segments: S1-Au nanoparticle (NP) inserting a telomerase strand primer (TSP) and S2-S4, three Cy5 dye modified DNA segments. Fluorescence of Cy5 at three vertices of the DNA tetrahedron is quenched by the Au NP at the other vertex due to the effective fluorescence resonance energy transfer (FRET) ("off" state). When the probe meets telomerase, the hairpin structure changes to rod-like through complementary hybridization with the telomerase-triggered stem elongation product, resulting in a large distance between the Au NP and Cy5 and the recovery of Cy5 fluorescence ("on" state). The molar ratio of 3 : 1 between the reporter (Cy5) and the target related TSP makes the probe show high sensitivity and recovery efficiency of Cy5 in the presence of telomerase extracted from HeLa cells. Given the functional and compact nanostructure, the mechanically stable and noncytotoxic nature of the DNA tetrahedron, this FRET-based probe provides more opportunities for biosensing, molecular imaging and drug delivery.

  17. Fluorescence Probe for Detecting CCG Trinucleotide Repeat DNA Expansion and Slip-Out.

    PubMed

    Shibata, Tomonori; Nakatani, Kazuhiko

    2016-09-15

    Trinucleotide repeat expansion in genomic DNA causes severe neurodegenerative diseases. Fluorescence probes that bind to trinucleotide repeats have potential as diagnostic tools of trinucleotide repeat disorders. Here, we report a novel tricyclic ligand that binds to CCG trinucleotide repeat DNA. The expansion of the aromatic ring system of the 2-amino-1,8-naphthyridine chromophore from the bicyclic to the tricyclic improved the binding ability to the CCG/CCG motif without losing the selectivity and emissive character. The fluorescence sensitively decreased in response to binding to the CCG trinucleotide repeat. The degree of quenching depended on the number of CCG repeats. In addition, the fluorescence detection was applicable to CCG slip-out DNA.

  18. Simultaneous detection of DNA from 10 food allergens by ligation-dependent probe amplification.

    PubMed

    Ehlert, Alexandra; Demmel, Anja; Hupfer, Christine; Busch, Ulrich; Engel, Karl-Heinz

    2009-04-01

    The simultaneous detection of DNA from different allergenic food ingredients by a ligation-dependent probe amplification (LPA) system is described. The approach allows detection of several targets in a one-tube assay. Synthetic oligonucleotides were designed to detect DNA from peanuts, cashews, pecans, pistachios, hazelnuts, sesame seeds, macadamia nuts, almonds, walnuts and brazil nuts. The specificity of the system was tested with DNA from more than 50 plant and animal species. The sensitivity of the method was suitable to detect allergenic ingredients in the low mg kg(-1) range. The limit of detection (LOD) for single allergens in different food matrices was 5 mg kg(-1). The novel analytical strategy represents a useful tool for the surveillance of established legislation on food allergens within the European Union.

  19. Resonance light scattering method for the determination of DNA with cationic methacrylate based polymer nanoparticle probes.

    PubMed

    Zou, Qi-Chao; Zhang, Jin-Zhi; Chai, Shi-Gan

    2011-11-01

    Narrowly distributed cationic poly (methyl methacrylate-co-diacetone acrylamide) (P(MMA-DAAM)) nanoparticles were successfully prepared by microemulsion polymerization. Photon correlation spectrometer (PCS) measurement and transmission electron microscope (TEM) observation revealed that z-average particle size of P(MMA-DAAM) is ∼27.5 nm. It was found that these cationic nanoparticles interact with DNA through electrostatic interaction to form P(MMA-DAAM)-DNA complex, which significantly enhances the resonance light scattering (RLS) signal. Therefore, a novel method using this polymer nanoparticle as a new probe for the detection of DNA by RLS technique is developed in this paper. The results showed this method is very convenient, sensitive, and reproducible.

  20. Using surface-enhanced Raman spectroscopy to probe for genetic markers on single-stranded DNA

    NASA Astrophysics Data System (ADS)

    Moody, Benjamin; Leotaud, John; McCarty, Gregory S.

    2010-03-01

    Methods capable of quickly and inexpensively collecting genetic information are of increasing importance. We report a method of using surface-enhanced Raman spectroscopy to probe single-stranded DNA for genetic markers. This unique approach is used to analyze unmodified genes of moderate length for genetic markers by hybridizing native test oligonucleotides into a surface-enhanced Raman complex, vastly increasing detection sensitivity as compared to traditional Raman spectroscopy. The Raman complex is formed by sandwiching the test DNA between 40-nm gold nanoparticles and a photolithographically defined gold surface. With this design, we are able to collect characteristic Raman spectra about the test DNA and to detect genetic markers such as single-nucleotide polymorphisms (SNPs) and polymorphic regions. Results show that strands containing one of three different types of polymorphism can be differentiated using statistically significant trends regarding Raman intensity.

  1. A review of scanning probe microscopy investigations of liposome-DNA complexes.

    PubMed

    Mozafari, M R; Reed, C J; Rostron, C; Hasirci, V

    2005-01-01

    Liposome-DNA complexes are one of the most promising systems for the protection and delivery of nucleic acids to combat neoplastic, viral, and genetic diseases. In addition, they are being used as models in the elucidation of many biological phenomena such as viral infection and transduction. In order to understand these phenomena and to realize the mechanism of nucleic acid transfer by liposome-DNA complexes, studies at the molecular level are required. To this end, scanning probe microscopy (SPM) is increasingly being used in the characterization of lipid layers, lipid aggregates, liposomes, and their complexes with nucleic acid molecules. The most attractive attributes of SPM are the potential to image samples with subnanometer spatial resolution under physiological conditions and provide information on their physical and mechanical properties. This review describes the application of scanning tunneling microscopy and atomic force microscopy, the two most commonly applied SPM techniques, in the characterisation of liposome-DNA complexes.

  2. Chromosomal DNA probes for the identification of Bacteroides tectum and Bacteroides fragilis from the oral cavity of cats.

    PubMed

    Love, D N; Bailey, G D

    1993-01-01

    A dot-blot hybridisation assay using high molecular weight DNA as whole chromosomal probes was used to differentiate Bacteroides tectum from Bacteroides fragilis. 32P-labelled probes were compared with digoxigenin (DIG)-labelled probes. The whole chromosomal probes were specific--differentiating B. tectum from B. fragilis and both from a variety of other species (including other members of the genera Bacteroides, Fusobacterium, Eubacterium, and Prevotella) found in normal and abnormal mouths of cats and horses. However, even at very high stringencies, B. tectum homology groups I, II and III were not distinguishable from one another using either 32P-labelled or DIG-labelled probes. Thus, DIG-labelled whole chromosome probes directed against cellular DNA released directly onto nitrocellulose membranes is considered a useful method for diagnostic veterinary laboratories wishing to identify B. tectum and distinguish it from B. fragilis and other oral anaerobic flora of cats.

  3. Ultrafast force-clamp spectroscopy to probe lac repressor-DNA interactions

    NASA Astrophysics Data System (ADS)

    Monico, Carina; Capitanio, Marco; Belcastro, Gionata; Vanzi, Francesco; Pavone, Francesco S.

    2013-06-01

    We recently developed an ultrafast force-clamp laser trap capable to probe, under controlled force, bimolecular interactions with unprecedented temporal resolution. Here we present the technique in the framework of protein-DNA interactions, specifically on Lactose repressor protein (LacI). The high temporal resolution of the method reveals the kinetics of both short- and long-lived interactions of LacI along the DNA template (from ˜100 μs to tens of seconds), as well the dependence on force of such interaction kinetics. The two kinetically well-distinct populations of interactions observed clearly represent specific interactions with the operator sequences and a fast scanning of LacI along non-cognate DNA. These results demonstrate the effectiveness of the method to study the sequence-dependent affinity of DNA-binding proteins along the DNA and the effects of force on a wide range of interaction durations, including μs time scales not accessible to other single-molecule methods. This improvement in time resolution provides also important means of investigation on the long-puzzled mechanism of target search on DNA and possible protein conformational changes occurring upon target recognition.

  4. Photocatalytic probing of DNA sequence by using TiO{sub 2}/dopamine-DNA triads.

    SciTech Connect

    Liu, J.; de la Garza, L.; Zhang, L.; Dimitrijevic, N. M.; Zuo, X.; Tiede, D. M.; Rajh, T.

    2007-10-15

    A method to control charge transfer reaction in DNA using hybrid nanometer-sized TiO{sub 2} nanoparticles was developed. In this system extended charge separation reflects the sequence of DNA and was measured using metallic silver deposition or by photocurrent response. Light-induced extended charge separation in these systems was found to be dependent on the DNA-bridge length and sequence. The yield of photocatalytic deposition of silver was studied in systems having GG accepting sites imbedded in AT runs at varying distances from the TiO{sub 2} nanoparticle surface. Weak distance dependence of charge separation indicative of a hole hopping through mediating adenine (A) sites was found. The quantum yield of silver deposition in the system having a GG accepting site placed 8.5 {angstrom} from the nanoparticle surface was found to be {Phi} = 0.70 (70%) and {Phi} = 0.56 (56%) for (A){sub n} and (AT){sub n/2} bridge, respectively. Hole injection to GG trapping sites as far as 70 {angstrom} from a nanoparticle surface in the absence of G hopping sites was measured. Introduction of G hopping sites increased the efficiency of hole injection. The efficiency of photocatalytic deposition of metallic silver was found to be sensitive to the presence of a single nucleobase mismatch in the DNA sequence.

  5. The Anopheles punctulatus complex: DNA probes for identifying the Australian species using isotopic, chromogenic, and chemiluminescence detection systems

    SciTech Connect

    Cooper, L.; Cooper, R.D.; Burkot, T.R. )

    1991-07-01

    Isotopic and enzyme-labeled species-specific DNA probes were made for the three known members of the Anopheles punctulatus complex of mosquitoes in Australia (Anopheles farauti Nos. 1, 2, and 3). Species-specific probes were selected by screening total genomic libraries made from the DNA of individual species with 32P-labeled DNA of homologous and heterologous mosquito species. The 32P-labeled probes for A. farauti Nos. 1 and 2 can detect less than 0.2 ng of DNA while the 32P-labeled probe for A. farauti No. 3 has a sensitivity of 1.25 ng of DNA. Probes were then enzyme labeled for chromogenic and chemiluminescence detection and compared to isotopic detection using 32P-labeled probes. Sequences of the probe repeat regions are presented. Species identifications can be made from dot blots or squashes of freshly killed mosquitoes or mosquitoes stored frozen, dried, and held at room temperature or fixed in isopropanol or ethanol with isotopic, chromogenic, or chemiluminescence detection systems. The use of nonisotopic detection systems will enable laboratories with minimal facilities to identify important regional vectors.

  6. High-performance analysis of single interphase cells with custom DNA probes spanning translocation break points

    NASA Astrophysics Data System (ADS)

    Weier, Heinz-Ulli G.; Munne, S.; Lersch, Robert A.; Marquez, C.; Wu, J.; Pedersen, Roger A.; Fung, Jingly

    1999-06-01

    The chromatin organization of interphase cell nuclei, albeit an object of intense investigation, is only poorly understood. In the past, this has hampered the cytogenetic analysis of tissues derived from specimens where only few cells were actively proliferating or a significant number of metaphase cells could be obtained by induction of growth. Typical examples of such hard to analyze cell systems are solid tumors, germ cells and, to a certain extent, fetal cells such as amniocytes, blastomeres or cytotrophoblasts. Balanced reciprocal translocations that do not disrupt essential genes and thus do not led to disease symptoms exit in less than one percent of the general population. Since the presence of translocations interferes with homologue pairing in meiosis, many of these individuals experience problems in their reproduction, such as reduced fertility, infertility or a history of spontaneous abortions. The majority of translocation carriers enrolled in our in vitro fertilization (IVF) programs carry simple translocations involving only two autosomes. While most translocations are relatively easy to spot in metaphase cells, the majority of cells biopsied from embryos produced by IVF are in interphase and thus unsuitable for analysis by chromosome banding or FISH-painting. We therefore set out to analyze single interphase cells for presence or absence of specific translocations. Our assay, based on fluorescence in situ hybridization (FISH) of breakpoint-spanning DNA probes, detects translocations in interphase by visual microscopic inspection of hybridization domains. Probes are prepared so that they span a breakpoint and cover several hundred kb of DNA adjacent to the breakpoint. On normal chromosomes, such probes label a contiguous stretch of DNA and produce a single hybridization domain per chromosome in interphase cells. The translocation disrupts the hybridization domain and the resulting two fragments appear as physically separated hybridization domains in

  7. Determination of mutated genes in the presence of wild-type DNA by using molecular beacons as probe

    NASA Astrophysics Data System (ADS)

    Zhang, Yonghua; Ai, Junjie; Gu, Qiaorong; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

    2017-03-01

    Low-abundance mutations in the presence of wild-type DNA can be determined using molecular beacon (MB) as probe. A MB is generally used as DNA probe because it can distinguish single-base mismatched target DNA from fully matched target DNA. However, the probe can not determine low-abundance mutations in the presence of wild-type DNA. In this study, this limitation is addressed by enhancing the stability of unpaired base-containing dsDNA with a hydrogen-bonding ligand, which was added after hybridization of the MB to the target DNA. The ligand formed hydrogen bonds with unpaired bases and stabilized the unpaired base-containing dsDNA if target DNA is mutated one. As a result, more MBs were opened by the mutant genes in the presence of the ligand and a further increase in the fluorescence intensity was obtained. By contrast, fluorescence intensity did not change if target DNA is wild-type one. Consequent increase in the fluorescence intensity of the MB was regarded as a signal derived from mutant genes. The proposed method was applied in synthetic template systems to determine point mutation in DNA obtained from PCR analysis. The method also allows rapid and simple discrimination of a signal if it is originated in the presence of mutant gene or alternatively by a lower concentration of wild gene.

  8. M13 repeat probe detects DNA minisatellite-like sequences in gymnosperms and angiosperms.

    PubMed

    Rogstad, S H; Patton, J C; Schaal, B A

    1988-12-01

    Several kinds of minisatellite DNA, all of which are composed of low to moderately repetitive DNA, have been identified in tetrapod genomes. While the repeating oligonucleotide elements (subrepeats) of a given minisatellite are virtually identical, subrepeat nucleotide composition differs between different minisatellites. Several minisatellites have exhibited moderate to high levels of restriction length polymorphism in a number of tetrapods. Such hypervariable markers provide powerful tools for genetic analyses in several fields of biology. Minisatellite applications have been restricted to tetrapods, but here we demonstrate that one probe, the M13 repeat probe previously used to detect minisatellites in humans and bovines, also reveals minisatellite-bearing endonuclease fragments in gymnosperms and angiosperms. While the plant minisatellites appear to be somatically stable within an individual, they often vary within species in potentially useful ways. These results demonstrate that minisatellite-like families may be distributed over a wide taxonomic range in eukaryotes, opening the possibility of a commensurately wide utility of minisatellite probes in genetic analyses.

  9. Construction and applications of DNA probes for detection of polychlorinated biphenyl-degrading genotypes in toxic organic-contaminated soil environments

    SciTech Connect

    Walia, S.; Khan, A.; Rosenthal, N. )

    1990-01-01

    Several DNA probes for polychlorinated biphenyl (PCB)-degrading genotypes were constructed from PCB-degrading bacteria. These laboratory-engineered DNA probes were used for the detection, enumeration, and isolation of specific bacteria degrading PCBs. Dot blot analysis of purified DNA from toxic organic chemical-contaminated soil bacterial communities showed positive DNA-DNA hybridization with a 32P-labeled DNA probe (pAW6194, cbpABCD). Less than 1% of bacterial colonies isolated from garden topsoil and greater than 80% of bacteria isolated from PCB-contaminated soils showed DNA homologies with 32P-labeled DNA probes. Some of the PCB-degrading bacterial isolates detected by the DNA probe method did not show biphenyl clearance. The DNA probe method was found to detect additional organisms with greater genetic potential to degrade PCBs than the biphenyl clearance method did. Results from this study demonstrate the usefulness of DNA probes in detecting specific PCB-degrading bacteria, abundance of PCB-degrading genotypes, and genotypic diversity among PCB-degrading bacteria in toxic chemical-polluted soil environments. We suggest that the DNA probe should be used with caution for accurate assessment of PCB-degradative capacity within soils and further recommend that a combination of DNA probe and biodegradation assay be used to determine the abundance of PCB-degrading bacteria in the soil bacterial community.

  10. Meat speciation by restriction fragment length polymorphism analysis using an α-actin cDNA probe.

    PubMed

    Fairbrother, K S; Hopwood, A J; Lockley, A K; Bardsley, R G

    1998-09-01

    Classical DNA fingerprinting is based on separation of DNA restriction fragments by electrophoresis and hybridisation to nucleic acid probes containing repetitive nucleotide sequences. The use of such mini- or micro-satellite probes tends to yield patterns specific to an individual rather than to a species, hence their value in forensic analysis but general unsuitability for meat speciation. In the present study, a cDNA probe based on conserved sequences contained in members of the actin multigene family has been evaluated for potential application in meat speciation. Genomic DNA was extracted from muscle and digested with BamHI before electrophoresis and hybridisation to a murine α-actin cDNA probe. Beef, pork, lamb, horse, chicken and fish DNA restriction fragments formed characteristic 'fingerprints' which were reproducible and varied sufficiently to allow discrimination even between closely-related species. However no major differences were seen between individuals of the same breed or between different breeds within a species. When DNA obtained from fresh tissue and also from meat heated at 120 °C was analysed, the gel patterns were essentially the same. An attractive feature of this approach is that it employs a single cross-reacting probe and set of conditions, and gives different patterns with all species so far studied. This simplicity suggests applications in meat speciation or related areas of biology.

  11. A electrogenerated chemiluminescence biosensor for Ramos cancer cell using DNA encapsulated Ru(bpy)₃Cl₂ as signal probe.

    PubMed

    Hun, Xu; Chen, Huaicheng; Wang, Wei

    2011-05-15

    A label-free sensing technology for detection of Ramos cell was developed based on a signal probe Ru(bpy)3Cl2 (Ru) encapsulated by DNA. Gold electrode or magnetic bead as the sensing surface was firstly modified with long-strand DNA with five repeating units. Then two kinds of short-strand DNA are grafted onto the long-strand DNA to form DNA strands A and B (L-A and L-B) through the hybridization, respectively. The addition of aptamer initiates hybridization of L-A and L-B with the aptamer sequence. As the hybridization proceeds, the four kinds of DNA would finally transform into a three-dimensional network structure and the signal probe Ru was encapsulated by DNA simultaneously. When Ramos cells are introduced to interact with the aptamer, the signal probe is released. In order to confirm the generality of this method the ferrocenecarboxylic acid and luminol selected as a signal probe mode were also tested. The Ru used as a signal probe for electrogenerated chemiluminescence (ECL) detection was detailedly studied. With this ECL biosensor, detection limit as low as 58 cells/mL was achieved for Ramos cell. The biosensor also exhibited excellent sensitivity and selectivity.

  12. [Genetic diversity of 3 DNA probes in the DNA fingerprinting of a Mexican population].

    PubMed

    Berumen-Campos, J; Casas-Avila, L; Hernández-Mendoza, A; Segura-Salinas, E; Medina-León, R; Larriva-Sahd, J

    1994-01-01

    Each individual may be identified by characterizing its genetic material by DNA fingerprinting technology. Its application in Mexico demands a knowledge of the allelic and genotypic diversity of the DNA markers and the probability that two individuals may have the same fingerprint. In the present study the allelic and genotypic diversities of the loci D12S11 (MS43A), D7S22 (g3) and D1S7 (MS1) were determined in 100 Mexican students of the military school of medicine (Escuela Médico Militar de México). The mean allelic frequency of the loci MS43A, g3, and MS1 was 0.01, 0.008 and 0.006, respectively. The heterozygosity of MS43A and g3 was 98 and 99% for MS1. The probability that two individuals might have the same genetic pattern was 2.0 x 10(-4), 1.3 x 10(-4) and 7.2 x 10(-5) for the loci MS43A, g3 and MS1, respectively, and as low as 1.9 x 10(-12) for the three taken together. These data indicate that the genetic diversity of these DNA fingerprinting markers in the Mexican population is high enough to warrant its use in paternity testing and in the identification of individuals in forensic medicine.

  13. Ultrafast excited-state dynamics at interfaces: fluorescent DNA probes at the dodecane/water interface

    NASA Astrophysics Data System (ADS)

    Licari, Giuseppe; Vauthey, Eric

    2015-08-01

    Although the interfaces between two isotropic media are of primary importance in many areas of science and technology, their properties are only partially understood. Our strategy to obtain an insight into these properties is to investigate the ultrafast excited-state dynamics of environment-sensitive molecular probes at liquid interfaces using time-resolved surface second harmonic generation, and to compare it with the dynamics of the same molecules in bulk solutions. Additionally, this approach gives rich information on how the chemical reactivity may change when going from the bulk phase to the interface. This is illustrated by an investigation performed with a series of fluorescent DNA probes at the dodecane/water interface without and with the presence of DNA in the aqueous phase. Substantial differences in the conformation of these cyanine dyes (aggregated or not) and in the excited-state dynamics are observed when going from bulk solutions to the interface. Moreover, the presence of double-stranded DNA in the aqueous phase induces some chirality at the interface.

  14. A DNA minor groove electronegative potential genome map based on photo-chemical probing.

    PubMed

    Lindemose, Søren; Nielsen, Peter Eigil; Hansen, Morten; Møllegaard, Niels Erik

    2011-08-01

    The double-stranded DNA of the genome contains both sequence information directly relating to the protein and RNA coding as well as functional and structural information relating to protein recognition. Only recently is the importance of DNA shape in this recognition process being fully appreciated, and it also appears that minor groove electronegative potential may contribute significantly in guiding proteins to their cognate binding sites in the genome. Based on the photo-chemical probing results, we have derived an algorithm that predicts the minor groove electronegative potential in a DNA helix of any given sequence. We have validated this model on a series of protein-DNA binding sites known to involve minor groove electrostatic recognition as well as on stable nucleosome core complexes. The algorithm allows for the first time a full minor groove electrostatic description at the nucleotide resolution of any genome, and it is illustrated how such detailed studies of this sequence dependent, inherent property of the DNA may reflect on genome organization, gene expression and chromosomal condensation.

  15. DNA-based stable isotope probing enables the identification of active bacterial endophytes in potatoes.

    PubMed

    Rasche, Frank; Lueders, Tillmann; Schloter, Michael; Schaefer, Sabine; Buegger, Franz; Gattinger, Andreas; Hood-Nowotny, Rebecca C; Sessitsch, Angela

    2009-03-01

    A (13)CO2 (99 atom-%, 350 ppm) incubation experiment was performed to identify active bacterial endophytes in two cultivars of Solanum tuberosum, cultivars Desirée and Merkur. We showed that after the assimilation and photosynthetic transformation of (13)CO2 into (13)C-labeled metabolites by the plant, the most directly active, cultivar specific heterotrophic endophytic bacteria that consume these labeled metabolite scan be identified by DNA stable isotope probing (DNA-SIP).Density-resolved DNA fractions obtained from SIP were subjected to 16S rRNA gene-based community analysis using terminal restriction fragment length polymorphism analysis and sequencing of generated gene libraries.Community profiling revealed community compositions that were dominated by plant chloroplast and mitochondrial 16S rRNA genes for the 'light' fractions of (13)CO2-incubated potato cultivars and of potato cultivars not incubated with (13)CO2. In the 'heavy' fractions of the (13)CO2-incubated endophyte DNA, a bacterial 492-bp terminal restriction fragment became abundant, which could be clearly identified as Acinetobacter and Acidovorax spp. in cultivars Merkur and Desirée,respectively, indicating cultivar-dependent distinctions in (13)C-label flow. These two species represent two common potato endophytes with known plant-beneficial activities.The approach demonstrated the successful detection of active bacterial endophytes in potato. DNA-SIP therefore offers new opportunities for exploring the complex nature of plant-microbe interactions and plant-dependent microbial metabolisms within the endosphere.

  16. Noncovalent attachment of psoralen derivatives with DNA: Hartree-Fock and density functional studies on the probes

    NASA Astrophysics Data System (ADS)

    El-Gogary, Tarek M.; El-Gendy, Eman M.

    2003-09-01

    Two psoralen derivatives (probes) were prepared. Their geometries were optimized at the Hartree-Fock (HF) and Density Functional (B3LYP) levels employing 6-31G** and cc-pVDZ basis sets. Their interaction with DNA was investigated using spectrophotometric and computational techniques. Both of them have shown strong binding to calf thymus DNA. The red-shift and hypochromism that detected in the spectrum were taken as an evidence for the strong interaction between these probes and DNA. The spectrophotometric DNA titration data were treated by two different methodologies to calculate the intercalation affinity. Half-reciprocal plots gave binding constants of 5.5065×10 4 and 6.4727×10 4 for 8-butoxypsoralen (8-BOP) and 8-hexoxypsoralen (8-HOP), respectively. Schatchard plots gave a comparable intercalation binding constants and also the surface binding constants along with the number of intercalated probe molecules per base pair. The interaction between these probes and DNA were studied theoretically. The energy of interaction was computed using molecular mechanics method. Strength of interaction of these probes with different types of DNA was computed and compared. Calculated energies of interaction were compared with the observed intercalation affinities. HOMO and LUMO energies were computed and used to account for the strength of interaction.

  17. Reactive Microcontact Printing of DNA Probes on (DMA-NAS-MAPS) Copolymer-Coated Substrates for Efficient Hybridization Platforms.

    PubMed

    Castagna, Rossella; Bertucci, Alessandro; Prasetyanto, Eko Adi; Monticelli, Marco; Conca, Dario Valter; Massetti, Matteo; Sharma, Parikshit Pratim; Damin, Francesco; Chiari, Marcella; De Cola, Luisa; Bertacco, Riccardo

    2016-04-05

    High-performing hybridization platforms fabricated by reactive microcontact printing of DNA probes are presented. Multishaped PDMS molds are used to covalently bind oligonucleotides over a functional copolymer (DMA-NAS-MAPS) surface. Printed structures with minimum width of about 1.5 μm, spaced by 10 μm, are demonstrated, with edge corrugation lower than 300 nm. The quantification of the immobilized surface probes via fluorescence imaging gives a remarkable concentration of 3.3 × 10(3) oligonucleotides/μm(2), almost totally active when used as probes in DNA-DNA hybridization assays. Indeed, fluorescence and atomic force microscopy show a 95% efficiency in target binding and uniform DNA hybridization over printed areas.

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

    PubMed Central

    Li, C; Ferguson, D A; Ha, T; Chi, D S; Thomas, E

    1993-01-01

    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. Images PMID:8370744

  19. DNA Modified Fe3O4@Au Magnetic Nanoparticles as Selective Probes for Simultaneous Detection of Heavy Metal Ions.

    PubMed

    Miao, Peng; Tang, Yuguo; Wang, Lei

    2017-02-01

    Driven by the urgent need to detect trace heavy metal ions in various real water samples, this article demonstrates for the first time an electrochemical biosensor based on DNA modified Fe3O4@Au magnetic nanoparticles (NPs). Three DNA probes are designed to contain certain mismatched base pairs. One is thiolated and modified on the surface of Fe3O4@Au NPs (DNA 1). The other two probes (DNA 2 and 3) are labeled with two independent electrochemical species. Stable structures of cytosine-Ag(+)-cytosine and thymine-Hg(2+)-thymine formed in the presence of Ag(+) and Hg(2+) can assist the hybridization of DNA 1/DNA 2 and DNA 1/DNA 3, which locate corresponding electrochemical species onto the surface of the magnetic NPs. The achieved nanocomposites are then used as selective electrochemical probes for the detection of heavy metal ions by recording the square wave voltammetry signals. Simultaneous detection of Ag(+) and Hg(2+) is demonstrated without significant interference, and their individual high sensitivities are fundamentally preserved, which meet the requirements of U.S. Environmental Protection Agency (USEPA). Furthermore, the proposed method has been challenged by various real water samples. The results confirm the DNA modified magnetic NPs based sensing method may have potential applications for the monitoring of heavy metal ions in real sample analysis.

  20. Dissecting the effect of anions on Hg2+ detection using a FRET based DNA probe.

    PubMed

    Kiy, Mehmet Murat; Zaki, Ahmed; Menhaj, Arsalsan Beg; Samadi, Azadeh; Liu, Juewen

    2012-08-07

    Many biosensors have been developed to detect Hg(2+) using thymine-rich DNA. While sensor response to various cations is often studied to demonstrate selectivity, the effect of anions has been largely overlooked. Anions may compete with DNA for metal binding and thus produce a false negative result. Anions cannot be added alone; the cation part of a salt may cause DNA compaction and other effects, obscuring the role of anions. We find that the sensitivity of a FRET-based Hg(2+) probe is independent of Na(+) concentration. Therefore, by using various sodium salts, any change in sensitivity can be attributed solely to the effect of anions. Halide salts, sulfides, and amines are strong inhibitors; anions containing oxo or hydroxyl groups (e.g. nitrate, sulfate, phosphate, carbonate, acetate, and citrate) do not interfere with Hg(2+) detection even at 100 mM concentration. Mercury hydrolysis and its diffusion into polypropylene containers can also strongly affect the detection results. We conclude that thymine-rich DNA should be useful for Hg(2+) detection in many environmental water samples.

  1. Ligand-incorporation site in 5-methylcytosine-detection probe modulating the site of osmium complexation with the target DNA.

    PubMed

    Sugizaki, Kaori; Nakamura, Akiko; Yanagisawa, Hiroyuki; Okamoto, Akimitsu

    2012-09-01

    ICON Probes, short DNA strands containing an adenine linked to a bipyridine ligand, formed an interstrand cross-link with 5-methylcytosine located opposite the modified adenine in the presence of an osmium oxidant. The location of a bipyridine-tethered adenine in the probes varied the selectivity of the reactive base. An ICON probe where the modified adenine was located at the probe center showed a 5-methylcytosine-selective osmium complexation, whereas an ICON probe with the modified adenine at the strand end exhibited high reactivity towards thymine as well as 5-methylcytosine. The modulation of reactive bases by the incorporation of a bipyridine-tethered adenine site made facilitates design of ICON probes for the fluorometric detection of 5-methylcytosine.

  2. LINE-1 repetitive DNA probes for species-specific cloning from Mus spretus and Mus domesticus genomes.

    PubMed

    Rikke, B A; Hardies, S C

    1991-12-01

    Mus domesticus and Mus spretus mice are closely related subspecies. For genetic investigations involving hybrid mice, we have developed a set of species-specific oligonucleotide probes based on the detection of LINE-1 sequence differences. LINE-1 is a repetitive DNA family whose many members are interspersed among the genes. In this study, library screening experiments were used to fully characterize the species specificity of four M. domesticus LINE-1 probes and three M. spretus LINE-1 probes. It was found that the nucleotide differences detected by the probes define large, species-specific subfamilies. We show that collaborative use of such probes can be employed to selectively detect thousands of species-specific library clones. Consequently, these probes could be exploited to monitor and access almost any given species-specific region of interest within hybrid genomes.

  3. Detection of hepatitis A virus in seeded estuarine samples by hybridization with cDNA probes

    SciTech Connect

    Jiang, X.; Estes, M.K.; Metcalf, T.G.; Melnick, J.L

    1986-10-01

    The development and trials of a nucleic acid hybridization test for the detection of hepatitis A virus (HAV) in estuarine samples within 48 h are described. Approximately 10/sup 4/ physical particlels of HAV per dot could be detected. Test sensitivity was optimized by the consideration of hydbridization stringency, /sup 32/P energy level, probe concentration, and nucleic acid binding to filters. Test specificity was shown by a lack of cross-hybridization with other enteroviruses and unrelated nucleic acids. Potential false-positive reactions between bacterial DNA in samples and residual vector DNA contamination of purified nucleotide sequences in probes were eliminated by DNase treatment of samples. Humic acid at concentrations of up to 100 mg/liter caused only insignificant decreases in test sensitivity. Interference with hybridization by organic components of virus-containing eluates was removed by proteinase K digestion followed by phenol extraction and ethanol precipitation. The test is suitable for detecting naturally occurring HAV in samples from polluted estuarine environments.

  4. Kinetics of Oligonucleotide Hybridization to DNA Probe Arrays on High-Capacity Porous Silica Substrates

    PubMed Central

    Glazer, Marc I.; Fidanza, Jacqueline A.; McGall, Glenn H.; Trulson, Mark O.; Forman, Jonathan E.; Frank, Curtis W.

    2007-01-01

    We have investigated the kinetics of DNA hybridization to oligonucleotide arrays on high-capacity porous silica films that were deposited by two techniques. Films created by spin coating pure colloidal silica suspensions onto a substrate had pores of ∼23 nm, relatively low porosity (35%), and a surface area of 17 times flat glass (for a 0.3-μm film). In the second method, latex particles were codeposited with the silica by spin coating and then pyrolyzed, which resulted in larger pores (36 nm), higher porosity (65%), and higher surface area (26 times flat glass for a 0.3-μm film). As a result of these favorable properties, the templated silica hybridized more quickly and reached a higher adsorbed target density (11 vs. 8 times flat glass at 22°C) than the pure silica. Adsorption of DNA onto the high-capacity films is controlled by traditional adsorption and desorption coefficients, as well as by morphology factors and transient binding interactions between the target and the probes. To describe these effects, we have developed a model based on the analogy to diffusion of a reactant in a porous catalyst. Adsorption values (ka, kd, and K) measured on planar arrays for the same probe/target system provide the parameters for the model and also provide an internally consistent comparison for the stability of the transient complexes. The interpretation of the model takes into account factors not previously considered for hybridization in three-dimensional films, including the potential effects of heterogeneous probe populations, partial probe/target complexes during diffusion, and non-1:1 binding structures. The transient complexes are much less stable than full duplexes (binding constants for full duplexes higher by three orders of magnitude or more), which may be a result of the unique probe density and distribution that is characteristic of the photolithographically patterned arrays. The behavior at 22°C is described well by the predictive equations for

  5. Kelvin probe force microscopy of DNA-capped nanoparticles for single-nucleotide polymorphism detection

    NASA Astrophysics Data System (ADS)

    Lee, Hyungbeen; Lee, Sang Won; Lee, Gyudo; Lee, Wonseok; Lee, Jeong Hoon; Hwang, Kyo Seon; Yang, Jaemoon; Lee, Sang Woo; Yoon, Dae Sung

    2016-07-01

    Kelvin probe force microscopy (KPFM) is a robust toolkit for profiling the surface potential (SP) of biomolecular interactions between DNAs and/or proteins at the single molecule level. However, it has often suffered from background noise and low throughput due to instrumental or environmental constraints, which is regarded as limiting KPFM applications for detection of minute changes in the molecular structures such as single-nucleotide polymorphism (SNP). Here, we show KPFM imaging of DNA-capped nanoparticles (DCNP) that enables SNP detection of the BRCA1 gene owing to sterically well-adjusted DNA-DNA interactions that take place within the confined spaces of DCNP. The average SP values of DCNP interacting with BRCA1 SNP were found to be lower than the DCNP reacting with normal (non-mutant) BRCA1 gene. We also demonstrate that SP characteristics of DCNP with different substrates (e.g., Au, Si, SiO2, and Fe) provide us with a chance to attenuate or augment the SP signal of DCNP without additional enhancement of instrumentation capabilities.Kelvin probe force microscopy (KPFM) is a robust toolkit for profiling the surface potential (SP) of biomolecular interactions between DNAs and/or proteins at the single molecule level. However, it has often suffered from background noise and low throughput due to instrumental or environmental constraints, which is regarded as limiting KPFM applications for detection of minute changes in the molecular structures such as single-nucleotide polymorphism (SNP). Here, we show KPFM imaging of DNA-capped nanoparticles (DCNP) that enables SNP detection of the BRCA1 gene owing to sterically well-adjusted DNA-DNA interactions that take place within the confined spaces of DCNP. The average SP values of DCNP interacting with BRCA1 SNP were found to be lower than the DCNP reacting with normal (non-mutant) BRCA1 gene. We also demonstrate that SP characteristics of DCNP with different substrates (e.g., Au, Si, SiO2, and Fe) provide us with a

  6. Pi30 DNA probe may be useful for the identification of Prevotella intermedia at the species or strain level.

    PubMed

    Shin, Yong Kook; Jeong, Seung-U; Yoo, So Young; Kim, Mi-Kwang; Kim, Hwa-Sook; Kim, Byung-Ock; Kim, Do Kyung; Hwang, Ho-Keel; Kook, Joong-Ki

    2004-01-01

    Recently, we introduced a new method for the rapid screening of bacterial species-or subspecies-specific DNA probes, named the "inverted dot blot hybridization screening method." This method has subsequently been then applied to develop species-or strain-specific DNA probes for Prevotella intermedia and Prevotella nigrescens. In a previous study, the inverted dot blot hybridization data showed that a probe, Pi30, was specific for P. intermedia. In this study, the DNA probe Pi30 was evaluated by Southern blot analysis to determine if it could distinguish P. intermedia from P. nigrescens. The data showed that the probe Pi30 reacted with the genomic DNAs from the reference strains and clinical isolates of both P. intermedia and P. nigrescens, but the size of the signal bands was different. In addition, the probe Pi30 reacted with a 1.4 kbp fragment from the genomic DNAs digested with Pst I of the P. intermedia strains but not with any fragments of P. nigrescens strains. The result indicates that the probe Pi30 could be useful for the identification of P. intermedia by restriction fragment length polymorphism (RFLP) at the species or strain level.

  7. Design and testing of a functional group-specific DNA probe for the study of natural populations of acetogenic bacteria.

    PubMed Central

    Lovell, C R; Hui, Y

    1991-01-01

    The acetogens, although phylogenetically diverse, can be characterized by their possession of the acetyl coenzyme A (acetyl-CoA) pathway for autotrophic CO2 fixation. The gene encoding formyltetrahydrofolate synthetase, a key enzyme of the acetyl-CoA pathway, was previously cloned from the thermophilic acetogen Clostridium thermoaceticum and has now been tested as a group-specific probe for acetogens. Stable hybrids were formed between the probe and single DNA fragments from eight known acetogens representing six genera. A hybrid was also formed between the probe and a DNA fragment from one sulfate reducer known to be capable of both autotrophic CO2 fixation and acetate catabolism. No such hybrid was formed between the probe and DNA from a homoacetate fermenter not known to use the acetyl-CoA pathway, with two known formyltetrahydrofolate synthetase-producing purine fermenters, or with DNA from 27 other species representing 16 genera of organisms that do not use the acetyl-CoA pathway. DNA purified from cells extracted from horse manure was also screened with the acetogen probe. Six hybrids, indicating at least six detectable acetogen "strains," were observed. Images PMID:1768134

  8. DNA Hybridization Probe for Use in Determining Restricted Nodulation among Bradyrhizobium japonicum Serocluster 123 Field Isolates

    PubMed Central

    Sadowsky, Michael J.; Cregan, Perry B.; Keyser, Harold H.

    1990-01-01

    Several soybean plant introduction (PI) genotypes have recently been described which restrict nodulation of Bradyrhizobium japonicum serocluster 123 in an apparently serogroup-specific manner. While PI 371607 restricts nodulation of strains in serogroup 123 and some in serogroup 127, those in serogroup 129 are not restricted. When DNA regions within and around the B. japonicum I-110 common nodulation genes were used as probes to genomic DNA from the serogroup strains USDA 123, USDA 127, and USDA 129, several of the probes differentially hybridized to the nodulation-restricted and -unrestricted strains. One of the gene regions, cloned in plasmid pMJS12, was subsequently shown to hybridize to 4.6-kilobase EcoRI fragments from DNAs from nodulation-restricted strains and to larger fragments in nodulation-unrestricted strains. To determine if the different hybridization patterns could be used to predict nodulation restriction, we hybridized pMJS12 to EcoRI-digested genomic DNAs from uncharacterized serocluster 123 field isolates. Of the 36 strains examined, 15 were found to have single, major, 4.6-kilobase hybridizing EcoRI fragments. When tested for nodulation, 80% (12 of 15) of the strains were correctly predicted to be restricted for nodulation of the PI genotypes. In addition, hybridization patterns obtained with pMJS12 and nodulation phenotypes on PI 371607 indicated that there are at least three types of serogroup 127 strains. Our results suggest that the pMJS12 gene probe may be useful in selecting compatible host-strain combinations and in determining the suitability of field sites for the placement of soybean genotypes containing restrictive nodulation alleles. Images PMID:16348217

  9. The interaction of taurine-salicylaldehyde Schiff base copper(II) complex with DNA and the determination of DNA using the complex as a fluorescence probe

    NASA Astrophysics Data System (ADS)

    Zhang, Xiaoyan; Wang, Yong; Zhang, Qianru; Yang, Zhousheng

    2010-09-01

    The interaction of taurine-salicylaldehyde Schiff base copper(II) (Cu(TSSB) 22+) complex with DNA was explored by using UV-vis, fluorescence spectrophotometry, and voltammetry. In pH 7.4 Tris-HCl buffer solution, the binding constant of the Cu(TSSB) 22+ complex interaction with DNA was 3.49 × 10 4 L mol -1. Moreover, due to the fluorescence enhancing of Cu(TSSB) 22+ complex in the presence of DNA, a method for determination of DNA with Cu(TSSB) 22+ complex as a fluorescence probe was developed. The fluorescence spectra indicated that the maximum excitation and emission wavelength were 389 nm and 512 nm, respectively. Under optimal conditions, the calibration graphs are linear over the range of 0.03-9.03 μg mL -1 for calf thymus DNA (CT-DNA), 0.10-36 μg mL -1 for yeast DNA and 0.01-10.01 μg mL -1 for salmon DNA (SM-DNA), respectively. The corresponding detection limits are 7 ng mL -1 for CT-DNA, 3 ng mL -1 for yeast DNA and 3 ng mL -1 for SM-DNA. Using this method, DNA in synthetic samples was determined with satisfactory results.

  10. Development of Prevotella intermedia-specific PCR primers based on the nucleotide sequences of a DNA probe Pig27.

    PubMed

    Kim, Min Jung; Hwang, Kyung Hwan; Lee, Young-Seok; Park, Jae-Yoon; Kook, Joong-Ki

    2011-03-01

    The aim of this study was to develop Prevotella intermedia-specific PCR primers based on the P. intermedia-specific DNA probe. The P. intermedia-specific DNA probe was screened by inverted dot blot hybridization and confirmed by Southern blot hybridization. The nucleotide sequences of the species-specific DNA probes were determined using a chain termination method. Southern blot analysis showed that the DNA probe, Pig27, detected only the genomic DNA of P. intermedia strains. PCR showed that the PCR primers, Pin-F1/Pin-R1, had species-specificity for P. intermedia. The detection limits of the PCR primer sets were 0.4pg of the purified genomic DNA of P. intermedia ATCC 49046. These results suggest that the PCR primers, Pin-F1/Pin-R1, could be useful in the detection of P. intermedia as well as in the development of a PCR kit in epidemiological studies related to periodontal diseases.

  11. A fluorescent aptasensor using double-stranded DNA/graphene oxide as the indicator probe.

    PubMed

    Xing, Xiao-Jing; Xiao, Wan-Lu; Liu, Xue-Guo; Zhou, Ying; Pang, Dai-Wen; Tang, Hong-Wu

    2016-04-15

    We developed a fluorescent aptasensor based on the making use of double-stranded DNA (dsDNA)/graphene oxide (GO) as the signal probe and the activities of exonuclease I (Exo I). This method takes advantage of the stronger affinity of the aptamer to its target rather than to its complementary sequence (competitor), and the different interaction intensity of dsDNA, mononucleotides with GO. Specifically, in the absence of target, the competitor hybridizes with the aptamer, preventing the digestion of the competitor by Exo I, and thus the formed dsDNA is adsorbed on GO surface, allowing fluorescence quenching. When the target is introduced, the aptamer preferentially binds with its target. Thereby, the corresponding nuclease reaction takes place, and slight fluorescence change is obtained after the introduction of GO due to the weak affinity of the generated mononucleotides to GO. Adenosine (AD) was chosen as a model system and tested in detail. Under the optimized conditions, smaller dissociation constant (Kd, 311.0 µM) and lower detection limit (LOD, 3.1 µM) were obtained in contrast with traditional dye-labeled aptamer/GO based platform (Kd=688.8 µM, LOD=21.2 µM). Satisfying results were still obtained in the evaluation of the specificity and the detection of AD in human serum, making it a promising tool for the diagnosis of AD-relevant diseases. Moreover, we demonstrated the effect of the competitor on the LOD, and the results reveal that the sensitivity could be enhanced by using the rational competitor. The present design not only constructs a label-free aptamer based platform but also extends the application of dsDNA/GO complex in biochemical and biomedical studies.

  12. Single-probe multistate detection of DNA via aggregation-induced emission on a graphene oxide platform.

    PubMed

    Tyagi, Abhishek; Chu, Kin Leung; Abidi, Irfan Haider; Cagang, Aldrine Abenoja; Zhang, Qicheng; Leung, Nelson L C; Zhao, Engui; Tang, Ben Zhong; Luo, Zhengtang

    2017-03-01

    Graphene and graphene oxides (GO), or their reduced forms, have been introduced in a variety of biosensing platforms and have exhibited enhanced performance levels in these forms. We herein report a DNA sensing platform consisting of aggregation-induced emission (AIE) molecules and complementary DNA (comDNA) adsorbed on GO. We experimentally turned the AIE molecule on and off by adjusting its distance, which correlates with DNA structures as shown in our computational results, from the GO sheet, which quenches depending on its distance from the graphene plane. The changes in florescence are reproducible, which demonstrates the probe's ability to identify the binding state of the DNA. Our molecular dynamics simulation results reveal strong π-π interactions between single-strand DNA (ssDNA) and GO, which enable the ssDNA molecule to move closer to the graphene oxide. This reduces the center of mass and binding free energies in the simulation. When hybridized with comDNA, the increased distance, evidenced by the reduced interaction, eliminates the quenching effect and turns on the AIE molecule. Our protocol use of the AIE molecule as a probe thus avoids the complicated steps involved in covalent functionalization and allows the rapid and label-free detection of DNA molecules.

  13. Quantum-dot-labeled DNA probes for fluorescence in situ hybridization (FISH) in the microorganism Escherichia coli.

    PubMed

    Wu, Sheng-Mei; Zhao, Xiang; Zhang, Zhi-Ling; Xie, Hai-Yan; Tian, Zhi-Quan; Peng, Jun; Lu, Zhe-Xue; Pang, Dai-Wen; Xie, Zhi-Xiong

    2006-05-12

    Semiconductor quantum dots (QDs) as a kind of nonisotopic biological labeling material have many unique fluorescent properties relative to conventional organic dyes and fluorescent proteins, such as composition- and size-dependent absorption and emission, a broad absorption spectrum, photostability, and single-dot sensitivity. These properties make them a promising stable and sensitive label, which can be used for long-term fluorescent tracking and subcellular location of genes and proteins. Here, a simple approach for the construction of QD-labeled DNA probes was developed by attaching thiol-ssDNA to QDs via a metal-thiol bond. The as-prepared QD-labeled DNA probes had high dispersivity, bioactivity, and specificity for hybridization. Based on such a kind of probe with a sequence complementary to multiple clone sites in plasmid pUC18, fluorescence in situ hybridization of the tiny bacterium Escherichia coli has been realized for the first time.

  14. Padlock probe-mediated qRT-PCR for DNA computing answer determination

    PubMed Central

    Xiong, Fusheng; Frasch, Wayne D.

    2011-01-01

    Padlock probe-mediated quantitative real time PCR (PLP-qRT-PCR) was adapted to quantify the abundance of sequential 10mer DNA sequences for use in DNA computing to identify optimal answers of traveling salesman problems. The protocol involves: (i) hybridization of a linear PLP with a target DNA sequence; (ii) PLP circularization through enzymatic ligation; and (iii) qRT-PCR amplification of the circularized PLP after removal of non-circularized templates. The linear PLP was designed to consist of two 10-mer sequence-detection arms at the 5′ and 3′ ends separated by a core sequence composed of universal PCR primers, and a qRT-PCR reporter binding site. Circularization of each PLP molecule is dependent upon hybridization with target sequence and high-fidelity ligation. Thus, the number of PLP circularized is determined by the abundance of target in solution. The amplification efficiency of the PLP was 98.7% within a 0.2 pg–20 ng linear detection range between thermal cycle threshold (Ct value) and target content. The Ct values derived from multiplex qRT-PCR upon three targets did not differ significantly from those obtained with singleplex assays. The protocol provides a highly sensitive and efficient means for the simultaneous quantification of multiple short nucleic acid sequences that has a wide range of applications in biotechnology. PMID:21691417

  15. Probing the active site tightness of DNA polymerase in subangstrom increments.

    PubMed

    Kim, Tae Woo; Delaney, James C; Essigmann, John M; Kool, Eric T

    2005-11-01

    We describe the use of a series of gradually expanded thymine nucleobase analogs in probing steric effects in DNA polymerase efficiency and fidelity. In these nonpolar compounds, the base size was increased incrementally over a 1.0-A range by use of variably sized atoms (H, F, Cl, Br, and I) to replace the oxygen molecules of thymine. Kinetics studies with DNA Pol I (Klenow fragment, exonuclease-deficient) in vitro showed that replication efficiency opposite adenine increased through the series, reaching a peak at the chlorinated compound. Efficiency then dropped markedly as a steric tightness limit was apparently reached. Importantly, fidelity also followed this trend, with the fidelity maximum at dichlorotoluene, the largest compound that fits without apparent repulsion. The fidelity at this point approached that of wild-type thymine. Surprisingly, the maximum fidelity and efficiency was found at a base pair size significantly larger than the natural size. Parallel bypass and mutagenesis experiments were then carried out in vivo with a bacterial assay for replication. The cellular results were virtually the same as those seen in solution. The results provide direct evidence for the importance of a tight steric fit on DNA replication fidelity. In addition, the results suggest that even high-fidelity replicative enzymes have more steric room than necessary, possibly to allow for an evolutionarily advantageous mutation rate.

  16. Novel Phenanthrene-Degrading Bacteria Identified by DNA-Stable Isotope Probing.

    PubMed

    Jiang, Longfei; Song, Mengke; Luo, Chunling; Zhang, Dayi; Zhang, Gan

    2015-01-01

    Microorganisms responsible for the degradation of phenanthrene in a clean forest soil sample were identified by DNA-based stable isotope probing (SIP). The soil was artificially amended with either 12C- or 13C-labeled phenanthrene, and soil DNA was extracted on days 3, 6 and 9. Terminal restriction fragment length polymorphism (TRFLP) results revealed that the fragments of 219- and 241-bp in HaeIII digests were distributed throughout the gradient profile at three different sampling time points, and both fragments were more dominant in the heavy fractions of the samples exposed to the 13C-labeled contaminant. 16S rRNA sequencing of the 13C-enriched fraction suggested that Acidobacterium spp. within the class Acidobacteria, and Collimonas spp. within the class Betaproteobacteria, were directly involved in the uptake and degradation of phenanthrene at different times. To our knowledge, this is the first report that the genus Collimonas has the ability to degrade PAHs. Two PAH-RHDα genes were identified in 13C-labeled DNA. However, isolation of pure cultures indicated that strains of Staphylococcus sp. PHE-3, Pseudomonas sp. PHE-1, and Pseudomonas sp. PHE-2 in the soil had high phenanthrene-degrading ability. This emphasizes the role of a culture-independent method in the functional understanding of microbial communities in situ.

