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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. DNA probe for lactobacillus delbrueckii

    SciTech Connect

    Delley, M.; Mollet, B.; Hottinger, H. )

    1990-06-01

    From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognized L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an {alpha}-{sup 32}P-labeled probe.

  3. Diagnostic applications of DNA probes.

    PubMed

    Pfaller, M A

    1991-02-01

    This review has described several of the most common molecular biologic techniques that are, or will be, employed in the diagnostic laboratory. The potential advantages of these DNA probe assays in the diagnosis of infectious diseases include: rapid detection and identification of infectious agents; the ability to screen selected specimens using batteries of probes; and the detection of nonviable or difficult-to-culture organisms. The potential disadvantages of DNA probe assays include: the use of isotopic detection methods for optimum sensitivity; limited diagnostic sensitivity of current assays; slow turna-round time for some assay formats; expense of current reagents; limited availability of many probes; lack of technical expertise in most diagnostic laboratories; and the requirement for antimicrobial susceptibility testing (requires culture). Given the above advantages and disadvantages, there are several key issues that must be considered before adopting DNA probe technology in the diagnostic laboratory; the cost of performing routine culture and identification versus the cost of screening with probes--both the number and type of specimens and the time savings that may be realized by eliminating routine cultures; the prevalence of the infectious agent--even the best DNA probe assay may not be useful or practical in a low-prevalence situation; the need for additional equipment and space; and the interpretation of false-positive and false-negative results--additional research is needed in this area. However, laboratories must consider these issues when using a test other than the current gold standard (i.e., culture). DNA probe technology is with us and expanding rapidly. The intelligent application of this new technology will require communication between laboratorians and clinicians and careful consideration of the many advantages and disadvantages discussed above.

  4. Immobilization of DNA for scanning probe microscopy.

    PubMed Central

    Allison, D P; Bottomley, L A; Thundat, T; Brown, G M; Woychik, R P; Schrick, J J; Jacobson, K B; Warmack, R J

    1992-01-01

    Reproducible scanning tunneling microscope and atomic force microscope images of entire molecules of uncoated plasmid DNA chemically bound to surfaces are presented. The chemically mediated immobilization of DNA to surfaces and subsequent scanning tunneling microscope imaging of DNA molecules demonstrate that the problem of molecular instability to forces exerted by the probe tip, inherent with scanning probe microscopes, can be prevented. Images PMID:1438201

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. DNA Binding Hydroxyl Radical Probes.

    PubMed

    Tang, Vicky J; Konigsfeld, Katie M; Aguilera, Joe A; Milligan, Jamie R

    2012-01-01

    The hydroxyl radical is the primary mediator of DNA damage by the indirect effect of ionizing radiation. It is a powerful oxidizing agent produced by the radiolysis of water and is responsible for a significant fraction of the DNA damage associated with ionizing radiation. There is therefore an interest in the development of sensitive assays for its detection. The hydroxylation of aromatic groups to produce fluorescent products has been used for this purpose. We have examined four different chromophores which produce fluorescent products when hydroxylated. Of these, the coumarin system suffers from the fewest disadvantages. We have therefore examined its behavior when linked to a cationic peptide ligand designed to bind strongly to DNA.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. Fluorescence studies with DNA probes: dynamic aspects of DNA structure and DNA-protein interactions

    NASA Astrophysics Data System (ADS)

    Millar, David P.; Carver, Theodore E.

    1994-08-01

    Time-resolved fluorescence measurements of optical probes incorporated at specific sites in DNA provides a new approach to studies of DNA structure and DNA:protein interactions. This approach can be used to study complex multi-state behavior, such as the folding of DNA into alternative higher order structures or the transfer of DNA between multiple binding sites on a protein. In this study, fluorescence anisotropy decay of an internal dansyl probe attached to 17/27-mer oligonucleotides was used to monitor the distribution of DNA 3' termini bound at either the polymerase of 3' to 5' exonuclease sites of the Klenow fragment of DNA polymerase I. Partitioning of the primer terminus between the two active sites of the enzyme resulted in a heterogeneous probe environment, reflected in the associative behavior of the fluorescence anisotropy decay. Analysis of the anisotropy decay with a two state model of solvent-exposed and protein-associated dansyl probes was used to determine the fraction of DNA bound at each site. We examined complexes of Klenow fragment with DNAs containing various base mismatches. Single mismatches at the primer terminus caused a 3-fold increase in the equilibrium partitioning of DNA into the exonuclease site, while two or more consecutive G:G mismatches caused the DNA to bind exclusively at the exonuclease site, with a partitioning constant at least 250- fold greater than that of the corresponding matched DNA sequence. Internal single mismatches located up to four bases from the primer terminus produced larger effects than the same mismatch at the primer terminus. These results provide insight into the recognition mechanisms that enable DNA polymerases to proofread misincorporated bases during DNA replication.

  16. DNA probe for the identification of Histoplasma capsulatum.

    PubMed Central

    Keath, E J; Spitzer, E D; Painter, A A; Travis, S J; Kobayashi, G S; Medoff, G

    1989-01-01

    A 1.85-kilobase HindIII nuclear DNA probe from Histoplasma capsulatum G217B detected polymorphic restriction fragments within whole-cell DNA from different clinical isolates of H. capsulatum, consistent with the previous system of classification. The probe failed to hybridize to DNA from Blastomyces dermatitidis, Candida spp., Saccharomyces cerevisiae, Sepedonium chrysospermum, and Chrysosporium keratinophilum under low-stringency conditions and therefore may have value as a diagnostic reagent to identify H. capsulatum. Images PMID:2573616

  17. 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.

  18. 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.

  19. The use of fluorescein for labeling genomic probes in the checkerboard DNA-DNA hybridization method.

    PubMed

    do Nascimento, Cássio; Santos Barbosa, Rodrigo Edson; Mardegan Issa, João Paulo; Watanabe, Evandro; Yoko Ito, Izabel; Monesi, Nadia; Albuquerque Junior, Rubens Ferreira de

    2008-01-01

    Molecular methods that permit the simultaneous detection and quantification of a large number of microbial species are currently employed in the evaluation of complex ecosystems. The checkerboard DNA-DNA hybridization technique enables the simultaneous identification of distinct bacterial species in a large number of dental samples. The original technique employed digoxigenin-labeled whole genomic DNA probes which were detected by chemiluminescence. In this study, we present an alternative protocol for labeling and detecting whole genomic DNA probes in the Checkerboard DNA-DNA hybridization method. Whole genomic DNA was extracted from five bacterial species and labeled with fluorescein. The fluorescein labeled whole genomic DNA probes were hybridized against whole genomic DNA or subgingival plaque samples in a checkerboard hybridization format, followed by chemiluminescent detection. Our results reveal that fluorescein is a viable and adequate alternative labeling reagent to be employed in the checkerboard DNA-DNA hybridization technique.

  20. Sensitive and specific DNA probe for detection of Plasmodium falciparum.

    PubMed Central

    Enea, V

    1986-01-01

    The isolation and some characteristics of a very sensitive DNA probe for the detection of Plasmodium falciparum are described. The probe is species specific and represents a large, albeit variable, fraction of the genome in all the strains tested. In addition to its immediate practical uses for the detection and quantitation of parasites, the probe defines an interesting family of repeated sequences. Images PMID:3023833

  1. Evaluation of reformulated chemiluminescent DNA probe (AccuProbe) for culture identification of Mycobacterium kansasii.

    PubMed Central

    Tortoli, E; Simonetti, M T; Lavinia, F

    1996-01-01

    A panel of 104 isolates belonging to the species Mycobacterium kansasii and 78 mycobacterial isolates belonging to other species was tested in parallel with the present commercially available DNA probe (AccuProbe; Gen-Probe) and with a new probe just developed by the same manufacturer. While the old version of the probe confirmed the previously reported low sensitivity (only 59% of the M. kansasii isolates reacted), the new one was 100% sensitive. Only two non-M. kansasii strains, both M. gastri isolates, gave false-positive hybridization results. PMID:8897195

  2. "DNA traffic lights": concept of wavelength-shifting DNA probes and application in an aptasensor.

    PubMed

    Holzhauser, Carolin; Wagenknecht, Hans-Achim

    2012-05-29

    Add it and see it: The concept of "DNA traffic lights" for wavelength-shifting DNA probes has a great potential in the application of biosensors, for example, in DNA aptamers. A visual color change in the DNA aptasensor fluorescence from green to red occurs after specific target binding. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Molecular probe for identification of Trichomonas vaginalis DNA.

    PubMed Central

    Rubino, S; Muresu, R; Rappelli, P; Fiori, P L; Rizzu, P; Erre, G; Cappuccinelli, P

    1991-01-01

    Trichomoniasis is one of the most widespread sexually transmitted diseases in the world. Diagnosis can be achieved by several methods, such as direct microscopic observation of vaginal discharge, cell culture, and immunological techniques. A 2.3-kb Trichomonas vaginalis DNA fragment present in strains from diverse geographic areas was cloned and used as a probe to detect T. vaginalis DNA in vaginal discharge by a dot blot hybridization technique. This probe was specific for T. vaginalis DNA. It recognized strains from two regions in Italy (Sardinia, Piemonte) and from Mozambique (Africa). In addition, our probe did not cross-react with bacterial (Escherichia coli, Enterococcus spp., group B streptococci, Gardnerella vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, and Lactobacillus spp.), viral (herpes simplex virus type 2), fungal (Candida albicans), protozoan (Entamoeba histolytica, Giardia lamblia, Plasmodium falciparum, Leishmania major, and Leishmania infantum), or human nucleic acids. The probe reacted with Pentatrichomonas hominis and Trichomonas foetus. The limit signal recognized by our probe corresponded to the DNA of 200 T. vaginalis isolates. The 2.3-kb probe was used in a clinical analysis of 98 samples. Of these, 20 samples were found to be positive both with the probe and by cell culture, and only 14 of these were positive by a standard wet mount method. Images PMID:1890171

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. Probing the mechanical unzipping of DNA

    NASA Astrophysics Data System (ADS)

    Voulgarakis, Nikos K.; Bishop, Alan R.; Rasmussen, Kim O.

    2006-03-01

    Recent advances in single-molecule force spectroscopy have made a systematic study of local melting in DNA possible. This provide new insight into important biological processes as replication and transcription. In this work, we present an extensive study of the micromechanical unzipping of DNA in the framework of the Peyrard-Bishop-Dauxois (PBD) model. The force required to separate the doubled strand is derived through analysis of the force-extension curve, while an estimation of the nucleation bubble size of the unzipping process is obtained by the distribution of the rapture force. Our findings are in very good agreement with existing experimental results; for example the force-temperature phase diagram obtained by the PBD model agrees excellently with recent constant-force experimental measurements of the lambda-phage DNA. Fundamental differences between the in vivo and vitro DNA unzipping, as predicted by the PBD model, are also discussed.

  9. 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

  10. 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.

  11. Probing the microscopic flexibility of DNA from melting temperatures

    NASA Astrophysics Data System (ADS)

    Weber, Gerald; Essex, Jonathan W.; Neylon, Cameron

    2009-10-01

    The microscopic flexibility of DNA is a key ingredient for understanding its interaction with proteins and drugs but is still poorly understood and technically challenging to measure. Several experimental methods probe very long DNA samples, but these miss local flexibility details. Others mechanically disturb or modify short molecules and therefore do not obtain flexibility properties of unperturbed and pristine DNA. Here, we show that it is possible to extract very detailed flexibility information about unmodified DNA from melting temperatures with statistical physics models. We were able to retrieve, from published melting temperatures, several established flexibility properties such as the presence of highly flexible TATA regions of genomic DNA and support recent findings that DNA is very flexible at short length scales. New information about the nanoscale Na+ concentration dependence of DNA flexibility was determined and we show the key role of ApT and TpA steps when it comes to ion-dependent flexibility and melting temperatures.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. 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. PMID:28335308

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. DNA Probe Pooling for Rapid Delineation of Chromosomal Breakpoints

    PubMed Central

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

    2009-01-01

    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 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 multiclone and multicolor 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 3 to 4 days. (J Histochem Cytochem 57:587–597, 2009) PMID:19223294

  11. DNA binding studies of a solvatochromic fluorescence probe 3-methoxybenzanthrone

    NASA Astrophysics Data System (ADS)

    Yang, Xin; Liu, Wan-Hui; Jin, Wei-Jun; Shen, Guo-Li; Yu, Ru-Qin

    1999-11-01

    A fluorescence probe of 3-methoxybenzanthrone (MBA) exhibits significant solvatochromic characteristics correlated with the polarity of solvents. The interaction of the solvatochromic fluorescence probe with calf thymus DNA (ct-DNA) has been investigated. In the presence of ct-DNA the fluorescence of MBA is strongly quenched with a blue-shift of emission peak and a hypochromism in absorption spectra. The absorption spectra, fluorescence quenching and fluorescence polarization experiments show that the MBA molecule as an intercalator is inserted into the base-stacking domain of the ct-DNA double helix, and the interaction of the nucleobases with the MBA molecule causes quenching of fluorescence and hypochromism in the absorption spectra. The intrinsic binding constant and the binding site number were determined to be 1.70×10 5 mol l -1 in base pairs and six, respectively. The I0/ I versus [ct-DNA] plot shows linear relationship in the range covering 4.3×10 -7-1.02×10 -4 mol l -1 in base pairs which can be used for ct-DNA determination. The limit of detection was found to be 4.3×10 -7 mol l -1 in base pairs (0.5 μg ml -1).

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. Probing the DNA structural requirements for facilitated diffusion.

    PubMed

    Hedglin, Mark; Zhang, Yaru; O'Brien, Patrick J

    2015-01-20

    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.

  19. [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.

  20. 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.

  1. Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

    PubMed Central

    Goda, Tatsuro; Singi, Ankit Balram; Maeda, Yasuhiro; Matsumoto, Akira; Torimura, Masaki; Aoki, Hiroshi; Miyahara, Yuji

    2013-01-01

    Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry. PMID:23435052

  2. Label-free potentiometry for detecting DNA hybridization using peptide nucleic acid and DNA probes.

    PubMed

    Goda, Tatsuro; Singi, Ankit Balram; Maeda, Yasuhiro; Matsumoto, Akira; Torimura, Masaki; Aoki, Hiroshi; Miyahara, Yuji

    2013-02-07

    Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry.

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. Simulation-Guided DNA Probe Design for Consistently Ultraspecific Hybridization

    PubMed Central

    Wang, J. Sherry; Zhang, David Yu

    2015-01-01

    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 for analyzing nucleic acids. Thermodynamics-guided probe design and empirical optimization of reaction conditions have been used to enable discrimination of single nucleotide variants, but typically these approaches provide only an approximate 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 different target single nucleotide variants sequences showed between 200- and 3000-fold (median 890) higher binding affinity than their corresponding wildtype sequences. As a demonstration of the usefulness of this simulation-guided design approach we developed probes which, in combination with PCR amplification, we use to detect low concentrations of variant alleles (1%) in human genomic DNA. PMID:26100802

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

    PubMed

    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.