  17. Detection of circovirus infection in pigeons by in situ hybridization using cloned DNA probes.

    PubMed

    Smyth, J A; Weston, J; Moffett, D A; Todd, D

    2001-11-01

    Degenerate primers were designed based on known sequence information for the circoviruses psittacine beak and feather disease virus and porcine circovirus and applied by polymerase chain reaction (PCR) to known virus-infected bursa of Fabricius (BF) from a pigeon. A 548-bp DNA fragment was amplified and shown to be specific to a novel circovirus, named pigeon circovirus (PiCV), and was used to produce sensitive and specific probes for detection of circovirus DNA by in situ hybridization (ISH). Using ISH on BF from 107 pigeons submitted for necropsy, infection was detected in 89%, compared with a histologic detection rate of 66%. Using the ISH technique, infected cells were also found in liver, kidney, trachea, lung, brain, crop, intestine, spleen, bone marrow, and heart of some birds. Large quantities of DNA were present in some of these tissues, and in the absence of BF, liver in particular is identified as a potentially useful organ to examine for presence of PiCV. This high prevalence of infection in diseased birds is noteworthy, emphasizing the need for studies to determine the precise role of this virus as a disease-producing agent.

  18. Identification of the autotrophic denitrifying community in nitrate removal reactors by DNA-stable isotope probing.

    PubMed

    Xing, Wei; Li, Jinlong; Cong, Yuan; Gao, Wei; Jia, Zhongjun; Li, Desheng

    2017-04-01

    Autotrophic denitrification has attracted increasing attention for wastewater with insufficient organic carbon sources. Nevertheless, in situ identification of autotrophic denitrifying communities in reactors remains challenging. Here, a process combining micro-electrolysis and autotrophic denitrification with high nitrate removal efficiency was presented. Two batch reactors were fed organic-free nitrate influent, with H(13)CO3(-) and H(12)CO3(-) as inorganic carbon sources. DNA-based stable-isotope probing (DNA-SIP) was used to obtain molecular evidence for autotrophic denitrifying communities. The results showed that the nirS gene was strongly labeled by H(13)CO3(-), demonstrating that the inorganic carbon source was assimilated by autotrophic denitrifiers. High-throughput sequencing and clone library analysis identified Thiobacillus-like bacteria as the most dominant autotrophic denitrifiers. However, 88% of nirS genes cloned from the (13)C-labeled "heavy" DNA fraction showed low similarity with all culturable denitrifiers. These findings provided functional and taxonomical identification of autotrophic denitrifying communities, facilitating application of autotrophic denitrification process for wastewater treatment.

  19. Novel Phenanthrene-Degrading Bacteria Identified by DNA-Stable Isotope Probing

    PubMed Central

    Luo, Chunling; Zhang, Dayi; Zhang, Gan

    2015-01-01

    Microorganisms responsible for the degradation of phenanthrene in a clean forest soil sample were identified by DNA-based stable isotope probing (SIP). The soil was artificially amended with either 12C- or 13C-labeled phenanthrene, and soil DNA was extracted on days 3, 6 and 9. Terminal restriction fragment length polymorphism (TRFLP) results revealed that the fragments of 219- and 241-bp in HaeIII digests were distributed throughout the gradient profile at three different sampling time points, and both fragments were more dominant in the heavy fractions of the samples exposed to the 13C-labeled contaminant. 16S rRNA sequencing of the 13C-enriched fraction suggested that Acidobacterium spp. within the class Acidobacteria, and Collimonas spp. within the class Betaproteobacteria, were directly involved in the uptake and degradation of phenanthrene at different times. To our knowledge, this is the first report that the genus Collimonas has the ability to degrade PAHs. Two PAH-RHDα genes were identified in 13C-labeled DNA. However, isolation of pure cultures indicated that strains of Staphylococcus sp. PHE-3, Pseudomonas sp. PHE-1, and Pseudomonas sp. PHE-2 in the soil had high phenanthrene-degrading ability. This emphasizes the role of a culture-independent method in the functional understanding of microbial communities in situ. PMID:26098417

  20. Interactions of L-Arg with calf thymus DNA using neutral red dye as a fluorescence probe

    NASA Astrophysics Data System (ADS)

    Lin, Jing; Liu, Rutao; Gao, Canzhu

    2012-11-01

    The interaction between L-Arg and calf thymus DNA (ctDNA) in sodium acetate-acetic acid buffer (pH = 4) was investigated with the use of neutral red (NR) dye as a spectral probe coupled with UV-vis absorption, fluorescence, and circular dichroism (CD) spectroscopy technique. The UV absorption spectroscopy indicated that L-Arg interacted with ctDNA via electrostatic force and the fluorescence enhancing of the DNA-NR system verified the electrostatic interaction. In addition, detectable changes in the CD spectrum of ctDNA in the presence of L-Arg indicated conformational changes in the DNA double helix after interaction with the drug. Docking studies were found to corroborate the experimental results. All these results prove that this drug interacts with ctDNA via an electrostatic binding mode.

  1. Interactions of L-Arg with calf thymus DNA using neutral red dye as a fluorescence probe.

    PubMed

    Lin, Jing; Liu, Rutao; Gao, Canzhu

    2012-11-01

    The interaction between l-Arg and calf thymus DNA (ctDNA) in sodium acetate-acetic acid buffer (pH=4) was investigated with the use of neutral red (NR) dye as a spectral probe coupled with UV-vis absorption, fluorescence, and circular dichroism (CD) spectroscopy technique. The UV absorption spectroscopy indicated that l-Arg interacted with ctDNA via electrostatic force and the fluorescence enhancing of the DNA-NR system verified the electrostatic interaction. In addition, detectable changes in the CD spectrum of ctDNA in the presence of l-Arg indicated conformational changes in the DNA double helix after interaction with the drug. Docking studies were found to corroborate the experimental results. All these results prove that this drug interacts with ctDNA via an electrostatic binding mode.

  2. Surface plasmon resonance technique for directly probing the interaction of DNA and graphene oxide and ultra-sensitive biosensing.

    PubMed

    Xue, Tianyu; Cui, Xiaoqiang; Guan, Weiming; Wang, Qiyu; Liu, Chang; Wang, Haitao; Qi, Kun; Singh, D J; Zheng, Weitao

    2014-08-15

    The binding of DNA with graphene oxide (GO) is important for applications in disease diagnosis, genetic screening, and drug discovery. The standard assay methods are mainly limited to indirect observation via fluorescence labeling. Here we report the use of surface plasmon resonance for direct sensing of DNA/GO binding. We show that this can be used for ultra-sensitive detection of single-stranded DNA (ssDNA). Furthermore, the results provide a more direct probe of DNA/GO binding abilities and confirm that hydrogen bonding plays a key role in the interaction between GO and ssDNA. This enables to a novel biosensor for highly sensitive and selective detection of ssDNA based on indirect competitive inhibition assay (ICIA). We report development of such a sensor with a linear dynamic range of 10(-14)-10(-6)M, a detection limit of 10fM and a high level of stability during repeated regeneration.

  3. Comparison of peroxidase-labeled DNA probes with radioactive RNA probes for detection of human papillomaviruses by in situ hybridization in paraffin sections

    SciTech Connect

    Park, J.S.; Kurman, R.J.; Kessis, T.D.; Shah, K.V. )

    1991-01-01

    A study comparing in situ hybridization using nonradioactive DNA probes directly conjugated with horseradish peroxidase (HRP), and {sup 35}S-labeled antisense RNA probes for human papillomavirus (HPV) types 6/11, 16, and 18 was performed on formalin-fixed, paraffin-embedded tissue from 34 lesions of the cervix and vulva. These lesions included exophytic condylomas and intraepithelial and invasive neoplasms. HPV 6/11 was detected in two of four condylomata acuminata by both in situ techniques. HPV 16 was detected in 13 of 30 cases of intraepithelial and invasive neoplasms by both methods. Discordance between the two methods occurred in two instances. The radiolabeled probe but not the HRP probe detected HPV 16 in one case of cervical intraepithelial neoplasia (CIN 3), whereas the converse occurred in one case of vulvar intraepithelial neoplasia (VIN 3). HPV 18 was not detected in any of the specimens by either method. This study demonstrates that nonradioactive HRP-labeled probes for the detection of specific HPV types are as sensitive as the more laborious and potentially hazardous radioactive probes.

  4. Rapid sex determination on buccal smears using DNA probes and fluorescence in situ hybridization

    SciTech Connect

    Giraldez, R.A.; Harris, C.

    1994-09-01

    Hybridization of dual-labeled DNA probes for the repetitive sequences on the X and Y chromosomes allows a fast, non-invasive, more reliable method for sex determination that current cytogenetic Barr body and Y chromatin assays. Scrapes of squamous epithelial cells were collected from the oral cavity of 14 subjects (5{male}, 9{female}) and smeared onto silanized slides. The smears were allowed to air dry. Samples were blinded and then fixed in 50% methanol/50% glacial acetic acid for 10 minutes, and allowed to dry. The slides were incubated in a pretreatment solution containing 30% sodium bisulfite at 45{degrees}C for 10 minutes. They were rinsed in 2XSSC pH 7.0 and then dehydrated through a series of 70%, 85%, and 100% ethanols at room temperature and allowed to air dry. A probe mixture (30 {mu}L containing 10 ng/{mu}L biotin-labeled DXZ1 and digoxigenin-labeled DYZ1/DYZ3 in 70% Formamide/2XSSC) was aliquoted onto each slide, coverslipped, and sealed with rubber cement. Probe and target DNA were simultaneously denatured at 72{degrees}C on a slide warmer for 6 minutes. Probe was allowed to hybridize overnight in a humidified chamber at 37{degrees}C. Slides were postwashed at 72{degrees}C in 0.5xSSC pH 7.0 for 5 minutes, then soaked at room temperature 1XPBD for 2 minutes, and detected with rhodamine/anti-digoxigenin-FITC/avidin for 15 minutes at 37{degrees}C. Slides were soaked 3X in 1XPBD and then counterstained with 15 {mu}L 0.05 {mu}g/mL DAP1/Antifade. 200 nuclei were scored for the presence of one green (X), two green (XX), one green and one red (XY), or a single red (Y) signal, using a fluorescent microscope equipped with a triple band pass filter. Greater than 90% of the hybridized nuclei from each of the 14 cases studied conformed to the sex chromosome pattern. The modal number in 9 cases showed two green signals (XX), and a green and a red signal (XY) in the other 5 cases; this was in complete agreement with the cytogenetic results.

  5. Detection of beer spoilage bacteria Pectinatus and Megasphaera with acridinium ester labelled DNA probes using a hybridisation protection assay.

    PubMed

    Paradh, A D; Hill, A E; Mitchell, W J

    2014-01-01

    DNA probes specific for rRNA of selected target species were utilised for the detection of beer spoilage bacteria of the genera Pectinatus and Megasphaera using a hybridisation protection assay (HPA). All the probes were modified during synthesis by addition of an amino linker arm at the 5' end or were internally modified by inserting an amine modified thymidine base. Synthesised probes then were labelled with acridinium ester (AE) and purified using reverse phase HPLC. The internally AE labelled probes were able to detect target RNA within the range of 0.016-0.0032pmol. All the designed probes showed high specificity towards target RNA and could detect bacterial contamination within the range of ca. 5×10(2)1×10(3) CFU using the HPA. The developed assay was also compatible with MRS, NBB and SMMP beer enrichment media, routinely used in brewing laboratories.

  6. Potential use of buccal smears for rapid diagnosis of autosomal trisomy or chromosomal sex in newborn infants using DNA probes

    SciTech Connect

    Harris, C.; Clark, K.; Lazarski, K.; Wilkerson, C.; Meisner, L. |

    1994-12-01

    Buccal smears from 3 women and 1 man were probed with alpha satellite DNA probes for chromosomes 8, 18, X, and Y. Buccal smears were also collected from an adolescent phenotypic female with uterine agenesis, as well as from newborn infants with suspected trisomy 18 and trisomy 21. The clinical cases were confirmed with conventional cytogenetic studies of peripheral lymphocytes. Overall probe efficiency at detecting expected chromosome number in interphase cells was found to be 71% {+-} 6.8%. Higher than expected n-1 signal numbers may be due to karyopyknotic intermediate epithelial cells present in all collected samples. Overall probe efficiency was found to be consistent using alpha satellite and cosmid probes, both of which accurately reflected the modal copy number of the target chromosomes. False trisomy was less than 1%. This study suggests DNA probes can be used in buccal smears for rapid diagnosis of trisomies and chromosomal sex in newborns, but because of high rates of false hydropoploid signals, probed buccal smear specimens may not be accurate at diagnosing mosaicism. 9 refs., 2 figs., 1 tab.

  7. The Preparation of Magnetic Silica Nanospheres and Incorporation of CdSe/ZnS Quantum Dots-DNA Probe.

    PubMed

    Do, Youngjin; Kim, Jongsung

    2016-03-01

    Silica nanospheres containing magnetic particles were prepared, and CdSe/ZnS QDs functionalized with carboxyl group were incorporated into the silica nanospheres by EDC/NHS coupling reaction. The silica nanospheres were prepared by a co-precipitation of ferrous and ferric solutions followed by the sol-gel reaction of TEOS (tetraethoxysilane) and APTES (3-aminopropyltriethoxysilane) using base catalyst. The size of magnetic silica nanospheres was confirmed by Transmission electron microscope (TEM). Thiol group modified single stranded oligonucleotides were immobilized on the surface of QDs and fluorescence quenching by intercalation dye (TOTO-3) after hybridization with target oligonucleotide was observed. The fluorescence from QDs could be quenched by intercalating dye (TOTO-3) after hybridization of target DNA to probe DNA. This shows that the magnetic silica-QD-DNA probe can be used to detect specific DNA.

  8. Chemical Methylation of RNA and DNA Viral Genomes as a Probe of In Situ Structure

    PubMed Central

    Yamakawa, Minoru; Shatkin, Aaron J.; Furuichi, Yasuhiro

    1981-01-01

    We used [methyl-3H] dimethyl sulfate to probe the genome structures of several RNA and DNA viruses. We compared sites of modification in nucleic acids that were methylated chemically before and after extraction from purified virions. With both single-stranded and double-stranded substrates alkylation occurred mainly at the N7 position of guanine. However, adenine N1 atoms were differentially accessible in single-stranded RNA and DNA. For example, the ratios of 1-methyladenosine to 7-methylguanosine for reovirus mRNA and deproteinized genome RNA were 0.43 and 0.03, respectively. Members of the Reoviridae methylated in situ yielded RNAs with ratios of 0.04 to 0.08, indicating that the intravirion genomes were double stranded. We obtained ratios of 0.26 and 0.35 for the RNAs of dimethyl sulfate-treated brome mosaic and avian sarcoma virions, respectively, which was consistent with partial protection of adenine N1 sites by structural proteins or genome conformation or both. The ratios of 1-methyladenosine to 7-methylguanosine for vaccinia virus DNAs methylated in situ (0.10) and after phenol extraction (0.14) were less than the ratios for φX174 and M13 DNAs (0.39 to 0.64) but considerably greater than the ratio observed with adenovirus DNA (0.002 to 0.02). The presence of a single-stranded region(s) in the vaccinia virus genome was confirmed by S1 nuclease digestion of [methyl-3H] DNA; the released radiolabeled fraction had a ratio of 0.41, compared with 0.025 for the residual duplex DNA. In addition to the structure-dependent accessibility of adenine N1, methylation of adenine N3 was severalfold lower in the intravirion genomes of vaccinia virus, φX174, and adenovirus than in the corresponding extracted DNAs. Chemical methylation of virions and subviral particles should be useful for in situ analyses of specific regions of RNA and DNA genomes, such as the sites of protein binding during virus maturation. PMID:6172596

  9. Oligonucleotide probes containing pyrimidine analogs reveal diminished hydrogen bonding capacity of the DNA adduct O⁶-methyl-G in DNA duplexes.

    PubMed

    Angelov, Todor; Dahlmann, Heidi A; Sturla, Shana J

    2013-10-15

    Oligonucleotide hybridization probes containing nucleoside analogs offer a potential strategy for binding specific DNA sequences that bear pro-mutagenic O(6)-G alkylation adducts. To optimize O(6)-Me-G-targeting probes, an understanding of how base pairs with O(6)-Me-G are stabilized is needed. In this study, we compared the ability of O(6)-Me-G and G to hydrogen bond with three pyrimidine-like nucleobases (Z, 4-thio-U, and 3-deaza-C) bearing varied hydrogen bond donor and acceptor groups. We found that duplexes containing the pyrimidine analog nucleoside:G pairs were more thermodynamically stable than those containing pyrimidine analog nucleoside:O(6)-alkyl-G pairs. Thus, hydrogen bonding alone was not sufficient to impart selectivity to probes that target O(6)-G alkylation adducts in DNA.

  10. Probing the Dynamic Interaction between Damaged DNA and a Cellular Responsive Protein Using a Piezoelectric Mass Biosensor.

    PubMed

    Jin, Yulong; Xie, Yunfeng; Wu, Kui; Huang, Yanyan; Wang, Fuyi; Zhao, Rui

    2017-03-15

    The binding events between damaged DNA and recognition biomolecules are of great interest for understanding the activity of DNA-damaging drugs and the related DNA repair networks. Herein, a simple and sensitive sensor system was tailored for real-time probing of the dynamic molecular recognition between cisplatin-damaged-DNA (cisPt-DNA) and a cellular responsive protein, high-mobility-group box 1 (HMGB1). By integration of flow injection analysis (FIA) with quartz crystal microbalance (QCM), the interaction time-course of cisPt-DNA and HMGB1 domain A (HMGB1a) was investigated. The highly specific sensing interface was carefully designed and fabricated using cisPt-DNA as recognition element. A hybrid self-assembled monolayer consisting of cysteamine and mercaptohexanol was introduced to resist nonspecific adsorption. The calculated kinetic parameters (kass and kdiss) and the dissociation constant (KD) demonstrated the rapid recognition and tight binding of HMGB1a toward cisPt-DNA. Molecular docking was employed to simulate the complex formed by cisPt-DNA and HMGB1a. The tight binding of such a DNA-damage responsive complex is appealing for the downstream molecular recognition event related to the resistance to DNA repair. This continuous-flow QCM biosensor is an ideal tool for studying specific interactions between drug-damaged-DNAs and their recognition proteins in a physiological-relevant environment, and will provide a potential sensor platform for rapid screening and evaluating metal anticancer drugs.

  11. Application of GelGreen™ in Cesium Chloride Density Gradients for DNA-Stable Isotope Probing Experiments

    PubMed Central

    Pan, Kailing; Li, Hongyu; Fan, Xiaoyan; Sun, Lixin; Zhang, Shujun; Gao, Yongqing

    2017-01-01

    In this study, GelGreen™ was investigated as a replacement for SYBR® Safe to stain DNA in cesium chloride (CsCl) density gradients for DNA-stable isotope probing (SIP) experiments. Using environmental DNA, the usage of GelGreen™ was optimized for sensitivity compared to SYBR® Safe, its optimal concentration, detection limit for environmental DNA and its application in environmental DNA-SIP assay. Results showed that GelGreen™ was more sensitive than SYBR® Safe, while the optimal dosage (15X concentration) needed was approximately one-third of SYBR® Safe, suggesting that its sensitivity was three times more superior than SYBR® Safe. At these optimal parameters, the detection limit of GelGreen™-stained environmental DNA was as low as 0.2 μg, but the usage of 0.5 μg environmental DNA was recommended to produce a more consistent DNA band. In addition, a modified needle extraction procedure was developed to withdraw DNA effectively by fractionating CsCl density gradients into four or five fractions. The successful application of GelGreen™ staining with 13C-labeled DNA from enriched activated sludge suggests that this stain was an excellent alternative of SYBR® Safe in CsCl density gradients for DNA-SIP assays. PMID:28056074

  12. GENETIC DIVERSITY OF TYPHA LATIFOLIA (TYPHACEAE) AND THE IMPACT OF POLLUTANTS EXAMINED WITH TANDEM-REPETITIVE DNA PROBES

    EPA Science Inventory

    Genetic diversity at variable-number-tandem-repeat (VNTR) loci was examined in the common cattail, Typha latifolia (Typhaceae), using three synthetic DNA probes composed of tandemly repeated "core" sequences (GACA, GATA, and GCAC). The principal objectives of this investigation w...

  13. Probing heterobivalent binding to the endocytic AP-2 adaptor complex by DNA-based spatial screening.

    PubMed

    Diezmann, F; von Kleist, L; Haucke, V; Seitz, O

    2015-08-07

    The double helical DNA scaffold offers a unique set of properties, which are particularly useful for studies of multivalency in biomolecular interactions: (i) multivalent ligand displays can be formed upon nucleic acid hybridization in a self-assembly process, which facilitates spatial screening (ii) valency and spatial arrangement of the ligand display can be precisely controlled and (iii) the flexibility of the ligand display can be adjusted by integrating nick sites and unpaired template regions. Herein we describe the use of DNA-based spatial screening for the characterization of the adaptor complex 2 (AP-2), a central interaction hub within the endocytic protein network in clathrin-mediated endocytosis. AP-2 is comprised of a core domain and two, so-called appendage domains, the α- and the β2-ear, which associate with cytoplasmatic proteins required for the formation or maturation of clathrin/AP-2 coated pits. Each appendage domain has two binding grooves which recognize distinct peptide motives with micromolar affinity. This provides opportunities for enhanced interactions with protein molecules that contain two (or more) different peptide motives. To determine whether a particular, spatial arrangement of binding motifs is required for high affinity binding we probed the distance-affinity relationships by means of DNA-programmed spatial screening with self-assembled peptide-DNA complexes. By using trimolecular and tetramolecular assemblies two different peptides were positioned in 2-22 nucleotide distance. The binding data obtained with both recombinant protein in well-defined buffer systems and native AP-2 in brain extract suggests that the two binding sites of the AP-2 α-appendage can cooperate to provide up to 40-fold enhancement of affinity compared to the monovalent interaction. The distance between the two recognized peptide motives was less important provided that the DNA duplex segments were connected by flexible, single strand segments. By

  14. Multicolor Gold-Silver Nano-Mushrooms as Ready-to-Use SERS Probes for Ultrasensitive and Multiplex DNA/miRNA Detection.

    PubMed

    Su, Jing; Wang, Dongfang; Nörbel, Lena; Shen, Jianlei; Zhao, Zhihan; Dou, Yanzhi; Peng, Tianhuan; Shi, Jiye; Mathur, Sanjay; Fan, Chunhai; Song, Shiping

    2017-02-21

    Uniform silver-containing metal nanostructures with strong and stable surface-enhanced Raman scattering (SERS) signals hold great promise for developing ultrasensitive probes for biodetection. Nevertheless, the direct synthesis of such ready-to-use nanoprobes remains extremely challenging. Herein we report a DNA-mediated gold-silver nanomushroom with interior nanogaps directly synthesized and used for multiplex and simultaneous SERS detection of various DNA and RNA targets. The DNA involved in the nanostructures can act as not only gap DNA (mediated DNA) but also probe DNA (hybridized DNA), and DNA's involvement enables the nanostructures to have the inherent ability to recognize DNA and RNA targets. Importantly, we were the first to establish a new method for the generation of multicolor SERS probes using two different strategies. First Raman-labeled alkanethiol probe DNA was assembled on gold nanoparticles, and second, thiol-containing Raman reporters were coassembled with the probe DNA. The ready-to-use probes also give great potential to develop ultrasensitive detection methods for various biological molecules.

  15. FISH analysis of the arrangement of chromosomes in interphase nuclei using telomeric, centromeric, and DNA painting probes

    NASA Astrophysics Data System (ADS)

    Monajembashi, Shamci; Schmitt, Eberhard; Dittmar, Heike; Greulich, Karl-Otto

    1999-01-01

    Fluorescence in situ hybridization is used to study the arrangement of chromosomes in interphase nuclei of unsynchronized human lymphocytes. DNA probes specific for telomeric DNA, centromeric (alpha) -satellite DNA and whole chromosomes 2, 7, 9 and X are employed. It is demonstrated that the shape of the chromosome territories is variable in cycling cells, for example, close to the metaphase chromosome homologues are arranged pairwise. Furthermore, the relative arrangement of chromosome homologues to each other is not spatially defined. Also, the relative orientation of centromeres and telomeres within a chromosome domain is variable.

  16. Ultrafast Hydration Dynamics Probed by Tryptophan at Protein Surface and Protein-DNA Interface

    NASA Astrophysics Data System (ADS)

    Qin, Yangzhong

    As we all live in a special water planet Earth, the significance of water to life has been universally recognized. The reason why water is so important to life has intrigued many researchers. This dissertation will focus on the ultrafast dynamics of protein surface water and protein-DNA interfacial water which have direct importance to the protein structure and function. Using tryptophan as an intrinsic fluorescence probe, combined with site-directed mutagenesis and ultrafast fluorescence up-conversion spectroscopy, we can achieve single residue spatial resolution and femtosecond temporal resolution. We can also precisely determine the local hydration water dynamics by monitoring the Stokes shift of tryptophan one at a time. Previously, the protein surface hydration has been extensively studied by our group. In this thesis, we will provide more details on the methods we are using to extract the hydration dynamics, and also validate our methods from both experimental and theoretical perspectives. To further interrogate the interfacial water hydration dynamics relative to the protein surface hydration, we studied two DNA polymerases: DNA Polymerase IV (Dpo4) and DNA Polymerase Beta (Pol beta). Both proteins show typical surface hydration pattern with three distinct time components including: (i) the ultrafast sub-picosecond component reflects the bulk type water motion; (ii) a few picoseconds component shows the inner water relaxation mainly corresponding to the local libration and reorientation; (iii) the tens to hundred picoseconds component represents the water-protein coupled motion involving the whole water network reorganization. Dpo4, a loosely DNA binding protein, exhibits very flexible interfacial water which resembles its surface water yet with a significantly reduced ultrafast component. Such dynamic interfacial water not only maintains interfacial flexibility, but also contributes to the low fidelity of the protein. In contrast to the Dpo4, pol beta

  17. Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA using boric acid-functionalized nano-microsphere fluorescent probes.

    PubMed

    Chen, Hua-Yan; Wei, Jing-Ru; Pan, Jiong-Xiu; Zhang, Wei; Dang, Fu-Quan; Zhang, Zhi-Qi; Zhang, Jing

    2017-05-15

    5-hydroxymethylcytosine (5hmC) is the sixth base of DNA. It is involved in active DNA demethylation and can be a marker of diseases such as cancer. In this study, we developed a simple and sensitive 2-(4-boronophenyl)quinoline-4-carboxylic acid modified poly (glycidyl methacrylate (PBAQA-PGMA) fluorescent probe to detect the 5hmC content of genomic DNA based on T4 β-glucosyltransferase-catalyzed glucosylation of 5hmC. The fluorescence-enhanced intensity recorded from the DNA sample was proportional to its 5-hydroxymethylcytosine content and could be quantified by fluorescence spectrophotometry. The developed probe showed good detection sensitivity and selectivity and a good linear relationship between the fluorescence intensity and the concentration of 5 hmC within a 0-100nM range. Compared with other fluorescence detection methods, this method not only could determine trace amounts of 5 hmC from genomic DNA but also could eliminate the interference of fluorescent dyes and the need for purification. It also could avoid multiple labeling. Because the PBAQA-PGMA probe could enrich the content of glycosyl-5-hydroxymethyl-2-deoxycytidine from a complex ground substance, it will broaden the linear detection range and improve sensitivity. The limit of detection was calculated to be 0.167nM after enrichment. Furthermore, the method was successfully used to detect 5-hydroxymethylcytosine from mouse tissues.

  18. Detection of human papillomavirus type 6/11 DNA in conjunctival papillomas by in situ hybridization with radioactive probes

    SciTech Connect

    McDonnell, P.J.; McDonnell, J.M.; Kessis, T.; Green, W.R.; Shah, K.V.

    1987-11-01

    Twenty-three conjunctival papillomas and 28 conjunctival dysplasias were examined for human papillomavirus (HPV)-DNA sequences by in situ hybridization with nick-translated /sup 35/S-labeled HPV probes. Adjacent paraffin sections were hybridized with HPV type 2, 6, 16, and 18 probes at Tm - 17 degrees C. Fifteen tissues, all papillomas, displayed positive hybridization with the HPV-6 probe. Infection with HPV-6 (or the closely related HPV-11) appeared to be responsible for most of the conjunctival papillomas of children and young adults. The presence of genital tract HPV-6 in these lesions suggests that some of the infections were acquired during passage through an infected birth canal. The lack of hybridization in adult conjunctival dysplasias indicates either that HPVs are not associated with this condition or that the probes and the technique utilized were not adequate for demonstration of this association.

  19. Salmonella detection using 16S ribosomal DNA/RNA probe-gold nanoparticles and lateral flow immunoassay.

    PubMed

    Liu, Cheng-Che; Yeung, Chun-Yan; Chen, Po-Hao; Yeh, Ming-Kung; Hou, Shao-Yi

    2013-12-01

    An ultrasensitive, simple, and fast lateral flow immunoassay for Salmonella detection using gold nanoparticles conjugated with a DNA probe, which is complementary to the 16S ribosomal RNA and DNA of Salmonella, has been developed. The detection limit is 5 fmol for the synthetic single-stranded DNA. For the Salmonella cultured samples, the nucleic acids from 10(7) bacteria were rapidly detected in 30 min. After silver enhancement, the detection limit was as low as 10(4) cells which is lower than 10(5) bacteria cells, the human infective dose of food-borne Salmonella. Furthermore, the probes used in this study are specific to Salmonella compared to several other Enterobacteriaceae. This approach would be a useful tool for microbial detection regarding food safety or clinical diagnosis. It is also suitable for large-scale screening in developing countries because it is low-cost, sensitive, specific and convenient.

  20. Thymine dimer formation as a probe of the path of DNA in and between nucleosomes in intact chromatin

    SciTech Connect

    Pehrson, J.R. )

    1989-12-01

    Photo-induced thymine dimer formation was used to probe nucleosome structure in nuclei. The distribution of thymine dimers in the nucleosome and recent studies of the structure of thymine dimer-containing DNA suggest that the rate of thymine dimer formation is affected by the direction and degree of DNA bending. This premise was used to construct a model of the path of DNA in the nucleosome, which has the following features. (i) There are four regions of sharp bending, two which have been seen previously by x-ray crystallography of the core particle. (ii) The DNA in H1-containing nucleosomes deviates from its superhelical path near the midpoint; this is not seen with H1-stripped chromatin. (iii) The internucleosomal (linker) DNA appears to be relatively straight.

  1. Real-time colorimetric detection of target DNA using isothermal target and signaling probe amplification and gold nanoparticle cross-linking assay.

    PubMed

    Jung, Cheulhee; Chung, Ji Won; Kim, Un Ok; Kim, Min Hwan; Park, Hyun Gyu

    2011-01-15

    We describe a facile gold nanoparticle (AuNP)-mediated colorimetric method for real-time detection of target DNA in conjugation with our unique isothermal target and signaling probe amplification (iTPA) method, comprising novel ICA (isothermal chain amplification) and CPT (cycling probe technology). Under isothermal conditions, the iTPA simultaneously amplifies the target and signaling probe through two displacement events induced by a combination of four specially designed primers, the strand displacement activity of DNA polymerase, and the RNA degrading activity of RNase H. The resulting target amplicons are hybridized with gold nanoparticle cross-linking assay (GCA) probes having a DNA-RNA-DNA chimeric form followed by RNA cleavage by RNase H in the CPT step. The intact GCA probes were designed to cross-link two sets of DNA-AuNPs conjugates in the absence of target DNA, inducing aggregation (blue color) of AuNPs. On the contrary, the presence of target DNA leads to cleavage of the GCA probes in proportion to the amount of amplified target DNA and the solution remains red in color without aggregation of AuNPs. Relying on this strategy, 10(2) copies of target Chlamydia trachomatis plasmid were successfully detected in a colorimetric manner. Importantly, all the procedures employed up to the final detection of the target DNA were performed under isothermal conditions without requiring any detection instruments. Therefore, this strategy would greatly benefit convenient, real-time monitoring technology of target DNA under restricted environments.

  2. Surface modification of poly(dimethylsiloxane) (PDMS) microchannels with DNA capture-probes for potential use in microfluidic DNA analysis systems

    NASA Astrophysics Data System (ADS)

    Khodakov, Dmitriy A.; Thredgold, Leigh D.; Lenehan, Claire E.; Andersson, Gunther A.; Kobus, Hilton; Ellis, Amanda V.

    2011-12-01

    Poly(dimethylsiloxane) (PDMS) is an elastomeric material used for microfluidic devices and is especially suited to medical and forensic applications. This is due to its relatively low cost, ease of fabrication, excellent optical transmission characteristics and its ability to support electroosmotic flow, required during electrophoretic separations. These aspects combined with its large range of surface modification chemistries, make PDMS an attractive substrate in microfluidic devices for, in particular, DNA separation. Here, we report the successful wet chemical surface modification of PDMS microchannels using a simple three step method to produce an isothiocyanate-terminated surface. Initially, PDMS was oxygen plasma treated to produce a silanol-terminated surface, this was then reacted with 3-aminopropyltriethoxysilane with subsequent reaction of the now amine-terminated surface with p-phenylenediisothiocyanate. Water contact angle measurements both before and after modification showed a reduction in hydrophobicity from 101o for native PDMS to 94o for the isothiocyante-terminated PDMS. The isothiocyanate-terminated surface was then coupled with an amineterminated single-stranded DNA (ssDNA) oligonucleotide capture probe via a thiourea linkage. Confirmation of capture probe attachment was observed using fluorescent microscopy after hybridization of the capture probes with fluorescently labeled complimentary ssDNA oligonucleotides.

  3. New Concepts of Fluorescent Probes for Specific Detection of DNA Sequences: Bis-Modified Oligonucleotides in Excimer and Exciplex Detection

    PubMed Central

    Gbaj, A; Bichenkova, EV; Walsh, L; Savage, HE; Sardarian, AR; Etchells, LL; Gulati, A; Hawisa, S; Douglas, KT

    2009-01-01

    The detection of single base mismatches in DNA is important for diagnostics, treatment of genetic diseases, and identification of single nucleotide polymorphisms. Highly sensitive, specific assays are needed to investigate genetic samples from patients. The use of a simple fluorescent nucleoside analogue in detection of DNA sequence and point mutations by hybridisation in solution is described in this study. The 5′-bispyrene and 3′-naphthalene oligonucleotide probes form an exciplex on hybridisation to target in water and the 5′-bispyrene oligonucleotide alone is an adequate probe to determine concentration of target present. It was also indicated that this system has a potential to identify mismatches and insertions. The aim of this work was to investigate experimental structures and conditions that permit strong exciplex emission for nucleic acid detectors, and show how such exciplexes can register the presence of mismatches as required in SNP analysis. This study revealed that the hybridisation of 5′-bispyrenyl fluorophore to a DNA target results in formation of a fluorescent probe with high signal intensity change and specificity for detecting a complementary target in a homogeneous system. Detection of SNP mutations using this split-probe system is a highly specific, simple, and accessible method to meet the rigorous requirements of pharmacogenomic studies. Thus, it is possible for the system to act as SNP detectors and it shows promise for future applications in genetic testing. PMID:21483539

  4. Fluorescent detection of single nucleotide polymorphism utilizing a hairpin DNA containing a nucleotide base analog pyrrolo-deoxycytidine as a fluorescent probe.

    PubMed

    Zhang, Hongge; Wang, Minjuan; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

    2011-05-15

    A novel fluorescent method for the detection of single nucleotide polymorphism (SNP) was developed using a hairpin DNA containing nucleotide base analog pyrrolo-deoxycytidine (P-dC) as a fluorescent probe. This fluorescent probe was designed by incorporating a fluorescent P-dC into a stem of the hairpin DNA, whose sequence of the loop moiety complemented the target single strand DNA (ss-DNA). In the absence of the target ss-DNA, the fluorescent probe stays a closed configuration in which the P-dC is located in the double strand stem of the fluorescent probe, such that there is weak fluorescence, attributed to a more efficient stacking and collisional quenching of neighboring bases. In the presence of target ss-DNA, upon hybridizing the ss-DNA to the loop moiety, a stem-loop of the fluorescent probe is opened and the P-dC is located in the ss-DNA, thus resulting in strong fluorescence. The effective discrimination of the SNP, including single base mismatch ss-DNA (A, T, G) and double mismatch DNA (C, C), against perfect complementary ss-DNA was achieved by increased fluorescence intensity, and verified by thermal denaturation and circular dichroism spectroscopy. Relative fluorescence intensity had a linear relationship with the concentration of perfect complementary ss-DNA and ranged from 50 nM to 3.0 μM. The linear regression equation was F/F(0)=2.73 C (μM)+1.14 (R=0.9961) and the detection limit of perfect complementary ss-DNA was 16 nM (S/N=3). This study demonstrates that a hairpin DNA containing nucleotide base analog P-dC is a promising fluorescent probe for the effective discrimination of SNP and for highly sensitive detection of perfect complementary DNA.

  5. Cultivation-independent detection of autotrophic hydrogen-oxidizing bacteria by DNA stable-isotope probing.

    PubMed

    Pumphrey, Graham M; Ranchou-Peyruse, Anthony; Spain, Jim C

    2011-07-01

    Knallgas bacteria are a physiologically defined group that is primarily studied using cultivation-dependent techniques. Given that current cultivation techniques fail to grow most bacteria, cultivation-independent techniques that selectively detect and identify knallgas bacteria will improve our ability to study their diversity and distribution. We used stable-isotope probing (SIP) to identify knallgas bacteria in rhizosphere soil of legumes and in a microbial mat from Obsidian Pool in Yellowstone National Park. When samples were incubated in the dark, incorporation of (13)CO(2) was H(2) dependent. SIP enabled the detection of knallgas bacteria that were not detected by cultivation, and the majority of bacteria identified in the rhizosphere soils were betaproteobacteria predominantly related to genera previously known to oxidize hydrogen. Bacteria in soil grew on hydrogen at concentrations as low as 100 ppm. A hydB homolog encoding a putative high-affinity NiFe hydrogenase was amplified from (13)C-labeled DNA from both vetch and clover rhizosphere soil. The results indicate that knallgas bacteria can be detected by SIP and populations that respond to different H(2) concentrations can be distinguished. The methods described here should be applicable to a variety of ecosystems and will enable the discovery of additional knallgas bacteria that are resistant to cultivation.

  6. A beta-mixture quantile normalization method for correcting probe design bias in Illumina Infinium 450 k DNA methylation data

    PubMed Central

    Teschendorff, Andrew E.; Marabita, Francesco; Lechner, Matthias; Bartlett, Thomas; Tegner, Jesper; Gomez-Cabrero, David; Beck, Stephan

    2013-01-01

    Motivation: The Illumina Infinium 450 k DNA Methylation Beadchip is a prime candidate technology for Epigenome-Wide Association Studies (EWAS). However, a difficulty associated with these beadarrays is that probes come in two different designs, characterized by widely different DNA methylation distributions and dynamic range, which may bias downstream analyses. A key statistical issue is therefore how best to adjust for the two different probe designs. Results: Here we propose a novel model-based intra-array normalization strategy for 450 k data, called BMIQ (Beta MIxture Quantile dilation), to adjust the beta-values of type2 design probes into a statistical distribution characteristic of type1 probes. The strategy involves application of a three-state beta-mixture model to assign probes to methylation states, subsequent transformation of probabilities into quantiles and finally a methylation-dependent dilation transformation to preserve the monotonicity and continuity of the data. We validate our method on cell-line data, fresh frozen and paraffin-embedded tumour tissue samples and demonstrate that BMIQ compares favourably with two competing methods. Specifically, we show that BMIQ improves the robustness of the normalization procedure, reduces the technical variation and bias of type2 probe values and successfully eliminates the type1 enrichment bias caused by the lower dynamic range of type2 probes. BMIQ will be useful as a preprocessing step for any study using the Illumina Infinium 450 k platform. Availability: BMIQ is freely available from http://code.google.com/p/bmiq/. Contact: a.teschendorff@ucl.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online PMID:23175756

  7. Time-resolved probes based on guanine/thymine-rich DNA-sensitized luminescence of terbium(III).

    PubMed

    Zhang, Min; Le, Huynh-Nhu; Jiang, Xiao-Qin; Yin, Bin-Cheng; Ye, Bang-Ce

    2013-12-03

    In this study, we have developed a novel strategy to highly sensitize the luminescence of terbium(III) (Tb(3+)) using a designed guanine/thymine-rich DNA (5'-[G3T]5-3') as an antenna ligand, in which [G3T]5 improved the luminescence of Tb(3+) by 3 orders of magnitude due to energy transfer from nucleic acids to Tb(3+) (i.e., antenna effect). Furthermore, label-free probes for the luminescent detection of biothiols, Ag(+), and sequence-specific DNA in an inexpensive, simple, and mix-and-read format are presented based on the [G3T]5-sensitized luminescence of Tb(3+) (GTSLT). The long luminescence lifetime of the probes readily enables time-resolved luminescence (TRL) experiments. Hg(2+) can efficiently quench the luminescence of Tb(3+) sensitized by [G3T]5 (Tb(3+)/[G3T]5); however, biothiols are readily applicable to selectively grab Hg(2+) for restoration of the luminescence of Tb(3+)/[G3T]5 initially quenched by Hg(2+), which can be used for "turn on" detection of biothiols. With the use of cytosine (C)-rich oligonucleotide c[G3T]5 complementary to [G3T]5, the formed [G3T]5/c[G3T]5 duplex cannot sensitize the luminescence of Tb(3+). However, in the presence of Ag(+), Ag(+) can combine the C base of c[G3T]5 to form C-Ag(+)-C complexes, leading to the split of the [G3T]5/c[G3T]5 duplex and then release of [G3T]5. The released [G3T]5 acts as an antenna ligand for sensitizing the luminescence of Tb(3+). Therefore, the Tb(3+)/[G3T]5/c[G3T]5 probe can be applied to detect Ag(+) in a "turn on" format. Moreover, recognition of target DNA via hybridization to a molecular beacon (MB)-like probe (MB-[G3T]5) can unfold the MB-[G3T]5 to release the [G3T]5 for sensitizing the luminescence of Tb(3+), producing a detectable signal directly proportional to the amount of target DNA of interest. This allows the development of a fascinating label-free MB probe for DNA sensing based on the luminescence of Tb(3+). Results and methods reported here suggest that a guanine/thymine-rich DNA

  8. Probing quantum coherence in a biological system by means of DNA amplification.

    PubMed

    Bieberich, E

    2000-07-01

    As a result of rapid decoherence, quantum effects in biological systems are usually confined to single electron or hydrogen delocalizations. In principle, molecular interactions at high temperatures can be guided by quantum coherence if embedded in a dynamics preventing decoherence. This was experimentally investigated by analyzing the thermodynamics, kinetics, and quantum mechanics of the primer/template duplex formation during DNA amplification by polymerase chain reaction. The structures of the two oligonucleotide primers used for amplification of a cDNA template were derived either from a repetitive motif or a fractal distribution of nucleotide residues. Contrary to the computer-based calculation of the primer melting temperatures (T(m)) that predicted a higher T(m) for the non-fractal primer due to nearest-neighbor effects, it was found that the T(m) of the non-fractal primer was actually 2 degrees C lower than that of its fractal counterpart. A thermodynamic analysis of the amplification reaction indicated that the primer annealing process followed Bose-Einstein instead of Boltzmann statistics, with an additional binding potential of mu=500 J/mol or 10(-21) J/molecule due to a superposition of binding states within the primer/template duplex. The temporal evolution of the Bose-Einstein state was determined by enzyme kinetic analysis of the association of the primer/template duplex to Taq polymerase. Assuming that collision with the enzyme interrupted the superposition, it was found that the Bose-Einstein state lasted for t(dec)=0.7x10(-12) s, corresponding to the energy dispersion (DeltaE) of quantum coherent states (mu=DeltaE>/=h/t(dec)). A quantum mechanical analysis revealed that the coherent state was stabilized by almost vanishing separation energies between distinct binding states during a temperature-driven shifting of the two DNA strands in the primer/template duplex. The additional binding potential is suggested to arise from a short-lived electron

  9. Multiple DNA extractions coupled with stable-isotope probing of anthracene-degrading bacteria in contaminated soil.

    PubMed

    Jones, Maiysha D; Singleton, David R; Sun, Wei; Aitken, Michael D

    2011-05-01

    In many of the DNA-based stable-isotope probing (SIP) studies published to date in which soil communities were investigated, a single DNA extraction was performed on the soil sample, usually using a commercial DNA extraction kit, prior to recovering the (13)C-labeled (heavy) DNA by density-gradient ultracentrifugation. Recent evidence suggests, however, that a single extraction of a soil sample may not lead to representative recovery of DNA from all of the organisms in the sample. To determine whether multiple DNA extractions would affect the DNA yield, the eubacterial 16S rRNA gene copy number, or the identification of anthracene-degrading bacteria, we performed seven successive DNA extractions on the same aliquot of contaminated soil either untreated or enriched with [U-(13)C]anthracene. Multiple extractions were necessary to maximize the DNA yield and 16S rRNA gene copy number from both untreated and anthracene-enriched soil samples. Sequences within the order Sphingomonadales, but unrelated to any previously described genus, dominated the 16S rRNA gene clone libraries derived from (13)C-enriched DNA and were designated "anthracene group 1." Sequences clustering with Variovorax spp., which were also highly represented, and sequences related to the genus Pigmentiphaga were newly associated with anthracene degradation. The bacterial groups collectively identified across all seven extracts were all recovered in the first extract, although quantitative PCR analysis of SIP-identified groups revealed quantitative differences in extraction patterns. These results suggest that performing multiple DNA extractions on soil samples improves the extractable DNA yield and the number of quantifiable eubacterial 16S rRNA gene copies but have little qualitative effect on the identification of the bacterial groups associated with the degradation of a given carbon source by SIP.

  10. Multiple DNA Extractions Coupled with Stable-Isotope Probing of Anthracene-Degrading Bacteria in Contaminated Soil▿†

    PubMed Central

    Jones, Maiysha D.; Singleton, David R.; Sun, Wei; Aitken, Michael D.

    2011-01-01

    In many of the DNA-based stable-isotope probing (SIP) studies published to date in which soil communities were investigated, a single DNA extraction was performed on the soil sample, usually using a commercial DNA extraction kit, prior to recovering the 13C-labeled (heavy) DNA by density-gradient ultracentrifugation. Recent evidence suggests, however, that a single extraction of a soil sample may not lead to representative recovery of DNA from all of the organisms in the sample. To determine whether multiple DNA extractions would affect the DNA yield, the eubacterial 16S rRNA gene copy number, or the identification of anthracene-degrading bacteria, we performed seven successive DNA extractions on the same aliquot of contaminated soil either untreated or enriched with [U-13C]anthracene. Multiple extractions were necessary to maximize the DNA yield and 16S rRNA gene copy number from both untreated and anthracene-enriched soil samples. Sequences within the order Sphingomonadales, but unrelated to any previously described genus, dominated the 16S rRNA gene clone libraries derived from 13C-enriched DNA and were designated “anthracene group 1.” Sequences clustering with Variovorax spp., which were also highly represented, and sequences related to the genus Pigmentiphaga were newly associated with anthracene degradation. The bacterial groups collectively identified across all seven extracts were all recovered in the first extract, although quantitative PCR analysis of SIP-identified groups revealed quantitative differences in extraction patterns. These results suggest that performing multiple DNA extractions on soil samples improves the extractable DNA yield and the number of quantifiable eubacterial 16S rRNA gene copies but have little qualitative effect on the identification of the bacterial groups associated with the degradation of a given carbon source by SIP. PMID:21398486

  11. Probe Lasso: a novel method to rope in differentially methylated regions with 450K DNA methylation data.

    PubMed

    Butcher, Lee M; Beck, Stephan

    2015-01-15

    The speed and resolution at which we can scour the genome for DNA methylation changes has improved immeasurably in the last 10years and the advent of the Illumina 450K BeadChip has made epigenome-wide association studies (EWAS) a reality. The resulting datasets are conveniently formatted to allow easy alignment of significant hits to genes and genetic features, however; methods that parse significant hits into discreet differentially methylated regions (DMRs) remain a challenge to implement. In this paper we present details of a novel DMR caller, the Probe Lasso: a flexible window based approach that gathers neighbouring significant-signals to define clear DMR boundaries for subsequent in-depth analysis. The method is implemented in the R package ChAMP (Morris et al., 2014) and returns sets of DMRs according to user-tuned levels of probe filtering (e.g., inclusion of sex chromosomes, polymorphisms) and probe-lasso size distribution. Using a sub-sample of colon cancer- and healthy colon-samples from TCGA we show that Probe Lasso shifts DMR calling away from just probe-dense regions, and calls a range of DMR sizes ranging from tens-of-bases to tens-of-kilobases in scale. Moreover, using TCGA data we show that Probe Lasso leverages more information from the array and highlights a potential role of hypomethylated transcription factor binding motifs not discoverable using a basic, fixed-window approach.

  12. Probe Lasso: A novel method to rope in differentially methylated regions with 450K DNA methylation data

    PubMed Central

    Butcher, Lee M.; Beck, Stephan

    2015-01-01

    The speed and resolution at which we can scour the genome for DNA methylation changes has improved immeasurably in the last 10 years and the advent of the Illumina 450K BeadChip has made epigenome-wide association studies (EWAS) a reality. The resulting datasets are conveniently formatted to allow easy alignment of significant hits to genes and genetic features, however; methods that parse significant hits into discreet differentially methylated regions (DMRs) remain a challenge to implement. In this paper we present details of a novel DMR caller, the Probe Lasso: a flexible window based approach that gathers neighbouring significant-signals to define clear DMR boundaries for subsequent in-depth analysis. The method is implemented in the R package ChAMP (Morris et al., 2014) and returns sets of DMRs according to user-tuned levels of probe filtering (e.g., inclusion of sex chromosomes, polymorphisms) and probe-lasso size distribution. Using a sub-sample of colon cancer- and healthy colon-samples from TCGA we show that Probe Lasso shifts DMR calling away from just probe-dense regions, and calls a range of DMR sizes ranging from tens-of-bases to tens-of-kilobases in scale. Moreover, using TCGA data we show that Probe Lasso leverages more information from the array and highlights a potential role of hypomethylated transcription factor binding motifs not discoverable using a basic, fixed-window approach. PMID:25461817

  13. Au-Ag template stripped pattern for scanning probe investigations of DNA arrays produced by dip pen nanolithography.

    PubMed

    Baserga, Andrea; Viganò, Marco; Casari, Carlo S; Turri, Stefano; Li Bassi, Andrea; Levi, Marinella; Bottani, Carlo E

    2008-11-18

    We report on DNA arrays produced by dip pen nanolithography (DPN) on a novel Au-Ag micropatterned template stripped surface. DNA arrays have been investigated by atomic force microscopy (AFM) and scanning tunneling microscopy (STM) showing that the patterned template stripped substrate enables easy retrieval of the DPN-functionalized zone with a standard optical microscope permitting multi-instrument and multitechnique local detection and analysis. Moreover the smooth surface of the Au squares ( approximately 5-10 A roughness) allows AFM/STM to be sensitive to the hybridization of the oligonucleotide array with label-free target DNA. Our Au-Ag substrates, combining the retrieving capabilities of the patterned surface with the smoothness of the template stripped technique, are candidates for the investigation of DPN nanostructures and for the development of label-free detection methods for DNA nanoarrays based on the use of scanning probes.

  14. Electrochemical DNA probe for Hg(2+) detection based on a triple-helix DNA and Multistage Signal Amplification Strategy.

    PubMed

    Wang, Huan; Zhang, Yihe; Ma, Hongmin; Ren, Xiang; Wang, Yaoguang; Zhang, Yong; Wei, Qin

    2016-12-15

    In this work, an ultrasensitive electrochemical sensor was developed for detection of Hg(2+). Gold nanoparticles decorated bovine serum albumin reduction of graphene oxide (AuNP-BSA-rGO) were used as subsurface material for the immobilization of triple-helix DNA. The triple-helix DNA containing a thiol labelled single-stranded DNA (sDNA) and a thymine-rich DNA (T-rich DNA), which could be unwinded in the present of Hg(2+) to form more stable thymine-Hg(2+)-thymine (T-Hg(2+)-T) complex. T-Hg(2+)-T complex was then removed and the sDNA was left on the electrode. At this time, gold nanoparticle carrying thiol labelled cytosine-rich complementary DNA (cDNA-AuNP) could bind with the free sDNA. Meanwhile, the other free cDNA on AuNP could bind with each other in the present of Ag(+) to form the stable cytosine-Ag(+)-cytosine (C-Ag(+)-C) complex and circle amplification. Plenty of C-Ag(+)-C could form silver nanoclusters by electrochemical reduction and the striping signal of Ag could be measured for purpose of the final electrochemical detection of Hg(2+). This sensor could detect Hg(2+) over a wide concentration range from 0.1 to 130nM with a detection limit of 0.03nM.

  15. Fluorescence imaging of single-copy DNA sequences within the human genome using PNA-directed padlock probe assembly

    PubMed Central

    Yaroslavsky, Anastasia I.; Smolina, Irina V.

    2013-01-01

    SUMMARY We present a novel approach for fluorescent in situ detection of short, single-copy sequences within genomic DNA in human cells. The single copy sensitivity and single base specificity of our method is achieved due to the combination of three components. First, a peptide nucleic acid (PNA) probe locally opens a chosen target site, which allows a padlock DNA probe to access the site and become ligated. Second, rolling circle amplification (RCA) generates thousands of single-stranded copies of the target sequence. Finally, fluorescent in situ hybridization (FISH) is used to visualize the amplified DNA. We validate this new technique by successfully detecting six unique target sites on human mitochondrial and autosomal DNA. We also demonstrate the high specificity of this method by detecting X- and Y- specific sequences on human sex chromosomes and by simultaneously detecting three unique target sites. Finally, we discriminate two target sites that differ by two nucleotides. The PNA-RCA-FISH approach is a unique in situ hybridization method capable of multi-target visualization within human chromosomes and nuclei that does not require DNA denaturation and is extremely sequence specific. PMID:23521801

  16. Histone Acetylation Induced Transformation of B-DNA to Z-DNA in Cells Probed through FT-IR Spectroscopy.

    PubMed

    Zhang, Fengqiu; Huang, Qing; Yan, Jingwen; Chen, Zhu

    2016-04-19

    A nucleosome is made up of DNA and histones, and acetylation of histones perturbs the interaction of DNA and histones and thus affects the chromatin conformation and function. However, whether or how acetylation induces DNA conformation changes is still elusive. In this work, we applied FT-IR spectroscopy to monitor the DNA signals in cells as the histone acetylation was regulated by trichostatin A (TSA), a reversible inhibitor to histone deacetylases (HDACs). Our results unambiguously demonstrate the significant transformation of B-DNA to Z-DNA upon histone acetylation in the TSA treated HeLa cells. This is the first report providing the explicit experimental evidence for such a B-Z transformation of DNA in the epigenetic states of cells.

  17. Method to detect the end-point for PCR DNA amplification using an ionically labeled probe and measuring impedance change

    DOEpatents

    Miles, Robin R.; Belgrader, Phillip; Fuller, Christopher D.

    2007-01-02

    Impedance measurements are used to detect the end-point for PCR DNA amplification. A pair of spaced electrodes are located on a surface of a microfluidic channel and an AC or DC voltage is applied across the electrodes to produce an electric field. An ionically labeled probe will attach to a complementary DNA segment, and a polymerase enzyme will release the ionic label. This causes the conductivity of the solution in the area of the electrode to change. This change in conductivity is measured as a change in the impedance been the two electrodes.

  18. TaqMan probes as blocking agents for enriched PCR amplification and DNA melting analysis of mutant genes.

    PubMed

    Botezatu, Irina V; Panchuk, Irina O; Stroganova, Anna M; Senderovich, Anastasia I; Kondratova, Valentina N; Shelepov, Valery P; Lichtenstein, Anatoly V

    2017-02-01

    Asymmetric PCR and DNA melting analysis with TaqMan probes applied for mutation detection is effectively used in clinical diagnostics. The method is simple, cost-effective, and carried out in a closed-tube format, minimizing time, labor, and risk of sample cross-contamination. Although DNA melting analysis is more sensitive than Sanger sequencing (mutation detection thresholds are ~5% and 15%-20%, respectively), it is less sensitive than more labor-intensive and expensive techniques such as pyrosequencing and droplet digital PCR. Here, we demonstrate that, under specially selected conditions of asymmetric PCR, TaqMan probes can play the role of blocking agents. Preferential blocking of the wild-type allele brings about enriched amplification of mutant alleles. As a result, an ~10-fold increase in the detection sensitivity for mutant BRAF and NRAS genes was achieved.

  19. Interfacing click chemistry with automated oligonucleotide synthesis for the preparation of fluorescent DNA probes containing internal xanthene and cyanine dyes.

    PubMed

    Astakhova, I Kira; Wengel, Jesper

    2013-01-14

    Double-labeled oligonucleotide probes containing fluorophores interacting by energy-transfer mechanisms are essential for modern bioanalysis, molecular diagnostics, and in vivo imaging techniques. Although bright xanthene and cyanine dyes are gaining increased prominence within these fields, little attention has thus far been paid to probes containing these dyes internally attached, a fact which is mainly due to the quite challenging synthesis of such oligonucleotide probes. Herein, by using 2'-O-propargyl uridine phosphoramidite and a series of xanthenes and cyanine azide derivatives, we have for the first time performed solid-phase copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click labeling during the automated phosphoramidite oligonucleotide synthesis followed by postsynthetic click reactions in solution. We demonstrate that our novel strategy is rapid and efficient for the preparation of novel oligonucleotide probes containing internally positioned xanthene and cyanine dye pairs and thus represents a significant step forward for the preparation of advanced fluorescent oligonucleotide probes. Furthermore, we demonstrate that the novel xanthene and cyanine labeled probes display unusual and very promising photophysical properties resulting from energy-transfer interactions between the fluorophores controlled by nucleic acid assembly. Potential benefits of using these novel fluorescent probes within, for example, molecular diagnostics and fluorescence microscopy include: Considerable Stokes shifts (40-110 nm), quenched fluorescence of single-stranded probes accompanied by up to 7.7-fold light-up effect of emission upon target DNA/RNA binding, remarkable sensitivity to single-nucleotide mismatches, generally high fluorescence brightness values (FB up to 26), and hence low limit of target detection values (LOD down to <5 nM).

  20. Clinical utility of a DNA probe to 17p11.2 in screening of patients with a peripheral neuropathy

    SciTech Connect

    Blancato, J.; Precht, K.; Meck, J.

    1994-09-01

    We assessed the usefulness of in situ hybridization with a DNA probe to the area of chromosome 17 at p11.2 as a diagnostic tool for screening for Charcot Marte Tooth 1A (CMT 1A). In situ hybridization with a probe to 17p11.2 was performed on fixed lymphocytes from the following groups of individuals: (1) normal controls; (2) patients evoking a strong clinical suspicion of CMT 1A; and (3) 3 families with an apparent autosomal dominant peripheral neuropathy of unknown diagnoses. Group 2 patients had evidence of demyelination as defined by nerve conduction of less that 50% of the normal mean or terminal latency greater than 50% of the normal mean in conduction studies. Analysis of interphase cells hybridized with a cosmid DNA probe to 17p11.2 requires inclusion of a normal control with each trial and masked observer. Due to the size of the target DNA and the nature of the centromeric heterochromatin, the scoring of this probe is more subjective than centromere probes. For example, if the two 17 chromosomes are decondensed as in interphase, two tandem signals may be visualized as one. Results from duplication positive patients demonstrate a large proportion of cells with two closely aligned, but separate, signals with an additional single signal. Normal results demonstrate a majority of cells with two separate signals representing both normal homologues. None of the 3 families with questionable diagnosis revealed a duplication at the region, reinforcing our belief that a clinical diagnosis is the most discriminating tool available for diagnosis of CMT 1A. We concur with Boylan that molecular analysis for CMT 1A is useful for establishing a diagnosis of CMT 1A, but is not a primary differential diagnostic test. The yield in screening patients without physiologic evidence of demyelination is likely to be low. We further find that the use of in situ hybridization is a simple method of performing the duplication analysis.

  1. Probing the interactions of the solvated electron with DNA by molecular dynamics simulations: II. bromodeoxyuridine-thymidine mismatched DNA.

    PubMed

    Gantchev, Tsvetan G; Hunting, Darel J

    2009-01-01

    The interaction of solvated electrons (e(-)(aq)) with DNA results in various types of DNA lesions. The in vitro and in vivo sensitisation of DNA to (e(-)(aq))-induced damage is achieved by incorporation of the electron-affinity radiosensitiser bromodeoxyuridine (BUdR) in place of thymidine. However, in DNA duplexes containing single-stranded regions (bulged BUdR-DNA), the type of lesion is different and the efficiency of damage is enhanced. In particular, DNA interstrand crosslinks (ICL) form at high efficiency in bulged DNA but are not detectable in completely duplex DNA. Knowledge about the processes and interactions leading to these differences is obscure. Previously, we addressed the problem by applying molecular modelling and molecular dynamics (MD) simulations to a system of normal (BUdR.A)-DNA and a hydrated electron, where the excess electron was modelled as a localised e(-)(H2O6) anionic cluster. The goal of the present study was to apply the same MD simulation to a wobble DNA-e(-)(aq) system, containing a pyrimidine-pyrimidine mismatched base pair, BUdR.T. The results show an overall dynamic pattern similar to that of the e(-)(aq) motion around normal DNA. However, the number of configuration states when e(-)(aq)) was particularly close to DNA is different. Moreover, in the (BUdR.T)-wobble DNA system, the electron frequently approaches the brominated strand, including BUdR, which was not observed with the normal (BUdR.A)-DNA. The structure and exchange of water at the sites of e(-)(aq) immobilisation near DNA were also characterised. The structural dynamics of the wobble DNA is prone to more extensive perturbations, including frequent formation of cross-strand (cs) interatomic contacts. The structural deviations correlated with e(-)(aq) approaching DNA from the major groove side, with sodium ions trapped deep in the minor groove. Altogether, the obtained results confirm and/or throw light on dynamic-structure determinants possibly responsible for the

  2. A comparative cytogenetic study of Drosophila parasitoids (Hymenoptera, Figitidae) using DNA-binding fluorochromes and FISH with 45S rDNA probe.

    PubMed

    Gokhman, Vladimir E; Bolsheva, Nadezhda L; Govind, Shubha; Muravenko, Olga V

    2016-06-01

    Karyotypes of Leptopilina boulardi (Barbotin, Carton et Keiner-Pillault, 1979) (n = 9), L. heterotoma (Thomson, 1862) (n = 10), L. victoriae Nordlander, 1980 (n = 10) and Ganaspis xanthopoda (Ashmead, 1896) (n = 9) (Hymenoptera, Figitidae) were studied using DNA-binding ligands with different base specificity [propidium iodide (PI), chromomycin A3 (CMA3) and 4',6-diamidino-2-phenylindole (DAPI)], and fluorescence in situ hybridization (FISH) with a 45S rDNA probe. Fluorochrome staining was similar between the different fluorochromes, except for a single CMA3- and PI-positive and DAPI-negative band per haploid karyotype of each species. FISH with 45S rDNA probe detected a single rDNA site in place of the bright CMA3-positive band, thus identifying the nucleolus organizing region (NOR). Chromosomal locations of NORs were similar for both L. heterotoma and L. victoriae, but strongly differed in L. boulardi as well as in G. xanthopoda. Phylogenetic aspects of NOR localization in all studied species are briefly discussed.

  3. Two-photon AgNP/DNA-TP dye nanosensing conjugate for biothiol probing in live cells.

    PubMed

    Liu, Mingli; Tang, Qiao; Deng, Ting; Yan, Huijuan; Li, Jishan; Li, Yinhui; Yang, Ronghua

    2014-12-07

    A novel silver nanoparticle (AgNP)/DNA-two-photon dye (TP dye) conjugate was fabricated as a two-photon nanoprobe for biothiol imaging in live cells. DNA-templated silver nanoparticles are efficient quenchers and also provide a biocompatible nanoplatform for facile delivery of DNA into living cells. In the presence of biothiols (Cys, Hcy, or GSH), the strong interaction between the thiol group and silver results in the release of TP dye-labeled single-stranded DNA (ssDNA) from the AgNP surface and the subsequent fluorescence emission of the TP dye, thus enabling biothiols to be assayed. Our results reveal that the AgNP/DNA-TP dye nanosensing conjugate not only is a robust, sensitive, and selective sensor for quantitative detection of biothiols in the complex biological environment but also can be efficiently delivered into live cells and act as a "signal-on" sensor for specific, high-contrast imaging of target biomolecules. Our design provides a methodology for the development of future DNA-templated silver nanoparticle-based two-photon fluorescent probes for use in vitro or in vivo as biomolecular sensors for live-cell imaging.

  4. Fluorescence determination of DNA with 1-pyrenebutyric acid nanoparticles coated with β-cyclodextrin as a fluorescence probe

    NASA Astrophysics Data System (ADS)

    Wang, Lun; Bian, Guirong; Wang, Leyu; Dong, Ling; Chen, Hongqi; Xia, Tingting

    2005-04-01

    A novel ultrasonication method has been successfully developed for the preparation of 1-pyrenebutyric acid (PBAC)/β-cyclodextrin(β-CD) complex nanoparticles. The as-prepared nanoparticles are characterized by transmission electron microscopy (TEM), fluorescence excitation and emission spectroscopy. Complex nanoparticles prepared with ultrasonication are smaller and better dispersed than single PBAC nanoparticles. At pH 3.0, the relative fluorescence intensity of complex nanoparticles of PBAC/β-CD can be quenched by the concentration of DNA. Based on this, a novel fluorimetric method has been developed for rapid determination of DNA. In comparison with single organic fluorophores, these nanoparticle probes are better water-solubility, more stable and do not suffer from blinking. Under optimum conditions, the calibration graphs are linear over the range 0.2-15 μg mL -1 for calf thymus DNA (ct-DNA) and 0.3-12 μg mL -1 for fish sperm DNA (fs-DNA). The corresponding detection limit is 0.01 μg mL -1 for ct-DNA and 0.02 μg mL -1 for fs-DNA. The relative standard deviation of seven replicate measurements is 1.2% for 2.0 μg mL -1 ct-DNA and 1.4% for 2.0 μg mL -1 fs-DNA, respectively. The method is simple and sensitive. The recovery and relative standard deviation are very satisfactory. A mechanism proposed to explain the process also has been studied.

  5. Comparative test of DNA probes for detection of Plasmodium vivax circumsporozoite protein polymorphs VK 247 and VK 210.

    PubMed

    Sattabongkot, J; Suwanabun, N; Rongnoparut, P; Wirtz, R A; Kain, K C; Rosenberg, R

    1994-02-01

    Oligonucleotide probes specific to the characteristic repeat sequences of two alleles of the circumsporozoite protein gene of Plasmodium vivax (VK 210 and VK 247) were selected, synthesized, and tested on matched blood and sporozoite DNA amplified by polymerase chain reaction from 182 cases naturally acquired in Thailand. Probe results were compared to those of circumsporozoite phenotype-specific ELISAs used to evaluate sporozoites from the same cases. There was a 96% agreement between probe results for blood and for sporozoites. Although there was also a nearly complete agreement between probe and ELISA results for cases producing only VK 210 or VK 247 sporozoites, the probes detected 45% more mixed infections than did the ELISAs when used to test specimens from western and southern Thailand; there was no discrepancy when mixed cases from Cambodia were tested. Examination of Southern blots from ambiguous mixed cases demonstrated the presence of both genes, suggesting suppression of VK 247 in some mixed cases to numbers below those detectable by the ELISA.

  6. Physical mapping of new DNA probes near the fragile X mutation (FRAXA) by using a panel of cell lines

    PubMed Central

    Suthers, G. K.; Hyland, V. J.; Callen, D. F.; Oberle, I.; Rocchi, M.; Thomas, N. S.; Morris, C. P.; Schwartz, C. E.; Schmidt, M.; Ropers, H. H.; Baker, E.; Oostra, B. A.; Dahl, N.; Wilson, P. J.; Hopwood, J. J.; Sutherland, G. R.

    1990-01-01

    The fragile X syndrome is a very common disorder, but there has been little progress toward isolating the fragile X mutation (FRAXA). We describe a panel of 14 somatic cell hybrid lines, lymphoblastoid cell lines, and peripheral lymphocytes with X-chromosome translocation or deletion breakpoints near FRAXA. The locations of the breakpoints were defined with 16 established probes between pX45d (DXS100) and St14–1 (DXS52). Seven of the cell lines had breakpoints between the probes RN1 (DXS369) and U6.2 (DXS304), which flank FRAXA at distances of 3–5 centimorgans. The panel of cell lines was used to localize 16 new DNA probes in this region. Six of the probes–VK16, VK18, VK23, VK24, VK37, and VK47–detected loci near FRAXA, and it was possible to order both the X-chromosome breakpoints and the probes in relation to FRAXA. The order of probes and loci near FRAXA is cen–RN1,VK24–VK47–VK23–VK16,FRAXA–VK21A–VK18–IDS–VK37–U6.2-qter. The breakpoints near FRAXA are sufficiently close together that probes localized with this panel can be linked on a large-scale restriction map by pulsed-field gel electrophoresis. This panel of cell lines will be valuable in rapidly localizing other probes near FRAXA. ImagesFigure 2 PMID:2378346

  7. Micron-sized surface enhanced Raman scattering reporter/fluorescence probe encoded colloidal microspheres for sensitive DNA detection.

    PubMed

    You, Lijun; Li, Ruimin; Dong, Xu; Wang, Fang; Guo, Jia; Wang, Changchun

    2017-02-15

    A new type of optical probes, featuring surface enhanced Raman scattering (SERS) and fluorescence spectra dual-mode encoding, has been reported in this article. Based on the uniform micrometer-sized melamine resin/Ag nanoparticles (MRM/Ag-NPs) composite microspheres, the SERS reporters and fluorescent probes were successfully fixed onto the different layers of the MEM/Ag-NPs microspheres, which supported the sensitive DNA detecton. The two spectroscopic methods commonly considered to be contradictive to each other, yet the optical signals were separable in the experiments. The dual-encoding strategy and single microsphere detecton method put the number of available independent codes to be rough the multiple of those available in the two optical detection channels, which increases far more rapidly than the summation of the two channels. As a proof of cencept, the utility of this dual spectrum mode SERS-fluoresecence encoded microsphere (SFEM) was demonstrated in a specific DNA detection using complimentary ssDNA functionalized magnetic beads as the DNA capturing and separation agents. Excellent encoding results were demonstrated from the decoding of the SERS and fluorescence signals of the SFEM. The method appears to be general in scope and we expect that the SERS-fluoresecence encoded microspheres system is applicable to multiplex bioassays of a variety of biomolecules.

  8. Use of a multiplexed CMOS microarray to optimize and compare oligonucleotide binding to DNA probes synthesized or immobilized on individual electrodes.

    PubMed

    Maurer, Karl; Yazvenko, Nina; Wilmoth, Jodi; Cooper, John; Lyon, Wanda; Danley, David

    2010-01-01

    The CombiMatrix microarray with 12,544 electrodes supports in situ electrochemical synthesis of user-defined DNA probes. As an alternative, we immobilized commercially synthesized DNA probes on individual electrodes coated with electropolymerized polypyrrole (Ppy). Hybridization was measured using a biotinylated target oligonucleotide and either Cy5-streptavidin and fluorescence detection or horseradish peroxidase-streptavidin and enzyme-enhanced electrochemical detection. Detection efficiencies were optimized by varying the deposition of the Ppy, the terminal groups on the DNA probes, and other factors that impacted fluorescence quenching and electrical conductivity. Optimized results were compared against those obtained using a microarray with the same DNA sequences synthesized in situ. Immobilized probes produced higher fluorescence signals, possibly by providing a greater stand off between the Cy5 on the target oligonucleotide and the quenching effects of the Ppy and the platinum electrode.

  9. Surface plasmon resonator using high sensitive resonance telecommunication wavelengths for DNA sensors of Mycobacterium tuberculosis with thiol-modified probes.

    PubMed

    Hsu, Shih-Hsiang; Hung, Shao-Chiang; Chen, Yu-Kun; Jian, Zhi-Hao

    2014-12-25

    Various analytes can be verified by surface plasmon resonance, thus continuous improvement of this sensing technology is crucial for better sensing selection and higher sensitivity. The SPR sensitivity on the wavelength modulation is enhanced with increasing wavelengths. The telecommunication wavelength range was then utilized to detect Mycobacterium tuberculosis (MTB) deoxyribonucleic acid (DNA) under two situations, without immobilization and with 5'-thiol end labeled IS6100 DNA probes, for SPR sensitivity comparison. The experimental data demonstrated that the SPR sensitivity increased more than 13 times with the wavelength modulation after immobilization. Since the operating wavelength accuracy of a tunable laser source can be controlled within 0.001 nm, the sensitivity and resolution on immobilized MTB DNA were determined as 1.04 nm/(μg/mL) and 0.9 ng/mL, respectively.

  10. A simple and cost-effective molecular diagnostic system and DNA probes synthesized by light emitting diode photolithography

    NASA Astrophysics Data System (ADS)

    Oleksandrov, Sergiy; Kwon, Jung Ho; Lee, Ki-chang; Sujin-Ku; Paek, Mun Cheol

    2014-09-01

    This work introduces a novel chip to be used in the future as a simple and cost-effective method for creating DNA arrays using light emission diode (LED) photolithography. The DNA chip platform contains 24 independent reaction sites, which allows for the testing of a corresponding amount of patients' samples in hospital. An array of commercial UV LEDs and lens systems was combined with a microfluidic flow system to provide patterning of 24 individual reaction sites, each with 64 independent probes. Using the LED array instead of conventional laser exposure systems or micro-mirror systems significantly reduces the cost of equipment. The microfluidic system together with microfluidic flow cells drastically reduces the amount of used reagents, which is important due to the high cost of commercial reagents. The DNA synthesis efficiency was verified by fluorescence labeling and conventional hybridization.

  11. DNA interstrand cross-links of an antitumor trinuclear platinum(II) complex: thermodynamic analysis and chemical probing.

    PubMed

    Malina, Jaroslav; Farrell, Nicholas P; Brabec, Viktor

    2011-06-06

    The trinuclear platinum compound [{trans-PtCl(NH(3))(2)}(2)(μ-trans-Pt(NH(3))(2){NH(2)(CH(2))(6)NH(2)}(2))](4+) (BBR3464) belongs to the polynuclear class of platinum-based anticancer agents. These agents form in DNA long-range (Pt,Pt) interstrand cross-links, whose role in the antitumor effects of BBR3464 predominates. Our results show for the first time that the interstrand cross-links formed by BBR3464 between two guanine bases in opposite strands separated by two base pairs (1,4-interstrand cross-links) exist as two distinct conformers, which are not interconvertible, not only if these cross-links are formed in the 5'-5', but also in the less-usual 3'-3' direction. Analysis of the conformers by differential scanning calorimetry, chemical probes of DNA conformation, and minor groove binder Hoechst 33258 demonstrate that each of the four conformers affects DNA in a distinctly different way and adopts a different conformation. The results also support the thesis that the molecule of antitumor BBR3464 when forming DNA interstrand cross-links may adopt different global structures, including different configurations of the linker chain of BBR3464 in the minor groove of DNA. Our findings suggest that the multiple DNA interstrand cross-links available to BBR3464 may all contribute substantially to its cytotoxicity.

  12. An atomic force microscopy study of DNA hairpin probes monolabelled with gold nanoparticle: Grafting and hybridization on oxide thin films

    NASA Astrophysics Data System (ADS)

    Lavalley, V.; Chaudouët, P.; Stambouli, V.

    2007-12-01

    First and original results are reported regarding the surface evolution of two kinds of oxide film after covalent grafting and hybridization of hairpin oligonucleotide probes. These hairpin probes were monolabelled with a 1.4 nm gold nanoparticle. One kind of oxide film was rough Sb doped SnO 2 oxide film and the other kind was smooth SiO 2 film. Same process of covalent grafting, involving a silanization step, was performed on both oxide surfaces. Atomic force microscopy (AFM) was used to study the evolution of each oxide surface after different steps of the process: functionalization, probe grafting and hybridization. In the case of rough SnO 2 films, a slight decrease of the roughness was observed after each step whereas in the case of smooth SiO 2 films, a maximum of roughness was obtained after probe grafting. Step height measurements of grafted probes could be performed on SiO 2 leading to an apparent thickness of around 3.7 ± 1.0 nm. After hybridization, on the granular surface of SnO 2, by coupling AFM with SEM FEG analyses, dispersed and well-resolved groups of gold nanoparticles linked to DNA duplexes could be observed. Their density varied from 6.6 ± 0.3 × 10 10 to 2.3 ± 0.3 × 10 11 dots cm -2. On the contrary, on smooth SiO 2 surface, the DNA duplexes behave like a dense carpet of globular structures with a density of 2.9 ± 0.5 × 10 11 globular structures cm -2.

  13. Hybridization-triggered fluorescence detection of DNA with minor groove binder-conjugated probes

    NASA Astrophysics Data System (ADS)

    Afonina, Irina A.; Lokhov, Sergey G.; Belousov, Yevheniy S.; Reed, Michael W.; Lukhtanov, Eugeny A.; Shishkina, Irina G.; Gorn, Vladimir V.; Sanders, Silvia M.; Walburger, David K.; Hoekstra, Merl F.; Vermeulen, Nicolaas M. J.

    2002-06-01

    Fluorogenic 2'-deoxynucleotide probes containing a minor groove binding-quencher compound at the 5'-end and a fluorophore at the 3'-end, were recently described. These probes fluoresce upon hybridization to the complementary target. The 5'-MGB-quencher group prevents 5'-nuclease digestion by Taq polymerase during homogeneous amplification. The 5'-MGB-quencher-oligonucleotide-fluor (MGB-Q-ODN-Fl) probes displayed a dynamic range of 7 order of magnitude, with an ultimate sensitivity of better than 5 copies per sample. The high sensitivity and specificity is illustrated by the application of the probes in single nucleotide polymorphism detection, final load determination and gene expression analyses. This paper summarizes new developments in sequence detection, gene expression and SNP analysis using new Tm prediction software to design robust 5'-MGB-Q-ODN-Fl probes. Furthermore, the software is capable of estimating the Tm of probes containing a modified base. Due to G:G self-association, many G-rich probes and primers are poor performers in amplification reactions. The software recognizes such sequences and substitution of G with 6-Amino-1,5-dihydro-pyrazolo(3,4- d)pyrimidin-4-one (PPG) is indicated, when necessary to eliminate G:G self-association. Examples of improved performance of PPG containing primers and probes is demonstrated.

  14. A Rotational BODIPY Nucleotide: An Environment-Sensitive Fluorescence-Lifetime Probe for DNA Interactions and Applications in Live-Cell Microscopy.

    PubMed

    Dziuba, Dmytro; Jurkiewicz, Piotr; Cebecauer, Marek; Hof, Martin; Hocek, Michal

    2016-01-04

    Fluorescent probes for detecting the physical properties of cellular structures have become valuable tools in life sciences. The fluorescence lifetime of molecular rotors can be used to report on variations in local molecular packing or viscosity. We used a nucleoside linked to a meso-substituted BODIPY fluorescent molecular rotor (dC(bdp)) to sense changes in DNA microenvironment both in vitro and in living cells. DNA incorporating dC(bdp) can respond to interactions with DNA-binding proteins and lipids by changes in the fluorescence lifetimes in the range 0.5-2.2 ns. We can directly visualize changes in the local environment of exogenous DNA during transfection of living cells. Relatively long fluorescence lifetimes and extensive contrast for detecting changes in the microenvironment together with good photostability and versatility for DNA synthesis make this probe suitable for analysis of DNA-associated processes, cellular structures, and also DNA-based nanomaterials.

  15. Use of RAPD-PCR to isolate a species specific DNA probe for Phytophthora cinnamomi.

    PubMed

    Dobrowolski, M P; O'Brien, P A

    1993-10-01

    The products of RAPD-PCR amplification of Phytophthora cinnamomi DNA were separated by electrophoresis in agarose. Parallel Southern blots of the gels were hybridized with nick translated DNA from different species of Phytophthora. Fragments that hybridized specifically to P. cinnamomi DNA were identified. These fragments were purified and cloned into pUC18. Their specificity for P. cinnamomi was confirmed.

  16. Application of DNA fingerprinting with digoxigenated oligonucleotide probe (CAC)5 to analysis of the genetic variation within Taenia taeniaeformis.

    PubMed

    Okamoto, M; Ueda, H; Hayashi, M; Oku, Y; Kurosawa, T; Kamiya, M

    1995-04-01

    DNA from T. taeniaeformis digested with the restriction endonuclease was hybridized with digoxigenated oligonucleotide probe (CAC)5. Metacestode and adult showed same clear multibanding patterns, which were characteristic of multilocus DNA fingerprinting. The fingerprinting patterns were quite different from those of the rodent hosts. Genetic variations in 4 laboratory-reared isolates of T. taeniaeformis, including 3 isolates which have been reported to be indistinguishable by infectivity, morphology and protein composition of metacestode, were investigated using this technique. Each of the 4 isolates exhibited isolate-specific fingerprinting patterns and were easily distinguished from one another, thus it was considered that (CAC)5 was a highly resolvable and informative probe for cestodes. However, it was also indicated that (CAC)5 was so sensitive that applying fingerprinting with (CAC)5 to taxonomical or phylogenetic analysis was limited where habitat of the host was restricted to the small area. In comparison to fingerprinting with 32P-labeled (CAC)5, fingerprinting with digoxigenated (CAC)5 represented more and sharper bands. It was considered that a digoxigenated probe was more useful for genetic analysis of cestodes.

  17. Real-time electrochemical LAMP: a rational comparative study of different DNA intercalating and non-intercalating redox probes.

    PubMed

    Martin, Alexandra; Bouffier, Laurent; Grant, Kathryn B; Limoges, Benoît; Marchal, Damien

    2016-06-20

    We present a comparative study of ten redox-active probes for use in real-time electrochemical loop-mediated isothermal amplification (LAMP). Our main objectives were to establish the criteria that need to be fulfilled for minimizing some of the current limitations of the technique and to provide future guidelines in the search for ideal redox reporters. To ensure a reliable comparative study, each redox probe was tested under similar conditions using the same LAMP reaction and the same entirely automatized custom-made real-time electrochemical device (designed for electrochemically monitoring in real-time and in parallel up to 48 LAMP samples). Electrochemical melt curve analyses were recorded immediately at the end of each LAMP reaction. Our results show that there are a number of intercalating and non-intercalating redox compounds suitable for real-time electrochemical LAMP and that the best candidates are those able to intercalate strongly into ds-DNA but not too much to avoid inhibition of the LAMP reaction. The strongest intercalating redox probes were finally shown to provide higher LAMP sensitivity, speed, greater signal amplitude, and cleaner-cut DNA melting curves than the non-intercalating molecules.

  18. Detection of DNA damage by using hairpin molecular beacon probes and graphene oxide.

    PubMed

    Zhou, Jie; Lu, Qian; Tong, Ying; Wei, Wei; Liu, Songqin

    2012-09-15

    A hairpin molecular beacon tagged with carboxyfluorescein in combination with graphene oxide as a quencher reagent was used to detect the DNA damage by chemical reagents. The fluorescence of molecular beacon was quenched sharply by graphene oxide; while in the presence of its complementary DNA the quenching efficiency decreased because their hybridization prevented the strong adsorbability of molecular beacon on graphene oxide. If the complementary DNA was damaged by a chemical reagent and could not form intact duplex structure with molecular beacon, more molecular beacon would adsorb on graphene oxide increasing the quenching efficiency. Thus, damaged DNA could be detected based on different quenching efficiencies afforded by damaged and intact complementary DNA. The damage effects of chlorpyrifos-methyl and three metabolites of styrene such as mandelieaeids, phenylglyoxylieaeids and epoxystyrene on DNA were studied as models. The method for detection of DNA damage was reliable, rapid and simple compared to the biological methods.

  19. rDNA-targeted PCR primers and FISH probe in the detection of Ophiocordyceps sinensis hyphae and conidia.

    PubMed

    Zhong, Xin; Peng, Qingyun; Qi, Lili; Lei, Wei; Liu, Xin

    2010-11-01

    Ophiocordyceps sinensis (Berk.) Sung, Sung, Hywel-Jones & Spatafora (syn. Cordyceps sinensis) one of the entomopathogenic fungi, is a rare Traditional Chinese Medicine (TCM) found in the Qinghai-Tibetan Plateau. Polymerase Chain Reaction (PCR) and Fluorescence in situ hybridization (FISH) methods are necessary to identify the mycelia or spores of O. sinensis from its habitat and to monitor its dispersal, colonization and infectivity. To develop both primers and probe specific to O. sinensis, ribosomal DNA (rDNA) amplified with universal primers from O. sinensis genomic DNA and seven closely related fungi were sequenced. According to these sequences, the upper and lower primers (OsT-F and OsT-R) were designed within internal transcribed spacer region 1 (ITS1) and ITS2 and flanked by universal primers ITS5 and ITS4, respectively. The designed primers were used for general PCR, touchdown PCR, or both together with the universal primers for nested-touchdown PCR. The results showed that only the extracted DNA of O. sinensis was specifically amplified. The sensitivity of nested-touchdown PCR with extracted DNA of O. sinensis is as low as 10(-14)g (10 fg) and at least 1000 times higher than the other PCR methods. In addition, Cy5-labeled probe (OsLSU) for cytoplasmic LSU rRNA was hybridized with the ascospores of O. sinensis. It showed a strong red fluorescence throughout the whole cell but did not cross-react with other entomopathogenic fungi. Taken together, these methods were useful for studying the biology and ecology of O. sinensis.

  20. Analysis of mtDNA HVRII in several human populations using an immobilised SSO probe hybridisation assay.

    PubMed

    Comas, D; Reynolds, R; Sajantila, A

    1999-01-01

    Several populations were typed for the hypervariable region II (HVRII) of the mitochondrial DNA (mtDNA) control region using immobilised sequence-specific oligonucleotide (SSO) probes. A total of 16 SSO probes was used to type 1081 individuals from eight different ethnic groups (African Americans, Somali, US Europeans, US Hispanics, Bosnians, Finns, Saami and Japanese). Data was compared with already published sequence data by analysis of principal components, genetic distances and analysis of the molecular variance (AMOVA). The analyses performed group the samples in several clusters according to their geographical origins. Most of the variability detected is assigned to differences between individuals and only 7% is assigned to differences among groups of populations within and between geographical regions. Several features are patent in the samples studied: Somali, as a representative East African population, seem to have experienced a detectable amount of Caucasoid maternal influence; different degrees of admixture in the US samples studied are detected; Finns and Saami belong to the European genetic landscape, although Saami present an outlier position attributable to a strong maternal founder effect. The technique used is a rapid and simple method to detect human variation in the mtDNA HVRII in a large number of samples, which might be useful in forensic and population genetic studies.

  1. Estimation of Bacterial Cell Numbers in Humic Acid-Rich Salt Marsh Sediments with Probes Directed to 16S Ribosomal DNA

    PubMed Central

    Edgcomb, Virginia P.; McDonald, John H.; Devereux, Richard; Smith, David W.

    1999-01-01

    The feasibility of using probes directed towards ribosomal DNAs (rDNAs) as a quantitative approach to estimating cell numbers was examined and applied to study the structure of a bacterial community in humic acid-rich salt marsh sediments. Hybridizations were performed with membrane-bound nucleic acids by using seven group-specific DNA oligonucleotide probes complementary to 16S rRNA coding regions. These included a general eubacterial probe and probes encompassing most members of the gram-negative, mesophilic sulfate-reducing bacteria (SRB). DNA was extracted from sediment samples, and contaminating materials were removed by a series of steps. Efficiency of DNA extraction was 48% based on the recovery of tritiated plasmid DNA added to samples prior to extraction. Reproducibility of the extraction procedure was demonstrated by hybridizations to replicate samples. Numbers of target cells in samples were estimated by comparing the amount of hybridization to extracted DNA obtained with each probe to that obtained with a standard curve of genomic DNA for reference strains included on the same membrane. In June, numbers of SRB detected with an SRB-specific probe ranged from 6.0 × 107 to 2.5 × 109 (average, 1.1 × 109 ± 5.2 × 108) cells g of sediment−1. In September, numbers of SRB detected ranged from 5.4 × 108 to 7.3 × 109 (average, 2.5 × 109 ± 1.5 × 109) cells g of sediment−1. The capability of using rDNA probes to estimate cell numbers by hybridization to DNA extracted from complex matrices permits initiation of detailed studies on community composition and changes in communities based on cell numbers in formerly intractable environments. PMID:10103245

  2. DNA-Directed Assembly of Nanogold Dimers: A Unique Dynamic Light Scattering Sensing Probe for Transcription Factor Detection

    PubMed Central

    Seow, Nianjia; Tan, Yen Nee; Yung, Lin-Yue Lanry; Su, Xiaodi

    2015-01-01

    We have developed a unique DNA-assembled gold nanoparticles (AuNPs) dimer for dynamic light scattering (DLS) sensing of transcription factors, exemplified by estrogen receptor (ER) that binds specifically to a double-stranded (ds) DNA sequence containing estrogen response element (ERE). Here, ERE sequence is incorporated into the DNA linkers to bridge the AuNPs dimer for ER binding. Coupled with DLS, this AuNP dimer-based DLS detection system gave distinct readout of a single ‘complex peak’ in the presence of the target molecule (i.e., ER). This unique signature marked the first time that such nanostructures can be used to study transcription factor-DNA interactions, which DLS alone cannot do. This was also unlike previously reported AuNP-DLS assays that gave random and broad distribution of particles size upon target binding. In addition, the ERE-containing AuNP dimers could also suppress the light-scattering signal from the unbound proteins and other interfering factors (e.g., buffer background), and has potential for sensitive detection of target proteins in complex biological samples such as cell lysates. In short, the as-developed AuNP dimer probe coupled with DLS is a simple (mix and test), rapid (readout in ~5 min) and sensitive (low nM levels of ER) platform to detect sequence-specific protein-DNA binding event. PMID:26678946

  3. DNA-Directed Assembly of Nanogold Dimers: A Unique Dynamic Light Scattering Sensing Probe for Transcription Factor Detection

    NASA Astrophysics Data System (ADS)

    Seow, Nianjia; Tan, Yen Nee; Yung, Lin-Yue Lanry; Su, Xiaodi

    2015-12-01

    We have developed a unique DNA-assembled gold nanoparticles (AuNPs) dimer for dynamic light scattering (DLS) sensing of transcription factors, exemplified by estrogen receptor (ER) that binds specifically to a double-stranded (ds) DNA sequence containing estrogen response element (ERE). Here, ERE sequence is incorporated into the DNA linkers to bridge the AuNPs dimer for ER binding. Coupled with DLS, this AuNP dimer-based DLS detection system gave distinct readout of a single ‘complex peak’ in the presence of the target molecule (i.e., ER). This unique signature marked the first time that such nanostructures can be used to study transcription factor-DNA interactions, which DLS alone cannot do. This was also unlike previously reported AuNP-DLS assays that gave random and broad distribution of particles size upon target binding. In addition, the ERE-containing AuNP dimers could also suppress the light-scattering signal from the unbound proteins and other interfering factors (e.g., buffer background), and has potential for sensitive detection of target proteins in complex biological samples such as cell lysates. In short, the as-developed AuNP dimer probe coupled with DLS is a simple (mix and test), rapid (readout in ~5 min) and sensitive (low nM levels of ER) platform to detect sequence-specific protein-DNA binding event.

  4. Probing the kinetic landscape of Hox transcription factor-DNA binding in live cells by massively parallel Fluorescence Correlation Spectroscopy.

    PubMed

    Papadopoulos, Dimitrios K; Krmpot, Aleksandar J; Nikolić, Stanko N; Krautz, Robert; Terenius, Lars; Tomancak, Pavel; Rigler, Rudolf; Gehring, Walter J; Vukojević, Vladana

    2015-11-01

    Hox genes encode transcription factors that control the formation of body structures, segment-specifically along the anterior-posterior axis of metazoans. Hox transcription factors bind nuclear DNA pervasively and regulate a plethora of target genes, deploying various molecular mechanisms that depend on the developmental and cellular context. To analyze quantitatively the dynamics of their DNA-binding behavior we have used confocal laser scanning microscopy (CLSM), single-point fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS) and bimolecular fluorescence complementation (BiFC). We show that the Hox transcription factor Sex combs reduced (Scr) forms dimers that strongly associate with its specific fork head binding site (fkh250) in live salivary gland cell nuclei. In contrast, dimers of a constitutively inactive, phospho-mimicking variant of Scr show weak, non-specific DNA-binding. Our studies reveal that nuclear dynamics of Scr is complex, exhibiting a changing landscape of interactions that is difficult to characterize by probing one point at a time. Therefore, we also provide mechanistic evidence using massively parallel FCS (mpFCS). We found that Scr dimers are predominantly formed on the DNA and are equally abundant at the chromosomes and an introduced multimeric fkh250 binding-site, indicating different mobilities, presumably reflecting transient binding with different affinities on the DNA. Our proof-of-principle results emphasize the advantages of mpFCS for quantitative characterization of fast dynamic processes in live cells.

  5. Isolates of viral hemorrhagic septicemia virus from North America and Europe can be detected and distinguished by DNA probes

    USGS Publications Warehouse

    Batts, W.N.; Arakawa, C.K.; Bernard, J.; Winton, J.R.

    1993-01-01

    Biotinylated DNA probes were constructed to hybndize with speclfic sequences within the messenger RNA (mRNA) of the nucleoprotein (N) gene of vlral hemorrhagic septicemia virus (VHSV) reference strains from Europe (07-71) and North Arnenca (Makah) Probes were synthesized that were complementary to (1) a 29-nucleotide sequence near the center of the N gene conlmon to both the 07-71 and Makah reference strains of the virus (2) a unique 28- nucleotide sequence that followed the open readng frame of the Makah N gene mRNA most of which was absent In the 07-71 strain, and (3) a 22-nucleobde sequence wthin the 07-71 N gene that had 6 nllsmatches \

  6. The ODN probes conjugating the Cu(II) complex enhance the luminol chemiluminescence by assembling on the DNA template.

    PubMed

    Taniguchi, Yosuke; Nitta, Akiko; Park, Sun Min; Kohara, Akiko; Uzu, Takahiro; Sasaki, Shigeki

    2010-12-15

    Potent peroxidase-like activity of the β-ketoenamine (1)-dicopper (II) complex (2) for the chemiluminescence (CL) of luminol either in the presence or absence of H(2)O(2) has been previously demonstrated by our group. In this study, the β-ketoenamine (1) as the ligand unit for copper(II) was incorporated into the oligonucleotide (ODN) probes. It has been shown that the catalytic activity of the ODN probes conjugating the ligand-Cu(II) complex is activated by hybridization with the target DNA with the complementary sequence. Thus, this study has successfully demonstrated the basic concept for the sensitive detection of nucleic acids by CL based on the template-inductive activation of the catalytic unit for CL.

  7. Methods of staining target chromosomal DNA employing high complexity nucleic acid probes

    DOEpatents

    Gray, Joe W.; Pinkel, Daniel; Kallioniemi, Ol'li-Pekka; Kallioniemi, Anne; Sakamoto, Masaru

    2006-10-03

    Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyses. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods and reagents are provided for the detection of genetic rearrangements. Probes and test kits are provided for use in detecting genetic rearrangements, particularly for use in tumor cytogenetics, in the detection of disease related loci, specifically cancer, such as chronic myelogenous leukemia (CML), retinoblastoma, ovarian and uterine cancers, and for biological dosimetry. Methods and reagents are described for cytogenetic research, for the differentiation of cytogenetically similar but genetically different diseases, and for many prognostic and diagnostic applications.

  8. Isolation and characterization of species-specific DNA probes from Taenia solium and Taenia saginata and their use in an egg detection assay.

    PubMed

    Chapman, A; Vallejo, V; Mossie, K G; Ortiz, D; Agabian, N; Flisser, A

    1995-05-01

    Cysticercosis results from ingestion of the eggs of the tapeworm Taenia solium. Reduction of the incidence of human and swine cysticercosis requires identification and treatment of individuals who carry the adult tapeworm. T. solium and Taenia saginata eggs cannot be differentiated on the basis of morphology; thus, in order to improve existing methods for the diagnosis of taeniasis, we have developed highly sensitive, species-specific DNA probes which differentiate T. solium and T. saginata. Recombinant clones containing repetitive DNA sequences which hybridize specifically with genomic DNAs from either species were isolated and characterized. T. solium-specific DNA sequences contained complete and truncated forms of a tandemly repeated 158-bp DNA sequence. An unrelated T. saginata DNA sequence was also characterized and shown to encode a portion of the mitochondrial cytochrome c oxidase I gene. T. solium- and T. saginata-specific DNA probes did not hybridize in dot blot assays either with genomic DNA from the platyhelminths Taenia hydatigena, Taenia pisiformis, Taenia taeniaeformis, Echinococcus granulosus, and Schistosoma mansoni or with genomic DNA from other eukaryotes, including Saccharomyces cerevisiae, Candida albicans, Cryptosporidium parvum, Entamoeba histolytica, Trypanosoma gambiense, Trypanosoma brucei, and Giardia lamblia, Caenorhabditis elegans, and human DNA. By using these T. solium and T. saginata DNA probes, a rapid, highly sensitive and specific dot blot assay for the detection of T. solium eggs was developed.

  9. DNA double helix unwinding triggers transcription block-dependent apoptosis: a semiquantitative probe of the response of ATM, RNAPII, and p53 to two DNA intercalators.

    PubMed

    Zhang, Zhichao; Wang, Yuanyuan; Song, Ting; Gao, Jin; Wu, Guiye; Zhang, Jing; Qian, Xuhong

    2009-03-16

    We have previously shown the binding modes of two DNA interacting analogues (1)a {3-(4-methyl-piperazin)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile} and (3)a {3-(3-dimethylamino-propylamino)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile} with the DNA double helix. In this study, we have determined the notably different DNA damage signal pathway elicited by (1)a and (3)a due to the different extents to which they unwind the DNA double helix. First, we have identified that ataxia-telangiectasia-mutated (ATM) protein kinase can respond to DNA double helix unwinding caused by both (1)a and (3)a. In addition, the amount of ATM activation is consistent with the degree to which the DNA double helix was unwound. Consequently, we used (1)a and (3)a to semiquantitatively probe the response of RNA polymerase II (RNAPII) and p53 toward DNA double helix unwinding in vivo. By means of flow cytometry, immunocytochemistry, ChIP, quantitative real-time polymerase chain reaction, and Western blot analyses, we measured the level of p53 and RNAPII phosphorylation, in addition to the dynamics of the RNAPII distribution along the c-Myc gene. These results provided novel evidence for the impact of subtle DNA structural changes on the activity of RNAPII and p53. Moreover, DNA double helix conformational damage-dependent apoptosis was studied for the first time. These results indicated that (1)a can induce transcriptional blockage following a shift of the unphosphorylated IIa form of RNAPII to the phosphorylated IIo form, while (3)a is unable to induce the same effect. Subsequently, p53 accumulation and phosphorylation events occur that lead to apoptosis in the case of (1)a exposure. This suggests that the transcriptional blockage is also correlated to the degree of double helix unwinding. Furthermore, we found that the degree of DNA conformational damage determines whether or not apoptosis occurs through transcriptional blockage. Under our experimental conditions, ATM does not

  10. Dynamics of the binding of acridine dyes to DNA investigated by triplet excited state probe techniques

    SciTech Connect

    Geacintov, N.E.; Waldmeyer, J.; Kuzmin, V.A.; Kolubayev, T.

    1981-11-26

    The binding of the polynuclear aromatic dyes acridine orange (AO) and proflavin (PF) to DNA in aqueous phosphate buffer solution at 25 +- 1/sup 0/C has been studied by measuring the properties of the triplet excited states of these dyes. The triplet lifetimes can be measured either by triplet-triplet absorption flash photolysis techniques or by delayed fluorescence methods. The triplet lifetimes of AO vary from about 0.5 ms with no DNA present to 20 to 35 ms at DNA concentration above 10/sup -3/M expressed in concentration of DNA phosphate (P), or at (P)/(D) ratios above 1000 ((D) is the dye concentration). At all DNA concentrations the decay profiles are exponential, except at high excitation intensities where nonexponentialities, attributed to triplet-triplet annihilation, become apparent. Similar results are observed with PF-DNA solutions. The exponentiality of the triplet decay at all DNA concentrations is attributed to rapid association and dissociation of the dye-DNA complexes on the time scales of the triplet lifetimes. A simplified one-step binding model is utilized to describe this effect. A dissociation rate of AO-DNA complexes greater than or equal to 10/sup 3/s/sup -1/ has been estimated from these results. It is shown that a detailed study of the triplet lifetime vs. DNA concentration provides a novel method for the estimation of the apparent equilibrium association constant K* for dye molecules in the triplet excited state and DNA. For AO, K* approx. = 10/sup 5/ M/sup -1/, while for PF it is approx. = 3 x 10/sup 4/ M/sup -1/. These values are of the same order of magnitude as the ground-state dye-DNA equilibrium association constants measured by others.

  11. Identification of PCR-amplified genetically modified organisms (GMOs) DNA by peptide nucleic acid (PNA) probes in anion-exchange chromatographic analysis.

    PubMed

    Rossi, Stefano; Lesignoli, Francesca; Germini, Andrea; Faccini, Andrea; Sforza, Stefano; Corradini, Roberto; Marchelli, Rosangela

    2007-04-04

    PCR products obtained by selective amplification of transgenic DNA derived from food samples containing Roundup Ready soybean or Bt-176 maize have been analyzed by anion-exchange HPLC. Peptide nucleic acids (PNAs), oligonucleotide analogues known to bind to complementary single-stranded DNA with high affinity and specificity, have been used as specific probes in order to assess the identity of the peaks observed. Two different protocols were adopted in order to obtain single-stranded DNA: amplification with an excess of one primer or digestion of one DNA strand. The single-stranded DNA was mixed with the PNA probe, and the presence of a specific sequence was revealed through detection of the corresponding PNA:DNA peak with significantly different retention time. Advantages and limits of this approach are discussed. The method was tested with reference materials and subsequently applied to commercial samples.

  12. Evaluation of binding selectivity of a polyamide probe to single base-pair different DNA in A.T-rich region by electrospray ionization mass spectrometry.

    PubMed

    Li, Huihui; Yuan, Gu

    2006-12-01

    In this study, electrospray ionization mass spectrometry (ESI-MS) was used for the evaluation of the binding selectivity of a polyamide probe to single-base pair different DNA in an A.T-rich region. In this procedure, DeltaIr(dsn) was introduced as a parameter to compare the binding affinities of the polyamides with the duplex DNA. The results show that ESI-MS is a very useful tool for analysis of binding selectivity of a polyamide probe to single-base pair different DNA.