  11. C2'-pyrene-functionalized triazole-linked DNA: universal DNA/RNA hybridization probes.

    PubMed

    Sau, Sujay P; Hrdlicka, Patrick J

    2012-01-06

    Development of universal hybridization probes, that is, oligonucleotides displaying identical affinity toward matched and mismatched DNA/RNA targets, has been a longstanding goal due to potential applications as degenerate PCR primers and microarray probes. The classic approach toward this end has been the use of "universal bases" that either are based on hydrogen-bonding purine derivatives or aromatic base analogues without hydrogen-bonding capabilities. However, development of probes that result in truly universal hybridization without compromising duplex thermostability has proven challenging. Here we have used the "click reaction" to synthesize four C2'-pyrene-functionalized triazole-linked 2'-deoxyuridine phosphoramidites. We demonstrate that oligodeoxyribonucleotides modified with the corresponding monomers display (a) minimally decreased thermal affinity toward DNA/RNA complements relative to reference strands, (b) highly robust universal hybridization characteristics (average differences in thermal denaturation temperatures of matched vs mismatched duplexes involving monomer W are <1.7 °C), and (c) exceptional affinity toward DNA targets containing abasic sites opposite of the modification site (ΔT(m) up to +25 °C). The latter observation, along with results from absorption and fluorescence spectroscopy, suggests that the pyrene moiety is intercalating into the duplex whereby the opposing nucleotide is pushed into an extrahelical position. These properties render C2'-pyrene-functionalized triazole-linked DNA as promising universal hybridization probes for applications in nucleic acid chemistry and biotechnology.

  12. Probing DNA hybridization efficiency and single base mismatch by X-ray photoelectron spectroscopy.

    PubMed

    Liu, Zheng-Chun; Zhang, Xin; He, Nong-Yue; Lu, Zu-Hong; Chen, Zhen-Cheng

    2009-07-01

    We demonstrated the use of X-ray photoelectron spectroscopy (XPS) to study DNA hybridization. Target DNA labeled with hexachloro-fluorescein (HEX) was hybridized to DNA arrays with four different probes. Each probe dot of the hybridized arrays was detected with XPS. The XPS Cl2p peak areas were found to decrease with an increase in mismatched bases in DNA probes. The Cl2p core-level peak area ratio of a probe perfectly matched to one, two and three base-mismatched probes accorded well with the results of conventional fluorescent imaging, which shows that XPS is a potential tool for analyzing DNA arrays. The DNA arrays' hybridization efficiency was assessed by the molar ratio of chlorine to phosphorus in a DNA strand, which was determined from the relevant XPS Cl2p and P2p core-level peak areas after hybridization. This could provide a new method to detect DNA hybridization efficiency.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. DNA Probes Using Fluorescence Resonance Energy Transfer (FRET): Designs and Applications

    PubMed Central

    Didenko, Vladimir V.

    2007-01-01

    Fluorescence resonance energy transfer (FRET) is widely used in biomedical research as a reporter method. Oligonucleotides with a DNA backbone and one or several chromophore tags have found multiple applications as FRET probes. They are especially advantageous for the real-time monitoring of biochemical reactions and in vivo studies. This paper reviews the design and applications of various DNA-based probes that use FRET. The approaches used in the design of new DNA FRET probes are discussed. PMID:11730017

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. Surface-enhanced Raman scattering based nonfluorescent probe for multiplex DNA detection.

    PubMed

    Sun, Lan; Yu, Chenxu; Irudayaraj, Joseph

    2007-06-01

    To provide rapid and accurate detection of DNA markers in a straightforward, inexpensive, and multiplex format, an alternative surface-enhanced Raman scattering based probe was designed and fabricated to covalently attach both DNA probing sequence and nonfluorescent 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 nonfluorescent 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 to its complementary targets was successfully accomplished with a long-term goal to use nonfluorescent RTags in a Raman-based DNA microarray platform.

  4. 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.

  5. 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

  6. Value of a DNA probe assay (Gen-Probe) compared with that of culture for diagnosis of gonococcal infection.

    PubMed

    Vlaspolder, F; Mutsaers, J A; Blog, F; Notowicz, A

    1993-01-01

    The Gen-Probe PACE 2 system for Neisseria gonorrhoeae (GP), which uses a chemiluminescently labeled DNA probe, was compared with conventional culture as the method of reference. A total of 1,750 specimens were collected from 496 females and 623 males visiting the outpatient clinic of the Sexually Transmitted Diseases Department of the Westeinde Hospital, The Hague, The Netherlands, during the year 1991. The prevalences of gonorrhea culture-positive men and women were 14.9 and 7.7%, respectively. The overall positive rate was 8.7%. Sensitivity, specificity, and positive and negative predictive values of GP were 97.1, 99.1, 90.6, and 99.8%, respectively. A total of 12 of 13 patients with positive GP results and negative cultures may have had a gonococcal infection, a conclusion based on clinical symptoms, positive methylene blue smears, and high relative light unit ratios. The DNA probe test can be useful as a suitable screening and diagnostic test for gonorrheal infection in men and women. An advantage of using this DNA probe technique is that simultaneous testing for Chlamydia trachomatis of the same specimen is possible. We also examined whether (all) rRNA had disappeared after adequate treatment for gonococcal and/or chlamydial infection in 30 patients. None of those positive patients showed a positive result in the DNA probe assay after treatment.

  7. 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.

  8. 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.

  9. Uropathogenic Escherichia coli virulence genes: invaluable approaches for designing DNA microarray probes.

    PubMed

    Jahandeh, Nadia; Ranjbar, Reza; Behzadi, Payam; Behzadi, Elham

    2015-01-01

    The pathotypes of uropathogenic Escherichia coli (UPEC) cause different types of urinary tract infections (UTIs). The presence of a wide range of virulence genes in UPEC enables us to design appropriate DNA microarray probes. These probes, which are used in DNA microarray technology, provide us with an accurate and rapid diagnosis and definitive treatment in association with UTIs caused by UPEC pathotypes. The main goal of this article is to introduce the UPEC virulence genes as invaluable approaches for designing DNA microarray probes. Main search engines such as Google Scholar and databases like NCBI were searched to find and study several original pieces of literature, review articles, and DNA gene sequences. In parallel with in silico studies, the experiences of the authors were helpful for selecting appropriate sources and writing this review article. There is a significant variety of virulence genes among UPEC strains. The DNA sequences of virulence genes are fabulous patterns for designing microarray probes. The location of virulence genes and their sequence lengths influence the quality of probes. The use of selected virulence genes for designing microarray probes gives us a wide range of choices from which the best probe candidates can be chosen. DNA microarray technology provides us with an accurate, rapid, cost-effective, sensitive, and specific molecular diagnostic method which is facilitated by designing microarray probes. Via these tools, we are able to have an accurate diagnosis and a definitive treatment regarding UTIs caused by UPEC pathotypes.

  10. A universal biosensor for multiplex DNA detection based on hairpin probe assisted cascade signal amplification.

    PubMed

    Liu, Jie; Chen, Lingbo; Lie, Puchang; Dun, Boying; Zeng, Lingwen

    2013-06-07

    A hairpin DNA probe mediated cascade signal amplification method was developed for visual and rapid DNA analysis with a detection limit of 100 aM. The implementation of tag/anti-tag DNA and gold nanoparticle reporters permits a universal platform for multiplex genotyping without instrumentation.

  11. Effect of probe characteristics on the subtractive hybridization efficiency of human genomic DNA

    PubMed Central

    2010-01-01

    Background The detection sensitivity of low abundance pathogenic species by polymerase chain reaction (PCR) can be significantly enhanced by removing host nucleic acids. This selective removal can be performed using a magnetic bead-based solid phase with covalently immobilized capture probes. One of the requirements to attain efficient host background nucleic acids subtraction is the capture probe characteristics. Findings In this study we investigate how various capture probe characteristics influence the subtraction efficiency. While the primary focus of this report is the impact of probe length, we also studied the impact of probe conformation as well as the amount of capture probe attached to the solid phase. The probes were immobilized on magnetic microbeads functionalized with a phosphorous dendrimer. The subtraction efficiency was assessed by quantitative real time PCR using a single-step capture protocol and genomic DNA as target. Our results indicate that short probes (100 to 200 bp) exhibit the best subtraction efficiency. Additionally, higher subtraction efficiencies with these probes were obtained as the amount of probe immobilized on the solid phase decreased. Under optimal probes condition, our protocol showed a 90 - 95% subtraction efficiency of human genomic DNA. Conclusions The characteristics of the capture probe are important for the design of efficient solid phases. The length, conformation and abundance of the probes determine the capture efficiency of the solid phase. PMID:20406484

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. Fluorescent silver nanocluster DNA probes for multiplexed detection using microfluidic capillary electrophoresis.

    PubMed

    Del Bonis-O'Donnell, Jackson Travis; Fygenson, Deborah K; Pennathur, Sumita

    2015-03-07

    DNA-stabilized fluorescent silver nanoclusters (AgNC DNA) are a new class of fluorophore that are formed by sequence specific interactions between silver and single-stranded DNA. By incorporating both target-binding and fluorescent-reporting sequences into a single synthetic DNA oligomer, AgNC DNA probes eliminate the need to conjugate dye or quencher molecules. In this study, we modify a AgNC DNA probe to demonstrate single-color multiplexed detection of DNA targets. We show that appending different lengths of poly-dT to the probe sequences tunes the electrophoretic mobility of AgNC DNA probes without affecting their fluorescence spectra. We use this to introduce a set of AgNC DNA probes selective for Hepatitis A, B and C target sequences that can be processed together in a simple, single-step protocol and distinguished with a resolution of 3.47 and signal to noise ratio of 17.23 in under 10 seconds by microfluidic capillary electrophoresis.

  17. 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.

  18. HBx-DNA probe preparation and its application in study of hepatocarcinogenesis

    PubMed Central

    Gao, Feng-Guang; Sun, Wen-Sheng; Cao, Ying-Lin; Zhang, Li-Ning; Song, Jing; Li, Hua-Fen; Yan, Shi-Kun

    1998-01-01

    AIM: To study the role of HBV especially HBx Open Reading Frame (ORF) in the development of hepatocellular carcinoma (HCC). METHODS: HBV 3.2 kb fragment was retrieved by digesting recombinant plasmid pBR322-2HBV with EcoR I, and HBx 0.59 kb fragments by digesting HBV-DNA with BamH I and Bgl II. These fragments were labelled with digoxigenin to get HBV-DNA and HBx-DNA probes. HBV-DNA was detected in HCC by dot blot and Southern blot hybridization with HBV-DNA probe, so the positive specimens in which HBV-DNA were integrated were selected. HBx-DNA was subsequently detected in the selected specimens with HBx-DNA probe. RESULTS: HBV-DNA was detected in 75% HCC, among which integrated type, integrated + free type covered 63.6% and 36.4%. There was no free type. HBx-DNA was detected in 90.5% specimens of integrated type. CONCLUSION: Hepatocarcinogenesis was highly related to HBV-DNA integration, and HBV-DNA mainly integrated into chromosome with incomplete virus DNA fragments among which HBx fragment was the predominant one. PMID:11819309

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Immobilization-free electrochemical DNA detection with anthraquinone-labeled pyrrolidinyl peptide nucleic acid probe.

    PubMed

    Kongpeth, Jutatip; Jampasa, Sakda; Chaumpluk, Piyasak; Chailapakul, Orawon; Vilaivan, Tirayut

    2016-01-01

    Electrochemical detection provides a simple, rapid, sensitive and inexpensive method for DNA detection. In traditional electrochemical DNA biosensors, the probe is immobilized onto the electrode. Hybridization with the DNA target causes a change in electrochemical signal, either from the intrinsic signal of the probe/target or through a label or a redox indicator. The major drawback of this approach is the requirement for probe immobilization in a controlled fashion. In this research, we take the advantage of different electrostatic properties between PNA and DNA to develop an immobilization-free approach for highly sequence-specific electrochemical DNA sensing on a screen-printed carbon electrode (SPCE) using a square-wave voltammetric (SWV) technique. Anthraquinone-labeled pyrrolidinyl peptide nucleic acid (AQ-PNA) was employed as a probe together with an SPCE that was modified with a positively-charged polymer (poly quaternized-(dimethylamino-ethyl)methacrylate, PQDMAEMA). The electrostatic attraction between the negatively-charged PNA-DNA duplex and the positively-charged modified SPCE attributes to the higher signal of PNA-DNA duplex than that of the electrostatically neutral PNA probe, resulting in a signal change. The calibration curve of this proposed method exhibited a linear range between 0.35 and 50 nM of DNA target with a limit of detection of 0.13 nM (3SD(blank)/Slope). The sub-nanomolar detection limit together with a small sample volume required (20 μL) allowed detection of <10 fmol (<1 ng) of DNA. With the high specificity of the pyrrolidinyl PNA probe used, excellent discrimination between complementary and various single-mismatched DNA targets was obtained. An application of this new platform for a sensitive and specific detection of isothermally-amplified shrimp's white spot syndrome virus (WSSV) DNA was successfully demonstrated. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. Probing Human Telomeric DNA and RNA Topology and Ligand Binding in a Cellular Model by Using Responsive Fluorescent Nucleoside Probes.

    PubMed

    Manna, Sudeshna; Panse, Cornelia H; Sontakke, Vyankat A; Sangamesh, Sarangamath; Srivatsan, Seergazhi G

    2017-08-17

    The development of biophysical systems that enable an understanding of the structure and ligand-binding properties of G-quadruplex (GQ)-forming nucleic acid sequences in cells or models that mimic the cellular environment would be highly beneficial in advancing GQ-directed therapeutic strategies. Herein, the establishment of a biophysical platform to investigate the structure and recognition properties of human telomeric (H-Telo) DNA and RNA repeats in a cell-like confined environment by using conformation-sensitive fluorescent nucleoside probes and a widely used cellular model, bis(2-ethylhexyl) sodium sulfosuccinate reverse micelles (RMs), is described. The 2'-deoxy and ribonucleoside probes, composed of a 5-benzofuran uracil base analogue, faithfully report the aqueous micellar core through changes in their fluorescence properties. The nucleoside probes incorporated into different loops of H-Telo DNA and RNA oligonucleotide repeats are minimally perturbing and photophysically signal the formation of respective GQ structures in both aqueous buffer and RMs. Furthermore, these sensors enable a direct comparison of the binding affinity of a ligand to H-Telo DNA and RNA GQ structures in the bulk and confined environment of RMs. These results demonstrate that this combination of a GQ nucleoside probe and easy-to-handle RMs could provide new opportunities to study and devise screening-compatible assays in a cell-like environment to discover GQ binders of clinical potential. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. 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

  14. 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.