  13. Detecting variants with Metabolic Design, a new software tool to design probes for explorative functional DNA microarray development

    PubMed Central

    2010-01-01

    Background Microorganisms display vast diversity, and each one has its own set of genes, cell components and metabolic reactions. To assess their huge unexploited metabolic potential in different ecosystems, we need high throughput tools, such as functional microarrays, that allow the simultaneous analysis of thousands of genes. However, most classical functional microarrays use specific probes that monitor only known sequences, and so fail to cover the full microbial gene diversity present in complex environments. We have thus developed an algorithm, implemented in the user-friendly program Metabolic Design, to design efficient explorative probes. Results First we have validated our approach by studying eight enzymes involved in the degradation of polycyclic aromatic hydrocarbons from the model strain Sphingomonas paucimobilis sp. EPA505 using a designed microarray of 8,048 probes. As expected, microarray assays identified the targeted set of genes induced during biodegradation kinetics experiments with various pollutants. We have then confirmed the identity of these new genes by sequencing, and corroborated the quantitative discrimination of our microarray by quantitative real-time PCR. Finally, we have assessed metabolic capacities of microbial communities in soil contaminated with aromatic hydrocarbons. Results show that our probe design (sensitivity and explorative quality) can be used to study a complex environment efficiently. Conclusions We successfully use our microarray to detect gene expression encoding enzymes involved in polycyclic aromatic hydrocarbon degradation for the model strain. In addition, DNA microarray experiments performed on soil polluted by organic pollutants without prior sequence assumptions demonstrate high specificity and sensitivity for gene detection. Metabolic Design is thus a powerful, efficient tool that can be used to design explorative probes and monitor metabolic pathways in complex environments, and it may also be used to

  14. Non-Natural Nucleotides as Probes for the Mechanism and Fidelity of DNA Polymerases

    PubMed Central

    Lee, Irene; Berdis, Anthony J.

    2009-01-01

    DNA is a remarkable macromolecule that functions primarily as the carrier of the genetic information of organisms ranging from viruses to bacteria to eukaryotes. The ability of DNA polymerases to efficiently and accurately replicate genetic material represents one of the most fundamental yet complex biological processes found in nature. The central dogma of DNA polymerization is that the efficiency and fidelity of this biological process is dependent upon proper hydrogen-bonding interactions between an incoming nucleotide and its templating partner. However, the foundation of this dogma has been recently challenged by the demonstration that DNA polymerases can effectively and, in some cases, selectively incorporate non-natural nucleotides lacking classic hydrogen-bonding capabilities into DNA. In this review, we describe the results of several laboratories that have employed a variety of non-natural nucleotide analogs to decipher the molecular mechanism of DNA polymerization. The use of various non-natural nucleotides has lead to the development of several different models that can explain how efficient DNA synthesis can occur in the absence of hydrogen-bonding interactions. These models include the influence of steric fit and shape complementarity, hydrophobicity and solvation energies, base-stacking capabilities, and negative selection as alternatives to rules invoking simple recognition of hydrogen bonding patterns. Discussions are also provided regarding how the kinetics of primer extension and exonuclease proofreading activities associated with high-fidelity DNA polymerases are influenced by the absence of hydrogen-bonding functional groups exhibited by non-natural nucleotides. PMID:19733263

  15. Hybridization probes for conventional DNA fingerprinting used as single primers in the polymerase chain reaction to distinguish strains of Cryptococcus neoformans.

    PubMed Central

    Meyer, W; Mitchell, T G; Freedman, E Z; Vilgalys, R

    1993-01-01

    In conventional DNA fingerprinting, hypervariable and repetitive sequences (minisatellite or microsatellite DNA) are detected with hybridization probes. As demonstrated here, these probes can be used as single primers in the polymerase chain reaction (PCR) to generate individual fingerprints. Several conventional DNA fingerprinting probes were used to prime the PCR, yielding distinctive, hypervariable multifragment profiles for different strains of Cryptococcus neoformans. PCR fingerprinting with the oligonucleotide primers (GTG)5, (GACA)4, and the phage M13 core sequence (GAGGGTGGXGGXTCT), but not with (CA)8 or (CT)8, generated DNA polymorphisms with all 42 strains of C. neoformans investigated. PCR fingerprints produced by priming with (GTG)5, (GACA)4, or the M13 core sequence differentiated the two varieties of C. neoformans, C. neoformans var. neoformans (serotypes A and D) and C. neoformans var. gattii (serotypes B and C). Furthermore, strains of serotypes A, D, and B or C could be distinguished from each other by specific PCR fingerprint patterns. These primers, which also successfully amplified hypervariable DNA segments from other species, provide a convenient method of identification at the species or individual level. Amplification of polymorphic DNA patterns by PCR with these primers offers several advantages over classical DNA fingerprinting techniques, appears to be more reliable than other PCR-based methods for detecting polymorphic DNA, such as analysis of random-amplified polymorphic DNA, and should be applicable to many other organisms. Images PMID:8408543

  16. A new system for the amplification of biological signals: RecA and complimentary single strand DNA probes on a leaky surface acoustic wave biosensor.

    PubMed

    Zhang, Liqun; Wang, Yunxia; Chen, Ming; Luo, Yang; Deng, Kun; Chen, Dong; Fu, Weiling

    2014-10-15

    This research describes a new amplification signals system of the leaky surface acoustic wave (LSAW) bis-peptide nucleic acid (bis-PNA) biosensor for the simple, sensitive and rapid detection of the target double-stranded DNA (dsDNA). The system consists of a RecA protein-coated complementary single-stranded DNA (cssDNA) probe complex that amplifies the biological signal to improve the sensitivity of the biosensor. The bis-PNA probe for detecting HPV was first immobilized on a gold surface membrane of the detection channel. After the probe was completely hybridized with the corresponding target DNA, different concentrations of the "RecA protein-complementary single strand DNA probe" were added to react with the bis-PNA/dsDNA complex. The phase shift of the LSAW biosensors, which was measured and found to be most significant when the RecA protein was 45 μg/mL and the ATPγS was 2.5 mmol/L. Compared with other concentrations (P<0.01) of RecA and ATPγS, the value of the phase shift was (11.74 ± 1.03) degrees and the ratio of the phase shift and hybridization time clearly outperformed that of the other concentrations. Compared to the direct hybridization of the bis-PNA probe and the target DNA sequence, the sensitivity was effectively improved and the detection time was significantly shortened. PNA binding adjacent to the area of the target sequence homologous to the probe significantly increased the yield of the hybridization reaction between the PNA/dsDNA complex and the RecA protein-coated cssDNA probe. In this condition, the phase shift was significantly obvious and the detection time was significantly shortened. In conclusion, the combination of the RecA protein-coated cssDNA probe and the LSAW bis-PNA biosensor provides sensitivity and simple and rapid detection of clinical trace pathogenic microorganisms.

  17. A lifetime-sensitive fluorescence anisotropy probe for DNA-based bioassays: The case of SYBR Green.

    PubMed

    Chovelon, Benoit; Fiore, Emmanuelle; Faure, Patrice; Peyrin, Eric; Ravelet, Corinne

    2017-04-15

    In standard steady-state fluorescence anisotropy (FA) DNA-based assays, the ligand binding to a given receptor is typically signalled by the rotational correlation time changes of the tracer. Herein, we report a radically different strategy that relies on the peculiar excited state lifetime features of the SYBR Green (SG) dye. This DNA-binding probe exhibits a drastically short lifetime in solution, leading to a high FA signal. Its complexation to oligonucleotides determines a singular and very large depolarization depending on the concerted effects of extreme lifetime enhancement and resonance energy homotransfer. On the basis of ligand-induced changes in the molar fractions of bound and free forms of SG, the approach provides an unprecedented means for the FA monitoring of the ligand binding to short DNA molecules, allowing the elaboration of a variety of intercalator displacement assays and label-free biosensors that involve diverse DNA structures (duplex, hairpin, G-quadruplex and single-stranded), ligand types (ion, small organic molecule and protein) and binding modes (intercalation, minor groove, allosteric switch). These findings open up promising avenues in the design of a new generation of FA assays.

  18. Non-Equilibrium DNA Dynamics Probed by Delayed Capture and Recapture by a Solid-State Nanopore

    NASA Astrophysics Data System (ADS)

    Mihovilovic, Mirna; Teich, Erin; Hagerty, Nicholas; Stein, Derek

    2012-02-01

    We studied the relaxation of λ-DNA following its translocation through a voltage-biased solid-state nanopore. The translocation process drives DNA into a non-equilibrium state because the ˜2 ms translocation time is roughly fifty times shorter that the polymer's characteristic (Zimm) relaxation time. By reversing the applied voltage at controlled delay times after a translocation event, the nanopore probed the configurations of recaptured molecules at various stages of relaxation. We monitored the disruptions of the ionic current through the nanopore and computed the integrated charge deficits (ECDs) resulting from DNA translocations. As the delay time between voltage reversals was decreased from 50 ms to 5 ms, the distribution of ECDs shifted to lower values. Furthermore, an increasing fraction of recapture events occurred in a shorter interval from the voltage reversal than the delay time. These observations are explained by the expansion of the DNA coil as it approaches equilibrium. Finally, we show that recapturing a molecule multiple times and averaging the ECDs reduces the measurement error, which is useful for molecular diagnostic applications. The variance decreases approximately as the inverse number of passes through the pore.

  19. 16S rDNA-based probes for two polycyclic aromatic hydrocarbon (PAH)-degrading soil Mycobacteria

    SciTech Connect

    Govindaswami, M.; Feldhake, D.J.; Loper, J.C.

    1994-12-31

    PAHs are a class of widespread pollutants, some of which have been shown to be genotoxic, hence the fate of these compounds in the environment is of considerable interest. Research on the biodegradation of 4 and 5 ring PAHs has been limited by the general lack of microbial isolates or consortia which can completely degrade these toxicants. Heitkamp and Cerniglia have described an oxidative soil Mycobacterium-strain PYR-1 that metabolizes pyrene and fluoranthene more rapidly than the 2 and 3 ring naphthalene and phenanthrene; although some metabolites of benzo-(a)-pyrene (BaP) were detected, no mineralization of BaP was observed. In 1991 Grosser et al. reported the isolation of a Mycobacterium sp. which mineralizes pyrene and also causing some mineralization of BaP. Their study describes a comparative analysis of these two strains, which show very similar colony morphology, growth rate and yellow-orange pigmentation. Genetic differences were shown by DNA amplification fingerprinting (DAF) using two arbitrary GC-rich octanucleotide primers, and by sequence comparison of PCR amplified 16S rDNA, although both strains show similarity closest to that of the genus Mycobacteria. These 16S rDNA sequences are in use for the construction of strain-specific DNA probes to monitor the presence, survival and growth of these isolates in PAH-contaminated soils in studies of biodegradation.

  20. Hybridization study of developmental plastid gene expression in mustard (Sinapsis alba L.) with cloned probes for most plastid DNA regions.

    PubMed

    Link, G

    1984-07-01

    An approach to assess the extent of developmental gene expression of various regions of plastid (pt)DNA in mustard (Sinapis alba L.) is described. It involves cloning of most ptDNA regions. The cloned regions then serve as hybridization probes to detect and assess the abundance of complementary RNA sequences represented in total plastid RNA. By comparison of the hybridization pattern observed with plastid RNA from either dark-grown or light-grown plants it was found that many ptDNA regions are constitutively expressed, while several 'inducible' regions account for much higher transcript levels in the chloroplast than in the etioplast stage. The reverse situation, i.e. 'repressed' regions which would account for higher transcript levels in the etioplast, was not observed. The hybridization results obtained with RNA from 'intermediatetype' plastids suggest that transient gene expression is a common feature during light-induced chloroplast development. The time-course of gene expression differs for various ptDNA regions.

  1. Absorption spectroscopic probe to investigate the interaction between Nd(III) and calf-thymus DNA

    NASA Astrophysics Data System (ADS)

    Devi, Ch. Victory; Singh, N. Rajmuhon

    2011-03-01

    The interaction between Nd(III) and Calf Thymus DNA (CT-DNA) in physiological buffer (pH 7.4) has been studied using absorption spectroscopy involving 4f-4f transition spectra in different aquated organic solvents. Complexation with CT-DNA is indicated by the changes in absorption intensity following the subsequent changes in the oscillator strengths of different 4f-4f bands and Judd-Ofelt intensity ( Tλ) parameters. The other spectral parameters namely Slator-Condon ( Fk's), nephelauxetic effect ( β), bonding ( b1/2) and percent covalency ( δ) parameters are computed to correlate with the binding of Nd(III) with DNA. The absorption spectra of Nd(III) exhibited hyperchromism and red shift in the presence of DNA. The binding constant, Kb has been determined by absorption measurement. The relative viscosity of DNA decreased with the addition of Nd(III). Thermodynamic parameters have been calculated according to relevant absorption data and Van't Hoff equation. The characterisation of bonding mode has been studied in detail. The results suggested that the major interaction mode between Nd(III) and DNA was external electrostatic binding.

  2. Electrochemical DNA Biosensor Based on a Tetrahedral Nanostructure Probe for the Detection of Avian Influenza A (H7N9) Virus.

    PubMed

    Dong, Shibiao; Zhao, Rongtao; Zhu, Jiangong; Lu, Xiao; Li, Yang; Qiu, Shaofu; Jia, Leili; Jiao, Xiong; Song, Shiping; Fan, Chunhai; Hao, RongZhang; Song, HongBin

    2015-04-29

    A DNA tetrahedral nanostructure-based electrochemical biosensor was developed to detect avian influenza A (H7N9) virus through recognizing a fragment of the hemagglutinin gene sequence. The DNA tetrahedral probe was immobilized onto a gold electrode surface based on self-assembly between three thiolated nucleotide sequences and a longer nucleotide sequence containing complementary DNA to hybridize with the target single-stranded (ss)DNA. The captured target sequence was hybridized with a biotinylated-ssDNA oligonucleotide as a detection probe, and then avidin-horseradish peroxidase was introduced to produce an amperometric signal through the interaction with 3,3',5,5'-tetramethylbenzidine substrate. The target ssDNA was obtained by asymmetric polymerase chain reaction (PCR) of the cDNA template, reversely transcribed from the viral lysate of influenza A (H7N9) virus in throat swabs. The results showed that this electrochemical biosensor could specifically recognize the target DNA fragment of influenza A (H7N9) virus from other types of influenza viruses, such as influenza A (H1N1) and (H3N2) viruses, and even from single-base mismatches of oligonucleotides. Its detection limit could reach a magnitude of 100 fM for target nucleotide sequences. Moreover, the cycle number of the asymmetric PCR could be reduced below three with the electrochemical biosensor still distinguishing the target sequence from the negative control. To the best of our knowledge, this is the first report of the detection of target DNA from clinical samples using a tetrahedral DNA probe functionalized electrochemical biosensor. It displays that the DNA tetrahedra has a great potential application as a probe of the electrochemical biosensor to detect avian influenza A (H7N9) virus and other pathogens at the gene level, which will potentially aid the prevention and control of the disease caused by such pathogens.

  3. DNA flexibility on short length scales probed by atomic force microscopy.

    PubMed

    Mazur, Alexey K; Maaloum, Mounir

    2014-02-14

    Unusually high bending flexibility has been recently reported for DNA on short length scales. We use atomic force microscopy (AFM) in solution to obtain a direct estimate of DNA bending statistics for scales down to one helical turn. It appears that DNA behaves as a Gaussian chain and is well described by the wormlike chain model at length scales beyond 3 helical turns (10.5 nm). Below this threshold, the AFM data exhibit growing noise because of experimental limitations. This noise may hide small deviations from the Gaussian behavior, but they can hardly be significant.

  4. A reagentless DNA-based electrochemical silver(I) sensor for real time detection of Ag(I) - the effect of probe sequence and orientation on sensor response.

    PubMed

    Wu, Yao; Lai, Rebecca Y

    2016-06-01

    Ag(I) is known to interact with cytosine (C) via the formation C-Ag(I)-C complexes. The authors have utilized this concept to design six electrochemical Ag(I) sensors using C-rich DNA probes. Alternating current voltammetry and cyclic voltammetry were used to analyze the sensors. The results show that the dual-probe sensors that require the use of both 5'- and 3'-thiolated DNA probes are not suitable for this application, the differences in probe orientation impedes formation of C-Ag(I)-C complexes. Sensors fabricated with DNA probes containing both thymine (T) and C, independent of the location of the alkanethiol linker, do not response to Ag(I) either; T-T mismatches destabilize the duplex even in the presence of Ag(I). However, sensors fabricated with DNA probes containing both adenine (A) and C are ideal for this application, owing to the formation of C-Ag(I)-C complexes, as well as other lesser known interactions between A and Ag(I). Both sensors are sensitive, specific and selective enough to be used in 50% human saliva. They can also be used to detect silver sulfadiazine, a commonly prescribed antimicrobial drug. With further optimization, this sensing strategy may offer a promising approach for detection of Ag(I) in environmental and clinical samples.

  5. Strain-specific differentiation of lactococci in mixed starter culture populations using randomly amplified polymorphic DNA-derived probes.

    PubMed Central

    Erlandson, K; Batt, C A

    1997-01-01

    A hydrophobic grid membrane filtration (HGMF) colony hybridization assay was developed that allows strain-specific differentiation of defined bacterial populations. The randomly amplified polymorphic DNA (RAPD) fingerprinting technique was used to identify potential signature nucleic acid sequences unique to each member of a commercial cheese starter culture blend. The blend consisted of two closely related Lactococcus lactis subsp. cremoris strains, 160 and 331, and one L. lactis subsp. lactis strain, 210. Three RAPD primers (OPX 1, OPX 12, and OPX 15) generated a total of 32 products from these isolates, 20 of which were potential strain-specific markers. Southern hybridization analyses revealed, that the RAPD-generated signature sequences OPX15-0.95 and a 0.36-kb HaeIII fragment of OPX1-1.0b were specific for strains 331 and 210, respectively, within the context of the test starter culture blend. These strain-specific probes were used in a HGMF colony hybridization assay. Colony lysis, hybridization, and nonradioactive detection parameters were optimized to allow specific differentiation and quantitation of the target strains in the mixed starter culture population. When the 210 and 331 probes were tested at their optimal hybridization temperatures against single cultures, they detected 100% of the target strain CFUs, without cross-reactivity to the other strains. The probes for strains 210 and 331 also successfully detected their targets in blended cultures even with a high background of the other two strains. PMID:9212417

  6. Strain-specific differentiation of lactococci in mixed starter culture populations using randomly amplified polymorphic DNA-derived probes.

    PubMed

    Erlandson, K; Batt, C A

    1997-07-01

    A hydrophobic grid membrane filtration (HGMF) colony hybridization assay was developed that allows strain-specific differentiation of defined bacterial populations. The randomly amplified polymorphic DNA (RAPD) fingerprinting technique was used to identify potential signature nucleic acid sequences unique to each member of a commercial cheese starter culture blend. The blend consisted of two closely related Lactococcus lactis subsp. cremoris strains, 160 and 331, and one L. lactis subsp. lactis strain, 210. Three RAPD primers (OPX 1, OPX 12, and OPX 15) generated a total of 32 products from these isolates, 20 of which were potential strain-specific markers. Southern hybridization analyses revealed, that the RAPD-generated signature sequences OPX15-0.95 and a 0.36-kb HaeIII fragment of OPX1-1.0b were specific for strains 331 and 210, respectively, within the context of the test starter culture blend. These strain-specific probes were used in a HGMF colony hybridization assay. Colony lysis, hybridization, and nonradioactive detection parameters were optimized to allow specific differentiation and quantitation of the target strains in the mixed starter culture population. When the 210 and 331 probes were tested at their optimal hybridization temperatures against single cultures, they detected 100% of the target strain CFUs, without cross-reactivity to the other strains. The probes for strains 210 and 331 also successfully detected their targets in blended cultures even with a high background of the other two strains.

  7. Time-Resolved Sequence Analysis on High Density Fiberoptic DNA Probe

    SciTech Connect

    Walt, D. R.; Lee, K-H

    2002-11-19

    A universal array format has been developed in which all possible n-mers of a particular oligonucleotide sequence can be represented. The ability to determine the sequence of the probes at every position in the array should enable unbiased gene expression as well as arrays for de novo sequencing.

  8. Role of the external NH2 linker on the conformation of surface immobilized single strand DNA probes and their SERS detection

    NASA Astrophysics Data System (ADS)

    He, Lijie; Langlet, Michel; Stambouli, Valerie

    2017-03-01

    The conformation and topological properties of DNA single strand probe molecules attached on solid surfaces are important, notably for the performances of devices such as biosensors. Commonly, the DNA probes are tethered to the surface using external linkers such as NH2. In this study, the role and influence of this amino-linker on the immobilization way and conformation of DNA probes on Ag nanoparticle surface is emphasized using Surface Enhanced Raman Spectroscopy (SERS). We compare the SERS spectra and their reproducibility in the case of two groups of DNA polybase probes which are polyA, polyC, polyT, and polyG. In the first group, the polybases exhibit an external NH2 functional linker while in the second group the polybases are NH2-free. The results show that the reproducibility of SERS spectra is enhanced in the case of the first group. It leads us to propose two models of polybase conformation on Ag surface according to the presence or the absence of the external NH2 linker. In the presence of the NH2 external linker, the latter would act as a major anchoring point. As a result, the polybases are much ordered with a less random orientation than in the case of NH2-free polybases. Consequently, in view of further in situ hybridization for biosensing applications, it is strongly recommended to use NH2 linker functionalized DNA probes.

  9. Detection and interrogation of biomolecules via nanoscale probes: From fundamental physics to DNA sequencing

    NASA Astrophysics Data System (ADS)

    Zwolak, Michael

    2013-03-01

    A rapid and low-cost method to sequence DNA would revolutionize personalized medicine, where genetic information is used to diagnose, treat, and prevent diseases. There is a longstanding interest in nanopores as a platform for rapid interrogation of single DNA molecules. I will discuss a sequencing protocol based on the measurement of transverse electronic currents during the translocation of single-stranded DNA through nanopores. Using molecular dynamics simulations coupled to quantum mechanical calculations of the tunneling current, I will show that the DNA nucleotides are predicted to have distinguishable electronic signatures in experimentally realizable systems. Several recent experiments support our theoretical predictions. In addition to their possible impact in medicine and biology, the above methods offer ideal test beds to study open scientific issues in the relatively unexplored area at the interface between solids, liquids, and biomolecules at the nanometer length scale. http://mike.zwolak.org

  10. Intercalation between antitumor anthracyclines and DNA as probed by resonance and surface-enhanced Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Smulevich, G.; Mantini, A. R.; Casu, M.; Marzocchi, M. P.

    1991-05-01

    The antiturnor anthracyclincs, idarubicin (IDA ), adrianiycin (ADM), epirubicin (EPI), carminomycin (CAR) and 1 1-deoxycarminornycin (DCM), whose siructural formula includes a substituted hydroxyanthraquirionc chrornophore and a sugar residue, form intercalation complexes with DNA. The stacking interaction between the chromophore and the base-pairs of DNA gives rise to noticeable ciTects on resonance Raman (RR) and surface-enhanced resonance Raman (SERRS) scattering as well as on the absorption (ABS), its second derivative (D2) and fluorescence emission (FEM) spectra.

  11. DNA mechanics as a tool to probe helicase and translocase activity.

    PubMed

    Lionnet, Timothée; Dawid, Alexandre; Bigot, Sarah; Barre, François-Xavier; Saleh, Omar A; Heslot, François; Allemand, Jean-François; Bensimon, David; Croquette, Vincent

    2006-01-01

    Helicases and translocases are proteins that use the energy derived from ATP hydrolysis to move along or pump nucleic acid substrates. Single molecule manipulation has proved to be a powerful tool to investigate the mechanochemistry of these motors. Here we first describe the basic mechanical properties of DNA unraveled by single molecule manipulation techniques. Then we demonstrate how the knowledge of these properties has been used to design single molecule assays to address the enzymatic mechanisms of different translocases. We report on four single molecule manipulation systems addressing the mechanism of different helicases using specifically designed DNA substrates: UvrD enzyme activity detection on a stretched nicked DNA molecule, HCV NS3 helicase unwinding of a RNA hairpin under tension, the observation of RecBCD helicase/nuclease forward and backward motion, and T7 gp4 helicase mediated opening of a synthetic DNA replication fork. We then discuss experiments on two dsDNA translocases: the RuvAB motor studied on its natural substrate, the Holliday junction, and the chromosome-segregation motor FtsK, showing its unusual coupling to DNA supercoiling.

  12. Highly sensitive detection of human papillomavirus type 16 DNA using time-resolved fluorescence microscopy and long lifetime probes

    NASA Astrophysics Data System (ADS)

    Wang, Xue F.; Periasamy, Ammasi; Wodnicki, Pawel; Siadat-Pajouh, M.; Herman, Brian

    1995-04-01

    We have been interested in the role of Human Papillomavirus (HPV) in cervical cancer and its diagnosis; to that end we have been developing microscopic imaging and fluorescent in situ hybridization (FISH) techniques to genotype and quantitate the amount of HPV present at a single cell level in cervical PAP smears. However, we have found that low levels of HPV DNA are difficult to detect accurately because theoretically obtainable sensitivity is never achieved due to nonspecific autofluorescence, fixative induced fluorescence of cells and tissues, and autofluorescence of the optical components in the microscopic system. In addition, the absorption stains used for PAP smears are intensely autofluorescent. Autofluorescence is a rapidly decaying process with lifetimes in the range of 1-100 nsec, whereas phosphorescence and delayed fluorescence have lifetimes in the range of 1 microsecond(s) ec-10 msec. The ability to discriminate between specific fluorescence and autofluorescence in the time-domain has improved the sensitivity of diagnostic test such that they perform comparably to, or even more sensitive than radioisotopic assays. We have developed a novel time-resolved fluorescence microscope to improve the sensitivity of detection of specific molecules of interest in slide based specimens. This time-resolved fluorescence microscope is based on our recently developed fluorescence lifetime imaging microscopy (FILM) in conjunction with the use of long lifetime fluorescent labels. By using fluorescence in situ hybridization and the long lifetime probe (europium), we have demonstrated the utility of this technique for detection of HPV DNA in cervicovaginal cells. Our results indicate that the use of time-resolved fluorescence microscopy and long lifetime probes increases the sensitivity of detection by removing autofluorescence and will thus lead to improved early diagnosis of cervical cancer. Since the highly sensitive detection of DNA in clinical samples using

  13. G-quadruplex formation in double strand DNA probed by NMM and CV fluorescence.

    PubMed

    Kreig, Alex; Calvert, Jacob; Sanoica, Janet; Cullum, Emily; Tipanna, Ramreddy; Myong, Sua

    2015-09-18

    G-quadruplexes (GQs) are alternative DNA secondary structures that can form throughout the human genome and control the replication and transcription of important regulatory genes. Here, we established an ensemble fluorescence assay by employing two GQ-interacting compounds, N-methyl mesoporphyrin IX (NMM) and Crystal Violet (CV). This enables quantitative measurement of the GQ folding propensity and conformation specificity in both single strand (ss) and double strand (ds) DNA. Our GQ mapping indicates that the likelihood of GQ formation is substantially diminished in dsDNA, likely due to the competition from the Watson-Crick base pairing. Unlike GQ folding sequence in ssDNA which forms both parallel and antiparallel GQs, dsDNA displays only parallel folding. Additionally, we employed single molecule FRET to obtain a direct quantitation of stably formed-, weakly folded and unfolded GQ conformations. The findings of this study and the method developed here will enable identifying and classifying potential GQ-forming sequences in human genome.

  14. Influence of the oxazole ring connection on the fluorescence of oxazoyl-triphenylamine biphotonic DNA probes.

    PubMed

    Dumat, Blaise; Faurel-Paul, Elodie; Fornarelli, Pauline; Saettel, Nicolas; Metgé, Germain; Fiorini-Debuisschert, Céline; Charra, Fabrice; Mahuteau-Betzer, Florence; Teulade-Fichou, Marie-Paule

    2016-01-07

    On the basis of our previous work on DNA fluorophores derived from vinylpyridinium-triphenylamine, we explored the structure space around the electron-rich triphenylamine (TP) core by changing the vinyl bond to an oxazole ring. As 2,5-diaryloxazoles are known to be highly fluorescent and efficient two photon absorbers, we synthesized analogues with two different connections of the oxazole to the triphenylamine core: TP-Ox2Py and TP-Ox5Py sets. Since the benzimidazolium group was proven to be more effective in the TP series than the pyridinium, we also synthesized a TP-Ox5Bzim set. The TP-Ox5Py series retains the TP-Py properties: on/off behavior on DNA, good two-photon cross-section and bright staining of nuclear DNA by microscopy under both one or two-photon excitation. On the other hand, the TP-Ox2Py series does not display fluorescence upon binding to DNA. The TP-Ox5Bzim set is fluorescent even in the absence of DNA and displays lower affinity than the corresponding TP-Ox5Py. CD experiments and docking were performed to understand these different behaviors.

  15. Small molecule probes finely differentiate between various ds- and ss-DNA and RNA by fluorescence, CD and NMR response.

    PubMed

    Crnolatac, Ivo; Rogan, Iva; Majić, Boris; Tomić, Sanja; Deligeorgiev, Todor; Horvat, Gordan; Makuc, Damjan; Plavec, Janez; Pescitelli, Gennaro; Piantanida, Ivo

    2016-10-12

    Two small molecules showed intriguing properties of analytical multipurpose probes, whereby one chromophore gives different signal for many different DNA/RNA by application of several highly sensitive spectroscopic methods. Dyes revealed pronounced fluorescence ratiomeric differentiation between ds-AU-RNA, AT-DNA and GC-DNA in approximate order 10:8:1. Particularly interesting, dyes showed specific fluorimetric response for poly rA even at 10-fold excess of any other ss-RNA, and moreover such emission selectivity is preserved in multicomponent ss-RNA mixtures. The dyes also showed specific chiral recognition of poly rU in respect to the other ss-RNA by induced CD (ICD) pattern in visible range (400-500 nm), which was attributed to the dye-side-chain contribution to binding (confirmed by absence of any ICD band for reference compound lacking side-chain). Most intriguingly, minor difference in the side-chain attached to dye chromophore resulted in opposite sign of dye-ICD pattern, whereby differences in NMR NOESY contacts and proton chemical shifts between two dye/oligo rU complexes combined with MD simulations and CD calculations attributed observed bisignate ICD to the dimeric dye aggregate within oligo rU.

  16. Identification of Benzo[a]pyrene-Metabolizing Bacteria in Forest Soils by Using DNA-Based Stable-Isotope Probing

    PubMed Central

    Song, Mengke; Jiang, Longfei; Zhang, Dayi; Wang, Yujie; Zhang, Gan

    2015-01-01

    DNA-based stable-isotope probing (DNA-SIP) was used in this study to investigate the uncultivated bacteria with benzo[a]pyrene (BaP) metabolism capacities in two Chinese forest soils (Mt. Maoer in Heilongjiang Province and Mt. Baicaowa in Hubei Province). We characterized three different phylotypes with responsibility for BaP degradation, none of which were previously reported as BaP-degrading microorganisms by SIP. In Mt. Maoer soil microcosms, the putative BaP degraders were classified as belonging to the genus Terrimonas (family Chitinophagaceae, order Sphingobacteriales), whereas Burkholderia spp. were the key BaP degraders in Mt. Baicaowa soils. The addition of metabolic salicylate significantly increased BaP degradation efficiency in Mt. Maoer soils, and the BaP-metabolizing bacteria shifted to the microorganisms in the family Oxalobacteraceae (genus unclassified). Meanwhile, salicylate addition did not change either BaP degradation or putative BaP degraders in Mt. Baicaowa. Polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHD) genes were amplified, sequenced, and quantified in the DNA-SIP 13C heavy fraction to further confirm the BaP metabolism. By illuminating the microbial diversity and salicylate additive effects on BaP degradation across different soils, the results increased our understanding of BaP natural attenuation and provided a possible approach to enhance the bioremediation of BaP-contaminated soils. PMID:26253666

  17. [Scanning for KRAS, NRAS, BRAF, and PIK3CA mutations by DNA melting analysis with TaqMan probes].

    PubMed

    Botezatu, I V; Panchuk, I O; Stroganova, A M; Senderovich, A I; Kondratova, V N; Shelepov, V P; Lichtenstein, A V

    2017-01-01

    Scanning for mutations by DNA melting analysis (DMA) is based on asymmetric PCR followed by the melting of duplexes formed by single-stranded amplicons with TaqMan probes. The method is optimally suited for clinical genetic testing; it is easy to perform, high-throughput, and sensitive. The detection limit of mutant alleles by the DMA method is about 3%, which is much higher than the sensitivity of Sanger sequencing. In addition, the DMA method is realized in a closed-tube format, while 2-h assay is carried out in a single tube without any intermediate or additional procedures thereby minimizing the risk of cross contamination of the samples. The validation of the DMA method was performed by scanning for mutations of clinically significant genes KRAS, NRAS, BRAF, and   PIK3CA in 324 DNA samples from tumors of patients with melanoma, colorectal and lung cancer. DNA was isolated either directly from tumor tissues, or from formalin-fixed paraffin-embedded tumor tissues. The detected mutations were verified by Sanger sequencing. The spectra of mutations identified in each tumor type correspond to the literature data and, thus, validate the use of DMA.

  18. Near IR-emitting DNA-probes exploiting stepwise energy transfer processes.

    PubMed

    Bodi, Andras; Borbas, K Eszter; Bruce, James I

    2007-10-10

    The synthesis and characterisation of two new cyclen-based near IR-emitting lanthanide complexes is reported; the lanthanides are sensitised by rhodamine, which in turn is excited by energy transfer from a coumarin 2 moiety. The three lumophores function as an energy transfer cascade spanning the UV-visible-near IR region of the spectrum, resulting in large Stokes shifts. Double stranded DNA selectively switches one of the two energy transfer processes off, enabling luminescent DNA-sensing in the near IR region. The regioselective di-alkylation of the cyclen scaffold is explained with the help of DFT calculations.

  19. An Ultrasensitive High Throughput Screen for DNA Methyltransferase 1-Targeted Molecular Probes

    PubMed Central

    Fagan, Rebecca L.; Wu, Meng; Chédin, Frédéric; Brenner, Charles

    2013-01-01

    DNA methyltransferase 1 (DNMT1) is the enzyme most responsible for epigenetic modification of human DNA and the intended target of approved cancer drugs such as 5-aza-cytidine and 5-aza-2′-deoxycytidine. 5-aza nucleosides have complex mechanisms of action that require incorporation into DNA, and covalent trapping and proteolysis of DNMT isozymes. Direct DNMT inhibitors are needed to refine understanding of the role of specific DNMT isozymes in cancer etiology and, potentially, to improve cancer prevention and treatment. Here, we developed a high throughput pipeline for identification of direct DNMT1 inhibitors. The components of this screen include an activated form of DNMT1, a restriction enzyme-coupled fluorigenic assay performed in 384 well plates with a z-factor of 0.66, a counter screen against the restriction enzyme, a screen to eliminate DNA intercalators, and a differential scanning fluorimetry assay to validate direct binders. Using the Microsource Spectrum collection of 2320 compounds, this screen identified nine compounds with dose responses ranging from 300 nM to 11 µM, representing at least two different pharmacophores with DNMT1 inhibitory activity. Seven of nine inhibitors identified exhibited two to four-fold selectivity for DNMT1 versus DNMT3A. PMID:24236046

  20. A Multiplexed, Probe-Based Quantitative PCR Assay for DNA of Phytophthora sojae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phytophthora sojae (Kaufm. & Gerd.) causes seed rot, pre- and post-emergence damping off, and sometimes foliar blight in soybean (Glycine max). Crop loss may approach 100% with susceptible cultivars. We report here the development of a unique quantitative PCR assay specific to DNA of P. sojae, and a...

  1. Probing the Absorption and Emission Transition Dipole Moment of DNA Stabilized Silver Nanoclusters.

    PubMed

    Hooley, Emma N; Carro-Temboury, Miguel R; Vosch, Tom

    2017-02-09

    Using single molecule polarization measurements, we investigate the excitation and emission polarization characteristics of DNA stabilized silver nanoclusters (C24-AgNCs). Although small changes in the polarization generally accompany changes to the emission spectrum, the emission and excitation transition dipoles tend to be steady over time and aligned in a similar direction, when immobilized in PVA. The emission transition dipole patterns, observed for C24-AgNCs in defocused wide field imaging, match that of a single emitter. The small changes to the polarization and spectral shifting that were observed could be due to changes to the conformation of the AgNC or the DNA scaffold. Although less likely, an alternative explanation could be that several well aligned spectrally similar emitters are present within the DNA scaffold which, due to Förster resonance energy transfer (FRET) processes such as energy hopping, energy transfer, and singlet-singlet annihilation, behave as a single emitter. The reported results can provide more insight in the structural and photophysical properties of DNA-stabilized AgNCs.

  2. A COMPARISON OF DNA DAMAGE PROBES IN TWO HMEC LINES WITH X-IRRADIATION

    SciTech Connect

    Wisnewski, C.L.; Bjornstad, K.A.; Rosen, C.J.; Chang, P.Y.; Blakely, E.A.

    2007-01-01

    In this study, we investigated γH2AXser139 and 53BP1ser25, DNA damage pathway markers, to observe responses to radiation insult. Two Human Mammary Epithelial Cell (HMEC) lines were utilized to research the role of immortalization in DNA damage marker expression, HMEC HMT-3522 (S1) with an infi nite lifespan, and a subtype of HMEC 184 (184V) with a fi nite lifespan. Cells were irradiated with 50cGy X-rays, fi xed with 4% paraformaldehyde after 1 hour repair at 37°C, and processed through immunofl uorescence. Cells were visualized with a fl uorescent microscope and images were digitally captured using Image-Pro Plus software. The 184V irradiated cells exhibited a more positive punctate response within the nucleus for both DNA damage markers compared to the S1 irradiated cells. The dose and time course will be expanded in future studies to augment the preliminary data from this research. It is important to understand whether the process of transformation to immortalization compromises the DNA damage sensor and repair process proteins of HMECs in order to understand what is “normal” and to evaluate the usefulness of cell lines as experimental models.

  3. A comparison of DNA damage probes in two HMEC lines withX-irradiation

    SciTech Connect

    Wisnewski, Christy L.; Bjornstad, Kathleen A.; Rosen, ChristoperJ.; Chang, Polly Y.; Blakely, Eleanor A.

    2007-01-19

    In this study, we investigated {gamma}H2AX{sup ser139} and 53BP1{sup ser25}, DNA damage pathway markers, to observe responses to radiation insult. Two Human Mammary Epithelial Cell (HMEC) lines were utilized to research the role of immortalization in DNA damage marker expression, HMEC HMT-3522 (S1) with an infinite lifespan, and a subtype of HMEC 184 (184V) with a finite lifespan. Cells were irradiated with 50 cGy X-rays, fixed with 4% paraformaldehyde after 1 hour repair at 37 C, and processed through immunofluorescence. Cells were visualized with a fluorescent microscope and images were digitally captured using Image-Pro Plus software. The 184V irradiated cells exhibited a more positive punctate response within the nucleus for both DNA damage markers compared to the S1 irradiated cells. We will expand the dose and time course in future studies to augment the preliminary data from this research. It is important to understand whether the process of transformation to immortalization compromises the DNA damage sensor and repair process proteins of HMECs in order to understand what is 'normal' and to evaluate the usefulness of cell lines as experimental models.

  4. Probing the mechanism of recognition of ssDNA by the Cdc13-DBD.

    PubMed

    Eldridge, Aimee M; Wuttke, Deborah S

    2008-03-01

    The Saccharomyces cerevisiae protein Cdc13 tightly and specifically binds the conserved G-rich single-stranded overhang at telomeres and plays an essential role in telomere end-protection and length regulation. The 200 residue DNA-binding domain of Cdc13 (Cdc13-DBD) binds an 11mer single-stranded representative of the yeast telomeric sequence [Tel11, d(GTGTGGGTGTG)] with a 3 pM affinity and specificity for three bases (underlined) at the 5' end. The structure of the Cdc13-DBD bound to Tel11 revealed a large, predominantly aromatic protein interface with several unusual features. The DNA adopts an irregular, extended structure, and the binding interface includes a long ( approximately 30 amino acids) structured loop between strands beta2-beta3 (L(2-3)) of an OB-fold. To investigate the mechanism of ssDNA binding, we studied the free and bound states of Cdc13-DBD using NMR spectroscopy. Chemical shift changes indicate that the basic topology of the domain, including L(2-3), is essentially intact in the free state. Changes in slow and intermediate time scale dynamics, however, occur in L(2-3), while conformational changes distant from the DNA interface suggest an induced fit mechanism for binding in the 'hot spot' for binding affinity and specificity. These data point to an overall binding mechanism well adapted to the heterogeneous nature of yeast telomeres.

  5. Evaluation of the biotinylated (Blugene) vs sup 32 P-labeled cDNA probes of beta-glucocerebrosidase: Relative sensitivities in genomic and other systems

    SciTech Connect

    Strasberg, P. )

    1989-07-01

    The sensitivity, rapidity, and ease of use of biotinylated (Blugene) and {sup 32}P cDNA probes have been compared, the probe being the cDNA for beta-glucocerebrosidase. With the Blugene kit I could detect 2 pg of biotinylated DNA on dot blots. However, under conditions of hybridization, the lower limit of detection for unlabeled cDNA (transblotted onto nitrocellulose) by its labeled counterpart was 5000-fold smaller (10 pg vs 50 ng) for the isotopically labeled probe. {sup 32}P- and Blugene-probes hybridized detectably with 0.5 and 10 micrograms, respectively, of transblotted EcoR 1-digested genomic DNA, making the radioactive method 20 times as sensitive. However, color development was complete within 30 min to 3 h, whereas radioautoradiography required 12 h to one week. Blugene was also safer, easy to use, and effective under appropriate conditions. The {sup 32}P method is expensive, hazardous, time-consuming, and technically difficult. This nonisotopic procedure represents a desirable improvement in biotechnology.

  6. Using DNA-Stable Isotope Probing to Identify MTBE- and TBA-Degrading Microorganisms in Contaminated Groundwater

    PubMed Central

    Key, Katherine C.; Sublette, Kerry L.; Duncan, Kathleen; Mackay, Douglas M.; Scow, Kate M.; Ogles, Dora

    2014-01-01

    Although the anaerobic biodegradation of methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA) has been documented in the laboratory and the field, knowledge of the microorganisms and mechanisms involved is still lacking. In this study, DNA-stable isotope probing (SIP) was used to identify microorganisms involved in anaerobic fuel oxygenate biodegradation in a sulfate-reducing MTBE and TBA plume. Microorganisms were collected in the field using Bio-Sep® beads amended with 13C5-MTBE, 13C1-MTBE (only methoxy carbon labeled), or13C4-TBA. 13C-DNA and 12C-DNA extracted from the Bio-Sep beads were cloned and 16S rRNA gene sequences were used to identify the indigenous microorganisms involved in degrading the methoxy group of MTBE and the tert-butyl group of MTBE and TBA. Results indicated that microorganisms were actively degrading 13C-labeled MTBE and TBA in situ and the 13C was incorporated into their DNA. Several sequences related to known MTBE- and TBA-degraders in the Burkholderiales and the Sphingomonadales orders were detected in all three13C clone libraries and were likely to be primary degraders at the site. Sequences related to sulfate-reducing bacteria and iron-reducers, such as Geobacter and Geothrix, were only detected in the clone libraries where MTBE and TBA were fully labeled with 13C, suggesting that they were involved in processing carbon from the tert-butyl group. Sequences similar to the Pseudomonas genus predominated in the clone library where only the methoxy carbon of MTBE was labeled with 13C. It is likely that members of this genus were secondary degraders cross-feeding on 13C-labeled metabolites such as acetate. PMID:25525320

  7. Detection of enterotoxigenic Clostridium perfringens in spices used in Mexico by dot blotting using a DNA probe.

    PubMed

    Rodríguez-Romo, L A; Heredia, N L; Labbé, R G; García-Alvarado, J S

    1998-02-01

    Several reports on the microbiology of spices and herbs indicate the presence of Clostridium perfringens, a spore-forming foodborne pathogen responsible for gastrointestinal disease. In the present study, a total of 380 samples of spices and herbs (cumin seed, black pepper, oregano, garlic powder, and bay leaves) widely used in Mexico were analyzed for the presence of C. perfringens, and the enterotoxigenicity of the isolates was determined by a dot-blot technique using an enterotoxin degoxigenin-labeled DNA probe. C. perfringens counts varied from <100 to 433 CFU/g in garlic powder, from <100 to 200 CFU/g in black pepper, from <100 to 433 CFU/g in cumin seed, from <100 to 340 CFU/g in oregano, and from < 100 to 450 CFU/g in bay leaves. The dot-blot technique detected the enterotoxin gene in 8 (4.25%) of 188 confirmed isolates of C. perfringens. dot-blot.

  8. Fluorescent C-linked C8-aryl-guanine probe for distinguishing syn from anti structures in duplex DNA.

    PubMed

    Manderville, Richard A; Omumi, Alireza; Rankin née Schlitt, Katherine M; Wilson, Katie A; Millen, Andrea L; Wetmore, Stacey D

    2012-06-18

    The synthesis and optical properties of the carbon (C)-linked C(8)-(2"-benzo[b]thienyl)-2'-deoxyguanosine ((Bth)dG), which acts as a fluorescent reporter of syn versus anti glycosidic conformations in duplex DNA, are described. In the syn-conformation, the probe stabilizes a G:G mismatch, emits at ∼385 nm (excitation ∼285 nm), and shows an induced circular dichroism (ICD) signal at ∼320 nm. Molecular dynamics (MD) simulations predict a wedge (W)-conformation for the mismatched duplex with the C(8)-benzo[b]thienyl moiety residing in the minor groove. In contrast, the probe destabilizes the duplex when base paired with its normal pyrimidine partner C. With flanking purine bases, a major groove B-type duplex is favored with (Bth)dG present in the anti-conformation emitting at ∼413 nm (excitation ∼326 nm) and no ICD signal. However, with flanking pyrimidine bases, (Bth)dG adopts the syn-conformation when base paired with C, and MD simulations predict a base-displaced stacked (S)-conformation, with the opposing C flipped out of the helix. The different duplex (B-, S-, and W-) conformers formed upon incorporation of (Bth)dG are known to play a critical role in the biological activity of N-linked C8-dG adducts formed by arylamine carcinogens. Bulky environment-sensitive fluorescent C(8)-dG adducts that mimic the duplex structures formed by carcinogens may be useful in luminescence-based DNA polymerase assays.

  9. Use of a plasmid DNA probe to monitor populations of Bacillus pumilus inoculant strains in hay

    SciTech Connect

    Hendrick, C.A.; Smiley, B.K.; Shelley, T.H.; Tomes, N.J. )

    1991-03-01

    The authors are evaluating naturally occurring isolates of Bacillus pumilus for use as microbial hay preservatives. Seven isolates of B, pumilus from hay contained a 42-kb cryptic plasmid (pMGD296). They wished to determine whether pMGD296 could be used as a molecular marker to follow populations of these isolates in hay over time. Southern blots and colony blots of 69 isolates of B. pumilus and other Bacillus spp. were probed with {sup 32}P-labeled pMGD296. Twenty-nine probe-positive isolates were identified; of these, 28 contained a plasmid with a restriction profile identical to that of pMGD296. One isolate from untreated hay contained a 40-kb plasmid (pMGD150) that was homologous to pMGD296 but had a different restriction fragment pattern. Regions of homology between the two plasmids were identified by Southern blotting, and a 1.9-kb HindIII-PstI fragment of pMGD296 lacking strong homology to pMGD150 was cloned in pUC18. The cloned fragment hybridized only with isolates containing pMGD296 and was used to estimate populations of these isolates in treated and untreated hay.

  10. Focused upon hybridization: rapid and high sensitivity detection of DNA using isotachophoresis and peptide nucleic acid probes.

    PubMed

    Ostromohov, Nadya; Schwartz, Ortal; Bercovici, Moran

    2015-09-15

    We present a novel assay for rapid and high sensitivity detection of nucleic acids without amplification. Utilizing the neutral backbone of peptide nucleic acids (PNA), our method is based on the design of low electrophoretic mobility PNA probes, which do not focus under isotachophoresis (ITP) unless bound to their target sequence. Thus, background noise associated with free probes is entirely eliminated, significantly improving the signal-to-noise ratio while maintaining a simple single-step assay requiring no amplification steps. We provide a detailed analytical model and experimentally demonstrate the ability to detect targets as short as 17 nucleotides (nt) and a limit of detection of 100 fM with a dynamic range of 5 decades. We also demonstrate that the assay can be successfully implemented for detection of DNA in human serum without loss of signal. The assay requires 15 min to complete, and it could potentially be used in applications where rapid and highly sensitive amplification-free detection of nucleic acids is desired.

  11. TaqMan probe real-time polymerase chain reaction assay for the quantification of canine DNA in chicken nugget.

    PubMed

    Rahman, Md Mahfujur; Hamid, Sharifah Bee Abd; Basirun, Wan Jefrey; Bhassu, Subha; Rashid, Nur Raifana Abdul; Mustafa, Shuhaimi; Mohd Desa, Mohd Nasir; Ali, Md Eaqub

    2016-01-01

    This paper describes a short-amplicon-based TaqMan probe quantitative real-time PCR (qPCR) assay for the quantitative detection of canine meat in chicken nuggets, which are very popular across the world, including Malaysia. The assay targeted a 100-bp fragment of canine cytb gene using a canine-specific primer and TaqMan probe. Specificity against 10 different animals and plants species demonstrated threshold cycles (Ct) of 16.13 ± 0.12 to 16.25 ± 0.23 for canine DNA and negative results for the others in a 40-cycle reaction. The assay was tested for the quantification of up to 0.01% canine meat in deliberately spiked chicken nuggets with 99.7% PCR efficiency and 0.995 correlation coefficient. The analysis of the actual and qPCR predicted values showed a high recovery rate (from 87% ± 28% to 112% ± 19%) with a linear regression close to unity (R(2) = 0.999). Finally, samples of three halal-branded commercial chicken nuggets collected from different Malaysian outlets were screened for canine meat, but no contamination was demonstrated.

  12. Probing the structure of long DNA molecules in solution using synchrotron radiation linear dichroism.

    PubMed

    Rittman, Martyn; Hoffmann, Søren V; Gilroy, Emma; Hicks, Matthew R; Finkenstadt, Bärbel; Rodger, Alison

    2012-01-07

    Linear dichroism (LD), a spectroscopic method for aligned samples, has been used with a synchrotron radiation source to reveal insights into the structure and stability of DNA with increasing salt concentrations (thus stabilizing the base pairing) and increasing temperature while remaining below the melting point (thus destabilizing the base pairing). Measurements have been made from 350 nm to 182 nm, and the spectral changes observed quantified using a Bayesian Markov chain Monte Carlo (MCMC) algorithm, which uses statistical methods to fit to experimental data. Based on literature H-D exchange experiments, we surmise that the cause of the spectral variations is the induction of transient single stranding of tracts in the DNA polymer, particularly those with significant content of the weaker AT base pairs. More detailed analysis of the LD data will require better nucleotide transition polarization assignments.

  13. A DNA-templated silver nanocluster probe for label-free, turn-on fluorescence-based screening of homo-adenine binding molecules.

    PubMed

    Park, Ki Soo; Park, Hyun Gyu

    2015-02-15

    A novel, label-free, turn-on fluorescence strategy to detect molecules that bind to adenine-rich DNA sequences has been developed. The probe employs DNA-templated silver nanoclusters (DNA-AgNCs) as the key detection component. The new strategy relies on the formation of non-Watson-Crick homo-adenine DNA duplex, triggered by strong interactions with homo-adenine binding molecules, which brings a guanine-rich sequence in one strand close to DNA-AgNCs located on the opposite strand. This phenomenon transforms weakly fluorescent AgNCs into highly emissive species that display bright red fluorescence. Finally, we have shown that the new fluorescence turn-on strategy can be employed to detect coralyne, the most representative homo-adenine binding molecule that triggers formation of a non-Watson-Crick homo-adenine DNA duplex.

  14. Please do not disturb: Destruction of chromatin structure by supravital nucleic acid probes revealed by a novel assay of DNA-histone interaction

    PubMed Central

    Wlodkowic, Donald; Darzynkiewicz, Zbigniew

    2008-01-01

    The biomarkers designed to be used supravitally are expected to have minimal effect on structure and function of the cell. Unfortunately nearly all fluorochromes developed to probe live cells interact in undesired way with cellular constituents and affect functional pathways. Herein we comment on potential applications of diverse DNA binding probes in view of the recent article by Wojcik & Dobrucki on DRAQ 5 and SYTO 17. The approach used by these authors to assess DNA-histone interactions using the cells having histones tagged with fluorescent proteins offers a valuable tool to study mechanism of action of antitumor drugs targeting DNA. While the effect of many intercalating drugs may be similar to that of DRAQ5, it may be of particular interest to observe the effects induced by intra-strand and inter-strand DNA crosslinking drugs, alkylating agents, histone deacetylase inhibitors or even anti-metabolites. The cells having histones tagged with fluorescent proteins thus may serve as biomarkers to probe mechanism of action of drugs targeting DNA or affecting chromatin structure. In fact, because such gross chromatin changes as revealed by dissociation and segregation of histones from DNA are most likely incompatible with long-term cell survival, the methodology may be applied for rapid screening of investigational antitumor agents. PMID:18671237

  15. Identification of human DNA in forensic evidence by loop-mediated isothermal amplification combined with a colorimetric gold nanoparticle hybridization probe.

    PubMed

    Watthanapanpituck, Khanistha; Kiatpathomchai, Wansika; Chu, Eric; Panvisavas, Nathinee

    2014-11-01

    A DNA test based on loop-mediated isothermal amplification (LAMP) and colorimetric gold nanoparticle (AuNP) hybridization probe to detect the presence of human DNA in forensic evidence was developed. The LAMP primer set targeted eight regions of the human cytochrome b, and its specificity was verified against the DNA of 11 animal species, which included animals closely related to humans, such as chimpanzee and orangutan. By using the AuNP probe, sequence-specific LAMP product could be detected and the test result could be visualized through the change in color. The limit of detection was demonstrated with reproducibility to be as low as 718 fg of genomic DNA, which is equivalent to approximately 100 plasmid DNA copies containing the cytochrome b DNA target region. A simple DNA extraction method for the commonly found forensic biological samples was also devised to streamline the test process. This LAMP-AuNP human DNA test showed to be a robust, specific, and cost-effective tool for the forensic identification of human specimens without requiring sophisticated laboratory instruments.

  16. Induction of DNA Damage Response by the Supravital Probes of Nucleic Acids

    PubMed Central

    Zhao, Hong; Traganos, Frank; Dobrucki, Jurek; Wlodkowic, Donald; Darzynkiewicz, Zbigniew

    2009-01-01

    The aim of this study was to assess the potential DNA damage response (DDR) to four supravitally used biomarkers Hoechst 33342 (Ho 42), DRAQ5, DyeCycle Violet (DCV) and SYTO 17. A549 cells were exposed to these biomarkers at concentrations generally applied to live cells and their effect on histone H2AX (Ser 139), p53 (Ser15), ATM (Ser1981) and Chk2 (Thr68) phosphorylation was assessed using phospho-specific Abs. Short-term treatment with Ho 42 led to modest degree of ATM activation with no evidence of H2AX, Chk2 or p53 phosphorylation. However, pronounced ATM, Chk2 and p53 phosphorylation and perturbed G2 progression were seen after 18 h. While short-term treatment with DRAQ5 induced ATM activation with no effect on H2AX, Chk2 and p53, dramatic changes marked by a high degree of H2AX, ATM, Chk2 and p53 phosphorylation, all occurring predominantly in S phase cells, and a block in cell cycle progression, were seen after 18 h exposure. These changes suggest that the DRAQ5-induced DNA lesions may become converted into double-strand DNA breaks during replication. Exposure to DCV also led to an increase in the level of activated ATM and Chk2 as well as of phosphorylated p53 and accumulation of cells in G2M and S phase. Exposure to SYTO 17 had no significant effect on any of the measured parameters. The data indicate that supravital use of Ho 42, DRAQ5 and DCV induces various degrees of DDR, including activation of ATM, Chk2 and p53, which may have significant consequences on regulatory cell cycle pathways and apoptosis. PMID:19373929

  17. DNA and protein change in tissues probed by Kubelka-Munk spectral function

    NASA Astrophysics Data System (ADS)

    Yang, Yuanlong; Celmer, Edward J.; Koutcher, Jason A.; Alfano, Robert R.

    2000-04-01

    Normal, fibroadenoma, malignant, and adipose breast tissues were investigated using Kubelka-Munk Spectral Function (KMSF). The spectral features in KMSF were identified and compared with absorption spectra determined by transmission measurements. A specified spectral feature measured in adipose tissue was assigned to (beta) -carotene, which can be used to separate fat form other molecular components in breast tissues. The peaks of (KMF) at 260nm and 280nm were attributed to DNA and proteins. The signal amplitude over 255nm to 265nm and 275nm to 285nm were found to be different for malignant fibroadenoma, and normal tissues.

  18. Electrochemiluminescence Biosensor Based on 3-D DNA Nanomachine Signal Probe Powered by Protein-Aptamer Binding Complex for Ultrasensitive Mucin 1 Detection.

    PubMed

    Jiang, Xinya; Wang, Haijun; Wang, Huijun; Zhuo, Ying; Yuan, Ruo; Chai, Yaqin

    2017-04-04

    Herein, we fabricated a novel electrochemiluminescence (ECL) biosensor for ultrasensitive detection of mucin 1 (MUC1) based on a three-dimensional (3-D) DNA nanomachine signal probe powered by protein-aptamer binding complex. The assembly of 3-D DNA nanomachine signal probe achieved the cyclic reuse of target protein based on the protein-aptamer binding complex induced catalyzed hairpin assembly (CHA), which overcame the shortcoming of protein conversion with enzyme cleavage or polymerization in the traditional examination of protein. In addition, CoFe2O4, a mimic peroxidase, was used as the nanocarrier of the 3-D DNA nanomachine signal probe to catalyze the decomposition of coreactant H2O2 to generate numerous reactive hydroxyl radical OH(•) as the efficient accelerator of N-(aminobutyl)-N-(ethylisoluminol) (ABEI) ECL reaction to amplify the luminescence signal. Simultaneously, the assembly of 3-D DNA nanomachine signal probe was executed in solution, which led to abundant luminophore ABEI be immobilized around the CoFe2O4 surface with amplified ECL signal output since the CHA reaction was occurred unencumberedly in all directions under homogeneous environment. The prepared ECL biosensor showed a favorable linear response for MUC1 detection with a relatively low detection limit of 0.62 fg mL(-1). With excellent sensitivity, the strategy may provide an efficient method for clinical application, especially in trace protein determination.

  19. GENETIC VARIATION IN RED RASPBERRIES (RUBUS IDAEUS L.; ROSACEAE) FROM SITES DIFFERING IN ORGANIC POLLUTANTS COMPARED WITH SYNTHETIC TANDEM REPEAT DNA PROBES

    EPA Science Inventory

    Two synthetic tandem repetitive DNA probes were used to compare genetic variation at variable-number-tandem-repeat (VNTR) loci among Rubus idaeus L. var. strigosus (Michx.) Maxim. (Rosaceae) individuals sampled at eight sites contaminated by pollutants (N = 39) and eight adjacent...

  20. Probing the Influence of Amino Acids on Photoluminescence from Carbon Nanotubes Suspended with DNA.

    PubMed

    Kurnosov, N V; Leontiev, V S; Karachevtsev, V A

    2016-11-01

    The quantitative analysis of amino acid levels in the human organism is required for the early clinical diagnosis of a variety of diseases. In this work the influence of 13 amino acid doping on the photoluminescence (PL) from the semiconducting single-walled carbon nanotubes (SWNTs) suspended with single-stranded DNA (ssDNA) in water has been studied. Amino acid doping leads to the PL enhancement and the strongest increase was found after cysteine doping of the nanotube suspension while addition of other amino acids yielded the significantly smaller effect. The emphasis of cysteine molecules is attributed to presence of the reactive thiol group that turns cysteine into reducing agent that passivates the p-defects on the nanotube sidewall and increases the PL intensity. The reasons of PL enhancement after doping with other amino acids are discussed. The response of nanotube PL to cysteine addition depends on the nanotube aqueous suspension preparation with tip or bath sonication treatment. The enhancement of the emission from different nanotube species after cysteine doping was analyzed too. It was shown that the increase of the carbon nanotube PL at addition of cysteine allows successful monitoring of the cysteine concentration in aqueous solution in the range of 50-1000 μM.

  1. Tuning the cellular uptake properties of luminescent heterobimetallic iridium(III)-ruthenium(II) DNA imaging probes.

    PubMed

    Wragg, Ashley; Gill, Martin R; Turton, David; Adams, Harry; Roseveare, Thomas M; Smythe, Carl; Su, Xiaodi; Thomas, Jim A

    2014-10-20

    The synthesis of two new luminescent dinuclear Ir(III)-Ru(II) complexes containing tetrapyrido[3,2-a:2',3'-c:3'',2''-h:2''',3'''-j]phenazine (tpphz) as the bridging ligand is reported. Unlike many other complexes incorporating cyclometalated Ir(III) moieties, these complexes display good water solubility, allowing the first cell-based study on Ir(III)-Ru(II) bioprobes to be carried out. Photophysical studies indicate that emission from each complex is from a Ru(II) excited state and both complexes display significant in vitro DNA-binding affinities. Cellular studies show that each complex is rapidly internalised by HeLa cells, in which they function as luminescent nuclear DNA-imaging agents for confocal microscopy. Furthermore, the uptake and nuclear targeting properties of the complex incorporating cyclometalating 2-(4-fluorophenyl)pyridine ligands around its Ir(III) centre is enhanced in comparison to the non-fluorinated analogue, indicating that fluorination may provide a route to promote cell uptake of transition-metal bioprobes.

  2. Probing diversity in freshwater fishes from Mexico and Guatemala with DNA barcodes.

    PubMed

    Valdez-Moreno, M; Ivanova, N V; Elías-Gutiérrez, M; Contreras-Balderas, S; Hebert, P D N

    2009-02-01

    The freshwater fish fauna of Mexico and Guatemala is exceptionally diverse with >600 species, many endemic. In this study, patterns of sequence divergence were analysed in representatives of this fauna using cytochrome c oxidase subunit 1 (COI) DNA barcodes for 61 species in 36 genera. The average divergence among conspecific individuals was 0.45%, while congeneric taxa showed 5.1% divergence. Three species of Poblana, each occupying a different crater lake in the arid regions of Central Mexico, have had a controversial taxonomic history but are usually regarded as endemics to a single lake. They possess identical COI barcodes, suggesting a very recent history of isolation. Representatives of the Cichlidae, a complex and poorly understood family, were well discriminated by barcodes. Many species of Characidae seem to be young, with low divergence values (<2%), but nevertheless, clear barcode clusters were apparent in the Bramocharax-Astyanax complex. The symbranchid, Opisthernon aenigmaticum, has been regarded as a single species ranging from Guatemala to Mexico, but it includes two deeply divergent barcode lineages, one a possible new endemic species. Aside from these special cases, the results confirm that DNA barcodes will be highly effective in discriminating freshwater fishes from Central America and that a comprehensive analysis will provide new important insights for understanding diversity of this fauna.

  3. Screening and development of DNA aptamers as capture probes for colorimetric detection of patulin.

    PubMed

    Wu, Shijia; Duan, Nuo; Zhang, Weixiao; Zhao, Sen; Wang, Zhouping

    2016-09-01

    Patulin (PAT) is a kind of mycotoxin that has serious harmful impacts on both food quality and human health. A high-affinity ssDNA aptamer that specifically binds to patulin was generated using systemic evolution of ligands by exponential enrichment (SELEX) assisted by graphene oxide (GO). After 15 rounds of positive and negative selection, a highly enriched ssDNA pool was sequenced and the representative sequences were subjected to binding assays to evaluate their affinity and specificity. Of the eight aptamer candidates tested, the sequence PAT-11 bound to patulin with high affinity and excellent selectivity with a dissociation constant (Kd) of 21.83 ± 5.022 nM. The selected aptamer, PAT-11, was subsequently used as a recognition element to develop a detection method for patulin based on an enzyme-chromogenic substrate system. The colorimetric aptasensor exhibited a linear range from 50 to 2500 pg mL(-1), and the limit of detection was found to be 48 pg mL(-1). The results indicated that GO-SELEX technology was appropriate for the screening of aptamers against small-molecule toxins, offering a promising application for aptamer-based biosensors.

  4. Chromatin Properties of Regulatory DNA Probed by Manipulation of Transcription Factors

    PubMed Central

    Sharov, Alexei A.; Nishiyama, Akira; Qian, Yong; Dudekula, Dawood B.; Longo, Dan L.; Schlessinger, David

    2014-01-01

    Abstract Transcription factors (TFs) bind to DNA and regulate the transcription of nearby genes. However, only a small fraction of TF binding sites have such regulatory effects. Here we search for the predictors of functional binding sites by carrying out a systematic computational screening of a variety of contextual factors (histone modifications, nuclear lamin-bindings, and cofactor bindings). We used regression analysis to test if contextual factors are associated with upregulation or downregulation of neighboring genes following the induction or knockdown of the 9 TFs in mouse embryonic stem (ES) cells. Functional TF binding sites appeared to be either active (i.e., bound by P300, CHD7, mediator, cohesin, and SWI/SNF) or repressed (i.e., with H3K27me3 histone marks and bound by Polycomb factors). Active binding sites mediated the downregulation of nearby genes upon knocking down the activating TFs or inducing repressors. Repressed TF binding sites mediated the upregulation of nearby genes (e.g., poised developmental regulators) upon inducing TFs. In addition, repressed binding sites mediated repressive effects of TFs, identified by the downregulation of target genes after the induction of TFs or by the upregulation of target genes after the knockdown of TFs. The contextual factors associated with functions of DNA-bound TFs were used to improve the identification of candidate target genes regulated by TFs. PMID:24918633

  5. Key Structural Elements of Unsymmetrical Cyanine Dyes for Highly Sensitive Fluorescence Turn-On DNA Probes.

    PubMed

    Uno, Kakishi; Sasaki, Taeko; Sugimoto, Nagisa; Ito, Hideto; Nishihara, Taishi; Hagihara, Shinya; Higashiyama, Tetsuya; Sasaki, Narie; Sato, Yoshikatsu; Itami, Kenichiro

    2017-01-17

    Unsymmetrical cyanine dyes, such as thiazole orange, are useful for the detection of nucleic acids with fluorescence because they dramatically enhance the fluorescence upon binding to nucleic acids. Herein, we synthesized a series of unsymmetrical cyanine dyes and evaluated their fluorescence properties. A systematic structure-property relationship study has revealed that the dialkylamino group at the 2-position of quinoline in a series of unsymmetrical cyanine dyes plays a critical role in the fluorescence enhancement. Four newly designed unsymmetrical cyanine dyes showed negligible intrinsic fluorescence in the free state and strong fluorescence upon binding to double-stranded DNA (dsDNA) with a quantum yield of 0.53 to 0.90, which is 2 to 3 times higher than previous unsymmetrical cyanine dyes. A detailed analysis of the fluorescence lifetime revealed that the dialkylamino group at the 2-position of quinoline suppressed nonradiative decay in favor of increased fluorescence quantum yield. Moreover, these newly developed dyes were able to stain the nucleus specifically in fixed HeLa cells examined by using a confocal laser-scanning microscope.

  6. Quantitation of fetal DNA in maternal serum during the first trimester of pregnancy by the use of a DAZ repetitive probe.

    PubMed

    Stanghellini, I; Bertorelli, R; Capone, L; Mazza, V; Neri, C; Percesepe, A; Forabosco, A

    2006-09-01

    Cell-free fetal DNA in maternal plasma or serum is at present widely investigated as a source of fetal genetic material, both in studies of pregnancy-related disorders and in planning strategies for non-invasive prenatal diagnosis. Despite the number of trials already performed on the quantitation of fetal DNA, data about the amount of DNA at the beginning of pregnancy, in particular in the first trimester, remain limited. A new probe mapping on the deleted in azoospermia (DAZ) repetitive region of the Yq chromosome was designed for an early assessment of fetal DNA concentration in maternal serum. Among 57 pregnant women prospectively studied in their first trimester, fetal DNA was detected already by the 5th gestational week, with the analysis becoming reliable by the 8th week of gestation when a 100% accuracy in fetal sex determination was achieved. Moreover, in the three cases of pregnancy ending in fetal loss, the amount of fetal DNA apparently decreased before the abortion was diagnosed, whereas it consistently showed an increasing trend in normal pregnancies. Real-time PCR with the use of DAZ multilocus probe can efficiently quantitate free fetal DNA in the maternal serum at the beginning of pregnancy.

  7. DNA-based probes for flow cytometry analysis of endocytosis and recycling.

    PubMed

    Dumont, Claire; Czuba, Ewa; Chen, Moore; Villadangos, Jose A; Johnston, Angus P R; Mintern, Justine D

    2016-12-26

    The internalization of proteins plays a key role in cell development, cell signaling and immunity. We have previously developed a specific hybridization internalization probe (SHIP) to quantitate the internalization of proteins and particles into cells. Herein, we extend the utility of SHIP to examine both the endocytosis and recycling of surface receptors using flow cytometry. SHIP was used to monitor endocytosis of membrane-bound transferrin receptor (TFR) and its soluble ligand transferrin (TF). SHIP enabled measurements of the proportion of surface molecules internalized, the internalization kinetics and the proportion and rate of internalized molecules that recycle to the cell surface with time. Using this method, we have demonstrated the internalization and recycling of holo-TF and an antibody against the TFR behave differently. This assay therefore highlights the implications of receptor internalization and recycling, where the internalization of the receptor-antibody complex behaves differently to the receptor-ligand complex. In addition, we observe distinct internalization patterns for these molecules expressed by different subpopulations of primary cells. SHIP provides a convenient and high throughput technique for analysis of trafficking parameters for both cell surface receptors and their ligands.

  8. Electrochemical detection of DNA binding by tumor suppressor p53 protein using osmium-labeled oligonucleotide probes and catalytic hydrogen evolution at the mercury electrode.

    PubMed

    Němcová, Kateřina; Sebest, Peter; Havran, Luděk; Orság, Petr; Fojta, Miroslav; Pivoňková, Hana

    2014-09-01

    In this paper, we present an electrochemical DNA-protein interaction assay based on a combination of protein-specific immunoprecipitation at magnetic beads (MBIP) with application of oligonucleotide (ON) probes labeled with an electroactive oxoosmium complex (Os,bipy). We show that double-stranded ONs bearing a dT20 tail labeled with Os,bipy are specifically recognized by the tumor suppressor p53 protein according to the presence or absence of a specific binding site (p53CON) in the double-stranded segment. We demonstrate the applicability of the Os,bipy-labeled probes in titration as well as competition MBIP assays to evaluate p53 relative affinity to various sequence-specific or structurally distinct unlabeled DNA substrates upon modulation of the p53-DNA binding by monoclonal antibodies used for the immunoprecipitation. To detect the p53-bound osmium-labeled probes, we took advantage of a catalytic peak yielded by Os,bipy-modified DNA at the mercury-based electrodes, allowing facile determination of subnanogram quantities of the labeled oligonucleotides. Versatility of the electrochemical MBIP technique and its general applicability in studies of any DNA-binding protein is discussed.

  9. Differentiation of Moraxella nonliquefaciens, M. lacunata, and M. bovis by using multilocus enzyme electrophoresis and hybridization with pilin-specific DNA probes.

    PubMed Central

    Tønjum, T; Caugant, D A; Bøvre, K

    1992-01-01

    Genetic relationships among strains of Moraxella nonliquefaciens, M. lacunata, and M. bovis were studied by using multilocus enzyme electrophoresis and DNA-DNA hybridization. The 74 isolates analyzed for electrophoretic variation at 12 enzyme loci were assigned to 59 multilocus genotypes. The multilocus genotypes were grouped in four major clusters, one representing strains of M. nonliquefaciens, two representing strains of M. lacunata, and one comprising strains of M. bovis and the single strain of M. equi analyzed. DNA-DNA hybridization with total genomic probes also revealed four major distinctive entities that corresponded to those identified by multilocus enzyme electrophoresis. The two distinct clusters recognized among the M. lacunata strains apparently corresponded to the species previously designated M. lacunata and M. liquefaciens. Distinction of the four entities was improved by hybridization with polymerase chain reaction products of nonconserved parts of pilin genes as DNA probes. With these polymerase chain reaction probes, new isolates of M. nonliquefaciens, M. lacunata, M. liquefaciens, and M. bovis can be identified easily by hybridization. PMID:1452691

  10. Phylogenetic relationships of annual and perennial wild rice: probing by direct DNA sequencing.

    PubMed

    Barbier, P; Morishima, H; Ishihama, A

    1991-05-01

    The phylogenetic relationships between Asian wild rice strains were analyzed by direct sequencing of PCR-amplified DNA fragments. The sequence of three introns located in the phytochrome gene was determined for eight strains of the Asian wild rice, Oryza rufipogon, and one strain of the related African species, Oryza longistaminata. The number of nucleotide substitutions per site between various strains within a single species, O. rufipogon, ranged between 0.0017 and 0.0050, while those between two related species, O. rufipogon and O. longistaminate, were 0.043-0.049 (23-26 within 532 bp). Taken together with the sequence differences of the 10-kDa prolamin gene, a model is proposed for the phylogenetic relationships and evolutionary history of annuals and perennials within O. rufipogon.

  11. PFGE-resolved RFLP analysis and long range restriction mapping of the DNA of Arabidopsis thaliana using whole YAC clones as probes.

    PubMed Central

    Bancroft, I; Westphal, L; Schmidt, R; Dean, C

    1992-01-01

    The cleavage patterns of 23 rare-cutting restriction endonucleases (rcREs) on high molecular weight DNA, isolated from leaves of Arabidopsis thaliana (Arabidopsis), have been analysed using pulsed field gel electrophoresis (PFGE). The DNA digested with rcREs can be used for restriction fragment length polymorphism (RFLP) analysis. We show that RFLPs are more readily identified in restriction fragments that require resolution by PFGE than in smaller restriction fragments. Taking advantage of the low dispersed repetitive DNA content of the Arabidopsis genome, whole yeast artificial chromosomes (YACs) were used as probes to PFGE resolved genomic DNA. This enabled whole YAC clones to be used as RFLP markers and long range restriction maps to be constructed. These techniques should enhance the analysis of regions of the genome of Arabidopsis (and other organisms with low levels of dispersed repetitive DNA) that are the subject of chromosome walking strategies to isolate particular loci. Images PMID:1361981

  12. Differential furanose selection in the active sites of archaeal DNA polymerases probed by fixed-conformation nucleotide analogues

    PubMed Central

    Ketkar, Amit; Zafar, Maroof K.; Banerjee, Surajit; Marquez, Victor E.; Egli, Martin; Eoff, Robert L.

    2012-01-01

    DNA polymerases select for the incorporation of deoxyribonucleotide triphosphates (dNTPs) using amino acid side-chains that act as a “steric-gate” to bar improper incorporation of rNTPs. An additional factor in the selection of nucleotide substrates resides in the preferred geometry for the furanose moiety of the incoming nucleotide triphosphate. We have probed the role of sugar geometry during nucleotide selection by model DNA polymerases from Sulfolobus solfataricus using fixed conformation nucleotide analogues. North-methanocarba-dATP (N-MC-dATP) locks the central ring into a RNA-type (C2′-exo, North) conformation near a C3′-endo pucker and South-methanocarba-dATP (S-MC-dATP) locks the central ring system into a (C3′-exo, South) conformation near a C2′-endo pucker. Dpo4 preferentially inserts N-MC-dATP and in the crystal structure of Dpo4 in complex with N-MC-dAMP, the nucleotide analogue superimposes almost perfectly with Dpo4 bound to unmodified dATP. Biochemical assays indicate that the S. solfataricus B-family DNA polymerase Dpo1 can insert and extend from both N-MC-dATP and S-MC-dATP. In this respect, Dpo1 is unexpectedly more tolerant of substrate conformation than Dpo4. The crystal structure of Dpo4 bound to S-MC-dADP shows that poor incorporation of the Southern pucker by the Y-family polymerase results from a hydrogen bond between the 3′-OH group of the nucleotide analogue and the OH group of the steric gate residue, Tyr12, shifting the S-MC-dADP molecule away from the dNTP binding pocket and distorting the base pair at the primer-template junction. These results provide insights into substrate specificity of DNA polymerases, as well as molecular mechanisms that act as a barrier against insertion of rNTPs. PMID:23050956

  13. CEP3 and CEP17 DNA probe potential in the genetic diagnosis and prognostic prediction of esophageal squamous cell cancer

    PubMed Central

    NIYAZ, MADINIYAT; ABDURAHMAN, ABLAJAN; TURGHUN, ABDUGHENI; AWUT, IDIRIS

    2016-01-01

    The aim of the present study was to investigate the clinical application of molecular pathological diagnosis for the prognosis of Kazakh patients with esophageal squamous cell carcinoma (ESCC) using centromere enumeration probes (CEPs) for chromosomes 3 and 17. A total of 40 patients with ESCC that had received radical surgical treatment and 10 healthy control participants were enrolled in the present study. Touch preparations of fresh cancerous and normal tissues were completed and fluorescence in situ hybridization (FISH) was performed to count the copy numbers of CEP 3 and 17, and abnormalities were analyzed, in comparison with routine pathological diagnoses. FISH analysis demonstrated that abnormal copy numbers of CEP 3 and 17 (aneuploidy) were detected in all 40 patients with ESCC. CEP 3 and 17 polyploidy rates differed significantly between poorly differentiated, moderately differentiated and well-differentiated ESCC groups (P<0.05): Well-differentiated, 35.38 and 30.92%; moderately differentiated, 55.81 and 44.43%; and poorly differentiated, 76.26 and 71.90%, respectively. Furthermore, polyploidy rates were significantly increased in the group with lymph node metastasis, as compared with the group without (CEP 3, P=0.0001; CEP 17, P=0.012). Variations in the copy numbers of CEP 3 and 17 were demonstrated to be correlated with the level of differentiation and lymph node metastasis in patients with ESCC. Therefore, the present results indicate that DNA probes may be used to predict prognostic factors in patients with ESCC. Furthermore, FISH technology is an objective and qualitative method that is capable of detecting variations in CEP 3 and 17; therefore, FISH may be used in the genetic diagnosis of ESCC in Kazakh patients. PMID:27073452

  14. Probing deactivation pathways of DNA nucleobases by two-dimensional electronic spectroscopy: first principles simulations.

    PubMed

    Nenov, Artur; Segarra-Martí, Javier; Giussani, Angelo; Conti, Irene; Rivalta, Ivan; Dumont, Elise; Jaiswal, Vishal K; Altavilla, Salvatore Flavio; Mukamel, Shaul; Garavelli, Marco

    2015-01-01

    The SOS//QM/MM [Rivalta et al., Int. J. Quant. Chem., 2014, 114, 85] method consists of an arsenal of computational tools allowing accurate simulation of one-dimensional (1D) and bi-dimensional (2D) electronic spectra of monomeric and dimeric systems with unprecedented details and accuracy. Prominent features like doubly excited local and excimer states, accessible in multi-photon processes, as well as charge-transfer states arise naturally through the fully quantum-mechanical description of the aggregates. In this contribution the SOS//QM/MM approach is extended to simulate time-resolved 2D spectra that can be used to characterize ultrafast excited state relaxation dynamics with atomistic details. We demonstrate how critical structures on the excited state potential energy surface, obtained through state-of-the-art quantum chemical computations, can be used as snapshots of the excited state relaxation dynamics to generate spectral fingerprints for different de-excitation channels. The approach is based on high-level multi-configurational wavefunction methods combined with non-linear response theory and incorporates the effects of the solvent/environment through hybrid quantum mechanics/molecular mechanics (QM/MM) techniques. Specifically, the protocol makes use of the second-order Perturbation Theory (CASPT2) on top of Complete Active Space Self Consistent Field (CASSCF) strategy to compute the high-lying excited states that can be accessed in different 2D experimental setups. As an example, the photophysics of the stacked adenine-adenine dimer in a double-stranded DNA is modeled through 2D near-ultraviolet (NUV) spectroscopy.

  15. Development of propidium iodide as a fluorescence probe for the on-line screening of non-specific DNA-intercalators in Fufang Banbianlian Injection.

    PubMed

    Niu, Yanyan; Li, Sensen; Lin, Zongtao; Liu, Meixian; Wang, Daidong; Wang, Hong; Chen, Shizhong

    2016-09-09

    Fufang Banbianlian Injection (FBI) has been widely used as an anti-inflammatory and anti-tumor prescription. To understand the relationships between its bioactive ingredients and pharmacological efficacies, our previous study has been successfully identified some DNA-binding compounds in FBI using an established on-line screening system, in which 4',6-diamidino-2-phenylindole (DAPI) was developed as a probe. However, DAPI can be only used to screen ATT-specific DNA minor groove binders, leaving the potential active intercalators unknown in FBI. As a continuation of our studies on FBI, here we present a sensitive analytical method for rapid identification and evaluation of DNA-intercalators using propidium iodide (PI) as a fluorescent probe. We have firstly established the technique of high-performance liquid chromatography-diode-array detector-multistage mass spectrometry-deoxyribonucleic acid-propidium iodide-fluorescence detector (HPLC-DAD-MS(n)-DNA-PI-FLD) system. As a result, 38 of 58 previously identified compounds in FBI were DNA-intercalation active. Interestingly, all previously reported DNA-binders also showed intercalative activities, suggesting they are dual-mode DNA-binders. Quantitative study showed that flavonoid glycosides and chlorogenic acids were the main active compounds in FBI, and displayed similar DNA-binding ability using either DAPI or PI. In addition, 13 active compounds were used to establish the structure-activity relationships. In this study, PI was developed into an on-line method for identifying DNA-intercalators for the first time, and thus it will be a useful high-throughput screening technique for other related samples.

  16. Adaptation of two commercially available DNA probes for the detection of E. coli and Staphylococcus aureus to selected fields of dairy hygiene--an exemplary study.

    PubMed

    Kneifel, W; Manafi, M; Breit, A

    1992-03-01

    The application of two commercially available colorimetric DNA hybridization tests (GENE-TRAK E. coli and Staphylococcus aureus) to selected aspects of dairy hygiene was investigated. Bacterial isolates of different origin, naturally contaminated cheese varieties, nonfat dry milk, milk concentrates, artificially contaminated milk and raw milks from udder quarters were examined. Based on the observation that the sensitivity of the E. coli DNA probe was comparable to that of the beta-D-glucuronidase-based fluorescence reaction (with 4-methyl-umbelliferyl-beta-D-glucuronide) of E. coli strains in Fluorocult lauryl sulfate broth, a Most Probable Number technique for enumerating E. coli in cheese using the DNA probe was developed. Another specific DNA probe was applied for the detection of S. aureus as a mastitis agent. By using a modified sample preparation, specific diagnosis of this microorganism in milk from udder quarters was enabled within 6 hours. This procedure is recommended to be used in screening tests. Based on the examples presented the potential of these tests in several fields of hygiene was illustrated.

  17. Positively charged polymer brush-functionalized filter paper for DNA sequence determination following Dot blot hybridization employing a pyrrolidinyl peptide nucleic acid probe.

    PubMed

    Laopa, Praethong S; Vilaivan, Tirayut; Hoven, Voravee P

    2013-01-07

    As inspired by the Dot blot analysis, a well known technique in molecular biology and genetics for detecting biomolecules, a new paper-based platform for colorimetric detection of specific DNA sequences employing peptide nucleic acid (PNA) as a probe has been developed. In this particular study, a pyrrolidinyl PNA bearing a conformationally rigid d-prolyl-2-aminocyclopentanecarboxylic acid backbone (acpcPNA) was used as a probe. The filter paper was modified to be positively charged with grafted polymer brushes of quaternized poly(dimethylamino)ethyl methacrylate (QPDMAEMA) prepared by surface-initiated polymerization of 2-(dimethylamino)ethyl methacrylate from the filter paper via ARGET ATRP followed by quaternization with methyl iodide. Following the Dot blot format, a DNA target was first immobilized via electrostatic interactions between the positive charges of the QPDMAEMA brushes and negative charges of the phosphate backbone of DNA. Upon hybridization with the biotinylated pyrrolidinyl peptide nucleic acid (b-PNA) probe, the immobilized DNA can be detected by naked eye observation of the yellow product generated by the enzymatic reaction employing HRP-labeled streptavidin. It has been demonstrated that this newly developed assay was capable of discriminating between complementary and single base mismatch targets at a detection limit of at least 10 fmol. In addition, the QPDMAEMA-grafted filter paper exhibited a superior performance to the commercial membranes, namely Nylon 66 and nitrocellulose.

  18. Flow Sorting and Molecular Cytogenetic Identification of Individual Chromosomes of Dasypyrum villosum L. (H. villosa) by a Single DNA Probe

    PubMed Central

    Grosso, Valentina; Farina, Anna; Gennaro, Andrea; Giorgi, Debora; Lucretti, Sergio

    2012-01-01

    Dasypyrum villosum (L.) Candargy (sin. Haynaldia villosa) is an annual wild diploid grass species (2n = 2x = 14; genome VV) belonging to the Poaceae family, which is considered to be an important source of biotic and abiotic stress resistance genes for wheat breeding. Enhanced characterization of D. villosum chromosomes can facilitate exploitation of its gene pool and its use in wheat breeding programs. Here we present the cytogenetic identification of D. villosum chromosomes on slide by fluorescent in situ hybridization (FISH), with the GAA simple sequence repeat (SSR) as a probe. We also describe the isolation and the flow cytometric analysis of D. villosum chromosomes in suspension, resulting in a distinguished flow karyotype. Chromosomes were flow sorted into three fractions, according their DNA content, one of which was composed of a single type of chromosome, namely 6 V, sorted with over 85% purity. Chromosome 6 V is known to carry genes to code for important resistance and seed storage characteristics, and its isolation represents a new source of genetic traits and specific markers useful for wheat improvement. PMID:23185561

  19. Identification of Mycobacterium avium and Mycobacterium intracellulare isolated in Puerto Rico from clinical samples by the use of a non-radioactive DNA probe.

    PubMed

    García, M T; Peña, I; Zlotnik, H

    1994-06-01

    The Mycobacterium avium complex (MAC), especially M. avium, is an important opportunistic pathogen of AIDS patients in the United States. In Puerto Rico, the incidence of infections caused by MAC has not been determined. This is due, in part, to the difficulties associated to the microbiological identification of the microorganisms. In this work, a commercially available kit (AccuProbe, Gen-Probe, Inc., San Diego, CA) utilizing a DNA probe complementary to rRNA of M. avium and M. intracellulare was used to identify seventeen MAC strains and one unknown atypical mycobacterium recovered in culture in Puerto Rico from clinical samples. The results obtained revealed that M. avium was the predominant species recovered (83% of isolates tested). Only two cultures were identified as M. intracellulare. The unknown culture, which did not react with either probe, turned out to be M. gordonae. The probe tests not only are simple to perform, but provide cultural identification results in as little as two hours. This study, the first one of its kind in Puerto Rico, demonstrates that the nucleic acid probes for the cultural identification of M. avium and M. intracellulare offer the potential of providing a prompt diagnosis and much needed data on the epidemiology of MAC infections in Puerto Rico.

  20. The internal dynamics of mini c TAR DNA probed by electron paramagnetic resonance of nitroxide spin-labels at the lower stem, the loop, and the bulge.

    PubMed

    Sun, Yan; Zhang, Ziwei; Grigoryants, Vladimir M; Myers, William K; Liu, Fei; Earle, Keith A; Freed, Jack H; Scholes, Charles P

    2012-10-30

    Electron paramagnetic resonance (EPR) at 236.6 and 9.5 GHz probed the tumbling of nitroxide spin probes in the lower stem, in the upper loop, and near the bulge of mini c TAR DNA. High-frequency 236.6 GHz EPR, not previously applied to spin-labeled oligonucleotides, was notably sensitive to fast, anisotropic, hindered local rotational motion of the spin probe, occurring approximately about the NO nitroxide axis. Labels attached to the 2'-aminocytidine sugar in the mini c TAR DNA showed such anisotropic motion, which was faster in the lower stem, a region previously thought to be partially melted. More flexible labels attached to phosphorothioates at the end of the lower stem tumbled isotropically in mini c TAR DNA, mini TAR RNA, and ψ(3) RNA, but at 5 °C, the motion became more anisotropic for the labeled RNAs, implying more order within the RNA lower stems. As observed by 9.5 GHz EPR, the slowing of nanosecond motions of large segments of the oligonucleotide was enhanced by increasing the ratio of the nucleocapsid protein NCp7 to mini c TAR DNA from 0 to 2. The slowing was most significant at labels in the loop and near the bulge. At a 4:1 ratio of NCp7 to mini c TAR DNA, all labels reported tumbling times of >5 ns, indicating a condensation of NCp7 and TAR DNA. At the 4:1 ratio, pulse dipolar EPR spectroscopy of bilabels attached near the 3' and 5' termini showed evidence of an NCp7-induced increase in the 3'-5' end-to-end distance distribution and a partially melted stem.

  1. The Internal Dynamics of Mini c TAR DNA Probed by EPR of Nitroxide Spin Labels at the Lower Stem, the Loop, and the Bulge †

    PubMed Central

    Sun, Yan; Zhang, Ziwei; Grigoryants, Vladimir M.; Myers, William K.; Liu, Fei; Earle, Keith A.; Freed, Jack H.; Scholes, Charles P.

    2012-01-01

    Electron paramagnetic resonance (EPR) at 236.6 GHz and 9.5 GHz probed the tumbling of nitroxide spin probes in the lower stem, the upper loop, and near the bulge of mini c TAR DNA. High frequency 236.6 GHz EPR, not previously applied to spin labeled oligonucleotides, was notably sensitive to fast, anisotropic, hindered local rotational motion of the spin probe, occurring approximately about the NO nitroxide axis. Labels attached to the 2′-amino cytidine sugar in the mini c TAR DNA showed such anisotropic motion, which was faster in the lower stem, a region previously suggested to be partially melted. More flexible labels attached to phosphorothioates at the end of the lower stem tumbled isotropically in mini c TAR DNA, mini TAR RNA, and ψ3 RNA, but at 5 °C the motion became more anisotropic for the labeled RNAs, implying more order within the RNA lower stems. As observed by 9.5 GHz EPR, the slowing of nanosecond motions of large segments of the oligonucleotide was enhanced by increasing the ratio of the nucleocapsid protein NCp7 to mini c TAR DNA from zero to two. The slowing was most significant at labels in the loop and near the bulge. At a 4:1 ratio of NCp7 to mini c TAR DNA all labels reported tumbling times > 5 ns, indicating a condensation of NCp7 and TAR DNA. At the 4:1 ratio, pulse dipolar EPR spectroscopy of bi-labels attached near the 3′ and 5′ terminals showed evidence for an NCp7-induced increase in the 3′ - 5 ′end-to-end distance distribution and a partially melted stem. PMID:23009298

  2. Probing the Interaction between a DNA Nucleotide (Adenosine-5'-Monophosphate Disodium) and Surface Active Ionic Liquids by Rotational Relaxation Measurement and Fluorescence Correlation Spectroscopy.

    PubMed

    Roy, Arpita; Banerjee, Pavel; Dutta, Rupam; Kundu, Sangita; Sarkar, Nilmoni

    2016-10-02

    This article demonstrates the interaction of a deoxyribonucleic acid (DNA) nucleotide, adenosine-5'-monophosphate disodium (AMP) with a cationic surface active ionic liquid (SAIL) 1-dodecyl-3-methylimidazoium chloride (C12mimCl) and an anionic SAIL, 1-butyl-3-methylimidazolium n-octylsulfate ([C4mim][C8SO4]). Dynamic light scattering (DLS) measurements and 1H NMR (nuclear magnetic resonance) studies indicate that substantial interaction is taking place among the DNA nucleotide, AMP and the SAILs. Moreover, cryogenic transmission electron microscopy (cryo-TEM) suggests that SAILs containing micellar assemblies are transformed into larger micellar assemblies in presence of DNA nucleotide. Additionally, the rotational motion of two oppositely charged molecules, Rhodamine 6G perchlorate (R6G) and Fluorescein sodium salt (Fl-Na) have been monitored in these aggregates. The rotational motion of R6G and Fl-Na differs significantly between SAILs micelles, and SAILs-AMP containing larger micellar aggregates. The effect of negatively charged DNA nucleotide (AMP) addition into the cationic and anionic SAILs is more prominent for the cationic charged molecule R6G than that of anionic probe Fl-Na due to the favourable electrostatic interaction between the AMP and cationic R6G. Moreover, the influence of the anionic DNA nucleotide on the cationic and anionic SAIL micelles is monitored through the variation of the lateral diffusion motion of oppositely charged probe molecules (R6G and Fl-Na) inside these aggregates. This variation in diffusion coefficient values also suggests that interaction pattern of these oppositely charged probes are different within the SAILs-nucleotide containing aggregates. Therefore, both rotational and translational diffusion measurements confirm that the DNA nucleotide (AMP) renders more rigid microenvironment within the micellar solution of SAILs.

  3. Direct immobilization of DNA probes on non-modified plastics by UV irradiation and integration in microfluidic devices for rapid bioassay.

    PubMed

    Sun, Yi; Perch-Nielsen, Ivan; Dufva, Martin; Sabourin, David; Bang, Dang Duong; Høgberg, Jonas; Wolff, Anders

    2012-01-01

    DNA microarrays have become one of the most powerful tools in the field of genomics and medical diagnosis. Recently, there has been increased interest in combining microfluidics with microarrays since this approach offers advantages in terms of portability, reduced analysis time, low consumption of reagents, and increased system integration. Polymers are widely used for microfluidic systems, but fabrication of microarrays on such materials often requires complicated chemical surface modifications, which hinders the integration of microarrays into microfluidic systems. In this paper, we demonstrate that simple UV irradiation can be used to directly immobilize poly(T)poly(C)-tagged DNA oligonucleotide probes on many different types of plastics without any surface modification. On average, five- and fourfold improvement in immobilization and hybridization efficiency have been achieved compared to surface-modified slides with aminated DNA probes. Moreover, the TC tag only costs 30% of the commonly used amino group modifications. Using this microarray fabrication technique, a portable cyclic olefin copolymer biochip containing eight individually addressable microfluidic channels was developed and used for rapid and parallel identification of Avian Influenza Virus by DNA hybridization. The one-step, cost-effective DNA-linking method on non-modified polymers significantly simplifies microarray fabrication procedures and permits great flexibility to plastic material selection, thus making it convenient to integrate microarrays into plastic microfluidic systems.

  4. Development of an on-site rapid real-time polymerase chain reaction system and the characterization of suitable DNA polymerases for TaqMan probe technology.

    PubMed

    Furutani, Shunsuke; Naruishi, Nahoko; Hagihara, Yoshihisa; Nagai, Hidenori

    2016-08-01

    On-site quantitative analyses of microorganisms (including viruses) by the polymerase chain reaction (PCR) system are significantly influencing medical and biological research. We have developed a remarkably rapid and portable real-time PCR system that is based on microfluidic approaches. Real-time PCR using TaqMan probes consists of a complex reaction. Therefore, in a rapid real-time PCR, the optimum DNA polymerase must be estimated by using actual real-time PCR conditions. In this study, we compared the performance of three DNA polymerases in actual PCR conditions using our rapid real-time PCR system. Although KAPA2G Fast HS DNA Polymerase has the highest enzymatic activity among them, SpeedSTAR HS DNA Polymerase exhibited better performance to rapidly increase the fluorescence signal in an actual real-time PCR using TaqMan probes. Furthermore, we achieved rapid detection of Escherichia coli in 7 min by using SpeedSTAR HS DNA Polymerase with the same sensitivity as that of a conventional thermal cycler.

  5. A novel snapback primer probe assay for the detection and discrimination of sympatric Haemonchus species using DNA melting analysis.

    PubMed

    Pichler, Rudolf; Silbermayr, Katja; Periasamy, Kathiravan

    2017-02-20

    Different sympatric species of Haemonchus parasites infecting ruminants and camels can be distinguished morphologically, but involves tedious microscopic examinations, measurements and several other limitations. Information on internal transcribed spacer-2 (ITS-2) sequence provides confirmatory differentiation of sympatric Haemonchus species. The present study introduces a novel, snapback primer probe based, real time PCR assay for the differentiation of three sympatric Haemonchus species, H. contortus (Hco), H. placei (Hpl) and H. longistipes (Hlo). The assay was designed to amplify a region of 130bp within the ITS-2 gene that included three diagnostic mutational sites capable of discriminating Hco, Hpl and Hlo. Following melt curve analysis, species-specific diagnostic melt peaks were obtained for Hco, Hpl and Hlo with a mean melting temperature of 56.6±0.3°C, 64.4±0.1°C and 54.4±0.1°C respectively. The test for analytical sensitivity revealed the ability of the assay to detect up to 5 copies per reaction. To evaluate the discriminating power of the assay, 174 samples from adult worms and 3rd stage larvae belonging to different Haemonchus species and various other nematode species including Cooperia curticei, Trichostrongylus axei, Trichostrongylus colubriformis, and Teladorsagia circumcincta were tested. Additionally, DNA extracted from 25 fecal egg samples was also tested and the specificity of the assay was verified by sequencing the ITS-2 gene of all the Haemonchus positive and non-Haemonchus samples. The assay worked accurately with 100% specificity in at least three real time PCR platforms. The assay is an effective alternative to the sequencing approach and is expected to be helpful for the screening of individual adult and larval Haemonchus parasites. However, caution needs to be applied while interpreting the results from fecal egg samples due to varying levels of sympatric co-infections from different Haemonchus species. The present study is the

  6. A ribosomal DNA fragment of Listeria monocytogenes and its use as a genus-specific probe in an aqueous-phase hybridization assay.

    PubMed Central

    Emond, E; Fliss, I; Pandian, S

    1993-01-01

    cDNAs were prepared from the total RNA of Listeria monocytogenes ATCC 19118 and used as probes to screen a genomic library of the same strain. Four clones were identified which contained ribosomal DNA fragments. Recombinant DNA from one of them was fractionated and differentially hybridized with the cDNA probes to RNA of L. monocytogenes and Kurthia zopfii. The resulting hybridization pattern revealed an HpaII fragment of 0.8 kb that was specific for the L. monocytogenes strain. The nucleotide sequence of this fragment showed 159 bases of the 3' end of the 16S rRNA gene, 243 bases of the spacer region, and 382 bases of the 5' end of the 23S rRNA gene. In dot blot hybridization assays, the 32P-labeled 784-bp fragment was specific only for Listeria species. Dot blot assays revealed that the 32P-labeled fragment can easily detect > or = 10 pg of total nucleic acids from pure cultures of L. monocytogenes, which corresponds to approximately 300 bacteria. This fragment was also used as a probe in an assay named the heteroduplex nucleic acid (HNA) enzyme-linked immunosorbent assay. In this system, the biotinylated DNA probe is hybridized in the aqueous phase with target RNA molecules and then specific HNAs are captured by HNA-specific antibodies. Captured HNA molecules are revealed with an enzyme conjugate of streptavidin. In a preliminary HNA enzyme-linked immunosorbent assay, the 784-bp fragment maintained its specificity for Listeria spp. and could detect 5 x 10(2) cells in artificially contaminated meat homogenate. Images PMID:8368854

  7. Facile construction of a highly sensitive DNA biosensor by in-situ assembly of electro-active tags on hairpin-structured probe fragment

    PubMed Central

    Wang, Qingxiang; Gao, Feng; Ni, Jiancong; Liao, Xiaolei; Zhang, Xuan; Lin, Zhenyu

    2016-01-01

    An ultrasensitive DNA biosensor has been developed through in-situ labeling of electroactive melamine-Cu2+ complex (Mel-Cu2+) on the end of hairpin-like probe using gold nanoparticles (AuNPs) as the signal amplification platform. The 3′-thiolated hairpin-like probe was first immobilized to the gold electrode surface by the Au-S bond. The AuNPs were then tethered on the free 5′-end of the immobilized probe via the special affinity between Au and the modified -NH2. Followed by, the Mel and Cu2+ were assembled on the AuNPs surface through Au-N bond and Cu2+-N bond, respectively. Due to the surface area and electrocatalytic effects of the AuNPs, the loading amount and electron transfer kinetic of the Mel-Cu2+ were enhanced greatly, resulting in significantly enhanced electrochemical response of the developed biosensor. Compared with the synthesis process of conventional electroactive probe DNA accomplished by homogeneous method, the method presented in this work is more reagent- and time-saving. The proposed biosensor showed high selectivity, wide linear range and low detection limit. This novel strategy could also be extended to the other bioanalysis platforms such as immunosensors and aptasensors. PMID:26931160

  8. Differentiation of toxigenic Staphylococcus aureus in staphylococcal isolates from prepared and frozen foods by combined arbitrarily primed polymerase chain reaction and DNA probe.

    PubMed

    Córdoba, Maria G; Jordano, Rafael; Aranda, Emilio; Benito, Maria J; Córdoba, Juan J

    2003-06-01

    In prepared and frozen flamenquín and hake fish fingers Staphylococcus aureus as sanitary hazards have been detected. In the present work, a combined method that includes an arbitrarily primed PCR (AP-PCR) and a mixed DNA probe hybridisation designed for the enterotoxigenic genes sea, seb, sec, and sed will be assayed to differentiate enterotoxigenic S. aureus from other staphylococcal species isolated during the processing of prepared and frozen foods. From the protocols tested for the AP-PCR, the highest number of amplification bands showing the best resolution was achieved at 30 degrees C annealing and 35 degrees C extension temperatures. Several staphylococci identified by a biochemical test as S. aureus showed in the AP-PCR analysis different banding patterns to the references S. aureus. The isolates, were investigated by slot blot hybridisation for genes encoding A, B, C, and D staphylococcal enterotoxins to determine their enterotoxigenic potential. Several isolates characterised by the AP-PCR analysis as S. aureus hybridised with the DNA probe mixture. The combined AP-PCR and DNA probe hybridisation assayed was able to differentiate toxigenic S. aureus from other staphylococcal species from prepared and frozen foods. This method could be considered as microbial quality assurance in these products.

  9. UniPROBE, update 2015: new tools and content for the online database of protein-binding microarray data on protein–DNA interactions

    PubMed Central

    Hume, Maxwell A.; Barrera, Luis A.; Gisselbrecht, Stephen S.; Bulyk, Martha L.

    2015-01-01

    The Universal PBM Resource for Oligonucleotide Binding Evaluation (UniPROBE) serves as a convenient source of information on published data generated using universal protein-binding microarray (PBM) technology, which provides in vitro data about the relative DNA-binding preferences of transcription factors for all possible sequence variants of a length k (‘k-mers’). The database displays important information about the proteins and displays their DNA-binding specificity data in terms of k-mers, position weight matrices and graphical sequence logos. This update to the database documents the growth of UniPROBE since the last update 4 years ago, and introduces a variety of new features and tools, including a new streamlined pipeline that facilitates data deposition by universal PBM data generators in the research community, a tool that generates putative nonbinding (i.e. negative control) DNA sequences for one or more proteins and novel motifs obtained by analyzing the PBM data using the BEEML-PBM algorithm for motif inference. The UniPROBE database is available at http://uniprobe.org. PMID:25378322

  10. Comparison of the hydrophobic grid-membrane filter DNA probe method and the Health Protection Branch standard method for the detection of Listeria monocytogenes in foods.