  15. [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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. Superior structure stability and selectivity of hairpin nucleic acid probes with an l-DNA stem

    PubMed Central

    Kim, Youngmi; Yang, Chaoyong James; Tan, Weihong

    2007-01-01

    Hairpin nucleic acid probes have been highly useful in many areas, especially for intracellular and in vitro nucleic acid detection. The success of these probes can be attributed to the ease with which their conformational change upon target binding can be coupled to a variety of signal transduction mechanisms. However, false-positive signals arise from the opening of the hairpin due mainly to thermal fluctuations and stem invasions. Stem invasions occur when the stem interacts with its complementary sequence and are especially problematic in complex biological samples. To address the problem of stem invasions in hairpin probes, we have created a modified molecular beacon that incorporates unnatural enantiomeric l-DNA in the stem and natural d-DNA or 2′-O-Me-modified RNA in the loop. l-DNA has the same physical characteristics as d-DNA except that l-DNA cannot form stable duplexes with d-DNA. Here we show that incorporating l-DNA into the stem region of a molecular beacon reduces intra- and intermolecular stem invasions, increases the melting temperature, improves selectivity to its target, and leads to enhanced bio-stability. Our results suggest that l-DNA is useful for designing functional nucleic acid probes especially for biological applications. PMID:17959649

  1. 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.

  2. 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.

  3. Rapid detection of Streptococcus pyogenes in pediatric patient specimens by DNA probe.

    PubMed Central

    Steed, L L; Korgenski, E K; Daly, J A

    1993-01-01

    A chemiluminescent DNA probe test (Group A Streptococcus Direct Test; Gen-Probe, Inc., San Diego, Calif.) for rapid, direct detection of cRNA of Streptococcus pyogenes in throat swabs was compared with conventional culture and identification techniques. Throat swabs from 277 patients suspected of having streptococcal pharyngitis were examined. By DNA probe alone, 10 specimens were positive, 51 were positive by both assays, and 8 were positive by culture alone. Thus, DNA probe sensitivity, specificity, and positive and negative predictive values were 86, 95, 84, and 96%, respectively. Including an indeterminate category, sensitivity, specificity, and positive and negative predictive values were 89, 96, 86, and 97%, respectively. After discrepancy testing, these values for the raw data improved to 90, 98, 93, and 97%, respectively. None of the 24 specimens that grew non-S. pyogenes beta-hemolytic streptococci in culture were positive by the DNA probe. Because mucoid S. pyogenes strains are more virulent than nonmucoid strains, 24 isolates were retrospectively tested with the DNA probe to ensure that both types would be detected equally well. Isolates were examined in pure cultures as well as mixed with representative normal oral flora. There was no statistical difference in detection of any of the four groups. Group A Streptococcus Direct Test is a rapid, sensitive, and specific test for S. pyogenes. PMID:8263185

  4. 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.

  5. Generation of digoxigenin-incorporated probes to enhance DNA detection sensitivity.

    PubMed

    Lai, Tsung-Po; Wright, Woodring E; Shay, Jerry W

    2016-01-01

    Telomere length in humans has been correlated with cancer and age-related diseases. The standard method to measure telomere length relies on Southern blot analysis with radioactively or non-radioactively labeled probes containing several telomeric DNA repeats. However, this approach requires relatively large amounts of genomic DNA, making it difficult to measure telomere length when a limited amount of sample is available. Here, we describe a non-radioactive labeling method that uses 3' fill-in combined with lambda exonuclease digestion to incorporate one or more digoxigenin (DIG) molecules into bridged nucleic acid (BNA)-containing oligonucleotides (ONTs). Using our method, we were able to generate probes to detect both C- and G-rich telomeric DNA strands. Compared with commercially available DIG-labeled telomere probes, probes generated using this new approach significantly enhance the sensitivity of telomere length measurements.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. Gold nanorods as multifunctional probes in a liquid crystalline DNA matrix.

    PubMed

    Olesiak-Banska, Joanna; Gordel, Marta; Matczyszyn, Katarzyna; Shynkar, Vasyl; Zyss, Joseph; Samoc, Marek

    2013-11-21

    We show how a single gold nanorod can serve as a multifunctional probe in an organized DNA matrix. Polarization analysis of two-photon luminescence excited with a femtosecond laser enables imaging of the orientation of a single nanorod, which reports the orientation of DNA strands. Carefully controlled photoinduced heating by the same laser is able to degrade the DNA matrix in a highly localized volume.

  12. 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

  13. Comprehensive characterization, annotation and innovative use of Infinium DNA methylation BeadChip probes

    PubMed Central

    Zhou, Wanding; Laird, Peter W.

    2017-01-01

    Abstract Illumina Infinium DNA Methylation BeadChips represent the most widely used genome-scale DNA methylation assays. Existing strategies for masking Infinium probes overlapping repeats or single nucleotide polymorphisms (SNPs) are based largely on ad hoc assumptions and subjective criteria. In addition, the recently introduced MethylationEPIC (EPIC) array expands on the utility of this platform, but has not yet been well characterized. We present in this paper an extensive characterization of probes on the EPIC and HM450 microarrays, including mappability to the latest genome build, genomic copy number of the 3΄ nested subsequence and influence of polymorphisms including a previously unrecognized color channel switch for Type I probes. We show empirical evidence for exclusion criteria for underperforming probes, providing a sounder basis than current ad hoc criteria for exclusion. In addition, we describe novel probe uses, exemplified by the addition of a total of 1052 SNP probes to the existing 59 explicit SNP probes on the EPIC array and the use of these probes to predict ethnicity. Finally, we present an innovative out-of-band color channel application for the dual use of 62 371 probes as internal bisulfite conversion controls. PMID:27924034

  14. 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

  15. 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.

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

    PubMed

    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. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. 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.

  18. 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.

  19. Selectivity of Hybridization Controlled by the Density of Solid Phase Synthesized DNA Probes on Glass Substrates

    NASA Astrophysics Data System (ADS)

    Song, Fayi; Krull, Ulrich J.

    Optical biochip design based on varying the density of immobilized single-stranded DNA (ssDNA) oligonucleotide probes was examined. A method of immobilization was developed to yield various densities of probe molecules using photochemical activation of surfaces and in situ solid phase synthesis for DNA immobilization. High surface density of ssDNA probe (up to 1 × 1013 probes/cm2) was obtained using the immobilization method. The densities and extent of hybridization of nucleic acids were determined using confocal fluorescence microscopy. Selective hybridization of targets associated with spinal muscular atrophy containing single nucleotide polymorphisms (SNP), and their thermal denaturation profiles were investigated to examine the sensitivity and selectivity for SNP detection. The detection limit was less than 16 pM at room temperature. Single base mismatch discrimination was demonstrated based on control of melt temperature by selection of probe density, and temperature differences of 12-15°C could be achieved for SNP determination. Importantly, the results demonstrate that poor control of probe density can result in significant variability of selectivity, as seen by melt temperature shifts of up to 5°C in the density range that was investigated.

  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. 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~ -

  2. Directly labeled DNA probes using fluorescent nucleotides with different length linkers.

    PubMed Central

    Zhu, Z; Chao, J; Yu, H; Waggoner, A S

    1994-01-01

    Directly labeled fluorescent DNA probes have been made by nick translation and PCR using dUTP attached to the fluorescent label, Cy3, with different length linkers. With preparation of probes by PCR we find that linker length affects the efficiency of incorporation of Cy3-dUTP, the yield of labeled probe, and the signal intensity of labeled probes hybridized to chromosome target sequences. For nick translation and PCR, both the level of incorporation and the hybridization fluorescence signal increased in parallel when the length of the linker arm is increased. Under optimal conditions, PCR yielded more densely labeled probes, however, the yield of PCR labeled probe decreased with greater linear density of labeling. By using a Cy3-modified dUTP with the longest linker under optimal conditions it was possible to label up to 28% of the possible substitution sites on the target DNA with reasonable yield by PCR and 18% by nick translation. A mechanism involving steric interactions between the polymerase, cyanine-labeled sites on template and extending chains and the modified dUTP substrate is proposed to explain the inverse correlation between the labeling efficiency and the yield of DNA probe synthesis by PCR. Images PMID:8078779

  3. Aptamer-based electrochemical sensors with aptamer-complementary DNA oligonucleotides as probe.

    PubMed

    Lu, Ying; Li, Xianchan; Zhang, Limin; Yu, Ping; Su, Lei; Mao, Lanqun

    2008-03-15

    This study describes a facile and general strategy for the development of aptamer-based electrochemical sensors with a high specificity toward the targets and a ready regeneration feature. Very different from the existing strategies for the development of electrochemical aptasensors with the aptamers as the probes, the strategy proposed here is essentially based on the utilization of the aptamer-complementary DNA (cDNA) oligonucleotides as the probes for electrochemical sensing. In this context, the sequences at both ends of the cDNA are tailor-made to be complementary and both the redox moiety (i.e., ferrocene in this study) and thiol group are labeled onto the cDNA. The labeled cDNA are hybridized with their respective aptamers (i.e., ATP- and thrombin-binding aptamers in this study) to form double-stranded DNA (ds-DNA) and the electrochemical aptasensors are prepared by self-assembling the labeled ds-DNA onto Au electrodes. Upon target binding, the aptamers confined onto electrode surface dissociate from their respective cDNA oligonucleotides into the solution and the single-stranded cDNA could thus tend to form a hairpin structure through the hybridization of the complementary sequences at both its ends. Such a conformational change of the cDNA resulting from the target binding-induced dissociation of the aptamers essentially leads to the change in the voltammetric signal of the redox moiety labeled onto the cDNA and thus constitutes the mechanism for the electrochemical aptasensors for specific target sensing. The aptasensors demonstrated here with the cDNA as the probe are readily regenerated and show good responses toward the targets. This study may offer a new and relatively general approach to electrochemical aptasensors with good analytical properties and potential applications.

  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. 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.

  6. 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.

  7. Comet-FISH with rDNA probes for the analysis of mutagen-induced DNA damage in plant cells.

    PubMed

    Kwasniewska, Jolanta; Grabowska, Marta; Kwasniewski, Miroslaw; Kolano, Bozena

    2012-06-01

    We used comet-fluorescence in situ hybridization (FISH) in the model plant species Crepis capillaris following exposure of seedlings to maleic hydrazide (MH). FISH with 5S and 25S rDNA probes was applied to comets obtained under alkaline conditions to establish whether these DNA regions were preferentially involved in comet tail formation. MH treatment induced significant fragmentation of nuclear DNA and of rDNA loci. A 24-h post-treatment recovery period allowed a partial reversibility of MH-induced damage on nuclear and rDNA regions. Analyses of FISH signals demonstrated that rDNA sequences were always involved in tail formation and that 5S rDNA was more frequently present in the tail than 25S rDNA, regardless of treatment. The involvement of 25S rDNA in nucleolus formation and differences in chromatin structure between the two loci may explain the different susceptibility of the 25S and 5S rDNA regions to migrate into the tail. This work is the first report on the application of FISH to comet preparations from plants to analyze the distribution and repair of DNA damage within specific genomic regions after mutagenic treatment. Moreover, our work suggests that comet-FISH in plants may be a useful tool for environmental monitoring assessment. Copyright © 2012 Wiley Periodicals, Inc.

  8. 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. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. 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.

  10. 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.

  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. 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.

  13. AuNP-CTG based probing system targeting CAG repeat DNA and RNA sequences.

    PubMed

    Le, Binh Huy; Joo, Han Na; Hwang, Do Won; Kim, Kyu Wan; Seo, Young Jun

    2017-08-15

    We have developed a AuNP-CTG based probing system that is applicable to the detection of many units of CAG repeat sequences which was synthesized by a rolling circle amplification (RCA) system with changes in fluorescence. We also demonstrate that our AuNP-CTG based probing system could transfect without using transfection reagent and detect target CAG repeat sequences in HeLa cells with dramatic changes in fluorescence. This AuNP-CTG based probing system could also be used, in conjunction with the CAG repeat RCA system, to detect target DNA. This system was so sensitive to the target DNA that it could detect even picomolar amounts with amplification of the fluorescence signal. Furthermore, we have used our gold-based CAG probing system for the detection of RNA CAG repeat sequences. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. 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

  15. Cyanine dye dUTP analogs for enzymatic labeling of DNA probes.

    PubMed Central

    Yu, H; Chao, J; Patek, D; Mujumdar, R; Mujumdar, S; Waggoner, A S

    1994-01-01

    Fluorescence in situ hybridization (FISH) has become and indispensable tool in a variety of areas of research and clinical diagnostics. Many applications demand an approach for simultaneous detection of multiple target sequences that is rapid and simple, yet sensitive. In this work, we describe the synthesis of two new cyanine dye-labeled dUTP analogs, Cy3-dUTP and Cy5-dUTP. They are efficient substrates for DNA polymerases and can be incorporated into DNA probes by standard nick translation, random priming and polymerase chain reactions. Optimal labeling conditions have been identified which yield probes with 20-40 dyes per kilobase. The directly labeled DNA probes obtained with these analogs offer a simple approach for multicolor multisequence analysis that requires no secondary detection reagents and steps. Images PMID:8065939

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. Microarray long oligo probe designing for Escherichia coli: an in-silico DNA marker extraction.

    PubMed

    Behzadi, Payam; Najafi, Ali; Behzadi, Elham; Ranjbar, Reza

    2016-01-01

    Urinary tract infections are predominant diseases which may be caused by different pathogenic microorganisms, particularly Escherichia coli (E.coli). DNA microarray technology is an accurate, rapid, sensitive, and specific diagnostic tool which may lead to definite diagnosis and treatment of several infectious diseases. DNA microarray is a multi-process method in which probe designing plays an important. Therefore, the authors of the present study have tried to design a range of effective and proper long oligo microarray probes for detection and identification of different strains of pathogenic E.coli and in particular, uropathogenic E.coli (UPEC). E.coli O26 H11 11368 uid41021 was selected as the standard strain for probe designing. This strain encompasses the largest nucleotide sequence and the most number of genes among other pathogenic strains of E.coli. For performing this in silico survey, NCBI database, GReview Server, PanSeq Server, Oligoanalyzer tool, and AlleleID 7.7 were used to design accurate, appropriate, effective, and flexible long oligo microarray probes. Moreover, the genome of E.coli and its closely related microorganisms were compared. In this study, 15 long oligo microarray probes were designed for detecting and identifying different strains of E.coli such as UPEC. These probes possessed the best physico-chemical characteristics. The functional and structural properties of the designed probes were recognized by practical tools and softwares. The use of reliable advanced technologies and methodologies for probe designing guarentees the high quality of microarray probes and makes DNA microarray technology more flexible and an effective diagnostic technique.

  3. 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.

  4. 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

  5. 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

  6. Use of DNA probes in the study of silage colonization by Lactobacillus and Pediococcus strains.

    PubMed

    Cocconcelli, P S; Triban, E; Basso, M; Bottazzi, V

    1991-10-01

    A technique to monitor lactic acid bacteria inoculants in silage, based on specific DNA probes, was developed and used to evaluate the colonization properties of two strains of Lactobacillus plantarum and one strain of Pediococcus pentosaceus which were used as maize silage inoculants in farm conditions. The results indicated that these three strains were able to dominate the natural microflora of the silage, representing more than the 95% of the bacterial biomass of the maize silage. These studies indicate that the colony hybridization with specific DNA probes may be an effective method for monitoring bacteria and evaluating the colonization properties of inoculants in maize silage.

  7. 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.

  8. Origin and molecular organization of supernumerary chromosomes of Prochilodus lineatus (characiformes, prochilodontidae) obtained by DNA probes.

    PubMed

    Voltolin, Tatiana Aparecida; Laudicina, Alejandro; Senhorini, José Augusto; Bortolozzi, Jehud; Oliveira, Cláudio; Foresti, Fausto; Porto-Foresti, Fábio

    2010-12-01

    In Prochilodus lineatus B-chromosomes are visualized as reduced size extra elements identified as microchromosomes and are variable in morphology and number. We describe the specific total probe (B-chromosome probe) in P. lineatus obtained by chromosome microdissection and a whole genomic probe (genomic probe) from an individual without B-chromosome. The specific B-chromosome was scraped and processed to obtain DNA with amplification by DOP-PCR, and so did the genomic probe DNA. Fluorescence in situ hybridization using the B-chromosome probe labeled with dUTP-Tetramethyl-rhodamine and the genomic probe labeled with digoxigenin-FITC permitted to establish that in this species supernumerary chromosomes with varying number and morphology had different structure of chromatin when compared to that of the regular chromosomes or A complement, since only these extra elements were labeled in the metaphases. The present findings suggest that modifications in the chromatin structure of B-chromosomes to differentiate them from the A chromosomes could occur along their dispersion in the individuals of the population.

  9. 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.

  10. 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.

  11. 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

  12. Probing the Electrostatic Shielding of DNA with Capillary Electrophoresis

    PubMed Central

    Stellwagen, Earle; Stellwagen, Nancy C.

    2003-01-01

    The free solution mobility of a 20-bp double-stranded DNA oligomer has been measured in diethylmalonate (DM) and Tris-acetate buffers, with and without added NaCl or TrisCl. DM buffers have the advantage that the buffering ion is anionic, so the cation composition in the solution can be varied at will. The results indicate that the free solution mobility of DNA decreases linearly with the logarithm of ionic strength when the ionic strength is increased by increasing the buffer concentration. The mobility also decreases linearly with the logarithm of ionic strength when NaCl is added to NaDM buffer or TrisCl is added to TrisDM buffer. Nonlinear effects are observed if the counterion in the added salt differs from the counterion in the buffer. The dependence of the mobility on ionic strength cannot be predicted using the Henry, Debye-Hückel-Onsager, or Pitts equations for electrophoresis. However, the mobilities observed in all buffer and buffer/salt solutions can be predicted within ∼20% by the Manning equation for electrophoresis, using no adjustable parameters. The results suggest that the electrostatic shielding of DNA is determined not only by the relative concentrations of the various ions in the solution, but also by their equivalent conductivities. PMID:12609887

  13. Probing evolutionary patterns in neotropical birds through DNA barcodes.

    PubMed

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

    2009-01-01

    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. 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. 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.

  14. 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

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

    PubMed Central

    Dillingham, Mark S.; Tibbles, Katherine L.; Hunter, Jackie L.; Bell, Jason C.; Kowalczykowski, Stephen C.; Webb, Martin R.

    2008-01-01

    The formation and maintenance of single-stranded DNA (ssDNA) are essential parts of many processes involving DNA. For example, strand separation of double-stranded DNA (dsDNA) is catalyzed by helicases, and this exposure of the bases on the DNA allows further processing, such as replication, recombination, or repair. Assays of helicase activity and probes for their mechanism are essential for understanding related biological processes. Here we describe the development and use of a fluorescent probe to measure ssDNA formation specifically and in real time, with high sensitivity and time resolution. The reagentless biosensor is based on the ssDNA binding protein (SSB) from Escherichia coli, labeled at a specific site with a coumarin fluorophore. Its use in the study of DNA manipulations involving ssDNA intermediates is demonstrated in assays for DNA unwinding, catalyzed by DNA helicases. PMID:18599625

  16. Quantitative, Fluorogenic Probe PCR Assay for Detection of Human Herpesvirus 8 DNA in Clinical Specimens

    PubMed Central

    Stamey, Felicia R.; Patel, Mitesh M.; Holloway, Brian P.; Pellett, Philip E.

    2001-01-01

    A quantitative, fluorescence-based PCR assay (TaqMan-based system) was developed for detection of human herpesvirus 8 (HHV-8) DNA in clinical specimens. Primers and probes chosen from each of five 10-kb segments from the unique region of the HHV-8 genome were evaluated for sensitivity with dilution series of DNA extracted from a cell line (BCBL-1) that harbors HHV-8 DNA. Although several of the primer-probe sets performed similarly with BCBL-1 DNA that had been diluted in water, their performance differed when target DNA was diluted in a constant background of uninfected cell DNA, an environment more relevant to their intended use. The two best primer-probe combinations were specific for HHV-8 relative to the other known human herpesviruses and herpesvirus saimiri, a closely related gammaherpesvirus of nonhuman primates. PCRs included an enzymatic digestion step to eliminate PCR carryover and an exogenous internal positive control that enabled discrimination of false-negative from true-negative reactions. The new assays were compared to conventional PCR assays for clinical specimens (saliva, rectal brushings, rectal swab specimens, peripheral blood lymphocytes, semen, and urine) from human immunodeficiency virus-positive patients with or without Kaposi's sarcoma. In all instances, the new assays agreed with each other and with the conventional PCR system. In addition, the quantitative results obtained with the new assays were in good agreement both for duplicate reactions in the same assay and between assays. PMID:11574569

  17. Quantitative, fluorogenic probe PCR assay for detection of human herpesvirus 8 DNA in clinical specimens.

    PubMed

    Stamey, F R; Patel, M M; Holloway, B P; Pellett, P E

    2001-10-01

    A quantitative, fluorescence-based PCR assay (TaqMan-based system) was developed for detection of human herpesvirus 8 (HHV-8) DNA in clinical specimens. Primers and probes chosen from each of five 10-kb segments from the unique region of the HHV-8 genome were evaluated for sensitivity with dilution series of DNA extracted from a cell line (BCBL-1) that harbors HHV-8 DNA. Although several of the primer-probe sets performed similarly with BCBL-1 DNA that had been diluted in water, their performance differed when target DNA was diluted in a constant background of uninfected cell DNA, an environment more relevant to their intended use. The two best primer-probe combinations were specific for HHV-8 relative to the other known human herpesviruses and herpesvirus saimiri, a closely related gammaherpesvirus of nonhuman primates. PCRs included an enzymatic digestion step to eliminate PCR carryover and an exogenous internal positive control that enabled discrimination of false-negative from true-negative reactions. The new assays were compared to conventional PCR assays for clinical specimens (saliva, rectal brushings, rectal swab specimens, peripheral blood lymphocytes, semen, and urine) from human immunodeficiency virus-positive patients with or without Kaposi's sarcoma. In all instances, the new assays agreed with each other and with the conventional PCR system. In addition, the quantitative results obtained with the new assays were in good agreement both for duplicate reactions in the same assay and between assays.

  18. 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.

  19. 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.

  20. 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. © 2017 Schneider and Meier; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  1. 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

  2. Bioorthogonal Labeling of 5-Hydroxymethylcytosine in Genomic DNA and Diazirine-Based DNA Photo-Cross-Linking Probes

    PubMed Central

    SONG, CHUN-XIAO; HE, CHUAN

    2013-01-01

    CONSPECTUS DNA is not merely a combination of four genetic codes, namely A, T, C, and G. It also contains minor modifications that play crucial roles throughout biology. For example, the fifth DNA base, 5-methylcytosine (5-mC), which accounts for ~1% of all the nucleotides in mammalian genomic DNA, is a vital epigenetic mark. It impacts a broad range of biological functions, from development to cancer. Recently, an oxidized form of 5-methylcytosine, 5-hydroxymethylcytosine (5-hmC), was found to constitute the sixth base in the mammalian genome; it was believed to be another crucial epigenetic mark. Unfortunately, further study of this newly discovered DNA base modification has been hampered by inadequate detection and sequencing methods, because current techniques fail to differentiate 5-hmC from 5-mC. The immediate challenge, therefore, is to develop robust methods for ascertaining the positions of 5-hmC within the mammalian genome. In this Account, we describe our development of the first bioorthogonal, selective labeling of 5-hmC to specifically address this challenge. We utilize β-glucosyltransferase (βGT) to transfer an azide-modified glucose onto 5-hmC in genomic DNA. The azide moiety enables further bioorthogonal click chemistry to install a biotin group, which allows for detection, affinity enrichment, and, most importantly, deep sequencing of the 5-hmC-containing DNA. With this highly effective and selective method, we revealed the first genome-wide distribution of 5-hmC in the mouse genome and began to shed further light on the biology of 5-hmC. The strategy lays the foundation for developing high-throughput, single-base-resolution sequencing methods for 5-hmC in mammalian genomes in the future. DNA and RNA are not static inside cells. They interact with protein and other DNA and RNA in fundamental biological processes such as replication, transcription, translation, and DNA and RNA modification and repair. The ability to investigate these interactions

  3. 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.

  4. 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

  5. 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.

  6. Fast, Background-Free DNA-PAINT Imaging Using FRET-Based Probes.

    PubMed

    Auer, Alexander; Strauss, Maximilian T; Schlichthaerle, Thomas; Jungmann, Ralf

    2017-10-11

    DNA point accumulation in nanoscale topography (DNA-PAINT) enables super-resolution microscopy by harnessing the predictable, transient hybridization between short dye-labeled "imager" and complementary target-bound "docking" strands. DNA-PAINT microscopy allows sub-5 nm spatial resolution, spectrally unlimited multiplexing, and quantitative image analysis. However, these abilities come at the cost of nonfluorogenic imager strands, also emitting fluorescence when not bound to their docking strands. This has thus far prevented rapid image acquisition with DNA-PAINT, as the blinking rate of probes is limited by an upper-bound of imager strand concentrations, which in turn is dictated by the necessity to facilitate the detection of single-molecule binding events over the background of unbound, freely diffusing probes. To overcome this limitation and enable fast, background-free DNA-PAINT microscopy, we here introduce FRET-based imaging probes, alleviating the concentration-limit of imager strands and speeding up image acquisition by several orders of magnitude. We assay two approaches for FRET-based DNA-PAINT (or FRET-PAINT) using either fixed or transient acceptor dyes in combination with transiently binding donor-labeled DNA strands and achieve high-quality super-resolution imaging on DNA origami structures in a few tens of seconds. Finally, we also demonstrate the applicability of FRET-PAINT in a cellular environment by performing super-resolution imaging of microtubules in under 30 s. FRET-PAINT combines the advantages of conventional DNA-PAINT with fast image acquisition times, facilitating the potential study of dynamic processes.

  7. Use of DNA probes to study tetracycline resistance determinants in gram-negative bacteria from swine

    SciTech Connect

    Lee, C.Y.

    1989-01-01

    Specific {sup 32}P-labeled DNA probes were prepared and used to evaluate the distribution of tetracycline resistance determinants carried by gram-negative enteric bacteria isolated from pigs in 3 swine herds with different histories of antibiotic exposure. Plasmid DNA, ranging in size from 2.1 to 186 Kb, was observed in over 84% of 114 isolates studied. Two of 78 tetracycline resistant strains did not harbor plasmids. The DNA probes were isolated from plasmids pSL18, pRT29/Tn10, pBR322 and pSL106, respectively, and they represented class A, B, C and D tetracycline resistance determinants. Hybridization conditions using 0.5X SSPE at 65{degrees}C minimize cross-hybridization between the different class of tetracycline resistance genes. Cross-hybridization between class A and class C determinants could be distinguished by simultaneous comparison of the intensity of their hybridization signals. Plasmids from over 44% of the tetracycline resistant isolates did not hybridize to DNA probes for the determinants tested. Class B determinant occurred more frequently than class A or C. None of the isolates hybridized with the class D probe.

  8. Using triplex-forming oligonucleotide probes for the reagentless, electrochemical detection of double-stranded DNA.

    PubMed

    Patterson, Adriana; Caprio, Felice; Vallée-Bélisle, Alexis; Moscone, Danila; Plaxco, Kevin W; Palleschi, Giuseppe; Ricci, Francesco

    2010-11-01

    We report a reagentless, electrochemical sensor for the detection of double-stranded DNA targets that employs triplex-forming oligonucleotides (TFOs) as its recognition element. These sensors are based on redox-tagged TFO probes strongly chemisorbed onto an interrogating gold electrode. Upon the addition of the relevant double-stranded DNA target, the probe forms a rigid triplex structure via reverse Hoogsteen base pairing in the major groove. The formation of the triplex impedes contact between the probe's redox moiety and the interrogating electrode, thus signaling the presence of the target. We first demonstrated the proof of principle of this approach by using a well-characterized 22-base polypurine TFO sequence that readily detects a synthetic, double-stranded DNA target. We then confirmed the generalizability of our platform with a second probe, a 19-base polypyrimidine TFO sequence that targets a polypurine tract (PPT) sequence conserved in all HIV-1 strains. Both sensors rapidly and specifically detect their double-stranded DNA targets at concentrations as low as ~10 nM and are selective enough to be employed directly in complex sample matrices such as blood serum. Moreover, to demonstrate real-world applicability of this new sensor platform, we have successfully detected unpurified, double-stranded PCR amplicons containing the relevant conserved HIV-1 sequence.

  9. 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.

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

    PubMed Central

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

    2014-01-01

    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

  11. 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

  12. 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

  13. 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

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

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

    Mechanical stimuli profoundly alter cell fate, yet the mechanisms underlying mechanotransduction remain obscure because of 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- to 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.

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

    PubMed

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

    2016-09-30

    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. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. 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 \

  17. DNA probes for monitoring dynamic and transient molecular encounters on live cell membranes

    NASA Astrophysics Data System (ADS)

    You, Mingxu; Lyu, Yifan; Han, Da; Qiu, Liping; Liu, Qiaoling; Chen, Tao; Sam Wu, Cuichen; Peng, Lu; Zhang, Liqin; Bao, Gang; Tan, Weihong

    2017-05-01

    Cells interact with the extracellular environment through molecules expressed on the membrane. Disruption of these membrane-bound interactions (or encounters) can result in disease progression. Advances in super-resolution microscopy have allowed membrane encounters to be examined, however, these methods cannot image entire membranes and cannot provide information on the dynamic interactions between membrane-bound molecules. Here, we show a novel DNA probe that can transduce transient membrane encounter events into readable cumulative fluorescence signals. The probe, which translocates from one anchor site to another, mimicking motor proteins, is realized through a toehold-mediated DNA strand displacement reaction. Using this probe, we successfully monitored rapid encounter events of membrane lipid domains using flow cytometry and fluorescence microscopy. Our results show a preference for encounters within the same lipid domains.