    PubMed

    Yan, W; Malik, M N; Peterkin, P I; Sharpe, A N

    1996-07-01

    The standard Health Protection Branch (HPB) method for the detection of L. monocytogenes in foods involves lengthy enrichment, selection and biochemical testing, requiring up to 8 days to complete. A hydrophobic grid-membrane filter (HGMF) method employing a digoxigenin-labelled listeriolysin O probe required 5 days to complete, and included an image-analysis system for electronic data acquisition. A total of 200 food samples encompassing 8 high-risk food groups (soft and semi-soft cheeses, packaged raw vegetables, frozen cooked shrimp, ground poultry, ground pork, ground beef, jellied meats, and pâté) were screened for the presence of L. monocytogenes by the two methods. Overall, 32 (16%) and 30 (15%) of the naturally-contaminated food samples tested positive for L. monocytogenes by the HPB and DNA methods, respectively. The DNA probe method was highly specific in discriminating L. monocytogenes from other Listeria spp. present in 50 of the samples tested. Results showed 94% sensitivity and 100% specificity between the two methods. The HGMF DNA probe method is an efficient and reliable alternative to the HPB standard method for detecting L. monocytogenes in foods.

  11. A novel homogenous detection method based on the self-assembled DNAzyme labeled DNA probes with SWNT conjugates and its application in detecting pathogen.

    PubMed

    Ding, Xinghua; Li, Hua; Deng, Le; Peng, Zhihui; Chen, Hui; Wang, Dan

    2011-07-15

    In this paper, a novel and cost-effective homogeneous detection method was constructed for the detection of genomic DNA and Staphylococcus aureus (S. aureus), based on the noncovalent assembly of DNAzyme-labeled detection probe and single-walled carbon nanotubes (SWNTs). When the target genomic DNA and hemin was existed in the detection solution, the detection probe wrapped on the SWNTs by π-stacking interactions would keep away from SWNTs and form a DNAzyme-self-assembly construction. This DNAzyme construction could catalyze 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS²⁻) and generate a colored product which could lead to the absorbance changes. Hence, according to its catalyzed capacity, the DNAzyme construction could amplify the detection signal. The concentration of target DNA could be quantified by exploiting their optical absorption changes at 414 nm and the concentration limit of detection of the method was 30 nM. And this detection method detected S. aureus quantitatively. In addition, this work proved that the method obtain higher detection sensitivity compared with the method without SWNTs because of the protection profile of SWNTs towards the detection probe.

  12. Conformation-sensitive nucleoside analogues as topology-specific fluorescence turn-on probes for DNA and RNA G-quadruplexes

    PubMed Central

    Tanpure, Arun A.; Srivatsan, Seergazhi G.

    2015-01-01

    Development of probes that can discriminate G-quadruplex (GQ) structures and indentify efficient GQ binders on the basis of topology and nucleic acid type is highly desired to advance GQ-directed therapeutic strategies. In this context, we describe the development of minimally perturbing and environment-sensitive pyrimidine nucleoside analogues, based on a 5-(benzofuran-2-yl)uracil core, as topology-specific fluorescence turn-on probes for human telomeric DNA and RNA GQs. The pyrimidine residues of one of the loop regions (TTA) of telomeric DNA and RNA GQ oligonucleotide (ON) sequences were replaced with 5-benzofuran-modified 2′-deoxyuridine and uridine analogues. Depending on the position of modification the fluorescent nucleoside analogues distinguish antiparallel, mixed parallel-antiparallel and parallel stranded DNA and RNA GQ topologies from corresponding duplexes with significant enhancement in fluorescence intensity and quantum yield. Further, these GQ sensors enabled the development of a simple fluorescence binding assay to quantify topology- and nucleic acid-specific binding of small molecule ligands to GQ structures. Together, our results demonstrate that these nucleoside analogues are useful GQ probes, which are anticipated to provide new opportunities to study and discover efficient G-quadruplex binders of therapeutic potential. PMID:26202965

  13. UniPROBE, update 2015: new tools and content for the online database of protein-binding microarray data on protein-DNA interactions.

    PubMed

    Hume, Maxwell A; Barrera, Luis A; Gisselbrecht, Stephen S; Bulyk, Martha L

    2015-01-01

    The Universal PBM Resource for Oligonucleotide Binding Evaluation (UniPROBE) serves as a convenient source of information on published data generated using universal protein-binding microarray (PBM) technology, which provides in vitro data about the relative DNA-binding preferences of transcription factors for all possible sequence variants of a length k ('k-mers'). The database displays important information about the proteins and displays their DNA-binding specificity data in terms of k-mers, position weight matrices and graphical sequence logos. This update to the database documents the growth of UniPROBE since the last update 4 years ago, and introduces a variety of new features and tools, including a new streamlined pipeline that facilitates data deposition by universal PBM data generators in the research community, a tool that generates putative nonbinding (i.e. negative control) DNA sequences for one or more proteins and novel motifs obtained by analyzing the PBM data using the BEEML-PBM algorithm for motif inference. The UniPROBE database is available at http://uniprobe.org.

  14. Solution structures of 2 : 1 and 1 : 1 DNA polymerase-DNA complexes probed by ultracentrifugation and small-angle X-ray scattering

    SciTech Connect

    Tang, Kuo-Hsiang; Niebuhr, Marc; Aulabaugh, Ann; Tsai, Ming-Daw

    2008-03-25

    We report small-angle X-ray scattering (SAXS) and sedimentation velocity (SV) studies on the enzyme-DNA complexes of rat DNA polymerase β (Pol β) and African swine fever virus DNA polymerase X (ASFV Pol X) with one-nucleotide gapped DNA. The results indicated formation of a 2 : 1 Pol β-DNA complex, whereas only 1 : 1 Pol X-DNA complex was observed. Three-dimensional structural models for the 2 : 1 Pol β-DNA and 1 : 1 Pol X-DNA complexes were generated from the SAXS experimental data to correlate with the functions of the DNA polymerases. The former indicates interactions of the 8 kDa 5'-dRP lyase domain of the second Pol β molecule with the active site of the 1 : 1 Pol β-DNA complex, while the latter demonstrates how ASFV Pol X binds DNA in the absence of DNA-binding motif(s). As ASFV Pol X has no 5'-dRP lyase domain, it is reasonable not to form a 2 : 1 complex. Based on the enhanced activities of the 2 : 1 complex and the observation that the 8 kDa domain is not in an optimal configuration for the 5'-dRP lyase reaction in the crystal structures of the closed ternary enzyme-DNA-dNTP complexes, we propose that the asymmetric 2 : 1 Pol β-DNA complex enhances the function of Pol β.

  15. Solution Structures of 2 : 1 And 1 : 1 DNA Polymerase - DNA Complexes Probed By Ultracentrifugation And Small-Angle X-Ray Scattering

    SciTech Connect

    Tang, K.H.; Niebuhr, M.; Aulabaugh, A.; Tsai, M.D.; /Ohio State U. /SLAC, SSRL

    2009-04-30

    We report small-angle X-ray scattering (SAXS) and sedimentation velocity (SV) studies on the enzyme-DNA complexes of rat DNA polymerase {beta} (Pol {beta}) and African swine fever virus DNA polymerase X (ASFV Pol X) with one-nucleotide gapped DNA. The results indicated formation of a 2 : 1 Pol {beta}-DNA complex, whereas only 1 : 1 Pol X-DNA complex was observed. Three-dimensional structural models for the 2 : 1 Pol {beta}-DNA and 1 : 1 Pol X-DNA complexes were generated from the SAXS experimental data to correlate with the functions of the DNA polymerases. The former indicates interactions of the 8 kDa 5{prime}-dRP lyase domain of the second Pol {beta} molecule with the active site of the 1 : 1 Pol {beta}-DNA complex, while the latter demonstrates how ASFV Pol X binds DNA in the absence of DNA-binding motif(s). As ASFV Pol X has no 5{prime}-dRP lyase domain, it is reasonable not to form a 2 : 1 complex. Based on the enhanced activities of the 2 : 1 complex and the observation that the 8 kDa domain is not in an optimal configuration for the 5{prime}-dRP lyase reaction in the crystal structures of the closed ternary enzyme-DNA-dNTP complexes, we propose that the asymmetric 2 : 1 Pol {beta}-DNA complex enhances the function of Pol {beta}.

  16. Enumeration of polysaccharide-degrading Bacteroides species in human feces by using species-specific DNA probes.

    PubMed Central

    Kuritza, A P; Shaughnessy, P; Salyers, A A

    1986-01-01

    DNA probes that are specific for each of five predominant species of human colonic Bacteroides (B. thetaiotaomicron, B. uniformis, B. distasonis, "Bacteroides group 3452-A", and B. ovatus) were used to detect and enumerate these species in fecal samples from two adult volunteers. These five species are capable of fermenting many of the complex polysaccharides that are thought to be sources of carbon and energy for bacteria in the colon. Estimates for the concentrations of some of these species in feces have not been previously available because of the difficulties in differentiating colonic Bacteroides spp. by conventional biochemical tests. Our results indicate that all the species except B. ovatus were present in high numbers (greater than 10(9)/g [dry weight]) in the feces of both volunteers. However, the concentrations of the more versatile polysaccharide-degrading species within this group of organisms (7.6 X 10(9) to 12.0 X 10(9)/g [dry weight] for B. thetaiotaomicron; 2.9 X 10(9) to 6.3 X 10(9)/g [dry weight] for "Bacteroides group 3452-A") did not differ significantly from the concentrations of less versatile polysaccharide-degrading species (1.2 X 10(10) to 2.0 X 10(10)/g [dry weight] for B. uniformis; 5.8 X 10(9) to 8.4 X 10(9)/g [dry weight] for B. distasonis). B. ovatus was not detectable by our method. Since our lower limit of detection is approximately 1 X 10(9) to 2 X 10(9)/g (dry weight) of feces, this is consistent with earlier estimates that indicated that the concentration of B. ovatus in feces is near or below this value.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3954350

  17. Enumeration of polysaccharide-degrading Bacteroides species in human feces by using species-specific DNA probes.

    PubMed

    Kuritza, A P; Shaughnessy, P; Salyers, A A

    1986-02-01

    DNA probes that are specific for each of five predominant species of human colonic Bacteroides (B. thetaiotaomicron, B. uniformis, B. distasonis, "Bacteroides group 3452-A", and B. ovatus) were used to detect and enumerate these species in fecal samples from two adult volunteers. These five species are capable of fermenting many of the complex polysaccharides that are thought to be sources of carbon and energy for bacteria in the colon. Estimates for the concentrations of some of these species in feces have not been previously available because of the difficulties in differentiating colonic Bacteroides spp. by conventional biochemical tests. Our results indicate that all the species except B. ovatus were present in high numbers (greater than 10(9)/g [dry weight]) in the feces of both volunteers. However, the concentrations of the more versatile polysaccharide-degrading species within this group of organisms (7.6 X 10(9) to 12.0 X 10(9)/g [dry weight] for B. thetaiotaomicron; 2.9 X 10(9) to 6.3 X 10(9)/g [dry weight] for "Bacteroides group 3452-A") did not differ significantly from the concentrations of less versatile polysaccharide-degrading species (1.2 X 10(10) to 2.0 X 10(10)/g [dry weight] for B. uniformis; 5.8 X 10(9) to 8.4 X 10(9)/g [dry weight] for B. distasonis). B. ovatus was not detectable by our method. Since our lower limit of detection is approximately 1 X 10(9) to 2 X 10(9)/g (dry weight) of feces, this is consistent with earlier estimates that indicated that the concentration of B. ovatus in feces is near or below this value.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Quenching of the electrochemiluminescence of RU-complex tagged shared-stem hairpin probes by graphene oxide and its application to quantitative turn-on detection of DNA.

    PubMed

    Huang, Xiang; Huang, Xiaopeng; Zhang, An; Zhuo, Bangrong; Lu, Fushen; Chen, Yaowen; Gao, Wenhua

    2015-08-15

    Efficient and stable quenching of electrochemiluminescence (ECL) of tris(2,2'-bipyridine)-ruthenium(II) (Ru(bpy)3(2+))/tri-n-propylamine (TPrA) system by graphene oxide (GO) at the glassy carbon electrode (GCE) was reported. For figuring out the possible reasons of the quenching mechanism, the electrochemical and ECL performance of GO, different reduction degree of reduced graphene oxide (RGOs) and polymer wrapped GO modified GCEs were systematacially investigated. The results demonstrated that the oxygen-containing groups and poor electrical conductivity of GO, along with the distance between GO and Ru(bpy)3(2+) was suggested as the reasons for quenching ECL. On the basis of this essential quenching mechanism, a novel "signal on" ECL DNA biosensor for ultrasensitive detection of specific DNA sequence was constructed by self-assembling the ECL probe of thiolated shared-stem hairpin DNA (SH-DNA) tagged with Ru complex (Ru(bpy)3(2+) derivatives) on the surface of GO/gold nanoparticles (AuNPs) modified GCE. The ECL probe sequences have their ECL signal efficiently quenched when they are self-assembled on the surface of GO unless they hybridizes with their target DNA (t-DNA) sequence. The designed ECL biosensor exhibited excellent stability and reproducibility, outstanding selectivity, and an extremely sensitive response to t-DNA in a wide linear range of 100 aM-10 pM with a low detection limit of 65 aM. Our findings and the design of biosensing switch would open a new avenue in the application of GO based ECL quenching strategy for ultrasensitive bioassays.

  19. Next-generation repeat-free FISH probes for DNA amplification in glioblastoma in vivo: Improving patient selection to MDM2-targeted inhibitors.

    PubMed

    Brunelli, Matteo; Eccher, Albino; Cima, Luca; Trippini, Tobia; Pedron, Serena; Chilosi, Marco; Barbareschi, Mattia; Scarpa, Aldo; Pinna, Giampietro; Cabrini, Giulio; Pilotto, Sara; Carbognin, Luisa; Bria, Emilio; Tortora, Giampaolo; Fioravanzo, Adele; Schiavo, Nicola; Meglio, Mario; Sava, Teodoro; Belli, Laura; Martignoni, Guido; Ghimenton, Claudio

    2017-01-01

    A next-generation FISH probe mapping to the MDM2 locus-specific region has recently been designed. The level of MDM2 gene amplification (high versus low) may allow selection of patients for cancer treatment with MDM2 inhibitors and may predict their responsiveness. We investigated the spectrum of MDM2 gene alterations using the new probes in vivo after visualizing single neoplastic cells in situ from a series of glioblastomas. Signals from next-generation repeat-free FISH interphase probes were identified in tissue microarrays that included 3 spots for each of the 48 cases. The murine double minutes (MDM2)-specific DNA probe and the satellite enumeration probe for chromosome 12 were used. Three cases (6%) showed more than 25 signals (high gene amplification), and 7 (15%) showed 3-10 signals (gains); among these, 4 cases (8%) had an equal number of MDM2 and centromeric signals on chromosome 12 (polyploidy). Genomic heterogeneity was observed only in 3 cases with low gene amplification. In our series, 6% of glioblastomas exhibited high MDM2 amplification (in vivo) with a pattern related to the known double minutes/chromothripsis phenomenon (in situ), and only cases with low amplification showed genomic heterogeneity. We concluded that the rate of MDM2 gene amplification can be a useful predictive biomarker to improve patient selection.

  20. Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes

    PubMed Central

    Ren, Xiaomei; El-Sagheer, Afaf H.; Brown, Tom

    2016-01-01

    A sterically undemanding azide analogue of dTTP (AHP dUTP) with an alkyl chain and ethynyl attachment to the nucleobase was designed and incorporated into DNA by primer extension, reverse transcription and polymerase chain reaction (PCR). An azide-modified 523 bp PCR amplicon with all 335 thymidines replaced by AHP dU was shown to be a perfect copy of the template from which it was amplified. Replacement of thymidine with AHP dU increases duplex stability, accounting in part for the high incorporation efficiency of the azide-modified triphosphate. Single-stranded azide-labelled DNA was conveniently prepared from PCR products by λ-exonuclease digestion and streptavidin magnetic bead isolation. Efficient fluorescent labelling of single and double-stranded DNA was carried out using dyes functionalized with bicyclo[6.1.0]non-4-yne (BCN) via the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. This revealed that the degree of labelling must be carefully controlled to achieve optimum fluorescence and avoid fluorescence quenching. Dual-coloured probes were obtained in a single tube fluorescent labelling reaction; and varying the ratios of the two dyes provides a simple method to prepare DNA probes with unique fluorescent signatures. AHP dUTP is a versatile clickable nucleotide with potentially wide applications in biology and nanotechnology including single molecule studies and synthesis of modified aptamer libraries via SELEX. PMID:26819406

  1. Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes.

    PubMed

    Ren, Xiaomei; El-Sagheer, Afaf H; Brown, Tom

    2016-05-05

    A sterically undemanding azide analogue of dTTP (AHP dUTP) with an alkyl chain and ethynyl attachment to the nucleobase was designed and incorporated into DNA by primer extension, reverse transcription and polymerase chain reaction (PCR). An azide-modified 523 bp PCR amplicon with all 335 thymidines replaced by AHP dU was shown to be a perfect copy of the template from which it was amplified. Replacement of thymidine with AHP dU increases duplex stability, accounting in part for the high incorporation efficiency of the azide-modified triphosphate. Single-stranded azide-labelled DNA was conveniently prepared from PCR products by λ-exonuclease digestion and streptavidin magnetic bead isolation. Efficient fluorescent labelling of single and double-stranded DNA was carried out using dyes functionalized with bicyclo[6.1.0]non-4-yne (BCN) via the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. This revealed that the degree of labelling must be carefully controlled to achieve optimum fluorescence and avoid fluorescence quenching. Dual-coloured probes were obtained in a single tube fluorescent labelling reaction; and varying the ratios of the two dyes provides a simple method to prepare DNA probes with unique fluorescent signatures. AHP dUTP is a versatile clickable nucleotide with potentially wide applications in biology and nanotechnology including single molecule studies and synthesis of modified aptamer libraries via SELEX.

  2. Use of a hypervariable minisatellite DNA probe (33.15) for evaluating engraftment two or more years after bone marrow transplantation for aplastic anaemia.

    PubMed

    Weitzel, J N; Hows, J M; Jeffreys, A J; Min, G L; Goldman, J M

    1988-09-01

    We have studied long-term engraftment in 24 multiply transfused patients transplanted for severe aplastic anaemia (SAA) 2-7 years previously from HLA identical sibling donors. All 24 patients had engrafted initially; nine (38%) developed grade II-IV a-GVHD, but only 5 (21%) developed chronic GVHD, which was mild, localized and transient. In 22 cases DNA 'fingerprint' analysis using a hypervariable minisatellite DNA probe (33.15) confirmed the donor/recipient origin of patient peripheral blood (PB) nucleated cells. Red cell antigens and PB lymphocyte chromosomes were also analysed in informative cases. In 19 patients (79%) PB cells were of donor origin confirming sustained engraftment, whereas five (21%) had PB cells of recipient origin. In four of these five cases complete autologous reconstitution was demonstrated. In one case DNA fingerprinting revealed mixed haemopoietic chimaerism. In three of the four cases of autologous reconstitution there had been a previous episode of late graft failure. The low incidence of chronic GVHD in the study group was not explained by autologous reconstitution or mixed chimaerism. We conclude that the hypervariable minisatellite probes are valuable in the study of engraftment after BMT, especially when patient and donor are HLA identical, of the same sex, and have the same ABO-Rh blood type. Pre-transplant specimens from the patient are not necessary for interpretation of the results provided that DNA from the donor is available.

  3. Evaluation of LNA, MGB and non-modified DNA probes to improve the detection limit of TaqMan real-time PCR assay for Pantoea stewartii subsp. stewartii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The goal of this study was to compare the sensitivity and amplification efficiency of the TaqMan assay using locked nucleic acid (LNA), minor groove binder (MGB) ligands and non-modified DNA probes. In monoplex or single target TaqMan assays for P. stewartii subsp. stewartii, LNA and MGB probes impr...

  4. Reverse Sample Genome Probing, a New Technique for Identification of Bacteria in Environmental Samples by DNA Hybridization, and Its Application to the Identification of Sulfate-Reducing Bacteria in Oil Field Samples

    PubMed Central

    Voordouw, Gerrit; Voordouw, Johanna K.; Karkhoff-Schweizer, Roxann R.; Fedorak, Phillip M.; Westlake, Donald W. S.

    1991-01-01

    A novel method for the identification of bacteria in environmental samples by DNA hybridization is presented. It is based on the fact that, even within a genus, the genomes of different bacteria may have little overall sequence homology. This allows the use of the labeled genomic DNA of a given bacterium (referred to as a “standard”) to probe for its presence and that of bacteria with highly homologous genomes in total DNA obtained from an environmental sample. Alternatively, total DNA extracted from the sample can be labeled and used to probe filters on which denatured chromosomal DNA from relevant bacterial standards has been spotted. The latter technique is referred to as reverse sample genome probing, since it is the reverse of the usual practice of deriving probes from reference bacteria for analyzing a DNA sample. Reverse sample genome probing allows identification of bacteria in a sample in a single step once a master filter with suitable standards has been developed. Application of reverse sample genome probing to the identification of sulfate-reducing bacteria in 31 samples obtained primarily from oil fields in the province of Alberta has indicated that there are at least 20 genotypically different sulfate-reducing bacteria in these samples. Images PMID:16348574

  5. Biofunction-assisted DNA detection through RNase H-enhanced 3' processing of a premature tRNA probe in a wheat germ extract.

    PubMed

    Ogawa, Atsushi; Tabuchi, Junichiro; Doi, Yasunori; Takamatsu, Masashi

    2016-08-01

    We have developed a novel type of biofunction-assisted, signal-turn-on sensor for simply and homogenously detecting DNA. This sensor system is composed of two types of in vitro-transcribed label-free RNAs (a 3' premature amber suppressor tRNA probe and an amber-mutated mRNA encoding a reporter protein), RNase H, and a wheat germ extract (WGE). A target DNA induces the 3' end maturation of the tRNA probe, which is enhanced by RNase H and leads to the expression of a full-length reporter protein through amber suppression in WGE, while there is almost no expression without the target due to the inactivity of the premature probe. Therefore, the target can be readily detected with the activity of the translated reporter. The catalytic reuse of the target with the help of RNase H in addition to various bioprocesses in WGE enables this sensor system to exhibit relatively high selectivity and sensitivity.

  6. Synthesis of circular double-stranded DNA having single-stranded recognition sequence as molecular-physical probe for nucleic acid hybridization detection based on atomic force microscopy imaging.

    PubMed

    Nakano, Koji; Matsunaga, Hideshi; Murata, Masaharu; Soh, Nobuaki; Imato, Toshihiko

    2009-08-01

    A new class of DNA probes having a mechanically detectable tag is reported. The DNA probe, which consists of a single-stranded recognition sequence and a double-stranded circular DNA entity, was prepared by polymerase reaction. M13mp18 single strand and a 32mer oligodeoxynucleotide whose 5'-end is decorated with the recognition sequence were used in combination as template and primer, respectively. We have successfully demonstrated that the DNA probe is useful for bioanalytical purposes: by deliberately attaching target DNA molecules onto Au(111) substrates and by mechanically reading out the tag-entity using a high-resolution microscopy including atomic force microscopy, visualization/detection of the individual target/probe DNA conjugate was possible simply yet straightforwardly. The present DNA probe can be characterized as a 100%-nucleic acid product material. It is simply available by one-pod synthesis. A surface topology parameter, image roughness, has witnessed its importance as a quantitative analysis index with particular usability in the present visualization/detection method.

  7. Site-specifically modified oligodeoxynucleotides as probes for the structural and biological effect of DNA-damaging agents

    SciTech Connect

    Basu, A.K.; Essigmann, J.M.

    1988-01-01

    This review critically analyzes the state of knowledge on the preparation, characterization, and uses of site-specifically modified DNA segments. Although these substrates have begun to have an impact upon several fields, the review focuses upon their applications in site-directed mutagenesis studies and for defining the effect of chemical damage upon DNA structure. Oligonucleotides have been synthesized containing alkylated DNA bases, aromatic amine adducts, base oxidation products, cyclic nucleic acid adducts, model apurinic/apyrimidinic sites, UV and psoralen photoadducts, and several antitumor drug-DNA covalent complexes. Below, the authors shall describe the progress to date on synthesis of site-specifically modified DNA segments and how these oligonucleotides have been used to further their understanding of the roles of individual DNA adducts in toxicology. The structures of the DNA adducts and adduct-derived products discussed in this review are presented. 168 references.

  8. Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

    SciTech Connect

    Chen, Kai; Roberts, Gareth A.; Stephanou, Augoustinos S.; Cooper, Laurie P.; White, John H.; Dryden, David T.F.

    2010-07-23

    Research highlights: {yields} Successful fusion of GFP to M.EcoKI DNA methyltransferase. {yields} GFP located at C-terminal of sequence specificity subunit does not later enzyme activity. {yields} FRET confirms structural model of M.EcoKI bound to DNA. -- Abstract: We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Foerster resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI.

  9. Methylation at the PW71 locus on chromosome 15 in DNA derived from CVS and from amniocytes; implications for the use of the PW71 probe in prenatal diagnosis of the Prader-Willi and Angleman syndromes

    SciTech Connect

    Telleria, P.; Yu, C.C.; Brown, S.

    1994-09-01

    The probe PW71 spans a HpaII site in the Prader-Willi/Angleman Syndrome critical region on chromosome 15. A single Southern blot with this probe can be used to detect deletion and uniparental disomy. We attempted to determine the methylation state of the PW71 locus in DNA derived from prenatal sources. Southern blots of HindIII and HindIII/HpaII double digests of DNA from cultured amniocytes and CVS specimens were prepared and probed with the PW71 probe. The results from 6 cultured CVS specimens indicate that several HPAII sites recognized by the PW71 probe are not methylated in trophoblast. Four amniotic fluid cultures gave results which were not different from lymphocyte-derived DNA; however, in several cases, amniotic fluid cultures resulted in Southern blots identical to those from CVS. Since we did not have verified prenatal cases of chromosome 15 uniparental disomy, we were unable to determine whether the parent-of-origin specific methylation present in lymphocyte DNA is also present in amniocyte DNA. We conclude that prenatal determination of chromosome 15 uniparental disomy with this probe will be unreliable.

  10. Development of a DNA probe for the myxosporean parasite, Ceratomyxa shasta, using the polymerase chain reaction with arbitrary primers

    USGS Publications Warehouse

    Bartholomew, Jerri L; Rodriguez, Rusty J.; Arakawa, Cindy K.

    1995-01-01

    The arbitrarily primed polymerase chain reaction (PCR) was used to generate a DNA marker specific for the myxosporean parasite Ceratomyxa shasta. The [32~]-labeled marker hybridized to purified C. shasta DNA and to parasite DNA combined with salmonid DNA in a dot blot assay, demonstrating its potential as a diagnostic tool. The amplified DNA segment was cloned and sequenced, and primers specific for the marker were designed. When these primers were used in a standard PCR assay, DNA was amplified from C. shasta and from infected fish tissues, but not from uninfected fish tissues or from 2 other myxosporean parasites. The sensitivity of the PCR assay will permit detection of low levels of C. shasta from infected fish or oligochaetes and will be useful in defining the parasite's life cycle as well as examining its impact on salmonid populatiosn

  11. Multicolor fluorescence in situ hybridization with centromeric DNA probes as a new approach to distinguish chromosome breakage from aneuploidy in interphase cells and micronuclei

    SciTech Connect

    Eastmond, D.A.; Rupa, D.S.; Chen, H.W.; Hasegawa, L.

    1993-12-31

    Chromosomal abnormalities are believed to contribute significantly to human reproductive failure, carcinogenesis and other pathophysiological conditions. For example, approximately 15% of recognized pregnancies terminate in spontaneous abortion, and of these approximately 30% have been shown to be chromosomally abnormal. The contribution of chromosomal abnormalities to early embryonic and fetal death appears to decrease with gestational age, suggesting that as many as 67% of the aborted embryos in early embryonic deaths are chromosomally abnormal. Furthermore, clinically significant chromosomal abnormalities can also be found to be present in approximately 0.58 to 0.67% of live births. These figures indicate that within a given year, hundreds of thousands of chromosomally abnormal babies will be born throughout the world and additional millions of chromosomally abnormal embryos will have been spontaneously aborted. For the past several years, our research has focused on utilizing new molecular cytogenetic techniques to develop assays for detecting aneuploidy-inducing agents in mammalian cells. One approach that we have sucessfully employed involves the use of fluorescence in situ hybridization with chromosome-specific DNA probes to determine the number of copies of a representative chromosome present within the nucleus following chemical exposure. DNA sequences (probes) which hybridize to blocks of repetitive centromeric DNA on specific chromosomes have been developed for most of the human chromosomes. In situ hybridization with these probes results in the staining of a compact chromosomal region which can be easily detected in interphase nuclei. The presence of 3 (or more) hybridization domains in an interphase nucleus indicates the presence of three centromeric regions and has been presumed to indicate that three copies of the entire chromosome were present in the nucleus.

  12. Probing radiation damage by alternated current conductivity as a method to characterize electron hopping conduction in DNA molecules

    SciTech Connect

    Gomes, Paulo J.; Coelho, Margarida; Antonio Ribeiro, Paulo; Raposo, Maria; Dionisio, Madalena

    2012-09-17

    Analysis of AC electrical conductivity of deoxyribonucleic acid (DNA) thin films, irradiated with ultraviolet (UV) light, revealed that electrical conduction arises from DNA chain electron hopping between base-pairs and phosphate groups. The hopping distance calculated from correlated barrier hopping model equals the distance between DNA base-pairs, which is consistent with the loss of conductivity with irradiation time arising from a decrease in phosphates groups. In the high frequency regime, at a given frequency, real part of conductivity strongly depends on irradiation time particularly for low dose levels suggesting the use of DNA based films for UV radiation sensors.

  13. Detection of Listeria monocytogenes by direct colony hybridization on hydrophobic grid-membrane filters by using a chromogen-labeled DNA probe.

    PubMed Central

    Peterkin, P I; Idziak, E S; Sharpe, A N

    1991-01-01

    A DNA probe specific for Listeria monocytogenes was isolated from a beta-hemolytic recombinant clone of an L. monocytogenes gene bank. It was labeled with horseradish peroxidase and used in a direct colony hybridization method on hydrophobic grid-membrane filters for the detection of the organism. Following color development of the chromogen, a commercial counter (HGMF Interpreter) was able to detect and count the organisms electronically. The method gave a positive reaction with 70 L. monocytogenes strains, while showing a negative reaction with 10 strains of other Listeria spp. and with 20 organisms of other genera. Images PMID:1901711

  14. Detection of Listeria monocytogenes by direct colony hybridization on hydrophobic grid-membrane filters by using a chromogen-labeled DNA probe.

    PubMed

    Peterkin, P I; Idziak, E S; Sharpe, A N

    1991-02-01

    A DNA probe specific for Listeria monocytogenes was isolated from a beta-hemolytic recombinant clone of an L. monocytogenes gene bank. It was labeled with horseradish peroxidase and used in a direct colony hybridization method on hydrophobic grid-membrane filters for the detection of the organism. Following color development of the chromogen, a commercial counter (HGMF Interpreter) was able to detect and count the organisms electronically. The method gave a positive reaction with 70 L. monocytogenes strains, while showing a negative reaction with 10 strains of other Listeria spp. and with 20 organisms of other genera.

  15. Tuning two-photon photoluminescence of gold nanoparticle aggregates with DNA and its application as turn-on photoluminescence probe for DNA sequence detection.

    PubMed

    Yuan, Peiyan; Ma, Rizhao; Guan, Zhenping; Gao, Nengyue; Xu, Qing-Hua

    2014-08-13

    Plasmon coupling between noble metal nanoparticles has been known to dramatically enhance linear and nonlinear optical properties of nearby chromophores and metal nanoparticles themselves. The interparticle distance is expected to have significant influence on the coupling strength. Here we have prepared DNA tuned Au nanoparticle assemblies with well controlled separation distances from 2.0 to 12.2 nm to investigate plasmon coupling strength and particle size effects on two-photon photoluminescence (TPPL) enhancement. TPPL intensities of these DNA coupled nanoassemblies were found to increase rapidly as the separation distance decreases. The largest TPPL enhancement factors of 115 and 265 were achieved at the shortest available separation distance of 2.0 nm for 21 and 41 nm Au NPs-dsDNA assemblies, respectively. We have further utilized DNA induced coupling of Au NPs and TPPL enhancement to develop a two-photon sensing scheme for detection of DNA sequences. This TPPL based method displayed high sensitivity with a limit of detection of 2.9 pM and excellent selectivity against ssDNA with mismatched bases. A single mismatch can be easily differentiated at room temperature. Taking the unique advantages of two-photon excitation, this method could be potentially further extended to DNA detection inside cells or even in vivo. These findings can provide important insight for fundamental understanding of plasmon-coupling enhanced TPPL and development of various two-photon excitation based applications.

  16. A triphenylamine-based colorimetric and fluorescent probe with donor–bridge–acceptor structure for detection of G-quadruplex DNA.

    PubMed

    Wang, Ming-Qi; Zhu, Wen-Xiang; Song, Zhuan-Zhuan; Li, Shuo; Zhang, Yong-Zhao

    2015-12-15

    In this Letter, three triphenylamine-based dyes (TPA-1, TPA-2a and TPA-2b) with donor–bridge–acceptor (D–p–A) structure were designed and synthesized for the purpose of G-quadruplexes recognition. In aqueous conditions, the interactions of the dyes with G-quadruplexes were studied with the aim to establish the influence of the geometry of the dyes on their binding and probing properties. Results indicate that TPA-2b displays significant selective colorimetric and fluorescent changes upon binding of G-quadruplex DNA. More importantly, its distinct color change enables visual detection and differentiation of G-quadruplexes from single and duplex DNA structures. CD titration date reveals that TPA-2b could induce and stabilize the formation of G-quadruplex structure. All these remarkable properties of TPA-2b suggest that it should have promising application in the field of G-quadruplexes research.

  17. Terminal protection of a small molecule-linked loop DNA probe for turn-on label-free fluorescence detection of proteins.

    PubMed

    Chen, Xuexu; Lin, Chunshui; Chen, Yiying; Luo, Feng; Wang, Yiru; Chen, Xi

    2016-09-15

    A novel label-free turn-on fluorescence biosensor for the determination of streptavidin (SA) was proposed. Using terminal protection of small molecule-linked DNA chimeras, which can protect DNA from degradation by various exonucleases when the small molecule moieties are bound to their protein target, we designed a loop probe, where the 3'-end was modified with biotin to resist digestion by exonucleases in the presence of target SA. Coupled with an intercalating dye, SYBR Green I, strong enhancement of the fluorescence signals was obtained compared with that in the absence of SA. A linear correlation equation was obtained for SA from 0 to 200nM with a limit detection of 0.4nM. This strategy holds great promise for practical applications with good specificity and sensitivity.

  18. Most frequent scenario for recurrent Candida vaginitis is strain maintenance with "substrain shuffling": demonstration by sequential DNA fingerprinting with probes Ca3, C1, and CARE2.

    PubMed Central

    Lockhart, S R; Reed, B D; Pierson, C L; Soll, D R

    1996-01-01

    The following three basic scenarios have emerged for the genetic relatedness of strains in recurrent vaginal candidiasis: strain maintenance without genetic variation, strain maintenance with minor genetic variation, and strain replacement. To test the frequency of each of the three scenarios, the genetic relatedness of Candida albicans isolates from each of 18 patients with recurrent infections was assessed by sequential DNA fingerprinting with the following three probes: the Ca3 probe; the C1 probe, a subfragment of the Ca3 probe which hybridizes to hypervariable genomic fragments; and the unrelated CARE2 probe. In each of the 18 patients with recurrent infections, the same strain was responsible for sequential infections, suggesting that the predominant scenario is strain maintenance. However, in 56% of these patients, the strain exhibited minor genetic variations in sequential infections. These changes were not found to be progressive. Rather, the changes suggest that substrains of an established infecting strain are shuffled in sequential infections. Results are also presented that in 45% of patients with recurrent infections, oral and vulvovaginal isolates were identical, in 35% they were highly related but not identical, and in 20% they were unrelated. These results differ markedly from those for commensal isolates simultaneously cultured from the oral cavity and vulvovaginal region of healthy individuals. Finally, it is demonstrated that in all eight cases in which C. albicans was isolated from both the male sexual partner of the patient with a recurrent infection and the patient, an isolate from the male partner was identical or highly related to the vulvovaginal strain. These results demonstrate that in patients with recurrent vulvovaginitis, a single strain usually dominates both in the different body locations of the patient and in the male partner and that it is maintained through sequential infections. However, in patients with recurrent infections

  19. Fluorescent probes based on side-chain chlorinated benzo[a]phenoxazinium chlorides: Studies of interaction with DNA

    NASA Astrophysics Data System (ADS)

    Raju, B. Rama; Gonçalves, M. Sameiro T.; Coutinho, Paulo J. G.

    2017-01-01

    The interaction of DNA with six water soluble benzo[a]phenoxazinium chlorides mono- or di-substituted with 3-chloropropyl groups at the O and N of 2- and 9-positions, along with methyl, hydroxyl and amine terminal groups at 5-positions, was investigated by photophysical techniques. The results indicated that almost all compounds intercalated in DNA base pairs at phosphate to dye ratio higher than 5. At lower values of this ratio, electrostatic binding mode with DNA was observed. Groove binding was detected mainly for the benzo[a]phenoxazinium dye with NH2·HBr terminal. The set of six benzo[a]phenoxazinium chlorides proved successful to label the migrating DNA in agarose gel electrophoresis assays. These finding proves the ability of these benzo[a]phenoxazinium dyes to strongly interact with DNA.

  20. Evaluation of the probe dihydrocalcein acetoxymethylester as an indicator of reactive oxygen species formation and comparison with oxidative DNA base modification determined by modified alkaline elution technique.

    PubMed

    Rohnstock, A; Lehmann, L

    2007-12-01

    Reactive oxygen species (ROS) play a predominant role in various diseases and the development of fast and easy methods for the quantification of intracellular ROS represents an important goal. Therefore, the aim of the present study was the evaluation of the fluorogenic probe dihydrocalcein acetoxymethylester (AM) for the detection of intracellular ROS. A flow cytometric method was developed using MCF-7 cells and the kinetics of ester hydrolysis and the cellular distribution and stability of calcein were characterized using calcein AM. Then, MCF-7 cells were challenged with model agents for the generation of singlet oxygen (illumination with visible light), peroxyl and hydroxyl radicals (tert-butylhydroperoxide, tBHP), superoxide anion radicals (potassium dioxide), and the intracellular formation of superoxide anion radicals by redox cycling (menadione) and the formation of calcein was compared with the induction of oxidative DNA base modifications assessed by modified alkaline elution technique. Every model agent significantly induced formamidopyrimidine-DNA glycosylase-sensitive sites (i.e. oxidative DNA base modifications) and most also induced DNA strand breaks. In contrast, exclusively tBHP and illumination with visible light induced the intracellular formation of calcein. In conclusion, though intracellular oxidation of dihydrocalcein represents a fast screening method, it detects a limited spectrum of ROS.

  1. Probing the structural dynamics of the CRISPR-Cas9 RNA-guided DNA-cleavage system by coarse-grained modeling.

    PubMed

    Zheng, Wenjun

    2017-02-01

    In the adaptive immune systems of many bacteria and archaea, the Cas9 endonuclease forms a complex with specific guide/scaffold RNA to identify and cleave complementary target sequences in foreign DNA. This DNA targeting machinery has been exploited in numerous applications of genome editing and transcription control. However, the molecular mechanism of the Cas9 system is still obscure. Recently, high-resolution structures have been solved for Cas9 in different structural forms (e.g., unbound forms, RNA-bound binary complexes, and RNA-DNA-bound tertiary complexes, corresponding to an inactive state, a pre-target-bound state, and a cleavage-competent or product state), which offered key structural insights to the Cas9 mechanism. To further probe the structural dynamics of Cas9 interacting with RNA and DNA at the amino-acid level of details, we have performed systematic coarse-grained modeling using an elastic network model and related analyses. Our normal mode analysis predicted a few key modes of collective motions that capture the observed conformational changes featuring large domain motions triggered by binding of RNA and DNA. Our flexibility analysis identified specific regions with high or low flexibility that coincide with key functional sites (such as DNA/RNA-binding sites, nuclease cleavage sites, and key hinges). We also identified a small set of hotspot residues that control the energetics of functional motions, which overlap with known functional sites and offer promising targets for future mutagenesis efforts to improve the specificity of Cas9. Finally, we modeled the conformational transitions of Cas9 from the unbound form to the binary complex and then the tertiary complex, and predicted a distinct sequence of domain motions. In sum, our findings have offered rich structural and dynamic details relevant to the Cas9 machinery, and will guide future investigation and engineering of the Cas9 systems. Proteins 2017; 85:342-353. © 2016 Wiley Periodicals

  2. Time-resolved luminescence biosensor for continuous activity detection of protein acetylation-related enzymes based on DNA-sensitized terbium(III) probes.

    PubMed

    Han, Yitao; Li, Hao; Hu, Yufang; Li, Pei; Wang, Huixia; Nie, Zhou; Yao, Shouzhuo

    2015-09-15

    Protein acetylation of histone is an essential post-translational modification (PTM) mechanism in epigenetic gene regulation, and its status is reversibly controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Herein, we have developed a sensitive and label-free time-resolved luminescence (TRL) biosensor for continuous detection of enzymatic activity of HATs and HDACs, respectively, based on acetylation-mediated peptide/DNA interaction and Tb(3+)/DNA luminescent probes. Using guanine (G)-rich DNA-sensitized Tb(3+) luminescence as the output signal, the polycationic substrate peptides interact with DNA with high affinity and subsequently replace Tb(3+), eliminating the luminescent signal. HAT-catalyzed acetylation remarkably reduces the positive charge of the peptides and diminishes the peptide/DNA interaction, resulting in the signal on detection via recovery of DNA-sensitized Tb(3+) luminescence. With this TRL sensor, HAT (p300) can be sensitively detected with a wide linear range from 0.2 to 100 nM and a low detection limit of 0.05 nM. The proposed sensor was further used to continuously monitor the HAT activity in real time. Additionally, the TRL biosensor was successfully applied to evaluating HAT inhibition by two specific inhibitors, anacardic acid and C464, and satisfactory Z'-factors above 0.73 were obtained. Moreover, this sensor is feasible to continuously monitor the HDAC (Sirt1)-catalyzed deacetylation with a linear range from 0.5 to 500 nM and a detection limit of 0.5 nM. The proposed sensor is a convenient, sensitive, and mix-and-read assay, presenting a promising platform for protein acetylation-targeted epigenetic research and drug discovery.

  3. Probing the role of interfacial waters in protein-DNA recognition using a hybrid implicit/explicit solvation model.

    PubMed

    Li, Shen; Bradley, Philip

    2013-08-01

    When proteins bind to their DNA target sites, ordered water molecules are often present at the protein-DNA interface bridging protein and DNA through hydrogen bonds. What is the role of these ordered interfacial waters? Are they important determinants of the specificity of DNA sequence recognition, or do they act in binding in a primarily nonspecific manner, by improving packing of the interface, shielding unfavorable electrostatic interactions, and solvating unsatisfied polar groups that are inaccessible to bulk solvent? When modeling details of structure and binding preferences, can fully implicit solvent models be fruitfully applied to protein-DNA interfaces, or must the individualistic properties of these interfacial waters be accounted for? To address these questions, we have developed a hybrid implicit/explicit solvation model that specifically accounts for the locations and orientations of small numbers of DNA-bound water molecules, while treating the majority of the solvent implicitly. Comparing the performance of this model with that of its fully implicit counterpart, we find that explicit treatment of interfacial waters results in a modest but significant improvement in protein side-chain placement and DNA sequence recovery. Base-by-base comparison of the performance of the two models highlights DNA sequence positions whose recognition may be dependent on interfacial water. Our study offers large-scale statistical evidence for the role of ordered water for protein-DNA recognition, together with detailed examination of several well-characterized systems. In addition, our approach provides a template for modeling explicit water molecules at interfaces that should be extensible to other systems.

  4. Improved testing for CMT1A and HNPP using multiplex ligation-dependent probe amplification (MLPA) with rapid DNA preparations: comparison with the interphase FISH method.

    PubMed

    Slater, Howard; Bruno, Damien; Ren, Hua; La, Phung; Burgess, Trent; Hills, Louise; Nouri, Sara; Schouten, Jan; Choo, K H Andy

    2004-08-01

    Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are the two most common peripheral neuropathies, with incidences of about 1 in 2,500. Several techniques can be used to detect the typical 1.5-Mb duplication or deletion associated with these respective conditions, but none combines simplicity with high sensitivity. MLPA is a new technique for measuring sequence dosage. We have assessed its performance for the detection of the specific 1.5-Mb duplication/deletion by prospectively testing 50 patients referred with differential diagnoses of CMT or HNPP. Probes were designed to evaluate the TEKT3, PMP22, and COX10 genes within the CMT1A/HNPP region. We have compared the results with our existing fluorescence in situ hybridization (FISH) assay, which was performed in parallel. There was concordance of results for 49 patients. Of note, one patient showed an intermediate multiplex ligation-dependent probe amplification (MLPA) result with an abnormal FISH result, which is consistent with mosaicism. The assay works equally well with either purified DNA or rapid DNA preparations made by direct cell lysis. The use of the latter significantly reduces the cost of the assay. MLPA is a sensitive, specific, robust, and cost-effective technique suitable for fast, high-throughput testing and offers distinct advantages over other testing methods.

  5. Label-free detection of specific DNA sequence-telomere using unmodified gold nanoparticles as colorimetric probes

    NASA Astrophysics Data System (ADS)

    Qi, Yingying; Li, Li; Li, Baoxin

    2009-09-01

    A simple and sensitive label-free colorimetric detection of telomere DNA has been developed. It was based on the color change of gold nanoparticles (AuNPs) due to DNA hybridization. UV-vis spectra and transmission electron microscopy (TEM) were used to investigate the change of AuNPs. Under the optimized conditions, the linear range for determination of telomere DNA was 5.7 × 10 -13 to 4.5 × 10 -6 mol/L. The detection limit (3 σ) of this method has decreased to pico-molar level.

  6. Screening of deletions in the dystrophin gene with the cDNA probes Cf23a, Cf56a, and Cf115.

    PubMed Central

    Passos-Bueno, M R; Rapaport, D; Love, D; Flint, T; Bortolini, E R; Zatz, M; Davies, K E

    1990-01-01

    We have analysed 38 DMD patients from 34 families and 30 BMD patients from 12 families using the cDNA probes Cf23a and Cf56a, which map near the centre of the dystrophin gene, and Cf115, which is close to the 3' end of this gene. Together, probes Cf23a and Cf56a detected deletions in 50% of the DMD families and 33% of the BMD families. Probe Cf115 detected a deletion in only one DMD patient, which has not been reported before in severe X linked myopathy. Most of the DMD deletions could be detected with Cf56a while all four BMD deletions were detected with Cf23a. The pattern of deletions could not be used to predict the precise clinical course of the disease and no correlation was found between the severity of the disease and the extent of the gene deletion. A higher frequency of deletions was observed in sporadic (73%) compared with familial DMD (28%) and BMD cases (33%). This result, if confirmed in a larger sample, would have important implications for genetic counselling. Images PMID:2182872

  7. Use of a Repetitive DNA Probe To Type Clinical and Environmental Isolates of Aspergillus flavus from a Cluster of Cutaneous Infections in a Neonatal Intensive Care Unit

    PubMed Central

    James, Michael J.; Lasker, Brent A.; McNeil, Michael M.; Shelton, Mark; Warnock, David W.; Reiss, Errol

    2000-01-01

    Aspergillus flavus is second to A. fumigatus as a cause of invasive aspergillosis, but no standard method exists for molecular typing of strains from human sources. A repetitive DNA sequence cloned from A. flavus and subcloned into a pUC19 vector, pAF28, was used to type 18 isolates from diverse clinical, environmental, and geographic sources. The restriction fragment length polymorphisms generated with EcoRI- or PstI-digested genomic DNA and probed with digoxigenin-labeled pAF28 revealed complete concordance between patterns. Eighteen distinct fingerprints were observed. The probe was used to investigate two cases of cutaneous A. flavus infection in low-birth-weight infants in a neonatal intensive care unit (NICU). Both infants were transported by the same ambulance and crew to the NICU on the same day. A. flavus strains of the same genotype were isolated from both infants, from a roll of tape used to fasten their umbilical catheters, from a canvas bag used to store the tape in the ambulance, and from the tape tray in the ambulance isolette. These cases highlight the need to consider exposures in critically ill neonates that might occur during their transport to the NICU and for stringent infection control practices. The hybridization profiles of strains from a second cluster of invasive A. flavus infections in two pediatric hematology-oncology patients revealed a genotype common to strains from a definite case patient and a health care worker. A probable case patient was infected with a strain with a genotype different from that of the strain from the definite case patient but highly related to that of an environmental isolate. The high degree of discrimination and reproducibility obtained with the pAF28 probe underscores its utility for typing clinical and environmental isolates of A. flavus. PMID:11015372

  8. Quantitative super-resolution localization microscopy of DNA in situ using Vybrant® DyeCycle™ Violet fluorescent probe

    PubMed Central

    Żurek-Biesiada, Dominika; Szczurek, Aleksander T.; Prakash, Kirti; Best, Gerrit; Mohana, Giriram K.; Lee, Hyun-Keun; Roignant, Jean-Yves; Dobrucki, Jurek W.; Cremer, Christoph; Birk, Udo

    2016-01-01

    Single Molecule Localization Microscopy (SMLM) is a recently emerged optical imaging method that was shown to achieve a resolution in the order of tens of nanometers in intact cells. Novel high resolution imaging methods might be crucial for understanding of how the chromatin, a complex of DNA and proteins, is arranged in the eukaryotic cell nucleus. Such an approach utilizing switching of a fluorescent, DNA-binding dye Vybrant® DyeCycle™ Violet has been previously demonstrated by us (Żurek-Biesiada et al., 2015) [1]. Here we provide quantitative information on the influence of the chemical environment on the behavior of the dye, discuss the variability in the DNA-associated signal density, and demonstrate direct proof of enhanced structural resolution. Furthermore, we compare different visualization approaches. Finally, we describe various opportunities of multicolor DNA/SMLM imaging in eukaryotic cell nuclei. PMID:27054149

  9. Quantitative super-resolution localization microscopy of DNA in situ using Vybrant® DyeCycle™ Violet fluorescent probe.

    PubMed

    Żurek-Biesiada, Dominika; Szczurek, Aleksander T; Prakash, Kirti; Best, Gerrit; Mohana, Giriram K; Lee, Hyun-Keun; Roignant, Jean-Yves; Dobrucki, Jurek W; Cremer, Christoph; Birk, Udo

    2016-06-01

    Single Molecule Localization Microscopy (SMLM) is a recently emerged optical imaging method that was shown to achieve a resolution in the order of tens of nanometers in intact cells. Novel high resolution imaging methods might be crucial for understanding of how the chromatin, a complex of DNA and proteins, is arranged in the eukaryotic cell nucleus. Such an approach utilizing switching of a fluorescent, DNA-binding dye Vybrant® DyeCycle™ Violet has been previously demonstrated by us (Żurek-Biesiada et al., 2015) [1]. Here we provide quantitative information on the influence of the chemical environment on the behavior of the dye, discuss the variability in the DNA-associated signal density, and demonstrate direct proof of enhanced structural resolution. Furthermore, we compare different visualization approaches. Finally, we describe various opportunities of multicolor DNA/SMLM imaging in eukaryotic cell nuclei.

  10. Construction and Use of a Nonradioactive DNA Hybridization Probe for Detection of Pseudomonas syringae pv. Tomato on Tomato Plants

    PubMed Central

    Cuppels, D. A.; Moore, R. A.; Morris, V. L.