  18. Sub-diffusion and trapped dynamics of neutral and charged probes in DNA-protein coacervates

    NASA Astrophysics Data System (ADS)

    Arfin, Najmul; Yadav, Avinash Chand; Bohidar, H. B.

    2013-11-01

    The physical mechanism leading to the formation of large intermolecular DNA-protein complexes has been studied. Our study aims to explain the occurrence of fast coacervation dynamics at the charge neutralization point, followed by the appearance of smaller complexes and slower coacervation dynamics as the complex experiences overcharging. Furthermore, the electrostatic potential and probe mobility was investigated to mimic the transport of DNA / DNA-protein complex in a DNA-protein complex coacervate medium [N. Arfin and H. B. Bohidar, J. Phys. Chem. B 116, 13192 (2012)] by assigning neutral, negative, or positive charge to the probe particle. The mobility of the neutral probe was maximal at low matrix concentrations and showed random walk behavior, while its mobility ceased at the jamming concentration of c = 0.6, showing sub-diffusion and trapped dynamics. The positively charged probe showed sub-diffusive random walk followed by trapped dynamics, while the negatively charged probe showed trapping with occasional hopping dynamics at much lower concentrations. Sub-diffusion of the probe was observed in all cases under consideration, where the electrostatic interaction was used exclusively as the dominant force involved in the dynamics. For neutral and positive probes, the mean square displacement ⟨R2⟩ exhibits a scaling with time as ⟨R2⟩ ˜ tα, distinguishing random walk and trapped dynamics at α = 0.64 ± 0.04 at c = 0.12 and c = 0.6, respectively. In addition, the same scaling factors with the exponent β = 0.64 ± 0.04 can be used to distinguish random walk and trapped dynamics for the neutral and positive probes using the relation between the number of distinct sites visited by the probe, S(t), which follows the scaling, S(t) ˜ tβ/ln (t). Our results established the occurrence of a hierarchy of diffusion dynamics experienced by a probe in a dense medium that is either charged or neutral.

  19. Sub-diffusion and trapped dynamics of neutral and charged probes in DNA-protein coacervates

    SciTech Connect

    Arfin, Najmul; Yadav, Avinash Chand; Bohidar, H. B.

    2013-11-15

    The physical mechanism leading to the formation of large intermolecular DNA-protein complexes has been studied. Our study aims to explain the occurrence of fast coacervation dynamics at the charge neutralization point, followed by the appearance of smaller complexes and slower coacervation dynamics as the complex experiences overcharging. Furthermore, the electrostatic potential and probe mobility was investigated to mimic the transport of DNA / DNA-protein complex in a DNA-protein complex coacervate medium [N. Arfin and H. B. Bohidar, J. Phys. Chem. B 116, 13192 (2012)] by assigning neutral, negative, or positive charge to the probe particle. The mobility of the neutral probe was maximal at low matrix concentrations and showed random walk behavior, while its mobility ceased at the jamming concentration of c = 0.6, showing sub-diffusion and trapped dynamics. The positively charged probe showed sub-diffusive random walk followed by trapped dynamics, while the negatively charged probe showed trapping with occasional hopping dynamics at much lower concentrations. Sub-diffusion of the probe was observed in all cases under consideration, where the electrostatic interaction was used exclusively as the dominant force involved in the dynamics. For neutral and positive probes, the mean square displacement 〈R{sup 2}〉 exhibits a scaling with time as 〈R{sup 2}〉 ∼ t{sup α}, distinguishing random walk and trapped dynamics at α = 0.64 ± 0.04 at c = 0.12 and c = 0.6, respectively. In addition, the same scaling factors with the exponent β = 0.64 ± 0.04 can be used to distinguish random walk and trapped dynamics for the neutral and positive probes using the relation between the number of distinct sites visited by the probe, S(t), which follows the scaling, S(t) ∼ t{sup β}/ln (t). Our results established the occurrence of a hierarchy of diffusion dynamics experienced by a probe in a dense medium that is either charged or neutral.

  20. Mapped DNA probes from Ioblolly pine can be used for restriction fragment length polymorphism mapping in other conifers

    Treesearch

    M.R. Ahuja; M.E. Devey; A.T. Groover; K.D. Jermstad; D.B Neale

    1994-01-01

    A high-density genetic map based on restriction fragment length polymorphisms (RFLPs) is being constructed for loblolly pine (Pinus taeda L.). Consequently, a large number of DNA probes from loblolly pine are potentially available for use in other species. We have used some of these DNA probes to detect RFLPs in 12 conifers and an angiosperm....

  1. 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.

  2. 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.

  3. Target-assembled ExciProbes: application to DNA detection at the level of PCR product and plasmid DNA.

    PubMed

    Walsh, Lindsey; Gbaj, Abdul; Savage, Hannah E; Bacigalupo, M Candelaria Rogert; Bichenkova, Elena V; Douglas, Kenneth T

    2007-12-01

    Recently, we introduced a novel exciplex-based approach for detection of nucleic acids using a model DNA-mounted exciplex system, consisting of two 8-mer ExciProbes hybridized to a complementary 16-mer DNA target. We now show, for the first time, that this approach can be used to detect DNA at the level of PCR product and plasmid, when the target sequence (5'-GCCAAACACAGAATCG-3') was embedded in long DNA molecules (PCR products and approximately 3 Kbp plasmid). A remarkably stringent demand is made of the solvent conditions for this exciplex emission to occur, viz., emission is optimal for DNA at 80% trifluoroethanol, even in the plasmid situations, raising the question of the molecular structural basis of this system. We show that a perfectly matched plasmid target can be differentiated from target containing single nucleotide substitutions; hence, ExciProbes could be applied to SNP analysis. The effect of counter cations (Na(+), K(+), and Mg(2+)) and PCR additives on exciplex emission has been also examined.

  4. DNA probe attachment on plastic surfaces and microfluidic hybridization array channel devices with sample oscillation.

    PubMed

    Liu, Yingjie; Rauch, Cory B

    2003-06-01

    DNA probe immobilization on plastic surfaces and device assembly are both critical to the fabrication of microfluidic hybridization array channel (MHAC) devices. Three oligonucleotide (oligo) probe immobilization procedures were investigated for attaching oligo probes on four different types of plastic surfaces (polystyrene, polycarbonate, poly(methylmethacrylate), and polypropylene). These procedures are the Surmodics procedure, the cetyltrimethylammonium bromide (CTAB) procedure, and the Reacti-Bind procedure. To determine the optimal plastic substrate and attachment chemistry for array fabrication, we investigated plastic hydrophobicity, intrinsic fluorescence, and oligo attachment efficiency. The Reacti-Bind procedure is least effective for attaching oligo probes in the microarray format. The CTAB procedure performs well enough to use in array fabrication, and the concentration of CTAB has a significant effect on oligo immobilization efficiency. We also found that use of amine-modified oligo probes resulted in better immobilization efficiency than use of unmodified oligos with the CTAB procedure. The oligo probe immobilization on plastic surfaces by the Surmodics procedure is the most effective with regard to probe spot quality and hybridization sensitivity. A DNA hybridization assay on such a device results in a limit of detection of 12pM. Utilizing a CO(2) IR laser machining and adhesive layer approach, we have developed an improved procedure for realizing a DNA microarray inside a microfluidic channel. This device fabrication procedure allows for more feasible spot placement in the channel and reduced sample adsorption by adhesive tapes used in the fabrication procedure. We also demonstrated improved hybridization kinetics and increased detection sensitivity in MHAC devices by implementing sample oscillation inside the channel. A limit of detection of 5pM has been achieved in MHAC devices with sample oscillation.

  5. 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

  6. 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

  7. Tetrahedral DNA probe coupling with hybridization chain reaction for competitive thrombin aptasensor.

    PubMed

    Chen, Ying-Xu; Huang, Ke-Jing; He, Liu-Liu; Wang, Yi-Han

    2017-09-15

    A novel competitive aptasensor for thrombin detection is developed by using a tetrahedral DNA (T-DNA) probe and hybridization chain reaction (HCR) signal amplification. Sulfur and nitrogen co-doped reduced graphene oxide (SN-rGO) is firstly prepared by a simple reflux method and used for supporting substrate of biosensor. Then, T-DNA probe is modified on the electrode by Au-S bond and a competition is happened between target thrombin and the complementary DNA (cDNA) of aptamer. The aptamer binding to thrombin forms an aptamer-target conjugate and make the cDNA remained, and subsequently hybridizes with the vertical domain of T-DNA. Finally, the cDNAs trigger HCR, which results in a great current response by the catalysis of horseradish peroxidase to the hydrogen peroxide + hydroquinone system. For thrombin detection, the proposed biosensor shows a wide linearity range of 10(-13)-10(-8)M and a low detection limit of 11.6fM (S/N = 3), which is hopeful to apply in biotechnology and clinical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. 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

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

    PubMed

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

    2016-08-14

    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.

  10. 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.

  11. A specific DNA probe for detecting Mycoplasma hyopneumoniae in experimentally infected piglets.

    PubMed

    Abiven, P; Blanchard, B; Saillard, C; Kobisch, M; Bove, J M

    1992-10-01

    Mycoplasma hyopneumoniae is the primary agent of swine enzootic pneumonia. Because of fastidious growth requirements and its serological cross-reactions with other porcine mycoplasmas, we developed a specific DNA probe for its detection. A partial genomic library of M. hyopneumoniae was constructed in plasmid pBR 322 using Hind III chromosomal fragments. The recombinant plasmids were screened by differential hybridization with M. flocculare and M. hyorhinis genomic DNA probes. One non-hybridizing recombinant plasmid was selected and its 1.65 kbp insert (designated I141) tested for specificity against genomic DNA from numerous mycoplasmas, other bacteria species and DNA from lung tissue of specific pathogen free (SPF) piglets. The 32P labelled I141 could detect specifically down to 400 pg of M. hyopneumoniae genomic DNA. To test the suitability of the I141 probe for the laboratory diagnosis of M. hyopneumoniae infections, we used clinical tracheobronchial specimens from piglets which were experimentally infected with M. hyopneumoniae. The results with hybridization on each specimen were compared to findings with an immunofluorescence test. Of the clinical specimen tested, there was agreement in the two tests of 63%.

  12. 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

  13. Molecular detection of bacterial pathogens using microparticle enhanced double-stranded DNA probes.

    PubMed

    Riahi, Reza; Mach, Kathleen E; Mohan, Ruchika; Liao, Joseph C; Wong, Pak Kin

    2011-08-15

    Rapid, specific, and sensitive detection of bacterial pathogens is essential toward clinical management of infectious diseases. Traditional approaches for pathogen detection, however, often require time-intensive bacterial culture and amplification procedures. Herein, a microparticle enhanced double-stranded DNA probe is demonstrated for rapid species-specific detection of bacterial 16S rRNA. In this molecular assay, the binding of the target sequence to the fluorophore conjugated probe thermodynamically displaces the quencher probe and allows the fluorophore to fluoresce. By incorporation of streptavidin-coated microparticles to localize the biotinylated probes, the sensitivity of the assay can be improved by 3 orders of magnitude. The limit of detection of the assay is as few as eight bacteria without target amplification and is highly specific against other common pathogens. Its applicability toward clinical diagnostics is demonstrated by directly identifying bacterial pathogens in urine samples from patients with urinary tract infections.

  14. 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.

  15. Probing the mechanics of the complete DNA transcription cycle in real-time using optical tweezers.

    PubMed

    Baumann, Christoph G; Cross, Stephen J

    2011-01-01

    RNA polymerase (RNAP) is a DNA-dependent motor protein that links ribonucleotide polymerization to force generation and DNA translocation through its active site, i.e., mechanical work. Single-molecule studies using optical tweezers have allowed researchers to probe the load-dependent ribonucleotide incorporation rate and processivity of both single-subunit viral and multisubunit prokaryotic and eukaryotic RNAPs engaged in transcription elongation. A single-molecule method is described here, which allows the complete transcription cycle (i.e., promoter binding, initiation, elongation and termination) to be followed in real-time using dual-trap optical tweezers and a unique "three-bead" geometry. This single-molecule transcription assay can be used to probe the mechanics of both stationary and moving RNAP-DNA complexes engaged in different stages of transcription.

  16. 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

  17. Demand for DNA probe testing in three genetic centres in Britain (August 1986 to July 1987).

    PubMed Central

    Rona, R J; Swan, A V; Beech, R; Prentice, L; Reynolds, A; Wilson, O; Mole, G; Vadera, P

    1989-01-01

    We report a preliminary analysis of the data collected during the first year of the evaluation of clinical genetics in the context of DNA probes in three genetic centres, to show the pattern of the demand for genetic services in the three centres and the services used in meeting that demand. The analysis includes information on 10,185 persons from 2852 families. The results are presented according to mode of inheritance and according to the most common disorders for which DNA probes have been used in the three centres. The results indicate that the use of DNA probes is now a major element of activity in genetic departments, and that as long as indirect DNA probe testing is the predominant manner of using recombinant technology, the clinical input will be an important element of the costs, probably more so than that of the DNA laboratories, as a large number of family members needs to be tested. In most cases centres have concentrated activity on DNA testing for common and severe genetic disorders. However, there are disorders, such as familial hypercholesterolaemia, which have not been part of the established pattern of services. Conversely, a relatively high number of families have been studied for some disorders of very low incidence. This suggests that the number of DNA laboratories should be limited. The precise arrangements will need to be established. With such services the distribution of DNA testing facilities for different disorders can be controlled to limit duplication. The model followed in Scotland based on collaboration between centres is worth considering. We have detected very large differences in take up rate of services within and between regions. Although many factors may contribute to these differences, ease of access and lay and professional awareness are probably the most important. This is supported by the fact that more patients from the same or neighbouring DHAs attend the genetic centre than from those further away. We also concluded that

  18. 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.

  19. 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.

  20. Real-time electrochemical detection of pathogen DNA using electrostatic interaction of a redox probe.

    PubMed

    Ahmed, Minhaz Uddin; Nahar, Sharifun; Safavieh, Mohammadali; Zourob, Mohammed

    2013-02-21

    Electrostatic redox probes interaction has been widely rendered for DNA quantification. We have established a proof-of-principle by using the ruthenium hexaamine molecule [Ru(NH(3))(6)](3+). We have applied this method for real-time electrochemical monitoring of a loop mediated isothermal amplification (LAMP) amplicon of target genes of Escherichia coli and Staphylococcus aureus by square wave voltammetry (SWV). Ruthenium hexaamine interaction with free DNAs in solution without being immobilized onto the biochip surface enabled us to discard the time-consuming overnight probe immobilization step in DNA quantification. We have measured the changes in the cathodic current signals using screen printed low-cost biochips both in the presence and the absence of LAMP amplicons of target DNAs in the solution-phase. By using this novel probe, we successfully carried out the real-time isothermal amplification and detection in less than 30 min for S. aureus and E. coli with a sensitivity up to 30 copies μL(-1) and 20 copies μL(-1), respectively. The cathode peak height of the current was related to the extent of amplicon formation and the amount of introduced template genomic DNA. Importantly, since laborious probe immobilization is not necessary at all, and both the in vitro amplification and real-time monitoring are performed in a single polypropylene tube using a single biochip, this novel approach could avoid all potential cross-contamination in the whole procedure.

  1. Novel DNA staining method and processing technique for the quantification of undamaged double-stranded DNA in epidermal tissue sections by PicoGreen probe staining and microspectrophotometry.

    PubMed

    Gagna, Claude E; Kuo, Hon-Reen; Chan, Norman J; Mitacek, Eugene J; Spivak, Alla; Pasquariello, Tiffany D; Balgobin, Chandrika; Mukhi, Ruhayna; Lambert, W Clark

    2007-10-01

    Histotechnological processing of DNA can cause damage to and loss of DNA and can change its structure. DNA probes have severe tissue-staining limitations. New DNA probes and improved histotechnology are needed to enhance the characterization of fixed tissue-bound DNA. Our team developed a novel DNA staining technique and histotechnological processing procedure that improves tissue-bound DNA retention and the qualification and quantification of intact double-stranded (ds)-B-DNA. We used the ultrasensitive PicoGreen ds-DNA probe for the histochemical characterization of ds-DNA. Fifteen fixatives were examined to determine which were best for preventing DNA denaturation and retaining original DNA content and structures. Our use of a microwave-vacuum oven reduced heating temperatures, shortened heating and processing times, and enhanced fixation. We achieved better qualitative and quantitative results by using superior tissue-acquisition techniques (e.g., reduced prefixation times) and improved histotechnology. We also compared our novel approach with archival tissues, delayed fixation, less sophisticated and conventional histological processing techniques, and by experimenting with preservation of tissue-bound ds-Z-DNA. Results demonstrate that our histotechnological procedure and nucleic acid staining method significantly improve the retention of intact, undamaged ds-DNA which, in turn, allows the investigator to more precisely quantify the content and structures of unaltered and undamaged tissue-bound ds-B-DNA.

  2. Probing the nature of hydrogen bonds in DNA base pairs.

    PubMed

    Mo, Yirong

    2006-07-01

    Energy decomposition analyses based on the block-localized wave-function (BLW-ED) method are conducted to explore the nature of the hydrogen bonds in DNA base pairs in terms of deformation, Heitler-London, polarization, electron-transfer and dispersion-energy terms, where the Heitler-London energy term is composed of electrostatic and Pauli-exchange interactions. A modest electron-transfer effect is found in the Watson-Crick adenine-thymine (AT), guanine-cytosine (GC) and Hoogsteen adenine-thymine (H-AT) pairs, confirming the weak covalence in the hydrogen bonds. The electrostatic attraction and polarization effects account for most of the binding energies, particularly in the GC pair. Both theoretical and experimental data show that the GC pair has a binding energy (-25.4 kcal mol(-1) at the MP2/6-31G** level) twice that of the AT (-12.4 kcal mol(-1)) and H-AT (-12.8 kcal mol(-1)) pairs, compared with three conventional N-H...O(N) hydrogen bonds in the GC pair and two in the AT or H-AT pair. Although the remarkably strong binding between the guanine and cytosine bases benefits from the opposite orientations of the dipole moments in these two bases assisted by the pi-electron delocalization from the amine groups to the carbonyl groups, model calculations demonstrate that pi-resonance has very limited influence on the covalence of the hydrogen bonds. Thus, the often adopted terminology "resonance-assisted hydrogen bonding (RHAB)" may be replaced with "resonance-assisted binding" which highlights the electrostatic rather than electron-transfer nature of the enhanced stabilization, as hydrogen bonds are usually regarded as weak covalent bonds.

  3. 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.

  4. Cloning and Characterization of a Complex DNA Fingerprinting Probe for Candida parapsilosis

    PubMed Central

    Enger, Lee; Joly, Sophie; Pujol, Claude; Simonson, Patricia; Pfaller, Michael; Soll, David R.

    2001-01-01

    Candida parapsilosis accounts for a significant number of nosocomial fungemias, but in fact, no effective and verified genetic fingerprinting method has emerged for assessing the relatedness of independent isolates for epidemiological studies. A complex 15-kb DNA fingerprinting probe, Cp3-13, was therefore isolated from a library of C. parapsilosis genomic DNA fragments. The efficacy of Cp3-13 for DNA fingerprinting was verified by a comparison of its clustering capacity with those of randomly amplified polymorphic DNA analysis and internally transcribed spacer region sequencing, by testing species specificity, and by assessing its capacity to identify microevolutionary changes both in vitro and in vivo. Southern blot hybridization of EcoRI/SalI-digested DNA with Cp3-13 provides a fingerprinting system that (i) identifies the same strain in independent isolates, (ii) discriminates between unrelated isolates, (iii) separates independent isolates into valid groups in a dendrogram, (iv) identifies microevolution in infecting populations, and (v) is amenable to automatic computer-assisted DNA fingerprint analysis. This probe is now available for epidemiological studies. PMID:11158125

  5. [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.

  6. 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.

  7. 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

  8. 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.

  9. Modulating molecular level space proximity: a simple and efficient strategy to design structured DNA probes.

    PubMed

    Zheng, Jing; Li, Jishan; Gao, Xiaoxia; Jin, Jianyu; Wang, Kemin; Tan, Weihong; Yang, Ronghua

    2010-05-01

    To construct efficient oligonucleotide probes, specific nucleic acid is designed as a conformationally constrained form based on the formation of a Watson-Crick-based duplex. However, instability of Watson-Crick hydrogen bonds in a complex biological environment usually leads to high background signal from the probe itself and false positive signal caused by nonspecific binding. To solve this problem, we propose a way to restrict the labeled-dyes in a hydrophobic cavity of cyclodextrin. This bounding, which acts like extra base pairs to form the Watson-Crick duplex, achieves variation of level of space proximity of the two labels and thus the degree of conformational constraint. To demonstrate the feasibility of the design, a stem-containing oligonucleotide probe (P1) for DNA hybridization assay and a stemless one (P2) for protein detection were examined as models. Both oligonucleotides were doubly labeled with pyrene at the 5'- and 3'- ends, respectively. It is the cyclodextrin/pyrene inclusion interaction that allows modulating the degree of conformational constraints of P1 and P2 and thus their background signals and selectivity. Under the optimal conditions, the ratio of signal-to-background of P1/gamma-CD induced by 1.0 equiv target DNA is near 174, which is 4-fold higher than that in the absence of gamma-CD. In addition, the usage of gamma-CD shifts the melting temperature of P1 from 57 to 68 degrees C, which is reasonable for improving target-binding selectivity. This approach is simple in design, avoiding any variation of the stem's length and sequences. Furthermore, the strategy is generalizable which is suited for not only the stem-containing probe but also the linear probe with comparable sensitivity and selectivity to conventional structured DNA probes.

  10. Probing DNA-DNA Interactions with a Combination of Quadruple-Trap Optical Tweezers and Microfluidics.

    PubMed

    Brouwer, Ineke; King, Graeme A; Heller, Iddo; Biebricher, Andreas S; Peterman, Erwin J G; Wuite, Gijs J L

    2017-01-01

    DNA metabolism and DNA compaction in vivo involve frequent interactions of remote DNA segments, mediated by proteins. In order to gain insight into such interactions, quadruple-trap optical tweezers have been developed. This technique provides an unprecedented degree of control through the ability to independently manipulate two DNA molecules in three dimensions. In this way, discrete regions of different DNA molecules can be brought into contact with one another, with a well-defined spatial configuration. At the same time, the tension and extension of the DNA molecules can be monitored. Furthermore, combining quadruple-trap optical tweezers with microfluidics makes fast buffer exchange possible, which is important for in situ generation of the dual DNA-protein constructs needed for these kinds of experiments. In this way, processes such as protein-mediated inter-DNA bridging can be studied with unprecedented control. This chapter provides a step-by-step description of how to perform a dual DNA manipulation experiment using combined quadruple-trap optical tweezers and microfluidics.

  11. Using Triplex-Forming Oligonucleotide Probes for the Reagentless, Electrochemical Detection of Double-Stranded DNA

    PubMed Central

    Patterson, Adriana; Caprio, Felice; Vallée-Bélisle, Alexis; Moscone, Danila; Plaxco, Kevin W.; Palleschi, Giuseppe; Ricci, Francesco

    2011-01-01

    We report a reagentless, electrochemical sensor for the detection of double-stranded DNA targets that employs triplex-forming oligonucleotides (TFOs) as its recognition element. These sensors are based on redox-tagged TFO probes strongly chemisorbed onto an interrogating gold electrode. Upon the addition of the relevant double-stranded DNA target, the probe forms a rigid triplex structure via reverse Hoogsteen base pairing in the major groove. The formation of the triplex impedes contact between the probe’s redox moiety and the interrogating electrode, thus signaling the presence of the target. We first demonstrated the proof of principle of this approach by using a well-characterized 22-base polypurine TFO sequence that readily detects a synthetic, double-stranded DNA target. We then confirmed the generalizability of our platform with a second probe, a 19-base polypyrimidine TFO sequence that targets a polypurine tract (PPT) sequence conserved in all HIV-1 strains. Both sensors rapidly and specifically detect their double-stranded DNA targets at concentrations as low as ~10 nM and are selective enough to be employed directly in complex sample matrices such as blood serum. Moreover, to demonstrate real-world applicability of this new sensor platform, we have successfully detected unpurified, double-stranded PCR amplicons containing the relevant conserved HIV-1 sequence. PMID:20936782

  12. Mapped DNA probes from loblolly pine can be used for restriction fragment length polymorphism mapping in other conifers.

    PubMed

    Ahuja, M R; Devey, M E; Groover, A T; Jermstad, K D; Neale, D B

    1994-06-01

    A high-density genetic map based on restriction fragment length polymorphisms (RFLPs) is being constructed for loblolly pine (Pinus taeda L.). Consequently, a large number of DNA probes from loblolly pine are potentially available for use in other species. We have used some of these DNA probes to detect RFLPs in 12 conifers and an angiosperm. Thirty complementary DNA and two genomic DNA probes from loblolly pine were hybridized to Southern blots containing DNA from five species of Pinus (P. elliottii, P. lambertiana, P. radiata, P. sylvestris, and P. taeda), one species from each of four other genera of Pinaceae (Abies concolor, Larix laricina, Picea abies, and Pseudotsuga menziesii), one species from each of three other families of Coniferales [Sequoia sempervirens (Taxodiaceae), Torreya californica (Taxaceae) and Calocedrus decurrens (Cupressaceae)], and to one angiosperm species (Populus nigra). Results showed that mapped DNA probes from lobolly pine will cross-hybridize to genomic DNA of other species of Pinus and some other genera of the Pinaceae. Only a small proportion of the probes hybridized to genomic DNA from three other families of the Coniferales and the one angiosperm examined. This study demonstrates that mapped DNA probes from loblolly pine can be used to construct RFLP maps for related species, thus enabling the opportunity for comparative genome mapping in conifers.

  13. 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

  14. 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.

  15. 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.

  16. DNA detection using water-soluble conjugated polymers and peptide nucleic acid probes

    PubMed Central

    Gaylord, Brent S.; Heeger, Alan J.; Bazan, Guillermo C.

    2002-01-01

    The light-harvesting properties of cationic conjugated polymers are used to sensitize the emission of a dye on a specific peptide nucleic acid (PNA) sequence for the purpose of homogeneous, “real-time” DNA detection. Signal transduction is controlled by hybridization of the neutral PNA probe and the negative DNA target. Electrostatic interactions bring the hybrid complex and cationic polymer within distances required for Förster energy transfer. Conjugated polymer excitation provides fluorescein emission >25 times higher than that obtained by exciting the dye, allowing detection of target DNA at concentrations of 10 pM with a standard fluorometer. A simple and highly sensitive assay with optical amplification that uses the improved hybridization behavior of PNA/DNA complexes is thus demonstrated. PMID:12167673

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

    PubMed

    Miotke, Laura; Maity, Arindam; Ji, Hanlee; Brewer, Jonathan; Astakhova, Kira

    2015-01-01

    Rapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence microscopy and nucleic acid analogues have been proposed so far. Here we report a novel enzyme-free approach to efficiently detect cancer mutations. This assay includes gene-specific target enrichment followed by annealing to oligonucleotides containing locked nucleic acids (LNAs) and finally, detection by fluorescence microscopy. The LNA containing probes display high binding affinity and specificity to DNA containing mutations, which allows for the detection of mutation abundance with an intercalating EvaGreen dye. We used a second probe, which increases the overall number of base pairs in order to produce a higher fluorescence signal by incorporating more dye molecules. Indeed we show here that using EvaGreen dye and LNA probes, genomic DNA containing BRAF V600E mutation could be detected by fluorescence microscopy at low femtomolar concentrations. Notably, this was at least 1000-fold above the potential detection limit. Overall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay, which allows for an accurate estimation of mutant abundance when the detection limit requirement is met. Using fluorescence microscopy, this approach presents the opportunity to detect DNA at single-molecule resolution and directly

  18. Investigation of paternity establishing without the putative father using hypervariable DNA probes.

    PubMed

    Yokoi, T; Odaira, T; Nata, M; Sagisaka, K

    1990-09-01

    Seven kinds of DNA probes which recognize hypervariable loci were applied for paternity test. The putative father was decreased and unavailable for the test. The two legitimate children and their mother (the deceased's wife) and the four illegitimate children and their mother (the deceased's kept mistress) were available for analysis. Paternity index of four illegitimate child was investigated. Allelic frequencies and their confidence intervals among unrelated Japanese individuals were previously reported from our laboratory, and co-dominant segregation of the polymorphism was confirmed in family studies. Cumulative paternity indices of four illegitimate children from 16 kinds of standard blood group markers were 165, 42, 0.09, and 36, respectively. On the other hand, cumulative paternity indices from 7 kinds of DNA probes are 2,363, 4,685, 57,678, and 54,994, respectively, which are 14, 113, 640, 864, and 1,509 times higher than that from standard blood group markers. The DNA analyses gave nearly conclusive evidence that the putative father was the biological father of the children. Especially, the paternity relation of the third illegitimate child could not be established without the DNA analyses. Accordingly, DNA polymorphism is considered to be informative enough for paternity test.