    1990-01-01

    Pseudomonas syringae pv. tomato, the causal agent for bacterial speck of tomato, produces the phytotoxin coronatine. A 5.3-kilobase XhoI fragment from the chromosomal region controlling toxin production was cloned into the plasmid pGB2, and the resulting recombinant plasmid, pTPR1, was tested for its ability to serve as a diagnostic probe for P. syringae pv. tomato. In a survey of 75 plant-associated bacteria, pTPR1 hybridized exclusively to those strains that produced coronatine. The detection limit for this probe, which was labeled with the Chemiprobe nonradioactive reporter system, was approximately 4 × 103 CFU of lesion bacteria. During the 1989 growing season, a total of 258 leaf and fruit lesions from nine tomato fields were screened for P. syringae pv. tomato by using pTPR1 and the culture method of detection. The best agreement between the two methods, 90%, occurred early in the season with samples taken from relatively young (5-week-old) plants. Young plants also had a higher percentage of P. syringae pv. tomato-positive lesions. P. syringae pv. tomato was the only coronatine producer recovered from the nine tomato fields. All 244 P. syringae pv. tomato strains isolated during this study reacted strongly with the probe. The P. syringae pv. tomato population of healthy field tomato leaves was determined by a pTPR1 colony hybridization procedure. Every probe-positive colony that was isolated and characterized was identified as P. syringae pv. tomato. The pTPR1 probe should expedite disease diagnosis and facilitate epidemiological studies of this pathogen. It also should aid in screening transplant seedlings for bacterial speck infestation. Images PMID:16348215

  11. Porphyrinic metal-organic framework as electrochemical probe for DNA sensing via triple-helix molecular switch.

    PubMed

    Ling, Pinghua; Lei, Jianping; Ju, Huangxian

    2015-09-15

    An electrochemical DNA sensor was developed based on the electrocatalysis of porphyrinic metal-organic framework (MOF) and triple-helix molecular switch for signal transduction. The streptavidin functionalized zirconium-porphyrin MOF (PCN-222@SA) was prepared as signal nanoprobe via covalent method and demonstrated high electrocatalysis for O2 reduction. Due to the large steric effect, the designed nanoprobe was blocked for the interaction with the biotin labeled triple-helix immobilized on the surface of glassy carbon electrode. In the presence of target DNA, the assistant DNA in triple-helix will hybridize with target DNA, resulting in the disassembly of triple-helix molecular. Consequently, the end biotin away from the electrode was ''activated'' for easy access to the signal nanoprobe, PCN-222@SA, on the basis of biotin-streptavidin biorecognition. The introduction of signal nanoprobe to a sensor surface led to a significantly amplified electrocatalytic current towards oxygen reduction. Integrating with DNA recycling amplification of Exonuclease III, the sensitivity of the biosensor was improved significantly with detection limit of 0.29 fM. Moreover, the present method has been successfully applied to detect DNA in complex serum matrix. This porphyrinic MOF-based strategy has promising application in the determination of various analytes for signal transduction and has great potential in bioassays.

  12. Thermodynamic and structural study of pyrene-1-carboxaldehyde/DNA interactions by molecular spectroscopy: Probing intercalation and binding properties

    NASA Astrophysics Data System (ADS)

    Grueso, E.; Prado-Gotor, R.

    2010-08-01

    The binding of pyrene-1-carboxaldehyde (1-PyCHO) with ctDNA was investigated through absorption, intrinsic and induced circular dichroism, viscosity measurements and steady-state fluorescence. The binding and the number of monomer units of the polymer involved in the binding of one dye molecule (site size) have been quantified. The results indicated that the 1-PyCHO molecule binds to the ctDNA in an intercalative mode. The spectroscopic evidence of this intercalation process is also corroborated by the effect of urea, iodide-induced fluorescence quenching of pyrene-1-carboxaldehyde and competitive binding using a fluorescent intercalator, SYBR Green I (SG). The induced circular dichroism (ICD) spectra of pyrene-1-carboxaldehyde complexed with ctDNA show that pyrene-1-carboxaldehyde intercalates into ctDNA and that the intercalation orientation of pyrene to the DNA base-pairs long axis is heterogeneous. On the other hand, the intrinsic circular dichroism (CD) spectra show a stabilization of the right-handed B form of ctDNA, due to the intercalation process.

  13. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  14. Thermodynamic and structural study of phenanthroline derivative ruthenium complex/DNA interactions: probing partial intercalation and binding properties.

    PubMed

    Grueso, E; López-Pérez, G; Castellano, M; Prado-Gotor, R

    2012-01-01

    The binding of [Ru(PDTA-H(2))(phen)]Cl (PDTA = propylene-1,2-diaminetetra-acetic acid; phen = 1,10 phenanthroline) with ctDNA (=calf thymus DNA) has been investigated through intrinsic and induced circular dichroism, UV-visible absorption and fluorescence spectroscopies, steady-state fluorescence, thermal denaturation technique, viscosity and electrochemical measurements. The latter indicate that the cathodic and anodic peak potentials of the ruthenium complex shift to more positive values on increasing the DNA concentration, this behavior being a direct consequence of the interaction of both the reduced and oxidized form with DNA binding. From spectrophotometric titration experiments, the equilibrium binding constant and the number of monomer units of the polymer involved in the binding of one ruthenium molecule (site size) have been quantified. The intrinsic circular dichroism (CD) spectra show an unwinding and a conformational change of the DNA helix upon interaction of the ruthenium complex. Quenching process, thermal denaturation experiments and induced circular dichroism (ICD) are consistent with a partial intercalative binding mode.

  15. Probing molecular pathways for DNA orientational trapping, unzipping and translocation in nanopores by using a tunable overhang sensor

    NASA Astrophysics Data System (ADS)

    Wang, Yong; Tian, Kai; Hunter, Lehr L.; Ritzo, Brandon; Gu, Li-Qun

    2014-09-01

    Nanopores provide a unique single-molecule platform for genetic and epigenetic detection. The target nucleic acids can be accurately analyzed by characterizing their specific electric fingerprints or signatures in the nanopore. Here we report a series of novel nanopore signatures generated by target nucleic acids that are hybridized with a probe. A length-tunable overhang appended to the probe functions as a sensor to specifically modulate the nanopore current profile. The resulting signatures can reveal multiple mechanisms for the orientational trapping, unzipping, escaping and translocation of nucleic acids in the nanopore. This universal approach can be used to program various molecular movement pathways, elucidate their kinetics, and enhance the sensitivity and specificity of the nanopore sensor for nucleic acid detection.Nanopores provide a unique single-molecule platform for genetic and epigenetic detection. The target nucleic acids can be accurately analyzed by characterizing their specific electric fingerprints or signatures in the nanopore. Here we report a series of novel nanopore signatures generated by target nucleic acids that are hybridized with a probe. A length-tunable overhang appended to the probe functions as a sensor to specifically modulate the nanopore current profile. The resulting signatures can reveal multiple mechanisms for the orientational trapping, unzipping, escaping and translocation of nucleic acids in the nanopore. This universal approach can be used to program various molecular movement pathways, elucidate their kinetics, and enhance the sensitivity and specificity of the nanopore sensor for nucleic acid detection. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr03195d

  16. Use of the Polymerase Chain Reaction and Complementary DNA Probes in the Detection of Duchenne Muscular Dystrophy Carriers

    DTIC Science & Technology

    1990-01-01

    provided by linkage analyses in which certain probes mapping to the short arm of the X chromosome (OTC [ ornithine transcarbamylase ] and L1.28...the additional X-linked disorders associated with DMD were glycerol kinase deficiency , adrenal hypoplasia, retinitis pigmentosa, the McLeod phenotype...deletion in patient T. M., who has multiple disorders including DMD, glycerol kinase deficiency and adrenal hypoplasia. There have been a number of reports

  17. Intra-molecular G-quadruplex structure generated by DNA-templated click chemistry: "turn-on" fluorescent probe for copper ions.

    PubMed

    Shen, Qinpeng; Zhou, Lifen; Yuan, Yijia; Huang, Yan; Xiang, Binbin; Chen, Chunyan; Nie, Zhou; Yao, Shouzhuo

    2014-05-15

    A novel homogenous fluorescent sensor for signal-on detection of Cu(2+) has been developed based on intra-molecular G-quadruplex formed by DNA-templated click reaction and crystal violet (CV) as label-free signal reporter. The clickable DNA probe consists of two G-rich strands (A and B) bearing azide and alkyne group, respectively, and a template strand (C) locating two proximate reactants by pairing with A and B. The sequences of A and B are derived from asymmetric split of the G-quadruplex sequence (TTAGGG)4. In the presence of Cu(2+), the whole G-quadruplex sequence A-B is generated by chemical ligation of A and B via copper ion-catalyzed alkyne-azide cycloaddition, then released from template by toehold strand displacement, and consequently forming a stable intra-molecular G-quadruplex, which binds with CV to generate a strong fluorescent signal. Oppositely, weak fluorescence was obtained without Cu(2+) because of unstable intermolecular G-quadruplex formed by A and B and lack of lateral loop connection. Therefore, the Cu(2+) can be sensitively and specifically detected by the fluorescence of the CV-stained G-quadruplex with a low detection limit of 65nM and a linear range of 0.1-3µM. This method rationally integrated the DNA-templated synthesis and G-quadruplex structure-switch, presenting a simple and promising approach for biosensor development.

  18. Evaluation of autotrophic growth of ammonia-oxidizers associated with granular activated carbon used for drinking water purification by DNA-stable isotope probing.

    PubMed

    Niu, Jia; Kasuga, Ikuro; Kurisu, Futoshi; Furumai, Hiroaki; Shigeeda, Takaaki

    2013-12-01

    Nitrification is an important biological function of granular activated carbon (GAC) used in advanced drinking water purification processes. Newly discovered ammonia-oxidizing archaea (AOA) have challenged the traditional understanding of ammonia oxidation, which considered ammonia-oxidizing bacteria (AOB) as the sole ammonia-oxidizers. Previous studies demonstrated the predominance of AOA on GAC, but the contributions of AOA and AOB to ammonia oxidation remain unclear. In the present study, DNA-stable isotope probing (DNA-SIP) was used to investigate the autotrophic growth of AOA and AOB associated with GAC at two different ammonium concentrations (0.14 mg N/L and 1.4 mg N/L). GAC samples collected from three full-scale drinking water purification plants in Tokyo, Japan, had different abundance of AOA and AOB. These samples were fed continuously with ammonium and (13)C-bicarbonate for 14 days. The DNA-SIP analysis demonstrated that only AOA assimilated (13)C-bicarbonate at low ammonium concentration, whereas AOA and AOB exhibited autotrophic growth at high ammonium concentration. This indicates that a lower ammonium concentration is preferable for AOA growth. Since AOA could not grow without ammonium, their autotrophic growth was coupled with ammonia oxidation. Overall, our results point towards an important role of AOA in nitrification in GAC filters treating low concentration of ammonium.

  19. Selective Luminescent Labeling of DNA and RNA Quadruplexes by π-Extended Ruthenium Light-Up Probes.

    PubMed

    Saadallah, Dounia; Bellakhal, Mehdi; Amor, Souheila; Lefebvre, Jean-François; Chavarot-Kerlidou, Murielle; Baussanne, Isabelle; Moucheron, Cécile; Demeunynck, Martine; Monchaud, David

    2017-01-26

    A series of Ru(II) complexes exhibiting π-extended, acridine-based ancillary chelating heterocycles display high affinity and selectivity for DNA and RNA quadruplexes. The most promising candidates (3, 4) possess remarkable light-up luminophore properties (up to 330-fold luminescence enhancement upon interaction with quadruplexes), enabling them to discriminate quadruplexes from genomic DNA owing to a photochemical mechanism involving DNA protection against non-radiative decay (DAND), thus deviating from the other complexes of this series of ligands that exhibit an excited-state intramolecular proton transfer (ESIPT) that quenches their luminescence. The in vitro and preliminary in cellulo results shown here confirm the interest of this new family of fluorophores as invaluable molecular tools to detect G-quadruplexes in cells.

  20. Screening cDNA Libraries Using Partial Probes to Isolate Full-Length cDNAs from Vascular Cells.

    PubMed

    Csortos, C; Lazar, V; Garcia, J G

    1999-01-01

    The purpose of screening cDNA libraries is to isolate a particular cDNA clone encoding a mRNA and by implication, a protein, of interest. The screening is based on identification of the desired clone among a large number of recombinant clones within the library selected (1,2). As an example of both the utility and power of library screening, we will relate our own library screening efforts utilized to isolate the nonmuscle high molecular weight myosin light chain kinase isoform from a human umbilical vein endothelial cell cDNA library (3). This unique nonmuscle myosin light chain kinase isoform phosphorylates myosin light chains, thereby playing an essential role in agonist-mediated endothelial cell contraction, paracellular gap formation and increased vascular permeability. We are hopeful that this step-by-step approach will help the reader to understand the discussed methods.

  1. Incorporation of nucleoside probes opposite O⁶-methylguanine by Sulfolobus solfataricus DNA polymerase Dpo4: importance of hydrogen bonding.

    PubMed

    Stornetta, Alessia; Angelov, Todor; Guengerich, F Peter; Sturla, Shana J

    2013-09-02

    O⁶-Methylguanine (O⁶-MeG) is a mutagenic DNA lesion, arising from the action of methylating agents on guanine (G) in DNA. Dpo4, an archaeal low-fidelity Y-family DNA polymerase involved in translesion DNA synthesis (TLS), is a model for studying how human Y-family polymerases bypass DNA adducts. Previous work showed that Dpo4-mediated dTTP incorporation is favored opposite O⁶-MeG rather than opposite G. However, factors influencing the preference of Dpo4 to incorporate dTTP opposite O⁶-MeG are not fully defined. In this study, we investigated the influence of structural features of incoming dNTPs on their enzymatic incorporation opposite O⁶-MeG in a DNA template. To this end, we utilized a new fluorescence-based primer extension assay to evaluate the incorporation efficiency of a panel of synthetic dNTPs opposite G or O⁶-MeG by Dpo4. In single-dNTP primer extension studies, the synthetic dNTPs were preferentially incorporated opposite G, relative to O⁶-MeG. Moreover, pyrimidine-based dNTPs were generally better incorporated than purine-based syn-conformation dNTPs. The results suggest that hydrophobicity of the incoming dNTP appears to have little influence on the process of nucleotide selection by Dpo4, with hydrogen bonding capacity being a major influence. Additionally, modifications at the C2-position of dCTP increase the selectivity for incorporation opposite O⁶-MeG without a significant loss of efficiency.

  2. Direct Detection of Mycobacterium tuberculosis Complex DNA and Rifampin Resistance in Clinical Specimens from Tuberculosis Patients by Line Probe Assay▿

    PubMed Central

    Traore, Hamidou; van Deun, Armand; Shamputa, Isdore Chola; Rigouts, Leen; Portaels, Françoise

    2006-01-01

    The INNO-LiPA.Rif TB test (LiPA) has only been applied to a limited number of clinical specimens. To assess the utility of this test for detecting Mycobacterium tuberculosis complex DNA and rifampin (RMP) resistance, 420 sputum samples comprising specimens from untreated (n = 160) and previously treated (n = 260) patients from 11 countries in Asia, Africa, Europe, and Latin America were tested. DNA was extracted from sputum samples by using a modification of the Boom's method, while the rpoB core region was amplified by nested PCR. The results were analyzed in conjunction with those obtained by Ziehl-Neelsen (ZN) microscopy and by culture on solid media. The LiPA test was positive for M. tuberculosis complex DNA in 389 (92.9%) specimens, including 92.0% (286 of 311) ZN-positive and 94.5% (103 of 109) ZN-negative specimens. Of these, 30.6% were RMP resistant. In contrast, 74.3% of the specimens were positive for M. tuberculosis by culture, and 30.8% of them were RMP resistant. LiPA detected M. tuberculosis complex DNA in 92.4% (110 of 119) of the culture-positive and 100.0% (41 of 41) of the culture-negative specimens from untreated patients. There was a 99.6% concordance between the RMP resistance as determined by culture and by the LiPA test. With an optimal DNA extraction method, LiPA allows rapid detection of M. tuberculosis complex DNA and RMP resistance directly from sputum specimens. LiPA can still provide useful information when culture fails for various reasons. The rapid availability of this information is necessary to adjust patient treatment and avoid the risk of amplification of drug resistance. PMID:17035487

  3. Are NORs always located on homeologous chromosomes? A FISH investigation with rDNA and whole chromosome probes in Gymnotus fishes (Gymnotiformes).

    PubMed

    Milhomem, Susana S R; Scacchetti, Priscilla C; Pieczarka, Julio C; Ferguson-Smith, Malcolm A; Pansonato-Alves, José C; O'Brien, Patricia C M; Foresti, Fausto; Nagamachi, Cleusa Y

    2013-01-01

    Gymnotus (Gymnotiformes, Gymnotidae) is the most diverse known Neotropical electric knife fish genus. Cytogenetic studies in Gymnotus demonstrate a huge karyotypic diversity for this genus, with diploid numbers ranging from 34 to 54. The NOR are also variable in this genus, with both single and multiple NORs described. A common interpretation is that the single NOR pair is a primitive trait while multiple NORs are derivative. However this hypothesis has never been fully tested. In this report we checked if the NOR-bearing chromosome and the rDNA site are homeologous in different species of the genus Gymnotus: G. carapo (2n = 40, 42, 54), G. mamiraua (2n = 54), G. arapaima (2n = 44), G. sylvius (2n = 40), G. inaequilabiatus (2n = 54) and G. capanema (2n = 34), from the monophyletic group G. carapo (Gymnotidae-Gymnotiformes), as well as G. jonasi (2n = 52), belonging to the G1 group. They were analyzed with Fluorescence in situ hybridization (FISH) using 18S rDNA and whole chromosome probes of the NOR-bearing chromosome 20 (GCA20) of G. carapo (cytotype 2n = 42), obtained by Fluorescence Activated Cell Sorting. All species of the monophyletic G. carapo group show the NOR in the same single pair, confirmed by hybridization with CGA20 whole chromosome probe. In G. jonasi the NORs are multiple, and located on pairs 9, 10 and 11. In G. jonasi the GCA20 chromosome probe paints the distal half of the long arm of pair 7, which is not a NOR-bearing chromosome. Thus these rDNA sequences are not always in the homeologous chromosomes in different species thus giving no support to the hypothesis that single NOR pairs are primitive traits while multiple NORs are derived. The separation of groups of species in the genus Gymnotus proposed by phylogenies with morphologic and molecular data is supported by our cytogenetic data.

  4. HU Binding to a DNA Four-Way Junction Probed by Főrster Resonance Energy Transfer‡

    PubMed Central

    Vitoc, Codruta Iulia; Mukerji, Ishita

    2016-01-01

    The Escherichia coli protein, HU, is a non sequence-specific DNA-binding protein that interacts with DNA primarily through electrostatic interactions. In addition to non-specific binding to linear DNA, HU has been shown to bind with nanomolar affinity to discontinuous DNA substrates, such as repair and recombination intermediates. This work specifically examines the HU-four way junction (4WJ) interaction using fluorescence spectroscopic methods. The conformation of the junction in the presence of different counterions was investigated by Förster resonance energy transfer (FRET) measurements, which revealed an ion-type conformational dependence, where Na+ yields the most stacked conformation followed by K+ and Mg2+. HU binding induces a greater degree of stacking in the Na+-stabilized and Mg2+-stabilized junctions but not the K+-stabilized junction, which is attributed to differences in the size of the ionic radii and potential differences in ion binding sites. Interestingly, junction conformation modulates binding affinity, where HU exhibits the lowest affinity for the Mg2+-stabilized form (24 μM−1), which is the least stacked conformation. Protein binding to a mixed population of open and stacked forms of the junction leads to near complete formation of a protein-stabilized stacked-X junction. These results strongly support a model in which HU binds to and stabilizes the stacked-X conformation. PMID:21230005

  5. Probing key DNA contacts in AraR-mediated transcriptional repression of the Bacillus subtilis arabinose regulon.

    PubMed

    Franco, Irina Saraiva; Mota, Luís Jaime; Soares, Cláudio Manuel; de Sá-Nogueira, Isabel

    2007-01-01

    In the absence of arabinose, the AraR transcription factor represses the expression of genes involved in the utilization of arabinose, xylose and galactose in Bacillus subtilis. AraR exhibits a chimeric organization: the N-terminal DNA-binding region belongs to the GntR family and the C-terminal effector-binding domain is homologous to the GalR/LacI family. Here, the AraR-DNA-binding interactions were characterized in vivo and in vitro. The effect of residue substitutions in the AraR N-terminal domain and of base-pair exchanges into an AraR-DNA-binding operator site were examined by assaying for AraR-mediated regulatory activity in vivo and DNA-binding activity in vitro. The results showed that residues K4, R45 and Q61, located in or near the winged-helix DNA-binding motif, were the most critical amino acids required for AraR function. In addition, the analysis of the various mutations in an AraR palindromic operator sequence indicated that bases G9, A11 and T16 are crucial for AraR binding. Moreover, an AraR mutant M34T was isolated that partially suppressed the effect of mutations in the regulatory cis-elements. Together, these findings extend the knowledge on the nature of AraR nucleoprotein complexes and provide insight into the mechanism that underlies the mode of action of AraR and its orthologues.

  6. Simulations of ordering and sequence reconstruction of random DNA clones hybridized with a small number of oligomeric probes

    SciTech Connect

    Labat, I.; Drmanac, R.

    1992-12-01

    The sequencing by hybridization (SBH) method has been developed for assaying millions of 0.5- to 2-kb-tong clones. This opens up an efficient way for defining the order of short clones and creating a physical map at 100-bp resolution. Moreover, complete sequences can be obtained using a modest number (about 3000) of probes if hybridization and gel sequence data from overlapped or similar sequences are used. In light of these possibilities, various heuristic algorithms have been developed and tested in simulation experiments. This approach can influence the interpretation of the intuitively obvious term, ``known sequence.``

  7. The allele-specific probe and primer amplification assay, a new real-time PCR method for fine quantification of single-nucleotide polymorphisms in pooled DNA.

    PubMed

    Billard, A; Laval, V; Fillinger, S; Leroux, P; Lachaise, H; Beffa, R; Debieu, D

    2012-02-01

    The evolution of fungicide resistance within populations of plant pathogens must be monitored to develop management strategies. Such monitoring often is based on microbiological tests, such as microtiter plate assays. Molecular monitoring methods can be considered if the mutations responsible for resistance have been identified. Allele-specific real-time PCR approaches, such as amplification refractory mutation system (ARMS) PCR and mismatch amplification mutation assay (MAMA) PCR, are, despite their moderate efficacy, among the most precise methods for refining SNP quantification. We describe here a new real-time PCR method, the allele-specific probe and primer amplification assay (ASPPAA PCR). This method makes use of mixtures of allele-specific minor groove binder (MGB) TaqMan probes and allele-specific primers for the fine quantification of SNPs from a pool of DNA extracted from a mixture of conidia. It was developed for a single-nucleotide polymorphism (SNP) that is responsible for resistance to the sterol biosynthesis inhibitor fungicide fenhexamid, resulting in the replacement of the phenylalanine residue (encoded by the TTC codon) in position 412 of the enzymatic target (3-ketoreductase) by a serine (TCC), valine (GTC), or isoleucine (ATC) residue. The levels of nonspecific amplification with the ASPPAA PCR were reduced at least four times below the level of currently available allele-specific real-time PCR approaches due to strong allele specificity in amplification cycles, including two allele selectors. This new method can be used to quantify a complex quadriallelic SNP in a DNA pool with a false discovery rate of less than 1%.

  8. β,γ-CHF- and β,γ-CHCl-dGTP diastereomers: synthesis, discrete 31P NMR signatures and absolute configurations of new stereochemical probes for DNA polymerases

    PubMed Central

    Wu, Yue; Zakharova, Valeria M.; Kashemirov, Boris A.; Goodman, Myron F.; Batra, Vinod K.; Wilson, Samuel H.; McKenna, Charles E.

    2012-01-01

    Deoxynucleoside 5′-triphosphate analogues in which the β,γ-bridging oxygen has been replaced with a CXY group are useful chemical probes to investigate DNA polymerase catalytic and base selection mechanisms. A limitation of such probes has been that conventional synthetic methods generate a mixture of diastereomers when the bridging carbon substitution is non-equivalent (X ≠ Y). We report here a general solution to this long-standing problem with four examples of individual β,γ-CXY dNTP diastereomers: (S)- and (R)-β,γ-CHCl dGTP (12a-1, 12a-2) and (S)- and (R)-β,γ-CHF dGTP (12b-1, 12b-2). Central to their preparation was conversion of the achiral parent bisphosphonic acids to P,C-dimorpholinamide derivatives (7) of their (R)-mandelic acid monoesters (6), which provided access to the individual diastereomers 7a-1, 7a-2, 7b-1, and 7b-2 by preparative HPLC. Selective acidic hydrolysis of the P-N bond then afforded the “ portal ” diastereomers 10, which were readily coupled to morpholine-activated dGMP. Removal of the chiral auxiliary by H2 (Pd/C) afforded the four individual diastereomeric nucleotides (12), which were characterized by 31P, 1H and 19F NMR, and by MS. After treatment with Chelex®-100 to remove traces of paramagnetic ions, at pH ~10 the diastereomer pairs 12a and 12b exhibit discrete Pα and Pβ 31P resonances. The more upfield Pα and more downfield Pβ resonances (and also the more upfield 19F NMR resonance in 12b) are assigned to the (R) configuration at the Pβ-CHX-Pγ carbons, based on the absolute configurations of the individual diastereomers as determined by X-ray crystallographic structures of their ternary complexes with DNA-pol β. PMID:22397499

  9. Sensitivity of a digoxigenin-labelled DNA probe in detecting Mikrocytos mackini, causative agent of Denman Island disease (mikrocytosis), in oysters.

    PubMed

    Meyer, Gary R; Bower, Susan M; Carnegie, Ryan B

    2005-02-01

    The protistan parasite Mikrocytos mackini, causative agent of Denman Island disease (mikrocytosis), induces mortality and reduces marketability in the Pacific oyster, Crassostrea gigas, in British Columbia, Canada. This parasite is a pathogen of international concern because it infects a range of oyster species, and because its life cycle and mode of transmission are unknown. A digoxigenin-labelled DNA probe in situ hybridisation technique (DIG-ISH) was developed, and its detection sensitivity was compared to standard histological sections stained with haematoxylin and eosin stain (H&E-histo). In H&E-histo preparations, the detection of M. mackini was certain only when the parasite occurred within the vesicular connective tissue of adult oysters. However, the DIG-ISH technique clearly demonstrated the presence of infection in all other host tissues as well as in juvenile oysters with poorly developed vesicular connective tissue. The probe hybridised strongly to M. mackini, did not hybridise to oyster tissues or with the other shellfish parasites tested, and was more sensitive for detecting infections when compared to H&E-histo.

  10. In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA.

    PubMed

    Choi, Yun Sik; Kim, Yong Cheol; Baek, Keum Jin; Choi, Youngnim

    2015-05-21

    The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

  11. DNA and protein changes caused by disease in human breast tissues probed by the Kubelka-Munk spectral functional.

    PubMed

    Yang, Yuanlong; Celmer, Edward J; Koutcher, Jason A; Alfano, R R

    2002-06-01

    Malignant, fibroadenoma, normal and adipose breast tissues were studied using diffuse reflectance spectroscopy. The absorption spectra of the breast tissues were extracted from the diffuse reflectance spectra using the Kubelka-Munk function (K-M function). The spectral features of the K-M function were identified and compared with those of the absorption spectra. The spectral features of the K-M function were assigned to DNA, protein, beta-carotene and hemoglobin (oxygenated and deoxygenated) molecules in the breast tissue. The amplitudes of the K-M function averaged from 275 to 285 nm and from 255 to 265 nm and were found to be different for malignant, fibroadenoma and normal tissues. These differences were attributed to changes in proteins and DNA. A set of critical parameters was determined for separating malignant tissues from fibroadenoma and normal tissues. This approach should hold for other tissue types such as cervix, uterus and colon.

  12. Specific and Sensitive Isothermal Electrochemical Biosensor for Plant Pathogen DNA Detection with Colloidal Gold Nanoparticles as Probes.

    PubMed

    Lau, Han Yih; Wu, Haoqi; Wee, Eugene J H; Trau, Matt; Wang, Yuling; Botella, Jose R

    2017-01-17

    Developing quick and sensitive molecular diagnostics for plant pathogen detection is challenging. Herein, a nanoparticle based electrochemical biosensor was developed for rapid and sensitive detection of plant pathogen DNA on disposable screen-printed carbon electrodes. This 60 min assay relied on the rapid isothermal amplification of target pathogen DNA sequences by recombinase polymerase amplification (RPA) followed by gold nanoparticle-based electrochemical assessment with differential pulse voltammetry (DPV). Our method was 10,000 times more sensitive than conventional polymerase chain reaction (PCR)/gel electrophoresis and could readily identify P. syringae infected plant samples even before the disease symptoms were visible. On the basis of the speed, sensitivity, simplicity and portability of the approach, we believe the method has potential as a rapid disease management solution for applications in agriculture diagnostics.

  13. Specific and Sensitive Isothermal Electrochemical Biosensor for Plant Pathogen DNA Detection with Colloidal Gold Nanoparticles as Probes

    NASA Astrophysics Data System (ADS)

    Lau, Han Yih; Wu, Haoqi; Wee, Eugene J. H.; Trau, Matt; Wang, Yuling; Botella, Jose R.

    2017-01-01

    Developing quick and sensitive molecular diagnostics for plant pathogen detection is challenging. Herein, a nanoparticle based electrochemical biosensor was developed for rapid and sensitive detection of plant pathogen DNA on disposable screen-printed carbon electrodes. This 60 min assay relied on the rapid isothermal amplification of target pathogen DNA sequences by recombinase polymerase amplification (RPA) followed by gold nanoparticle-based electrochemical assessment with differential pulse voltammetry (DPV). Our method was 10,000 times more sensitive than conventional polymerase chain reaction (PCR)/gel electrophoresis and could readily identify P. syringae infected plant samples even before the disease symptoms were visible. On the basis of the speed, sensitivity, simplicity and portability of the approach, we believe the method has potential as a rapid disease management solution for applications in agriculture diagnostics.

  14. Specific and Sensitive Isothermal Electrochemical Biosensor for Plant Pathogen DNA Detection with Colloidal Gold Nanoparticles as Probes

    PubMed Central

    Lau, Han Yih; Wu, Haoqi; Wee, Eugene J. H.; Trau, Matt; Wang, Yuling; Botella, Jose R.

    2017-01-01

    Developing quick and sensitive molecular diagnostics for plant pathogen detection is challenging. Herein, a nanoparticle based electrochemical biosensor was developed for rapid and sensitive detection of plant pathogen DNA on disposable screen-printed carbon electrodes. This 60 min assay relied on the rapid isothermal amplification of target pathogen DNA sequences by recombinase polymerase amplification (RPA) followed by gold nanoparticle-based electrochemical assessment with differential pulse voltammetry (DPV). Our method was 10,000 times more sensitive than conventional polymerase chain reaction (PCR)/gel electrophoresis and could readily identify P. syringae infected plant samples even before the disease symptoms were visible. On the basis of the speed, sensitivity, simplicity and portability of the approach, we believe the method has potential as a rapid disease management solution for applications in agriculture diagnostics. PMID:28094255

  15. Molecular Mechanism of Dioxin Action: Molecular Cloning of the Ah Receptor Using a DNA Recognition Site Probe

    DTIC Science & Technology

    1992-01-13

    analysis of AhR binding to the DRE (see attached manuscript an the following brief description of these results) and have bequn the library screening . Although...relatively rapidly as to whether they represent AhR clones or not. As mentioned above, we have only recently begun the library screening . We have obtained a...DNA oligonucleotides, identify the DRE oligonucleotide with the highest binding affinity, optimize the screening protocol and begin the actual library

  16. Bacteria capable of degrading anthracene, phenanthrene, and fluoranthene as revealed by DNA based stable-isotope probing in a forest soil.

    PubMed

    Song, Mengke; Jiang, Longfei; Zhang, Dayi; Luo, Chunling; Wang, Yan; Yu, Zhiqiang; Yin, Hua; Zhang, Gan

    2016-05-05

    Information on microorganisms possessing the ability to metabolize different polycyclic aromatic hydrocarbons (PAHs) in complex environments helps in understanding PAHs behavior in natural environment and developing bioremediation strategies. In the present study, stable-isotope probing (SIP) was applied to investigate degraders of PAHs in a forest soil with the addition of individually (13)C-labeled phenanthrene, anthracene, and fluoranthene. Three distinct phylotypes were identified as the active phenanthrene-, anthracene- and fluoranthene-degrading bacteria. The putative phenanthrene degraders were classified as belonging to the genus Sphingomona. For anthracene, bacteria of the genus Rhodanobacter were the putative degraders, and in the microcosm amended with fluoranthene, the putative degraders were identified as belonging to the phylum Acidobacteria. Our results from DNA-SIP are the first to directly link Rhodanobacter- and Acidobacteria-related bacteria with anthracene and fluoranthene degradation, respectively. The results also illustrate the specificity and diversity of three- and four-ring PAHs degraders in forest soil, contributes to our understanding on natural PAHs biodegradation processes, and also proves the feasibility and practicality of DNA-based SIP for linking functions with identity especially uncultured microorganisms in complex microbial biota.

  17. Karyotype stability in the family Issidae (Hemiptera, Auchenorrhyncha) revealed by chromosome techniques and FISH with telomeric (TTAGG)n and 18S rDNA probes

    PubMed Central

    Maryańska-Nadachowska, Anna; Anokhin, Boris A.; Gnezdilov, Vladimir M.; Kuznetsova, Valentina G.

    2016-01-01

    Abstract We report several chromosomal traits in 11 species from 8 genera of the planthopper family Issidae, the tribes Issini, Parahiraciini and Hemisphaeriini. All species present a 2n = 27, X(0) chromosome complement known to be ancestral for the family. The karyotype is conserved in structure and consists of a pair of very large autosomes; the remaining chromosomes gradually decrease in size and the X chromosome is one of the smallest in the complement. For selected species, analyses based on C-, AgNOR- and CMA3-banding techniques were also carried out. By fluorescence in situ hybridization, the (TTAGG)n probe identified telomeres in all species, and the major rDNA loci were detected on the largest pair of autosomes. In most species, ribosomal loci were found in an interstitial position while in two species they were located in telomeric regions suggesting that chromosomal rearrangements involving the rDNA segments occurred in the evolution of the family Issidae. Furthermore, for 8 species the number of testicular follicles is provided for the first time. PMID:27830046

  18. The characterization of silicon nanowire modified surface with multiwall carbon nanotube (MWCNT) and chitosan (CS) for perfect attachment of ssDNA-probe

    NASA Astrophysics Data System (ADS)

    Adam, Tijjani; Hashim, U.

    2017-03-01

    The study presents an investigation into the effect of chitosan-CNT modification of silicon nanowire on DNA binding chemistry for the perfect attachment of ssDNA adhesion. MWCNT were purified through refluxing in concentrated HNO3 (V/V, 1:1) mixture for 2 hours at 55°C mixture of MWCNT-chitosan solution was prepared first immobilized on the Si surface. The surface was characterized by UV-vis measurement, Scanning Electrons microscope (SEM) and Fourier transform infrared spectroscopy (FTIRs) and series of electrical test were conducted to investigate the effect of MWCNT-chitosan concentration on the performance of the surface binding chemistry with Si-wire and chitosan-CNT modified. The results showed that the chitosan-CNT modification produced good probe adhesion and improve electrical properties. the took the advantage of CNTs with -COOH groups on silicon nanowire dispersed among chitosan containing -NH2 groups due to the peptide bonds formed between -COOH and -NH2 and due nanowire large surface area-to-volume ratio promises high sensitivity. We have established reliable procedure for the functionalization and is tapproach will allow a variety of species to be sensed on nanowire device.

  19. Synthesis and characterization of 8-methoxy-2'- deoxyadenosine-containing oligonucleotides to probe the syn glycosidic conformation of 2'-deoxyadenosine within DNA.

    PubMed Central

    Eason, R G; Burkhardt, D M; Phillips, S J; Smith, D P; David, S S

    1996-01-01

    The synthesis of 8-methoxy-2'-deoxyadenosine (moA) protected at N6 as an N,N-dimethylformamidine derivative and incorporation of the modified nucleoside into oligodeoxynucleotides via the phosphoramidite method are described. UV thermal denaturation studies were conducted on duplexes containing moA:G, moA:C and moA:T base pairs to determine the thermodynamic stability of duplexes containing moA relative to their adenosine (A)-containing counterparts. In the case of moA:G base pairs the effect of moA substitution is sequence dependent. In A:G mismatch-containing sequences, which have been shown by structural characterization to have a syn conformational preference at the glycosidic bond of A, moA substitution results in stabilization of the duplex. In contrast, in sequences where the A in the A:G mismatch has been shown to prefer the anti conformation moA substitution is destabilizing to the duplex. Thus moA may be a useful probe for investigating the conformational preferences of the N-glycosidic bond of adenosine within DNA. In addition, moA nucleoside is more resistant to acid-catalyzed depurination than previously described 8-bromo-2'-deoxyadenosine, allowing for facile incorporation into oligonucleotides via automated solid phase DNA synthesis. PMID:8600457

  20. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  1. Development of a Single Stranded DNA Aptamer as a Molecular Probe for LNCap Cells Using Cell-SELEX

    PubMed Central

    Almasi, Faezeh; Mousavi Gargari, Seyed Latif; Bitaraf, Fatemeh; Rasoulinejad, Samaneh

    2016-01-01

    Background: Nowadays, highly specific aptamers generated by cell SELEX technology (systematic evolution of ligands by exponential enrichment) are being applied for early detection of cancer cells. Prostate Specific Membrane Antigen (PSMA), over expressed in prostate cancer, is a highly specific marker and therefore can be used for diagnosis of the prostate cancer cells. The aim of the present study was to select single-stranded DNA aptamers against LNCap cells highly expressing PSMA, using cell–SELEX method which can be used as a diagnostic tool for the detection of prostate cancer cells. Methods: After 10 rounds of cell-SELEX, DNA aptamers were isolated against PSMA using LNCaP cells as a target and PC-3 cell lines for counter SELEX. Five DNA aptamers with more than 70% affinity were selected up on flow cytometry analysis of positive clones. Results: Dissociation constants of two selected sequences (A12-B1) were estimated in the range of 33.78±3.77 and 57.49±2.214 pmol, respectively. Conserved secondary structures of A12 and B1 sequences suggest the necessity of these structures for binding with high affinity to native PSMA. Comparison of the secondary structures of our isolated aptamers and aptamer A10 obtained by protein SELEX showed similar stem-loop structures which could be responsible for the recognition of PSMA on LNCap cell surface. Conclusion: Our results indicated that selected aptamers may turn out to be ideal candidates for the development of a detection tool and also can be used in targeted drug delivery for future smart drugs. PMID:27563422

  2. Sensitive Visual Detection of AHPND Bacteria Using Loop-Mediated Isothermal Amplification Combined with DNA-Functionalized Gold Nanoparticles as Probes

    PubMed Central

    Arunrut, Narong; Kampeera, Jantana; Sirithammajak, Sarawut; Sanguanrut, Piyachat; Proespraiwong, Porranee; Suebsing, Rungkarn; Kiatpathomchai, Wansika

    2016-01-01

    Acute hepatopancreatic necrosis disease (AHPND) is a component cause of early mortality syndrome (EMS) of shrimp. In 2013, the causative agent was found to be unique isolates of Vibrio parahaemolyticus (VPAHPND) that contained a 69 kbp plasmid (pAP1) carrying binary Pir-like toxin genes PirvpA and PirvpB. In Thailand, AHPND was first recognized in 2012, prior to knowledge of the causative agent, and it subsequently led to a precipitous drop in shrimp production. After VPAHPND was characterized, a major focus of the AHPND control strategy was to monitor broodstock shrimp and post larvae for freedom from VPAHPND by nucleic acid amplification methods, most of which required use of expensive and sophisticated equipment not readily available in a shrimp farm setting. Here, we describe a simpler but equally sensitive approach for detection of VPAHPND based on loop-mediated isothermal amplification (LAMP) combined with unaided visual reading of positive amplification products using a DNA-functionalized, ssDNA-labled nanogold probe (AuNP). The target for the special set of six LAMP primers used was the VPAHPND PirvpA gene. The LAMP reaction was carried out at 65°C for 45 min followed by addition of the red AuNP solution and further incubation at 65°C for 5 min, allowing any PirvpA gene amplicons present to hybridize with the probe. Hybridization protected the AuNP against aggregation, so that the solution color remained red upon subsequent salt addition (positive test result) while unprotected AuNP aggregated and underwent a color change from red to blue and eventually precipitated (negative result). The total assay time was approximately 50 min. The detection limit (100 CFU) was comparable to that of other commonly-used methods for nested PCR detection of VPAHPND and 100-times more sensitive than 1-step PCR detection methods (104 CFU) that used amplicon detection by electrophoresis or spectrophotometry. There was no cross reaction with DNA templates derived from non

  3. Evaluation of the New BD Max GC Real-Time PCR Assay, Analytically and Clinically as a Supplementary Test for the BD ProbeTec GC Qx Amplified DNA Assay, for Molecular Detection of Neisseria gonorrhoeae

    PubMed Central

    Golparian, Daniel; Boräng, Stina; Sundqvist, Martin

    2015-01-01

    The new BD Max GC real-time PCR assay showed high clinical and analytical sensitivity and specificity. It can be an effective and accurate supplementary test for the BD ProbeTec GC Qx amplified DNA assay, which had suboptimal specificity, and might also be used for initial detection of Neisseria gonorrhoeae. PMID:26468501

  4. Complexities of bloom dynamics in the toxic dinoflagellate Alexandrium fundyense revealed through DNA measurements by imaging flow cytometry coupled with species-specific rRNA probes

    NASA Astrophysics Data System (ADS)

    Brosnahan, Michael L.; Farzan, Shahla; Keafer, Bruce A.; Sosik, Heidi M.; Olson, Robert J.; Anderson, Donald M.

    2014-05-01

    Measurements of the DNA content of different protist populations can shed light on a variety of processes, including cell division, sex, prey ingestion, and parasite invasion. Here, we modified an Imaging FlowCytobot (IFCB), a custom-built flow cytometer that records images of microplankton, to measure the DNA content of large dinoflagellates and other high-DNA content species. The IFCB was also configured to measure fluorescence from Cy3-labeled rRNA probes, aiding the identification of Alexandrium fundyense (syn. A. tamarense Group I), a photosynthetic dinoflagellate that causes paralytic shellfish poisoning (PSP). The modified IFCB was used to analyze samples from the development, peak and termination phases of an inshore A. fundyense bloom (Salt Pond, Eastham, MA, USA), and from a rare A. fundyense ‘red tide’ that occurred in the western Gulf of Maine, offshore of Portsmouth, NH (USA). Diploid or G2 phase (‘2C’) A. fundyense cells were frequently enriched at the near-surface, suggesting an important role for aggregation at the air-sea interface during sexual events. Also, our analysis showed that large proportions of A. fundyense cells in both the Salt Pond and red tide blooms were planozygotes during bloom decline, highlighting the importance of sexual fusion to bloom termination. At Salt Pond, bloom decline also coincided with a dramatic rise in infections by the parasite genus Amoebophrya. The samples that were most heavily infected contained many large cells with higher DNA-associated fluorescence than 2C vegetative cells, but these cells' nuclei were also frequently consumed by Amoebophrya trophonts. Neither large cell size nor increased DNA-associated fluorescence could be replicated by infecting an A. fundyense culture of vegetative cells. Therefore, we attribute these characteristics of the large Salt Pond cells to planozygote maturation rather than Amoebophrya infection, though an interaction between infection and planozygote maturation may

  5. Detection of {open_quotes}cryptic{close_quotes}karyotypic rearrangements in closely related primate species by fluorescence in situ hybridization (FISH) using human subtelomeric DNA probes

    SciTech Connect

    Youngblom, J.J.; Trask, B.J.; Friedman, C.

    1994-09-01

    Specific human subtelomeric DNA probes were used to reveal cryptic chromosomal rearrangements that cannot be detected by conventional high resolution cytogenetic techniques, or by chromosomal in situ suppression hybridization using whole chromosome paint analysis. Two cosmids containing different subtelomeric DNA sequences were derived from human chromosome 19 and designated as 7501 and 16432. Cosmid 7501 was hybridized to chromosomes from humans, chimpanzee, gorilla and orangutan. In humans, 7501 consistently labeled chromosomes 3q, 15q, and 19p. Additional chromosomes were labeled in different individuals, indicating a polymorphic distribution of this sequence in the human genome. In contrast, 7501 consistently and strongly labeled only the q arm terminus of chromosome 3 in both chimp and gorilla. The identification of the chromosome was made by two-color FISH analysis using human chromosome 4-specific paint and homologous to human chromosome 4. None of the human subjects showed labeling of chromosome 4 with 7501. This finding suggests that in the course of human evolution, subsequent to the divergence of humans and African apes, a cryptic translocation occurred between the ancestral human chromosome 4 and one or more of the other human chromosomes that now contain this DNA segment. In orangutan, 7501 labeled a single acrocentric chromosome pair, a distinctly different chromosome than that labeled in chimp and gorilla. Comparison of chromosome sites labeled with cosmid 16432 showed the distribution of signals on chromosome 1q arm is the same for humans and chimp, but different in the gorilla. Humans and chimps show distinct labeling on sites 1q terminus and 1q41-42. In gorilla, there is instead a large cluster of intense signal near the terminus of 1q that clearly does not extend all the way to the terminus. A paracentric inversion or an unequal cross-over event may account for the observed difference between these species.

  6. Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher

    NASA Astrophysics Data System (ADS)

    Normanno, Davide; Boudarène, Lydia; Dugast-Darzacq, Claire; Chen, Jiji; Richter, Christian; Proux, Florence; Bénichou, Olivier; Voituriez, Raphaël; Darzacq, Xavier; Dahan, Maxime

    2015-07-01

    Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein-DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells.

  7. Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher

    PubMed Central

    Normanno, Davide; Boudarène, Lydia; Dugast-Darzacq, Claire; Chen, Jiji; Richter, Christian; Proux, Florence; Bénichou, Olivier; Voituriez, Raphaël; Darzacq, Xavier; Dahan, Maxime

    2015-01-01

    Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein–DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells. PMID:26151127

  8. Probing the Ion Binding Site in a DNA Holliday Junction Using Förster Resonance Energy Transfer (FRET)

    PubMed Central

    Litke, Jacob L.; Li, Yan; Nocka, Laura M.; Mukerji, Ishita

    2016-01-01

    Holliday Junctions are critical DNA intermediates central to double strand break repair and homologous recombination. The junctions can adopt two general forms: open and stacked-X, which are induced by protein or ion binding. In this work, fluorescence spectroscopy, metal ion luminescence and thermodynamic measurements are used to elucidate the ion binding site and the mechanism of junction conformational change. Förster resonance energy transfer measurements of end-labeled junctions monitored junction conformation and ion binding affinity, and reported higher affinities for multi-valent ions. Thermodynamic measurements provided evidence for two classes of binding sites. The higher affinity ion-binding interaction is an enthalpy driven process with an apparent stoichiometry of 2.1 ± 0.2. As revealed by Eu3+ luminescence, this binding class is homogeneous, and results in slight dehydration of the ion with one direct coordination site to the junction. Luminescence resonance energy transfer experiments confirmed the presence of two ions and indicated they are 6–7 Å apart. These findings are in good agreement with previous molecular dynamics simulations, which identified two symmetrical regions of high ion density in the center of stacked junctions. These results support a model in which site-specific binding of two ions in close proximity is required for folding of DNA Holliday junctions into the stacked-X conformation. PMID:26978349

  9. Micro-RNA detection based on fluorescence resonance energy transfer of DNA-carbon quantum dots probes.

    PubMed

    Khakbaz, Faeze; Mahani, Mohamad

    2017-04-15

    Carbon quantum dots have been proposed as an effective platform for miRNA detection. Carbon dots were synthesized by citric acid. The synthesized dots were characterized by dynamic light scattering, UV-Vis spectrophotometry, spectrofluorimetry, transmission electron microscopy and FT-IR spectrophotometry. The fluorescence quantum yield of the synthesized dots was determined using quinine sulfate as the standard. The FAM-labeled single stranded DNA, as sensing element, was adsorbed on dots by π-π interaction. The quenching of the dots fluorescence due to fluorescence resonance energy transfer (FRET) was used for mir 9-1 detection. In the presence of the complementary miRNA, the FRET did not take place and the fluorescence was recovered.

  10. Use of an antikinetochore antibody and DNA probes to measure aneuploidy induction in interphase human lymphocytes and Chinese hamster ovary cells

    SciTech Connect

    Eastmond, D.A.; Tucker, J.D.; Pinkel, D.

    1988-12-05

    Aneuploidy in germ cells is associated with birth defects, spontaneous abortions, and infertility, whereas in somatic cells aneuploidy may lead to cell death and carcinogenesis. The nonrandom numerical chromosomal changes that are often observed in tumors or transformed cells suggest that aneuploidy induction by chemicals may be involved in carcinogenesis. The identification of aneuploidy inducing agents (aneuploidogens) and studies into the mechanisms by which aneuploidy may be involved in carcinogenesis are currently limited in that standard cytogenetic techniques are time consuming, require highly skilled personnel and are prone to technical artifacts. Recent developments in immunology and molecular biology have resulted in new techniques which may allow simple and rapid identification of aneuploidogens. We report the development of two new approaches to determine the aneuploidy-inducing potential of chemicals. The first approach involves the induction of micronuclei in human lymphocytes and Chinese hamster ovary (CHO) cells and the use of an antikinetochore antibody to determine whether micronuclei contain centromeres---a condition indicating potential aneuploidy. The second approach involves the use of in situ hybridization with fluorescently labeled chromosome-specific DNA probes and the subsequent counting of the number of copies of that chromosome in the interphase nuclei of human lymphocytes. 8 refs., 1 fig., 1 tab.

  11. DNA stable-isotope probing of oil sands tailings pond enrichment cultures reveals different key players for toluene degradation under methanogenic and sulfidogenic conditions.

    PubMed

    Laban, Nidal Abu; Dao, Anh; Foght, Julia

    2015-05-01

    Oil sands tailings ponds are anaerobic repositories of fluid wastes produced by extraction of bitumen from oil sands ores. Diverse indigenous microbiota biodegrade hydrocarbons (including toluene) in situ, producing methane, carbon dioxide and/or hydrogen sulfide, depending on electron acceptor availability. Stable-isotope probing of cultures enriched from tailings associated specific taxa and functional genes to (13)C6- and (12)C7-toluene degradation under methanogenic and sulfate-reducing conditions. Total DNA was subjected to isopycnic ultracentrifugation followed by gradient fraction analysis using terminal restriction fragment length polymorphism (T-RFLP) and construction of 16S rRNA, benzylsuccinate synthase (bssA) and dissimilatory sulfite reductase (dsrB) gene clone libraries. T-RFLP analysis plus sequencing and in silico digestion of cloned taxonomic and functional genes revealed that Clostridiales, particularly Desulfosporosinus (136 bp T-RF) contained bssA genes and were key toluene degraders during methanogenesis dominated by Methanosaeta. Deltaproteobacterial Desulfobulbaceae (157 bp T-RF) became dominant under sulfidogenic conditions, likely because the Desulfosporosinus T-RF 136 apparently lacks dsrB and therefore, unlike its close relatives, is presumed incapable of dissimilatory sulfate reduction. We infer incomplete oxidation of toluene by Desulfosporosinus in syntrophic association with Methanosaeta under methanogenic conditions, and complete toluene oxidation by Desulfobulbaceae during sulfate reduction.