  19. 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.

  20. 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.

  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. 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.

  3. 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.

  4. Detection of bovine trichomoniasis with a specific DNA probe and PCR amplification system.

    PubMed

    Ho, M S; Conrad, P A; Conrad, P J; LeFebvre, R B; Perez, E; BonDurant, R H

    1994-01-01

    Trichomoniasis is a widespread, economically important venereal disease of cattle which causes infertility and abortion. Effective control of trichomoniasis has been impeded by the insensitivity of traditional diagnostic procedures, which require the isolation and cultivation of the parasite, Tritrichomonas foetus, from infected cattle. We developed a 0.85-kb T. foetus DNA probe by identifying conserved sequences in DNAs from T. foetus that were isolated from cattle in California, Idaho, Nevada, and Costa Rica. The probe hybridized specifically to DNAs of T. foetus isolates from different geographic areas but not to DNA preparations of Trichomonas vaginalis, bovine cells, or a variety of bacteria from cattle. The probe detected DNA from a minimum of 10(5) T. foetus organisms. To improve sensitivity, a partial sequence of the probe was used to identify oligonucleotide primers (TF1 and TF2) which could be used to amplify a 162-bp product from T. foetus DNAs by PCR. A chemiluminescent internal T. foetus sequence probe was hybridized to Southern blots of the amplification product. This system detected as few as one T. foetus organism in culture media or 10 parasites in samples containing bovine preputial smegma. Analysis of 52 clinical samples showed that 47 (90.4%) of the 52 samples were correctly identified, with no false-positive reactions. In comparison, the traditional cultivation method detected 44 (84.6%) of the 52 samples from T. foetus-infected and uninfected bulls. These results indicate that the PCR-based amplification system could be a useful alternative method for the diagnosis of bovine trichomoniasis.

  5. 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.

  6. 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.

  7. DNA quantification via ICP-MS using lanthanide-labeled probes and ligation-mediated amplification.

    PubMed

    Brückner, Kathrin; Schwarz, Kathleen; Beck, Sebastian; Linscheid, Michael W

    2014-01-07

    The combination of lanthanide-tagged oligonucleotide probes with inductively coupled plasma mass spectrometry (ICP-MS) as the detection technique is a novel labeling and analysis strategy for heterogeneous nucleic acid quantification assays. We describe a hybridization assay based on biotin-streptavidin affinity using lanthanide-labeled reporter probes and biotinylated capture probes. For the basic sandwich type assay, performed in streptavidin-coated microtitration wells, the limit of detection (LOD) was 7.2 fmol of DNA target, corresponding to a final concentration of 6 pM terbium-labeled probes detectable by ICP-MS after elution from the solid support. To improve the sensitivity and sequence specificity of the approach, it was combined with established molecular biological techniques, i.e., elution with a restriction endonuclease and signal and target amplification by the ligase detection reaction (LDR) and ligase chain reaction (LCR), respectively. Initial experiments showed that the enzymes facilitated the discrimination of single-base mismatches within the recognition or ligation site. Furthermore, LCR as a target amplification step resulted in a 6000-fold increase of sensitivity, and finally an LOD of 2.6 amol was achieved with an artificial double-stranded DNA target.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. 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

  14. 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.

  15. [Identification of chromosomal aberration in esophageal cancer cells by mixed BAC DNA probes of chromosome arms and regions].

    PubMed

    Jiajie, Hao; Chunli, Wang; Wenyue, Gu; Xiaoyu, Cheng; Yu, Zhang; Xin, Xu; Yan, Cai; Mingrong, Wang

    2014-06-01

    Chromosomal aberration is an important genetic feature of malignant tumor cells. This study aimed to clarify whether BAC DNA could be used to identify chromosome region and arm alterations. For each chromosome region, five to ten 1 Mb BAC DNA clones were selected to construct mixed BAC DNA clones for the particular region. All of the mixed clones from regions which could cover the whole chromosome arm were then mixed to construct mixed BAC DNA clones for the arms. Mixed BAC DNA probes of arms and regions were labeled by degenerate oligonucleotide primed PCR (DOP-PCR) and Nick translation techniques, respectively. The specificities of these probes were validated by fluorescence in situ hybridization (FISH) on the metaphase chromosomes of normal human peripheral blood lymphocytes. FISH with arm-specific mixed BAC DNA probes showed that chromosomal rearrangements and involved chromosome arms were confirmed in several esophageal cancer cells. By using region-specific mixed probes, the breakpoint on 1q from the derivative chromosome t(1q;7q) was identified in 1q32-q41 in esophageal KYSE140 cells. In conclusion, we established an effective labeling method for 1 Mb BAC DNA mixed clone probes, and chromosome arm and region rearrangements could be identified in several esophageal cancer cells by using these probes. Our study provides a more precise method for identification of chromosomal aberration by M-FISH, and the established method may also be applied to the karyotype analysis of hematological malignancies and prenatal diagnosis.

  16. 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

  17. Modeling Hybridization Kinetics of Gene Probes in a DNA Biochip Using FEMLAB.

    PubMed

    Munir, Ahsan; Waseem, Hassan; Williams, Maggie R; Stedtfeld, Robert D; Gulari, Erdogan; Tiedje, James M; Hashsham, Syed A

    2017-05-29

    Microfluidic DNA biochips capable of detecting specific DNA sequences are useful in medical diagnostics, drug discovery, food safety monitoring and agriculture. They are used as miniaturized platforms for analysis of nucleic acids-based biomarkers. Binding kinetics between immobilized single stranded DNA on the surface and its complementary strand present in the sample are of interest. To achieve optimal sensitivity with minimum sample size and rapid hybridization, ability to predict the kinetics of hybridization based on the thermodynamic characteristics of the probe is crucial. In this study, a computer aided numerical model for the design and optimization of a flow-through biochip was developed using a finite element technique packaged software tool (FEMLAB; package included in COMSOL Multiphysics) to simulate the transport of DNA through a microfluidic chamber to the reaction surface. The model accounts for fluid flow, convection and diffusion in the channel and on the reaction surface. Concentration, association rate constant, dissociation rate constant, recirculation flow rate, and temperature were key parameters affecting the rate of hybridization. The model predicted the kinetic profile and signal intensities of eighteen 20-mer probes targeting vancomycin resistance genes (VRGs). Predicted signal intensities and hybridization kinetics strongly correlated with experimental data in the biochip (R² = 0.8131).

  18. Real-time electrochemical PCR with a DNA intercalating redox probe.

    PubMed

    Deféver, Thibaut; Druet, Michel; Evrard, David; Marchal, Damien; Limoges, Benoit

    2011-03-01

    The proof-of-principle of a nonoptical real-time PCR method based on the electrochemical monitoring of a DNA intercalating redox probe that becomes considerably less easily electrochemically detectable once intercalated to the amplified double-stranded DNA is demonstrated. This has been made possible thanks to the finding of a redox intercalator that (i) strongly and specifically binds to the amplified double-stranded DNA, (ii) does not significantly inhibit PCR, (iii) is chemically stable under PCR cycling, and (iv) is sensitively detected by square wave voltammetry during PCR cycling. Among the different DNA intercalating redox probes that we have investigated, namely, methylene blue, Os[(bpy)(2)phen](2+), Os[(bpy)(2)DPPZ](2+), Os[(4,4'-dimethyl-bpy)(2)DPPZ](2+) and Os[(4,4'-diamino-bpy)(2)DPPZ](2+) (with bpy = 2,2'-bipyridine, phen = phenanthroline, and DPPZ = dipyrido[3,2-a:2',3'-c]phenazine), the one and only compound with which it has been possible to demonstrate the proof-of-concept is the Os[(bpy)(2)DPPZ](2+). In terms of analytical performances, the methodology described here compares well with optical-based real-time PCRs, offering finally the same advantages than the popular and routinely used SYBR Green-based real-time fluorescent PCR, but with the additional incomes of being potentially much cheaper and easier to integrate in a hand-held miniaturized device.

  19. 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.

  20. 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.

  1. Monitoring triplex DNA formation with fluorescence resonance energy transfer between a fluorophore-labeled probe and intercalating dyes.

    PubMed

    Chiou, Chiuan-Chian; Chen, Shiau-Wen; Luo, Ji-Dung; Chien, Yu-Tzu

    2011-09-01

    Triplex-forming oligonucleotides (TFOs) are sequence-dependent DNA binders that may be useful for DNA targeting and detection. A sensitive and convenient method to monitor triplex formation by a TFO and its target DNA duplex is required for the application of TFO probes. Here we describe a novel design by which triplex formation can be monitored homogeneously without prelabeling the target duplex. The design uses a TFO probe tagged with a fluorophore that undergoes fluorescence resonance energy transfer with fluorescent dyes that intercalate into the target duplex. Through color compensation analysis, the specific emission of the TFO probe reveals the status of the triple helices. We used this method to show that triple helix formation with TFOs is magnesium dependent. We also demonstrated that the TFO probe can be used for detection of sequence variation in melting analysis and for DNA quantitation in real-time polymerase chain reaction. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Detection of adenovirus in nasopharyngeal specimens by radioactive and nonradioactive DNA probes

    SciTech Connect

    Hyypiae, T.

    1985-05-01

    The presence of adenovirus DNA in clinical specimens was analyzed by nucleic acid hybridization assays by both radioactive and enzymatic detection systems. The sensitivity of the hybridization tests was in the range of 10 to 100 pg of homologous adenovirus DNA. Minimal background was noticed with unrelated viral and nonviral DNA. Twenty-four nasopharyngeal mucus aspirate specimens, collected from children with acute respiratory infection, were assayed in the hybridization tests and also by an enzyme immunoassay for adenovirus hexon antigen which was used as a reference test. Sixteen specimens positive by the enzyme immunoassay also were positive in the two nucleic acid hybridization tests, and the remaining eight specimens were negative in all of the tests. The results indicate that nucleid acid hybridization tests with both radioactive and nonradioactive probes can be used for diagnosis of microbial infections.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. DNA binding studies of hematoxylin-Dy(ш) complex by spectrometry using acridine orange as a probe.

    PubMed

    Xiong, Xiaoli; Huang, Jianhang; Wang, Xingming

    2014-01-01

    The interaction of a hematoxylin(HE)-Dy(Ш) complex with herring sperm DNA(hsDNA) was studied using acridine orange(AO) as a probe by UV-vis absorption, circular dichroism(CD), fluorescence spectroscopy and viscosity measurements. From the results of the probe experiment, we found that the HE-Dy(Ш) complex could compete with AO for intercalating into hsDNA. The binding constants of the HE-Dy(Ш) complex to hsDNA was obtained by the double reciprocal method and indicated that the affinity between hsDNA and the complex is weaker than that between hsDNA and classical intercalators. The thermodynamic parameters(ΔH°, ΔG°, ΔS°) were calculated from the UV-vis absorption data measured at two different temperatures. Further experimental results suggested that there exist groove binding and partial intercalation binding between hsDNA and HE-Dy(Ш) complex.

  14. 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

  15. Development and Characterization of Complex DNA Fingerprinting Probes for the Infectious Yeast Candida dubliniensis

    PubMed Central

    Joly, Sophie; Pujol, Claude; Rysz, Michal; Vargas, Kaaren; Soll, David R.

    1999-01-01

    Using a strategy to clone large genomic sequences containing repetitive elements from the infectious yeast Candida dubliniensis, the three unrelated sequences Cd1, Cd24, and Cd25, with respective molecular sizes of 15,500, 10,000, and 16,000 bp, were cloned and analyzed for their efficacy as DNA fingerprinting probes. Each generated a complex Southern blot hybridization pattern with endonuclease-digested genomic DNA. Cd1 generated an extremely variable pattern that contained all of the bands of the pattern generated by the repeat element RPS of Candida albicans. We demonstrated that Cd1 does not contain RPS but does contain a repeat element associated with RPS throughout the C. dubliniensis genome. The Cd1 pattern was the least stable over time both in vitro and in vivo and for that reason proved most effective in assessing microevolution. Cd24, which did not exhibit microevolution in vitro, was highly variable in vivo, suggesting in vivo-dependent microevolution. Cd25 was deemed the best probe for broad epidemiological studies, since it was the most stable over time, was the only truly C. dubliniensis-specific probe of the three, generated the most complex pattern, was distributed throughout all C. dubliniensis chromosomes, and separated a worldwide collection of 57 C. dubliniensis isolates into two distinct groups. The presence of a species-specific repetitive element in Cd25 adds weight to the already substantial evidence that C. dubliniensis represents a bona fide species. PMID:10074523

  16. Rapid detection of chromosome 18 copy number in buccal smears using DNA probes and FISH

    SciTech Connect

    Harris, C.; Nunez, M.; Giraldez, R.

    1994-09-01

    Rapid diagnosis of trisomy 18 in newborns is often critical to clinical management decisions that must be made in a minimum of time. DNA probes combined with FISH can be used to accurately to determine the copy number of chromosome 18 in interphase cells. We have used the D18Z1 alpha satellite DNA probe to determine signal frequency in normal, previously karyotyped subjects, 12 females and 6 males. We also present one clinical case of trisomy 18, confirmed by karyotype, for comparison to the results obtained from normal subjects. Buccal smears, unlike cytogenetic preparations from peripheral blood, are quite resistant to penetration of probes and detection reagents resulting in higher levels of false monosomy. We have studied 19 individuals and have obtained consistent FISH results, ranging from 64 to 90% disomy. False monosomy rates ranged from 10 to 36%, while false trisomy or tetrasomy was less than 1% in all samples. High rates of false monosomy make this test questionable for detection of low order mosaicism for monosomy, but the extremely low false hyperploidy rate suggests that this is a dependable procedure for detection of trisomy 18, enabling the use of buccal epithelium which can be collected easily from even premature and tiny infants.

  17. 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.

  18. 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.

  19. B- to Z-DNA transition probed by oligonucleotides containing methylphosphonates.

    PubMed Central

    Callahan, L; Han, F S; Watt, W; Duchamp, D; Kézdy, F J; Agarwal, K

    1986-01-01

    The simulation of the B--Z-DNA transition by using space-filling models of the dimer d(C-G) shows the possibility of hydrogen-bond formation between the N-2 amino group of the partially rotated guanine and one of the 5'-phosphate oxygens of deoxyguanylic acid. To probe the importance of this postulated interaction, analogs of the hexamer d(C-G)3 were synthesized. These analogs contained a methylphosphonate linkage, of distinct stereochemistry, which replaced the first 5'-phosphate linkage of deoxyguanosine. The CD spectra in high salt concentration showed that the hexamer containing a methylphosphonate linkage with the RP stereochemistry formed Z-DNA to the same extent as d(C-G)3, whereas the hexamer containing a methylphosphonate linkage with the SP stereochemistry did not form Z-DNA. These results are consistent with a mechanism in which an interaction between the N-2 amino group of guanine and the prochiral SP oxygen of deoxyguanosine 5'-phosphate kinetically controls the formation of Z-DNA. A water bridge between the N-2 amino group of guanine and the 3'-phosphate oxygen of deoxyguanylic acid has been implicated in the stabilization of Z-DNA. To probe the importance of this water bridge, two additional analogs of the hexamer d(C-G)3 were synthesized. These analogs contained a methylphosphonate linkage, of distinct stereochemistry, that replaced the first deoxyguanosine 3'-phosphate. The CD spectra showed that the hexamer containing a methylphosphonate linkage of the RP stereochemistry underwent the transition to Z-DNA to the same extent as d(C-G)3, whereas the hexamer containing a methylphosphonate linkage of the SP stereochemistry underwent the transition to Z-DNA to a 35% lesser extent. Thus the water bridge involving the prochiral SP oxygen provides modest stabilization energy for Z-DNA. These studies, therefore, suggest that the B--Z-DNA transition is regulated both thermodynamically and kinetically through hydrogen-bond interactions involving phosphate

  20. 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.