  12. Identification of biomass utilizing bacteria in a carbon-depleted glacier forefield soil by the use of 13C DNA stable isotope probing.

    PubMed

    Zumsteg, Anita; Schmutz, Stefan; Frey, Beat

    2013-06-01

    As Alpine glaciers are retreating rapidly, bare soils with low organic C and N contents are becoming exposed. Carbon availability is a key factor regulating microbial diversity and ecosystem functioning in these soils. The aim of this study was to investigate how bacterial activity, community structure and composition are influenced by organic carbon availability. Bare soils were supplied with (13)C-labelled fungal (Penicillium sp.) and green algal (Chlorella sp.) biomass and the CO2 evolution and its δ(13)C signature were monitored up to 60 days. These organisms have previously been isolated near the glacier terminus. DNA stable isotope probing followed by T-RFLP profiling and sequencing of 16S rRNA genes was employed to identify consumers able to assimilate carbon from these biomass amendments. Higher respiration and higher bacterial activity indicated a more efficient utilization of algal cells than fungal cells. Flavobacterium sp. predominantly incorporated fungal-derived C, whereas the algal-derived C was mainly incorporated by Acidobacteria and Proteobacteria. This study emphasizes the important role of both fungal and algal biomass in increasing the carbon pool in recently deglaciated bare soils, as only 20% of the added C was respired as CO2, and the rest, we presume, remained in the soil.

  13. Molecular DNA switches and DNA chips

    NASA Astrophysics Data System (ADS)

    Sabanayagam, Chandran R.; Berkey, Cristin; Lavi, Uri; Cantor, Charles R.; Smith, Cassandra L.

    1999-06-01

    We present an assay to detect single-nucleotide polymorphisms on a chip using molecular DNA switches and isothermal rolling- circle amplification. The basic principle behind the switch is an allele-specific oligonucleotide circularization, mediated by DNA ligase. A DNA switch is closed when perfect hybridization between the probe oligonucleotide and target DNA allows ligase to covalently circularize the probe. Mismatches around the ligation site prevent probe circularization, resulting in an open switch. DNA polymerase is then used to preferentially amplify the closed switches, via rolling-circle amplification. The stringency of the molecular switches yields 102 - 103 fold discrimination between matched and mismatched sequences.

  14. Synthesis of PET probe O(6)-[(3-[(11)C]methyl)benzyl]guanine by Pd(0)-mediated rapid C-[(11)C]methylation toward imaging DNA repair protein O(6)-methylguanine-DNA methyltransferase in glioblastoma.

    PubMed

    Koyama, Hiroko; Ikenuma, Hiroshi; Toda, Hiroshi; Kondo, Goro; Hirano, Masaki; Kato, Masaya; Abe, Junichiro; Yamada, Takashi; Wakabayashi, Toshihiko; Ito, Kengo; Natsume, Atsushi; Suzuki, Masaaki

    2017-03-18

    O(6)-Benzylguanine (O(6)-BG) is a substrate of O(6)-methylguanine-DNA methyltransferase (MGMT), which is involved in drug resistance of chemotherapy in the majority of glioblastoma multiform. For clinical diagnosis, it is hoped that the MGMT expression level could be determined by a noninvasive method to understand the detailed biological properties of MGMT-specific tumors. We synthesized (11)C-labeled O(6)-[(3-methyl)benzyl]guanine ([(11)C]mMeBG) as a positron emission tomography probe. Thus, a mixed amine-protected stannyl precursor, N(9)-(tert-butoxycarbonyl)-O(6)-[3-(tributylstannyl)benzyl]-N(2)-(trifluoroacetyl)guanine, was subjected to rapid C-[(11)C]methylation under [(11)C]CH3I/[Pd2(dba)3]/P(o-CH3C6H4)3/CuCl/K2CO3 in NMP, followed by quick deprotection with LiOH/H2O, giving [(11)C]mMeBG with total radioactivity of 1.34GBq and ≥99% radiochemical and chemical purities.

  15. Characterization of Growing Soil Bacterial Communities across a pH gradient Using H218O DNA-Stable Isotope Probing

    NASA Astrophysics Data System (ADS)

    Welty-Bernard, A. T.; Schwartz, E.

    2014-12-01

    Recent studies have established consistent relationships between pH and bacterial diversity and community structure in soils from site-specific to landscape scales. However, these studies rely on DNA or PLFA extraction techniques from bulk soils that encompass metabolically active and inactive, or dormant, communities, and loose DNA. Dormant cells may comprise up to 80% of total live cells. If dormant cells dominate a particular environment, it is possible that previous interpretations of the soil variables assumed to drive communities could be profoundly affected. We used H218O stable isotope probing and bar-coded illumina sequencing of 16S rRNA genes to monitor the response of actively growing communities to changes in soil pH in a soil microcosm over 14 days. This substrate-independent approach has several advantages over 13C or 15N-labelled molecules in that all growing bacteria should be able to make use of water, allowing characterization of whole communities. We hypothesized that Acidobacteria would increasingly dominate the growing community and that Actinobacteria and Bacteroidetes would decline, given previously established responses by these taxa to soil pH. Instead, we observed the reverse. Actinobacteria abundance increased three-fold from 26 to 76% of the overall community as soil pH fell from pH 5.6 to pH 4.6. Shifts in community structure and decreases in diversity with declining soil pH were essentially driven by two families, Streptomyceaca and Microbacteracea, which collectively increased from 2 to 40% of the entire community. In contrast, Acidobacteria as a whole declined although numbers of subdivision 1 remained stable across all soil pH levels. We suggest that the brief incubation period in this SIP study selected for growth of acid-tolerant Actinobacteria over Acidobacteria. Taxa within Actinomycetales have been readily cultured over short time frames, suggesting rapid growth patterns. Conversely, taxa within Acidobacteria have been

  16. High Resolution DNA Stable Isotope Probing Reveals that Root Exudate Addition to Soil Changes the Identity of the Microbes that Degrade Cellulose but not the Rate of Degradation

    NASA Astrophysics Data System (ADS)

    Campbell, A.; Pepe-Ranney, C. P.; Nguyen, A. V. T.; Buckley, D. H.

    2015-12-01

    Plant roots release compounds, such as root exudates, which can alter soil organic matter (SOM) decomposition and have large impacts on soil carbon (C) retention. The changes in SOM turnover resulting from the addition of organic and/or inorganic substrates are termed 'priming effects'. In this study we examine the effects of root exudates on the priming of cellulose added as particulate organic matter. We amended soil microcosms with 13C-cellulose in the presence or absence of artificial root exudate additions and incubated over time for 45 days. Soils receiving the root exudate (RE) were given either one large dose or multiple, small doses of RE. In each treatment we tracked operational taxonomic units (OTUs) assimilating 13C from cellulose (herein, known as a 'responder') over time using DNA stable isotope probing coupled with next generation sequencing. In all treatments the same amount of cellulose-13C was respired indicating the addition of RE did not result in the priming of cellulose decomposition. However, cellulose responders were different depending on treatment and time of sampling (days 14, 28 and 45). We identified a total of 10,361 OTUs, of which there were 369 cellulose responders in the cellulose only treatment, 273 in the repeated, small dose RE treatment, and 358 in the RE single, large dose treatment. Most of the cellulose responders found in all treatments belonged to phyla Bacteroidetes, Planctomycetes, Proteobacteria, Verrucomicrobia, and Chloroflexi. The response time of phyla varies; for instance, more OTUs in Bacteroidetes were observed on day 14 and diminish with each subsequent sampling time. On the other hand, OTUs in Verrucomicrobia increased in response over time. Our study shows no priming effect resulting from the addition of root exudates, although the identity of the microbial mediators of cellulose decomposition varies in each treatment.

  17. Androgen receptor location in the dark-eyed junco using a probe for in situ hybridization histochemistry generated from zebra finch cDNA.

    PubMed

    Satre, Danielle; Kim, Yong-Hwan; Corbitt, Cynthia

    2011-09-30

    Due to the role of sex steroids, namely testosterone (T), in the development and production of song in songbirds, androgen receptor (AR) densities in the brain regions controlling this behavior (i.e., the song control system) have long been studied. Many methods have been used to determine AR density and location to investigate the functional role of T in song development and production; however, a riboprobe developed from zebra finch (Taeniopygia guttata) cDNA was shown to be much more sensitive than previous methods. The zebra finch is a common model for song development and is sexually dimorphic, but does not breed seasonally or display seasonal changes in song control region volume. In this study, we used this riboprobe for in situ hybridization histochemistry (ISHH) to describe AR mRNA location in the brain of the dark-eyed junco (Junco hyemalis), a seasonally breeding model for which T has been shown to be important. Additionally, we provide a detailed comparison of AR mRNA location between these species. We found that this probe is indeed highly sensitive. We detected AR mRNA in four major regions of the song control system (HVC, MAN, RA and Area X). Additionally, we found that the location of AR mRNA in other regions varied only slightly between these two species. These findings suggest that this method is suitable for use across songbirds and it could be useful in the ongoing attempts to elucidate the roles of sex steroid hormones on the development of this and other sex steroid dependent behaviors in songbirds.

  18. A novel probe density controllable electrochemiluminescence biosensor for ultra-sensitive detection of Hg2+ based on DNA hybridization optimization with gold nanoparticles array patterned self-assembly platform.

    PubMed

    Gao, Wenhua; Zhang, An; Chen, Yunsheng; Chen, Zixuan; Chen, Yaowen; Lu, Fushen; Chen, Zhanguang

    2013-11-15

    Biosensor based on DNA hybridization holds great potential to get higher sensitivity as the optimal DNA hybridization efficiency can be achieved by controlling the distribution and orientation of probe strands on the transducer surface. In this work, an innovative strategy is reported to tap the sensitivity potential of current electrochemiluminescence (ECL) biosensing system by dispersedly anchoring the DNA beacons on the gold nanoparticles (GNPs) array which was electrodeposited on the glassy carbon electrode surface, rather than simply sprawling the coil-like strands onto planar gold surface. The strategy was developed by designing a "signal-on" ECL biosensing switch fabricated on the GNPs nanopatterned electrode surface for enhanced ultra-sensitivity detection of Hg(2+). A 57-mer hairpin-DNA labeled with ferrocene as ECL quencher and a 13-mer DNA labeled with Ru(bpy)3(2+) as reporter were hybridized to construct the signal generator in off-state. A 31-mer thymine (T)-rich capture-DNA was introduced to form T-T mismatches with the loop sequence of the hairpin-DNA in the presence of Hg(2+) and induce the stem-loop open, meanwhile the ECL "signal-on" was triggered. The peak sensitivity with the lowest detection limit of 0.1 nM was achieved with the optimal GNPs number density while exorbitant GNPs deposition resulted in sensitivity deterioration for the biosensor. We expect the present strategy could lead the renovation of the existing probe-immobilized ECL genosensor design to get an even higher sensitivity in ultralow level of target detection such as the identification of genetic diseases and disorders in basic research and clinical application.

  19. Biocompatible core-shell nanoparticle-based surface-enhanced Raman scattering probes for detection of DNA related to HIV gene using silica-coated magnetic nanoparticles as separation tools.

    PubMed

    Liang, Yi; Gong, Ji-Lai; Huang, Yong; Zheng, Yue; Jiang, Jian-Hui; Shen, Guo-Li; Yu, Ru-Qin

    2007-04-30

    A novel, highly selective DNA hybridization assay has been developed based on surface-enhanced Raman scattering (SERS) for DNA sequences related to HIV. This strategy employs the Ag/SiO(2) core-shell nanoparticle-based Raman tags and the amino group modified silica-coated magnetic nanoparticles as immobilization matrix and separation tool. The hybridization reaction was performed between Raman tags functionalized with 3'-amino-labeled oligonucleotides as detection probes and the amino group modified silica-coated magnetic nanoparticles functionalized with 5'-amino-labeled oligonucleotides as capture probes. The Raman spectra of Raman tags can be used to monitor the presence of target oligonucleotides. The utilization of silica-coated magnetic nanoparticles not only avoided time-consuming washing, but also amplified the signal of hybridization assay. Additionally, the results of control experiments show that no or very low signal would be obtained if the hybridization assay is conducted in the presence of DNA sequences other than complementary oligonucleotides related to HIV gene such as non-complementary oligonucleotides, four bases mismatch oligonucleotides, two bases mismatch oligonucleotides and even single base mismatch oligonucleotides. It was demonstrated that the method developed in this work has high selectivity and sensitivity for DNA detection related to HIV gene.

  20. Long-Term Prognostic Effects of Plasma Epstein-Barr Virus DNA by Minor Groove Binder-Probe Real-Time Quantitative PCR on Nasopharyngeal Carcinoma Patients Receiving Concurrent Chemoradiotherapy

    SciTech Connect

    Lin, J.-C. . E-mail: jclin@vghtc.gov.tw; Wang, W.-Y.; Liang, W.-M.; Chou, H.-Y.; Jan, J.-S.; Jiang, R.-S.; Wang, J.-Y.; Twu, C.-W.; Liang, K.-L.; Chao, Jeffrey; Shen, W.-C.

    2007-08-01

    Purpose: To evaluate the long-term prognostic impact of plasma Epstein-Barr virus (EBV) DNA concentration measured by real-time quantitative polymerase chain reaction (RTQ-PCR) in nasopharyngeal carcinoma (NPC) patients receiving concurrent chemoradiotherapy (CCRT). Methods and Materials: Epstein-Barr virus DNA was retrospectively measured from stock plasma of 152 biopsy-proven NPC patients with Stage II-IV (M0) disease with a RTQ-PCR using the minor groove binder-probe. All patients received CCRT with a median follow-up of 78 months. We divided patients into three subgroups: (1) low pretreatment EBV DNA (<1,500 copies/mL) and undetectable posttreatment EBV DNA (pre-L/post-U) (2) high pretreatment EBV DNA ({>=}1,500 copies/mL) and undetectable posttreatment EBV DNA (pre-H/post-U), and (3) low or high pretreatment EBV DNA and detectable posttreatment EBV DNA (pre-L or H/post-D) for prognostic analyses. Results: Epstein-Barr virus DNA (median concentration, 573 copies/mL; interquartile range, 197-3,074) was detected in the pretreatment plasma of 94.1% (143/152) of patients. After treatment, plasma EBV DNA decreased or remained 0 for all patients and was detectable in 31 patients (20.4%) with a median concentration 0 copy/mL (interquartile range, 0-0). The 5-year overall survival rates of the pre-L/post-U, pre-H/post-U, and pre-L or H/post-D subgroups were 87.2%, 71.0%, and 38.7%, respectively (p < 0.0001). The relapse-free survival showed similar results with corresponding rates of 85.6%, 75.9%, and 26.9%, respectively (p < 0.0001). Multivariate Cox analysis confirmed the superior effects of plasma EBV DNA compared to other clinical parameters in prognosis prediction. Conclusion: Plasma EBV DNA is the most valuable prognostic factor for NPC. More chemotherapy should be considered for patients with persistently detectable EBV DNA after CCRT.

  1. Spectrophotometric probe

    DOEpatents

    Prather, W.S.; O'Rourke, P.E.

    1994-08-02

    A support structure is described bearing at least one probe for making spectrophotometric measurements of a fluid using a source of light and a spectrophotometer. The probe includes a housing with two optical fibers and a planoconvex lens. A sleeve bearing a mirror surrounds the housing. The lens is separated from the mirror by a fixed distance, defining an interior space for receiving a volume of the fluid sample. A plurality of throughholes extending through the sleeve communicate between the sample volume and the exterior of the probe, all but one hole bearing a screen. A protective jacket surrounds the probe. A hollow conduit bearing a tube is formed in the wall of the probe for venting any air in the interior space when fluid enters. The probe is held at an acute angle so the optic fibers carrying the light to and from the probe are not bent severely on emergence from the probe. 3 figs.

  2. Spectrophotometric probe

    DOEpatents

    Prather, William S.; O'Rourke, Patrick E.

    1994-01-01

    A support structure bearing at least one probe for making spectrophotometric measurements of a fluid using a source of light and a spectrophotometer. The probe includes a housing with two optical fibers and a planoconvex lens. A sleeve bearing a mirror surrounds the housing. The lens is separated from the mirror by a fixed distance, defining an interior space for receiving a volume of the fluid sample. A plurality of throughholes extending through the sleeve communicate between the sample volume and the exterior of the probe, all but one hole bearing a screen. A protective jacket surrounds the probe. A hollow conduit bearing a tube is formed in the wall of the probe for venting any air in the interior space when fluid enters. The probe is held at an acute angle so the optic fibers carrying the light to and from the probe are not bent severely on emergence from the probe.

  3. Probing the Salt Concentration Dependent Nucelobase Distribution in a Single-Stranded DNA-Single-Walled Carbon Nanotube Hybrid with Molecular Dynamics.

    PubMed

    Ghosh, Soumadwip; Patel, Nisheet; Chakrabarti, Rajarshi

    2016-01-28

    The hybrids of single-walled carbon nanotube (SWCNT) and single stranded DNA (ssDNA) are novel nanoscale materials having remarkable applications in nanotechnology. The absorption of nucleobases on the surface of a SWCNT depends strongly on the ionic strength of the medium. In this paper, using atomistic molecular dynamics we have shown that at low salt concentration ssDNA wraps on the surface of SWCNT through hydrophobic π-π stacking between the DNA bases and the sp(2)-hybridized carbon atoms of the carbon nanotube. At high salt concentration, however, the DNA molecule adopts a partially folded structure and the ssDNA-SWCNT wrapping gets weakened significantly due to the self-stacking of the DNA bases. Our study can find relevance in CNT mediated gene delivery processes where subsequent unwrapping of the gene from its carrier is anticipated across the cell membrane regulated by an existing salt concentration gradient.

  4. Mono and trimethine cyanines Cyan 40 and Cyan 2 as probes for highly selective fluorescent detection of non-canonical DNA structures.

    PubMed

    Kovalska, Vladyslava B; Losytskyy, Mykhaylo Yu; Yarmoluk, Sergiy M; Lubitz, Irit; Kotlyar, Alexander B

    2011-01-01

    Two of earlier reported dsDNA sensitive cyanine dyes-monomethine Cyan 40 and meso-substituted trimethine Cyan 2 were studied for their ability to interact with non-canonical DNA conformations. These dyes were characterized by spectral-luminescent methods in the presence of G-quadruplex, triplex and dsDNA motifs. We have demonstrated that Cyan 2 binds strongly and preferentially to triple- and quadruple-stranded DNA forms that results in a strong enhancement of the dye fluorescence, as compared to dsDNA, while Cyan 40 form fluorescent complexes preferentially only with the triplex form. Highly fluorescent complexes of Cyan 2 with DNA triplexes and G-quadruplexes and Cyan 40 with DNA triplexes are very stable and do not dissociate during gel electrophoresis, leading to preferential staining of the above DNA forms in gels. The data presented point to the intercalation mechanism of the Cyan 2 binding to G4-DNA, while the complexes of Cyan 40 and Cyan 2 with triplex DNA are believed to be formed via groove binding mode. The Cyan dyes can provide a highly sensitive method for detection and quantification of non-canonical structures in genome.

  5. Pushing the limit: synthesis, photophysical and DNA binding studies of a NIR-emitting Ru(II)-polypyridyl probe with 'light switch' behaviour.

    PubMed

    Elmes, Robert B P; Kitchen, Jonathan A; Williams, D Clive; Gunnlaugsson, Thorfinnur

    2012-06-14

    The new Ru(II) polypyridyl complex 1 was synthesised using microwave irradiation from the new polypyridyl ligand 2'DipyTAP', and its photophysical properties, and DNA binding abilities were investigated using various spectroscopic techniques; and 1 was shown to act as a 'NIR molecular light switch' for DNA with an emission window between 680 and 860 nm.

  6. Two-color spectroscopy of UV excited ssDNA complex with a single-wall nanotube (SWNT) probe: Fast nucleobase autoionization mechanism

    NASA Astrophysics Data System (ADS)

    Rotkin, Slava V.; Ignatova, Tetyana; Balaeff, Alexander; Zheng, Ming; Blades, Michael; Stoeckl, Peter

    DNA autoionization is a fundamental process wherein UV-photoexcited nucleobases dissipate energy to the environment without undergoing chemical damage. SWNT is shown to serve as a photoluminescent reporter for studying the mechanism and rates of DNA autoionization. Two-color photoluminescence (PL) spectroscopy revealed a strong SWNT PL quenching when the UV pump is resonant with the DNA absorption [Nano Research, 2015]. Semiempirical calculations of the DNA-SWNT electronic structure, combined with a Green's function theory for charge transfer, show a 20 fs autoionization rate, dominated by the hole transfer. Rate-equation analysis of the spectroscopy data confirms that the quenching rate is limited by the thermalization of the free charge carriers transferred to the nanotube reservoir. The developed approach has a great potential for monitoring DNA excitation, autoionization, and chemical damage both in vivo and in vitro. NSF ECCS-1509786 (S.V.R.,T.I.) and PHY-1359195 (P.S.), NIST and UCF facilities.

  7. Synthesis of a novel 4H-pyran analog as minor groove binder to DNA using ethidium bromide as fluorescence probe

    NASA Astrophysics Data System (ADS)

    Ramana, M. M. V.; Betkar, Rahul; Nimkar, Amey; Ranade, Prasanna; Mundhe, Balaji; Pardeshi, Sachin

    2016-01-01

    In the present work, isopropyl-6-amino-4-(3,5-bis(trifluoromethyl)phenyl)-5-cyano-2-methyl-4H-pyran-3-carboxylate (4H-pyran analog) has been synthesized by a three component reaction catalyzed by CsOH/γ-Al2O3 and characterized. The interaction of 4H-pyran analog with herring sperm DNA (hs DNA) under physiological conditions (phosphate buffer of pH 7.2) was investigated by UV absorption, FT-IR, fluorescence, 31P NMR and circular dichroism (CD) spectroscopy. Fluorescence quenching results reveal that static quenching mechanism is involved in binding between 4H-pyran analog and hs DNA. The calculated thermodynamic parameters (ΔH° and ΔS°) indicate that hydrogen bonding plays a major role in binding between them. UV absorption and fluorescence shows the binding mode of 4H-pyran analog with hs DNA as non-intercalative. According to the IR spectroscopy, 4H-pyran analog binds to guanine, thymine, adenine bases of hs DNA but not to phosphate backbone of hs DNA which is also in good agreement with 31P NMR results. CD and competitive binding experiment results confirms the minor groove binding of 4H-pyran analog to hs DNA.

  8. Synthesis of a novel 4H-pyran analog as minor groove binder to DNA using ethidium bromide as fluorescence probe.

    PubMed

    Ramana, M M V; Betkar, Rahul; Nimkar, Amey; Ranade, Prasanna; Mundhe, Balaji; Pardeshi, Sachin

    2016-01-05

    In the present work, isopropyl-6-amino-4-(3,5-bis(trifluoromethyl)phenyl)-5-cyano-2-methyl-4H-pyran-3-carboxylate (4H-pyran analog) has been synthesized by a three component reaction catalyzed by CsOH/γ-Al2O3 and characterized. The interaction of 4H-pyran analog with herring sperm DNA (hs DNA) under physiological conditions (phosphate buffer of pH 7.2) was investigated by UV absorption, FT-IR, fluorescence, (31)P NMR and circular dichroism (CD) spectroscopy. Fluorescence quenching results reveal that static quenching mechanism is involved in binding between 4H-pyran analog and hs DNA. The calculated thermodynamic parameters (ΔH° and ΔS°) indicate that hydrogen bonding plays a major role in binding between them. UV absorption and fluorescence shows the binding mode of 4H-pyran analog with hs DNA as non-intercalative. According to the IR spectroscopy, 4H-pyran analog binds to guanine, thymine, adenine bases of hs DNA but not to phosphate backbone of hs DNA which is also in good agreement with (31)P NMR results. CD and competitive binding experiment results confirms the minor groove binding of 4H-pyran analog to hs DNA.

  9. Comparison of the benzoyl-DL-arginine-naphthylamide (BANA) test, DNA probes, and immunological reagents for ability to detect anaerobic periodontal infections due to Porphyromonas gingivalis, Treponema denticola, and Bacteroides forsythus.

    PubMed Central

    Loesche, W J; Lopatin, D E; Giordano, J; Alcoforado, G; Hujoel, P

    1992-01-01

    Most forms of periodontal disease are associated with the presence or overgrowth of anaerobic species that could include Treponema denticola, Porphyromonas gingivalis, and Bacteroides forsythus among others. These three organisms are among the few cultivable plaque species that can hydrolyze the synthetic trypsin substrate benzoyl-DL-arginine-naphthylamide (BANA). In turn, BANA hydrolysis by the plaque can be associated with periodontal morbidity and with the presence of these three BANA-positive organisms in the plaque. In this investigation, the results of the BANA test, which simultaneously detects one or more of these organisms, were compared with the detection of these organisms by (i) highly specific antibodies to P. gingivalis, T. denticola, and B. forsythus; (ii) whole genomic DNA probes to P. gingivalis and T. denticola; and (iii) culturing or microscopic procedures. The BANA test, the DNA probes, and an enzyme-linked immunosorbent assay or an indirect immunofluorescence assay procedure exhibited high sensitivities, i.e., 90 ot 96%, and high accuracies, i.e., 83 to 92%, in their ability to detect combinations of these organisms in over 200 subgingival plaque samples taken from the most periodontally diseased sites in 67 patients. This indicated that if P. gingivalis, T. denticola, and B. forsythus are appropriate marker organisms for an anaerobic periodontal infection, then the three detection methods are equally accurate in their ability to diagnose this infection. The same statement could not be made for the culturing approach, where accuracies of 50 to 62% were observed. PMID:1311335

  10. Construction of genome-wide physical BAC contigs using mapped cDNA as probes: Toward an integrated BAC library resource for genome sequencing and analysis. Annual report, July 1995--January 1997

    SciTech Connect

    Mitchell, S.C.; Bocskai, D.; Cao, Y.

    1997-12-31

    The goal of human genome project is to characterize and sequence entire genomes of human and several model organisms, thus providing complete sets of information on the entire structure of transcribed, regulatory and other functional regions for these organisms. In the past years, a number of useful genetic and physical markers on human and mouse genomes have been made available along with the advent of BAC library resources for these organisms. The advances in technology and resource development made it feasible to efficiently construct genome-wide physical BAC contigs for human and other genomes. Currently, over 30,000 mapped STSs and 27,000 mapped Unigenes are available for human genome mapping. ESTs and cDNAs are excellent resources for building contig maps for two reasons. Firstly, they exist in two alternative forms--as both sequence information for PCR primer pairs, and cDoreen genomic libraries efficiently for large number of DNA probes by combining over 100 cDNA probes in each hybridization. Second, the linkage and order of genes are rather conserved among human, mouse and other model organisms. Therefore, gene markers have advantages over random anonymous STSs in building maps for comparative genomic studies.

  11. DNA Investigations.

    ERIC Educational Resources Information Center

    Mayo, Ellen S.; Bertino, Anthony J.

    1991-01-01

    Presents a simulation activity that allow students to work through the exercise of DNA profiling and to grapple with some analytical and ethical questions involving a couple arranging with a surrogate mother to have a baby. Can be used to teach the principles of restriction enzyme digestion, gel electrophoresis, and probe hybridization. (MDH)

  12. ESTIMATION OF BACTERIAL CELL NUMBERS IN HUMIC ACID-RICH SALT MARSH SEDIMENTS WITH PROBES DIRECTED TO 16S RIBOSOMAL DNA

    EPA Science Inventory

    The feasibility of using probes directed towards ribosomal DNAs (rDNAs) as a quantitative approach to estimating cell numbers was examined and applied to study the structure of a bacterial community in humic acid-rich salt marsh sediments. Hybridizations were performed with membr...

  13. Hairpin DNA probe with 5'-TCC/CCC-3' overhangs for the creation of silver nanoclusters and miRNA assay.

    PubMed

    Xia, Xiaodong; Hao, Yuanqiang; Hu, Shengqiang; Wang, Jianxiu

    2014-01-15

    A facile strategy for the assay of target miRNA using fluorescent silver nanoclusters (AgNCs) has been described. Due to the preferable interaction between cytosine residues and Ag(+), a short cytosine-rich oligonucleotide (ODN) with only six bases 5'-TCCCCC-3' served as an efficient scaffold for the creation of the AgNCs. The AgNCs displayed a bright red emission when excited at 545nm. Such ODN base-stabilized AgNCs have been exploited for miRNA sensing. Overhangs of TCC at the 5' end (5'-TCC) and CCC at the 3' end (CCC-3') (denoted as 5'-TCC/CCC-3') appended to the hairpin ODN probe which also contains recognition sequences for target miRNA were included. Interestingly, the AgNCs/hairpin ODN probe showed similar spectral properties as that templated by 5'-TCCCCC-3'. The formation of the hairpin ODN probe/miRNA duplex separated the 5'-TCC/CCC-3' overhangs, thus disturbing the optical property or structure of the AgNCs. As a result, fluorescence quenching of the AgNCs/hairpin ODN probe was obtained, which allows for facile determination of target miRNA. The proposed method is simple and cost-effective, holding great promise for clinical applications.

  14. Probing the relation between protein-protein interactions and DNA binding for a linker mutant of the bacterial nucleoid protein H-NS.

    PubMed

    Giangrossi, Mara; Wintraecken, Kathelijne; Spurio, Roberto; de Vries, Renko

    2014-02-01

    We have investigated the relationship between oligomerization in solution and DNA binding for the bacterial nucleoid protein H-NS. This was done by comparing oligomerization and DNA binding of H-NS with that of a H-NS D68V-D71V linker mutant. The double linker mutation D68V-D71V, that makes the linker significantly more hydrophobic, leads to a dramatically enhanced and strongly temperature-dependent H-NS oligomerization in solution, as detected by dynamic light scattering. The DNA binding affinity of H-NS D68V-D71V for the hns promoter region is lower and has stronger temperature dependence than that of H-NS. DNase I footprinting experiments show that at high concentrations, regions protected by H-NS D68V-D71V are larger and less defined than for H-NS. In vitro transcription assays show that the enhanced protection also leads to enhanced transcriptional repression. Whereas the lower affinity of the H-NS D68V-D71V for DNA could be caused by competition between oligomerization in solution and oligomerization on DNA, the larger size of protected regions clearly confirms the notion that cooperative binding of H-NS to DNA is related to protein-protein interactions. These results emphasize the relative contributions of protein-protein interactions and substrate-dependent oligomerization in the control of gene repression operated by H-NS.

  15. Peptide nucleic acid probes with charged photocleavable mass markers

    PubMed Central

    Ball, Rachel J; Green, Philip S; Gale, Nittaya; Langley, G John

    2010-01-01

    Halogen-labelled peptide organic acid (HPOA) monomers have been synthesised and incorporated into sequence-specific peptide nucleic acid (PNA) probes. Three different types of probe have been prepared; the unmodified PNA probe, the PNA probe with a mass marker, and the PNA probe with photocleavable mass marker. All three types of probe have been used in model studies to develop a mass spectrometry-based hybridisation assay for detection of point mutations in DNA. PMID:21687524

  16. Electroactive crown ester-Cu(2+) complex with in-situ modification at molecular beacon probe serving as a facile electrochemical DNA biosensor for the detection of CaMV 35s.

    PubMed

    Zhan, Fengping; Liao, Xiaolei; Gao, Feng; Qiu, Weiwei; Wang, Qingxiang

    2017-06-15

    A novel electrochemical DNA biosensor has been facilely constructed by in-situ assembly of electroactive 4'-aminobenzo-18-crown-6-copper(II) complex (AbC-Cu(2+)) on the free terminal of the hairpin-structured molecule beacon. The 3'-SH modified molecule beacon probe was first immobilized on the gold electrode (AuE) surface through self-assembly chemistry of Au-S bond. Then the crow ester of AbC was covalently coupled with 5'-COOH on the molecule beacon, and served as a platform to attach the Cu(2+) by coordination with ether bond (-O-) of the crown cycle. Thus, an electroactive molecule beacon-based biosensing interface was constructed. In comparison with conventional methods for preparation of electroactive molecule beacon, the approach presented in this work is much simpler, reagent- and labor-saving. Selectivity study shows that the in-situ fabricated electroactive molecule beacon remains excellent recognition ability of pristine molecule beacon probe to well differentiate various DNA fragments. The target DNA can be quantatively determined over the range from 0.10pM to 0.50nM. The detection limit of 0.060pM was estimated based on signal-to-noise ratio of 3. When the biosensor was applied for the detection cauliflower mosaic virus 35s (CaMV 35s) in soybean extraction samples, satisfactory results are achieved. This work opens a new strategy for facilely fabricating electrochemical sensing interface, which also shows great potential in aptasensor and immurosensor fabrication.

  17. Structure-based design of platinum(II) complexes as c-myc oncogene down-regulators and luminescent probes for G-quadruplex DNA.

    PubMed

    Wang, Ping; Leung, Chung-Hang; Ma, Dik-Lung; Yan, Siu-Cheong; Che, Chi-Ming

    2010-06-18

    A series of platinum(II) complexes with tridentate ligands was synthesized and their interactions with G-quadruplex DNA within the c-myc gene promoter were evaluated. Complex 1, which has a flat planar 2,6-bis(benzimidazol-2-yl)pyridine (bzimpy) scaffold, was found to stabilize the c-myc G-quadruplex structure in a cell-free system. An in silico G-quadruplex DNA model has been constructed for structure-based virtual screening to develop new Pt(II)-based complexes with superior inhibitory activities. By using complex 1 as the initial structure for hit-to-lead optimization, bzimpy and related 2,6-bis(pyrazol-3-yl)pyridine (dPzPy) scaffolds containing amine side-chains emerge as the top candidates. Six of the top-scoring complexes were synthesized and their interactions with c-myc G-quadruplex DNA have been investigated. The results revealed that all of the complexes have the ability to stabilize the c-myc G-quadruplex. Complex 3 a ([Pt(II)L2R](+); L2=2,6-bis[1-(3-piperidinepropyl)-1H-enzo[d]imidazol-2-yl]pyridine, R=Cl) displayed the strongest inhibition in a cell-free system (IC(50)=2.2 microM) and was 3.3-fold more potent than that of 1. Complexes 3 a and 4 a ([Pt(II)L3R](+); L3=2,6-bis[1-(3-morpholinopropyl)-1H-pyrazol-3-yl]pyridine, R=Cl) were found to effectively inhibit c-myc gene expression in human hepatocarcinoma cells with IC(50) values of approximately 17 microM, whereas initial hit 1 displayed no significant effect on gene expression at concentrations up to 50 microM. Complexes 3 a and 4 a have a strong preference for G-quadruplex DNA over duplex DNA, as revealed by competition dialysis experiments and absorption titration; 3 a and 4 a bind G-quadruplex DNA with binding constants (K) of approximately 10(6)-10(7) dm(3) mol(-1), which are at least an order of magnitude higher than the K values for duplex DNA. NMR spectroscopic titration experiments and molecular modeling showed that 4 a binds c-myc G-quadruplex DNA through an external end-stacking mode at

  18. Determination of cell metabolite VEGF₁₆₅ and dynamic analysis of protein-DNA interactions by combination of microfluidic technique and luminescent switch-on probe.

    PubMed

    Lin, Xuexia; Leung, Ka-Ho; Lin, Ling; Lin, Luyao; Lin, Sheng; Leung, Chung-Hang; Ma, Dik-Lung; Lin, Jin-Ming

    2016-05-15

    In this paper, we rationally design a novel G-quadruplex-selective luminescent iridium (III) complex for rapid detection of oligonucleotide and VEGF165 in microfluidics. This new probe is applied as a convenient biosensor for label-free quantitative analysis of VEGF165 protein from cell metabolism, as well as for studying the kinetics of the aptamer-protein interaction combination with a microfluidic platform. As a result, we have successfully established a quantitative analysis of VEGF165 from cell metabolism. Furthermore, based on the principles of hydrodynamic focusing and diffusive mixing, different transient states during kinetics process were monitored and recorded. Thus, the combination of microfluidic technique and G-quadruplex luminescent probe will be potentially applied in the studies of intramolecular interactions and molecule recognition in the future.

  19. Lab in a Tube: Sensitive Detection of MicroRNAs in Urine Samples from Bladder Cancer Patients Using a Single-Label DNA Probe with AIEgens.

    PubMed

    Min, Xuehong; Zhuang, Yuan; Zhang, Zhenyu; Jia, Yongmei; Hakeem, Abdul; Zheng, Fuxin; Cheng, Yong; Tang, Ben Zhong; Lou, Xiaoding; Xia, Fan

    2015-08-05

    We demonstrate an ultrasensitive microRNA detection method based on an extremely simple probe with only fluorogens but without quencher groups. It avoids complex and difficult steps to accurately design the relative distance between the fluorogens and quencher groups in the probes. Furthermore, the assay could accomplish various detection limits by tuning the reaction temperature due to the different activity of exonuclease III corresponding to the diverse temperature. Specifically, 1 pM miR-21 can be detected in 40 min at 37 °C, and 10 aM (about 300 molecules in 50 μL) miR-21 could be discriminated in 7 days at 4 °C. The great specificity of the assay guarantees that the real 21 urine samples from the bladder cancer patients are successfully detected by our method.

  20. Differential Incorporation of Carbon Substrates among Microbial Populations Identified by Field-Based, DNA Stable-Isotope Probing in South China Sea

    PubMed Central

    Xie, Xiabing; Jiao, Nianzhi

    2016-01-01

    To determine the adapted microbial populations to variant dissolved organic carbon (DOC) sources in the marine environment and improve the understanding of the interaction between microorganisms and marine DOC pool, field-based incubation experiments were carried out using supplemental 13C-labeled typical substrates D-glucose and D-glucosamine (D-Glc and D-GlcN, respectively), which are two important components in marine DOC pool in the South China Sea. 13C- and 12C-DNA were then fractionated by ultracentrifugation and the microbial community was analyzed by terminal-restriction fragment length polymorphism and 454 pyrosequencing of 16S rRNA gene. 12C-DNA-based communities showed relatively high similarities with their corresponding in situ communities, and their bacterial diversities were generally higher than 13C-DNA-based counterparts. Distinct differences in community composition were found between 13C- and 12C-DNA-based communities and between two substrate-supplemented 13C-DNA-based communities; these differences distinctly varied with depth and site. In most cases, there were more genera with relative abundances of >0.1% in D-Glc-incorporating communities than in D-GlcN-incorporating communities. The Roseobacter clade was one of the prominent actively substrate-incorporating bacterial populations in all 13C-DNA-based communities. Vibrio was another prominent actively D-GlcN-incorporating bacterial population in most incubations. However notably, different OTUs dominated this clade or genus in different treatments at different depths. Altogether, these results suggested that there were taxa-specific differences in DOC assimilations and, moreover, their differences varied among the typical water masses, which could have been caused by the variant compositions of original bacterial communities from different hydrological environments. This implies that ecologically, the levels of labile or recalcitrance of DOC can be maintained only in a specific environmental

  1. Site-specific insertion of nitroxide-spin labels into DNA probes by click chemistry for structural analyses by ELDOR spectroscopy.

    PubMed

    Flaender, M; Sicoli, G; Fontecave, Th; Mathis, G; Saint-Pierre, C; Boulard, Y; Gambarelli, S; Gasparutto, D

    2008-01-01

    A new approach is described for the insertion of nitroxide spin-labels at specific positions within DNA oligomers. The latter bioconjugaison strategy is based on a click chemistry 1,3-dipolar cycloaddition between a spin-labeling reagent, namely the 4-azido-TEMPO, and alkyne modified uridine-containing oligonucleotides. This highly efficient labeling method was applied for site-specific incorporation of two TEMPO units within a set of double-stranded DNA constructs. Then the determination of the inter-nitroxide distances was achieved by using a four-pulses DEER technique that successfully validates the new site-directed spin labeling strategy.

  2. Optical probe

    DOEpatents

    Hencken, Kenneth; Flower, William L.

    1999-01-01

    A compact optical probe is disclosed particularly useful for analysis of emissions in industrial environments. The instant invention provides a geometry for optically-based measurements that allows all optical components (source, detector, rely optics, etc.) to be located in proximity to one another. The geometry of the probe disclosed herein provides a means for making optical measurements in environments where it is difficult and/or expensive to gain access to the vicinity of a flow stream to be measured. Significantly, the lens geometry of the optical probe allows the analysis location within a flow stream being monitored to be moved while maintaining optical alignment of all components even when the optical probe is focused on a plurality of different analysis points within the flow stream.

  3. The 8, 5’-Cyclopurine-2’-Deoxynucleosides: Candidate Neurodegenerative DNA Lesions in Xeroderma Pigmentosum, and Unique Probes of Transcription and Nucleotide Excision Repair

    PubMed Central

    Brooks, P. J.

    2008-01-01

    It is a commonly held view that oxidatively-induced DNA lesions are repaired by the base excision repair (BER) pathway, whereas DNA lesions induced by UV light and other “bulky” chemical adducts are repaired by the nucleotide excision repair (NER) pathway. While this distinction is generally accurate, the 8,5’ cyclopurine deoxynucleosides represent an important exception, in that they are formed in DNA by the hydroxyl radical, but are specifically repaired by NER, not by BER. They are also strong blocks to nucleases and polymerases, including RNA polymerase II in human cells. In this review, I will discuss the evidence that these lesions are in part responsible for the neurodegeneration that occurs in some XP patients, and what additional evidence would be necessary to prove such a role. I will also consider other DNA lesions that might be involved in XP neurologic disease. Finally, I will also discuss how our recent studies of these lesions have generated novel insights into the process of transcriptional mutagenesis in human cells, as well as the value of studying these lesions not only for a better understanding of NER, but also for other aspects of human health and disease. PMID:18495558

  4. A label-free fluorescence DNA probe based on ligation reaction with quadruplex formation for highly sensitive and selective detection of nicotinamide adenine dinucleotide.

    PubMed

    Zhao, Jingjin; Zhang, Liangliang; Jiang, Jianhui; Shen, Guoli; Yu, Ruqin

    2012-05-11

    A simple label-free fluorescent sensing scheme for sensitive and selective detection of nicotinamide adenine dinucleotide (NAD(+)) has been developed based on DNA ligation reaction with ligand-responsive quadruplex formation. This approach can detect 0.5 nM NAD(+) with high selectivity against other NAD(+) analogs.

  5. Origins of sequence selectivity in homologous genetic recombination: insights from rapid kinetic probing of RecA-mediated DNA strand exchange.

    PubMed

    Lee, Andrew M; Xiao, Jie; Singleton, Scott F

    2006-07-07

    Despite intense effort over the past 30 years, the molecular determinants of sequence selectivity in RecA-mediated homologous recombination have remained elusive. Here, we describe when and how sequence homology is recognized between DNA strands during recombination in the context of a kinetic model for RecA-mediated DNA strand exchange. We characterized the transient intermediates of the reaction using pre-steady-state kinetic analysis of strand exchange using oligonucleotide substrates containing a single fluorescent G analog. We observed that the reaction system was sensitive to heterology between the DNA substrates; however, such a "heterology effect" was not manifest when functional groups were added to or removed from the edges of the base-pairs facing the minor groove of the substrate duplex. Hence, RecA-mediated recombination must occur without the involvement of a triple helix, even as a transient intermediate in the process. The fastest detectable reaction phase was accelerated when the structure or stability of the substrate duplex was perturbed by internal mismatches or the replacement of G.C by I.C base-pairs. These findings indicate that the sequence specificity in recombination is achieved by Watson-Crick pairing in the context of base-pair dynamics inherent to the extended DNA structure bound by RecA during strand exchange.

  6. Probing the rate-limiting step for intramolecular transfer of a transcription factor between specific sites on the same DNA molecule by (15)Nz-exchange NMR spectroscopy.

    PubMed

    Ryu, Kyoung-Seok; Tugarinov, Vitali; Clore, G Marius

    2014-10-15

    The kinetics of translocation of the homeodomain transcription factor HoxD9 between specific sites of the same or opposite polarities on the same DNA molecule have been studied by (15)Nz-exchange NMR spectroscopy. We show that exchange occurs by two facilitated diffusion mechanisms: a second-order intermolecular exchange reaction between specific sites located on different DNA molecules without the protein dissociating into free solution that predominates at high concentrations of free DNA, and a first-order intramolecular process involving direct transfer between specific sites located on the same DNA molecule. Control experiments using a mixture of two DNA molecules, each possessing only a single specific site, indicate that transfer between specific sites by full dissociation of HoxD9 into solution followed by reassociation is too slow to measure by z-exchange spectroscopy. Intramolecular transfer with comparable rate constants occurs between sites of the same and opposing polarity, indicating that both rotation-coupled sliding and hopping/flipping (analogous to geminate recombination) occur. The half-life for intramolecular transfer (0.5-1 s) is many orders of magnitude larger than the calculated transfer time (1-100 μs) by sliding, leading us to conclude that the intramolecular transfer rates measured by z-exchange spectroscopy represent the rate-limiting step for a one-base-pair shift from the specific site to the immediately adjacent nonspecific site. At zero concentration of added salt, the intramolecular transfer rate constants between sites of opposing polarity are smaller than those between sites of the same polarity, suggesting that hopping/flipping may become rate-limiting at very low salt concentrations.

  7. Probing DNA interactions with proteins using a single-molecule toolbox: inside the cell, in a test tube and in a computer.

    PubMed

    Wollman, Adam J M; Miller, Helen; Zhou, Zhaokun; Leake, Mark C

    2015-04-01

    DNA-interacting proteins have roles in multiple processes, many operating as molecular machines which undergo dynamic meta-stable transitions to bring about their biological function. To fully understand this molecular heterogeneity, DNA and the proteins that bind to it must ideally be interrogated at a single molecule level in their native in vivo environments, in a time-resolved manner, fast enough to sample the molecular transitions across the free-energy landscape. Progress has been made over the past decade in utilizing cutting-edge tools of the physical sciences to address challenging biological questions concerning the function and modes of action of several different proteins which bind to DNA. These physiologically relevant assays are technically challenging but can be complemented by powerful and often more tractable in vitro experiments which confer advantages of the chemical environment with enhanced detection signal-to-noise of molecular signatures and transition events. In the present paper, we discuss a range of techniques we have developed to monitor DNA-protein interactions in vivo, in vitro and in silico. These include bespoke single-molecule fluorescence microscopy techniques to elucidate the architecture and dynamics of the bacterial replisome and the structural maintenance of bacterial chromosomes, as well as new computational tools to extract single-molecule molecular signatures from live cells to monitor stoichiometry, spatial localization and mobility in living cells. We also discuss recent developments from our laboratory made in vitro, complementing these in vivo studies, which combine optical and magnetic tweezers to manipulate and image single molecules of DNA, with and without bound protein, in a new super-resolution fluorescence microscope.

  8. Repeated probing of Southwestern blots using alkaline phosphatase stripping.

    PubMed

    Jia, Yinshan; Jiang, Daifeng; Jarrett, Harry W

    2010-11-05

    Southwestern blotting is when a DNA sequence is used to probe DNA-binding proteins on an electrophoretic gel blot. It would be highly desirable to be able to probe a blot repeatedly with different DNA sequences. Alkaline phosphatase can remove 5'-phosphoryl groups from DNA and radiolabeled 5'-(32)P-DNA probes are commonly used in Southwestern blotting. Here is shown that once probed, the radioisotope signal on the blot can be effectively removed by brief digestion with alkaline phosphatase, and the blot can then be repeatedly probed at least six times with different DNA probes. This exceeds the repetitions possible with another commonly used method using SDS. The technique can be used with either one-dimensional or multi-dimensional Southwestern blots and does not have a large effect on the phosphorylation state of the blotted proteins. An alternative method using T4 polynucleotide kinase stripping is also introduced but was less well characterized.

  9. Salt Dependence of the Radius of Gyration and Flexibility of Single-stranded DNA in Solution probed by Small-angle X-ray Scattering

    SciTech Connect

    Sim, Adelene Y.L.; Lipfert, Jan; Herschlag, Daniel; Doniach, Sebastian

    2012-07-06

    Short single-stranded nucleic acids are ubiquitous in biological processes and understanding their physical properties provides insights to nucleic acid folding and dynamics. We used small angle x-ray scattering to study 8-100 residue homopolymeric single-stranded DNAs in solution, without external forces or labeling probes. Poly-T's structural ensemble changes with increasing ionic strength in a manner consistent with a polyelectrolyte persistence length theory that accounts for molecular flexibility. For any number of residues, poly-A is consistently more elongated than poly-T, likely due to the tendency of A residues to form stronger base-stacking interactions than T residues.

  10. Unusual DNA structures

    SciTech Connect

    Wells, R.D.; Harvey, S.C.

    1988-01-01

    The contents of this book are: Unusual DNS Structures and the Probes Used for Their Detection; The Specificity of Single Strand Specific Endonucleases; Chromatin STructure and DNA Structure at the hsp 26 Locus of Drosophilia; Cruciform Extrusion in Supercoiled DNA-Mechanisms and Contextual Influence; Torsional Stress, Unusual DNA Structures, and Eukaryotic Gene Expression; DNA Sequence and Structure: Bending to Biology. Cruciform Transitions Assayed Using a Psoralen Cross-linking Method: Applications to Measurements of DNA Torisonal Tension; NMR-Distance Geometry Studies of Helical Errors and Sequence Dependent Conformations of DNA in Solution; Hyperreactivity of the B-Z Junctions Probed by Two Aromatic Chemical Carcinogens; Inherently Curved DNA and Its Structural Elements; and DNA Flexibility Under Control: The Juma Algorithm and its Application to BZ Junctions.

  11. Quaternized chitosan particles as ion exchange supports for label-free DNA detection using PNA probe and MALDI-TOF mass spectrometry.

    PubMed

    Meebungpraw, Jittima; Wiarachai, Oraphan; Vilaivan, Tirayut; Kiatkamjornwong, Suda; Hoven, Voravee P

    2015-10-20

    Quaternized chitosan particles are introduced as anion-exchanged captures to be used with a conformationally constrained pyrrolidinyl peptide nucleic acid (acpcPNA) and MALDI-TOF mass spectrometry for DNA sequence analysis. Methylated chitosan (MC) and methylated N-benzyl chitosan (MBzC) particles were obtained by heterogeneous chemical modification of ionically cross-linked chitosan particles via direct methylation and reductive benzylation/methylation, respectively. N,N,N-trimethylchitosan (TMC) and N-[(2-hydroxyl-3-trimethylammonium)propyl]chitosan chloride (HTACC) particles were prepared by ionic cross-linking of quaternized chitosan derivatives, homogeneously modified from chitosan, namely TMC and HTACC, respectively. The particles formed had a size in a sub-micrometer range and possessed positive charge. Investigation by MALDI-TOF mass spectrometry suggested that some quaternized particles in combination with acpcPNA were capable of detecting a single mismatched base out of 9-14 base DNA sequences. Potential application of this technique for the detection of wild-type and mutant K-ras DNA, a gene that mutation is associated with certain cancers, has also been demonstrated.

  12. Photophysical and structural investigation of a (Py)A-modified adenine cluster: its potential use for fluorescent DNA probes exhibiting distinct emission color changes.

    PubMed

    Kim, Ki Tae; Heo, Wooseok; Joo, Taiha; Kim, Byeang Hyean

    2015-08-21

    In this study, we found a (Py)A-modified adenine cluster (A-cluster), a minimum fluorescent unit for significant emission wavelength changes, and investigated its photophysical and structural properties. The basic A-cluster unit was an adenine-pentad duplex containing stacked (Py)A pairs in the center aligned in an antiparallel manner. Spectral analysis of the A-cluster revealed remarkable reddish fluorescence with a large Stokes shift (∼195 nm) and a long life-ti