  1. 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.

  2. 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.

  3. [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.

  4. Base sequence- and T m-dependent DNA oligomer separation by open tubular capillary columns carrying complementary DNA oligomers as probes.

    PubMed

    Devarayapalli, Kamakshaiah Charyulu; Pack, Seung Pil; Kamisetty, Nagendra Kumar; Nonogawa, Mitsuru; Watanabe, Seiya; Kodaki, Tsutomu; Makino, Keisuke

    2007-06-01

    DNA chips prepared on a flat glass surface have unavoidable drawbacks when used for quantitative analysis. In an attempt to overcome this problem, we constructed an HPLC-type system suitable for quantitative analysis that enables base sequence- and T (m)-dependent DNA oligomer separation in a flow system. A small open tubular capillary column (300-mm x 100-microm I.D.) was used. The DNA oligomers used as probes had an amino group at the 5'-end and were immobilized on the inner silica surface of the capillary column which had been sequentially treated with 3-aminopropyltriethoxysilane, butyltrimethoxysilane, and disuccinimidylglutarate. Using the combination of probe-immobilized column placed in a column oven equipped with temperature gradient function, a nano-flow-controllable pump, a small sample-loading injector, and a capillary-fitted UV detector, we succeeded in separating complementary and non-complementary DNA oligomers in specific and quantitative modes. We also designed a temperature gradient strategy for efficient separation of target DNA oligomers in DNA mixture samples. Using a column carrying two different probes with similar T (m) values, their complementary target DNA oligomers were also separated and detected. The developed DNA open tubular capillary column system investigated in the present study could be further improved as an alternative tool to DNA chips to be used for the quantitative analysis of DNA or mRNA samples.

  5. 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.

  6. 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.

  7. [Principle and application of DNA-based stable isotope probing---a review].

    PubMed

    Jia, Zhongjun

    2011-12-01

    Microbial communities are the engines that drive the global biogeochemical cycle of carbon and nitrogen essential for life on Earth. However, microorganisms have evolved as a result of complex interactions with other organisms and environments. Deciphering the metabolism of microorganisms at the community level in nature will be crucial for a better understanding of the mechanisms that lead to the enormous divergence of microbial ecophysiology. Due to the immense number of uncultivated microbial species and the complexity of microbial communities, delineating community metabolism proves a virtually insurmountable hurdle. By tracing the heavy isotope flow of key elements such as carbon and nitrogen, DNA-based stable isotope probing (DNA-SIP) can provide unequivocal evidence for substrate assimilation by microorganisms in complex environments. The essential prerequisite for a successful DNA-SIP is the identification, with confidence, of isotopically enriched 13C-DNA, of which the amount is generally too low to allow the direct measurement of 13C atom percent of nucleic acid. The methodological considerations for obtaining unambiguous DNA highly enriched in heavy isotope are presented with emphasis on next-generation sequencing technology and metagenomics.

  8. 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.

  9. A method for generating selective DNA probes for the analysis of C-negative regions in human chromosomes.

    PubMed

    Morozkin, E S; Loseva, E M; Karamysheva, T V; Babenko, V N; Laktionov, P P; Vlassov, V V; Rubtsov, N B

    2011-01-01

    Linker-adapter polymerase chain reaction (LA-PCR) is among the most efficient techniques for whole genome DNA amplification. The key stage in LA-PCR is the hydrolysis of a DNA sample with restriction endonucleases, and the choice of a restriction endonuclease (or several endonucleases) determines the composition of DNA probes generated in LA-PCR. Computer analysis of the localization of the restriction sites in human genome has allowed us to propose an efficient technique for generating DNA probes by LA-PCR using the restriction endonucleases HaeIII and RsaI. In silico hydrolysis of human genomic DNA with endonucleases HaeIII and RsaI demonstrate that 100- to 1,000-bp DNA fragments are more abundant in the gene-rich regions. Applying in situ hybridization to metaphase chromosomes, we demonstrated that the produced DNA probes predominantly hybridized to the C-negative chromosomal regions, whereas the FISH signal was almost absent in the C-positive regions. The described protocol for generating DNA probes may be successfully used in subsequent cytogenetic analysis of the C-negative chromosomal regions. Copyright © 2011 S. Karger AG, Basel.

  10. Hemoglobin Pasadena: identification of the gene mutant by DNA analysis using synthetic DNA probes.

    PubMed

    Rahbar, S; Rosen, R; Nozari, G; Lee, T D; Asmerom, Y; Wallace, R B

    1988-03-01

    Hemoglobin Pasadena [beta 75(E19)Leu----Arg] was found in a boy who had an acute episode of anemia and rapid splenic enlargement. His father was the only other member of a large family with this hemoglobinopathy. We have used gene mapping techniques for direct identification of the beta-globin gene mutation. To correlate the DNA findings with the structural identification of this variant, we have also performed globin chain separation and analysis of the tryptic peptides using high performance liquid chromatography and secondary ion mass spectral analysis.

  11. Investigation into the effect that probe immobilisation method type has on the analytical signal of an EIS DNA biosensor.

    PubMed

    Lillis, Brian; Manning, Mary; Hurley, Eileen; Berney, Helen; Duane, Russell; Mathewson, Alan; Sheehan, Michelle M

    2007-02-15

    The analytical performance of an enhanced surface area electrolyte insulator semiconductor (EIS) device was investigated for DNA sensor development. The work endeavored to advance EIS performance by monitoring the effect of DNA probe layers have on the impedimetric signal during target hybridisation detection. Two universally employed covalent chemistries, direct and spacer-mediated attachment of amino modified probe molecules to amino-functionalised surfaces were investigated. Relative areal densities of immobilised probe were measured on planar and enhanced surface area substrates using epi-fluorescence microscopy. The reproducibility of the each immobilisation method was seen to have a direct effect on the reproducibility of the impedimetric signal. The sensitivity and selectivity was seen to be dependent on the type of immobilisation method. Real time, impedimetric detection of target DNA hybridisation concentrations as low as 25 and 1 nM were possible. The impact that probe concentration had on the impedimetric signal for selective and non-selective interactions was also investigated.

  12. Multiple components in restriction enzyme digests of mammalian (insectivore), avian and reptilian genomic DNA hybridize with murine immunoglobulin VH probes.

    PubMed

    Litman, G W; Berger, L; Jahn, C L

    1982-06-11

    High molecular weight genomic DNAs isolated from an insectivore, Tupaia, and a representative reptilian, Caiman, and avian, Gallus, were digested with restriction endonucleases transferred to nitrocellulose and hybridized with nick-translated probes of murine VH genes. The derivations of the probes designated S107V (1) and mu 107V (2,3) have been described previously. Under conditions of reduced stringency, multiple hybridizing components were observed with Tupaia and Caiman; only mu mu 107V exhibited significant hybridization with the separated fragments of Gallus DNA. The nick-translated S107V probe was digested with Fnu4H1 and subinserts corresponding to the 5' and 3' regions both detected multiple hybridizing components in Tupaia and Caiman DNA. A 5' probe lacking the leader sequence identified the same components as the intact 5' probe, suggesting that VH coding regions distant as the reptilians may possess multiple genetic components which exhibit significant homology with murine immunoglobulin in VH regions.

  13. 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.

  14. The attachment and characterization of DNA probes on gallium arsenide-based semiconductor surfaces

    NASA Astrophysics Data System (ADS)

    Yang, Joonhyuk

    2007-12-01

    Immobilization of nucleic acid molecules on solid surfaces is the core of numerous important technologies in the genomics, disease diagnostics and biosensors applications. The architecture and density of immobilized probe molecules depend on the type of the solid surface on which they are anchored. Even though many different types of surfaces have been studied as substrates for deoxyribonucleic acid (DNA) attachment, the development of a new type of substrate, which is reproducible, stable, highly controlled and easily transferred to practical applications, is still needed. Recent studies have shown that As terminated GaAs-based semiconductors can be used as substrates for immobilized DNA layers. In this study, I aim to understand the attachment of nucleic acid onto the surfaces of As-terminated GaAs-based semiconductors and focus on improving the "brush-structure", which is essential for high quality of biochip based on a DNA layer. Attachment of 8-base and 100-base thiolated ssDNA layers on arsenic terminated GaAs(001) was achieved and characterized. The covalent bonds between the thiolated oligonucleotides with As atoms on the GaAs surface were investigated using x-ray photoelectron spectroscopy (XPS), and the surface morphology was obtained using atomic force microscopy (AFM) and field emission scanning electron microscopy (FESEM). In addition, I studied the effect of DNA length and the presence of a good solvent, such as water, on the oligonucleotides on a GaAs surface. I also investigated the effects of the thiol-based spacer and electrolyte concentration to improve the brush-like structure of the DNA layer. Finally, irradiation effects and AlGaAs resonators have been studied for the applications of DNA brush layer on GaAs as biosensor during the change of attachment probe DNA and hybridization to target DNA. For the 8-base thiolated ssDNA case, AFM results showed that the layer thickness was about ˜2.2 nm in dry mode and increased in wet mode. Replacement

  15. Individual identification by VNTR analysis using minisatellite DNA probe to distinguish variable alleles at a single locus.

    PubMed

    Akasaka, H; Nonomura, S

    1991-04-01

    Variable number of tandem repeat analysis using a ministatellite DNA probe pYNH24 to distinguish variable alleles at a single locus was applied to forensic analysis of DNA extracted from a murder and body abandonment specimen. Two bands detected by pYNH24 correspond to identical DNA from both the upper and lower halves of a separated body, suggesting that these halves were from the same body.

  16. Fast quantitative PCR, locked nucleic acid probes and reduced volume reactions are effective tools for detecting Batrachochytrium dendrobatidis DNA.

    PubMed

    Ruthig, Gregory R; Deridder, Benjamin P

    2012-01-24

    The fungal pathogen Batrachochytrium dendrobatidis threatens amphibian populations around the world. The ability to detect this pathogen on infected animals and in the environment is critical for understanding and controlling this pandemic. We tested several advances in quantitative PCR (qPCR) techniques to detect B. dendrobatidis DNA. We used a fast PCR thermocycler and enzymes that reduced the volume and the duration of the reaction. We also compared a conventional TaqMan minor groove binding (MGB) probe to an identical locked nucleic acid (LNA) counterpart. The fast qPCR reaction had a high degree of sensitivity to B. dendrobatidis DNA. The LNA probe was effective for detecting B. dendrobatidis DNA and produced results -similar to those of the MGB probe. The modifications that we tested can improve the cost, time efficiency and specificity of quantitative PCR as a tool for detecting pathogen DNA.

  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-templated Ag nanoclusters as fluorescent probes for sensing and intracellular imaging of hydroxyl radicals.

    PubMed

    Zhang, Li; Liang, Ru-Ping; Xiao, Sai-Jin; Bai, Jian-Mei; Zheng, Lin-Ling; Zhan, Lei; Zhao, Xi-Juan; Qiu, Jian-Ding; Huang, Cheng-Zhi

    2014-01-01

    We have developed a simple, rapid and label-free sensor for the essential biological OH radicals based on the fluorescence quenching of DNA-templated Ag nanoclusters (DNA-Ag NCs). The OH radicals generated from the Fenton reagent attack and cleave the DNA template, which disturbs the microenvironments around Ag NCs, resulting in spontaneous aggregation due to the lack of stabilization and further the quenching of the Ag NCs fluorescence. These changes in fluorescence intensity allow sensing of OH radicals with good sensitivity and selectivity under optimal conditions. The sensor can be also applied for quantifying the radical scavenging action of antioxidants. Various characterizations including absorption spectra, fluorescence lifetimes, light scattering (LS) spectra, transmission electron microscopy (TEM), dark field light scattering imaging, and circular dichroism (CD) spectrometry have been employed to illustrate the proposed sensing mechanism. Further investigations demonstrate that the fluorescent probe could penetrate into intact cell membranes to selectively detect intracellular OH radicals induced by the phorbol myristate acetate (PMA) stimulation. These advantageous characteristics make the fluorescent DNA-Ag NCs potentially useful as a new candidate to monitor OH in broad biosystems. © 2013 Published by Elsevier B.V.

  20. Serum Albumin Binding Inhibits Nuclear Uptake of Luminescent Metal-Complex-Based DNA Imaging Probes.

    PubMed

    Wragg, Ashley; Gill, Martin R; McKenzie, Luke; Glover, Caroline; Mowll, Rachel; Weinstein, Julia A; Su, Xiaodi; Smythe, Carl; Thomas, Jim A

    2015-08-10

    The DNA binding and cellular localization properties of a new luminescent heterobimetallic Ir(III) Ru(II) tetrapyridophenazine complex are reported. Surprisingly, in standard cell media, in which its tetracationic, isostructural Ru(II) Ru(II) analogue is localized in the nucleus, the new tricationic complex is poorly taken up by live cells and demonstrates no nuclear staining. Consequent cell-free studies reveal that the Ir(III) Ru(II) complex binds bovine serum albumin, BSA, in Sudlow's Site I with a similar increase in emission and binding affinity to that observed with DNA. Contrastingly, in serum-free conditions the complex is rapidly internalized by live cells, where it localizes in cell nuclei and functions as a DNA imaging agent. The absence of serum proteins also greatly alters the cytotoxicity of the complex, where high levels of oncosis/necrosis are observed due to this enhanced uptake. This suggests that simply increasing the lipophilicity of a DNA imaging probe to enhance cellular uptake can be counterproductive as, due to increased binding to serum albumin protein, this strategy can actually disrupt nuclear targeting. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. 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.

  2. Probing Minor Groove Hydrogen-bonding Interactions Between RB69 DNA Polymerase and DNA†

    PubMed Central

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

    2012-01-01

    Minor groove hydrogen bonding (HB) interactions between DNA polymerases 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. Since 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 analog 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/template (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 102–103 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 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 102–103 fold decrease in nucleotide incorporation. The minor groove HB interactions between the n-2 position of the primer strand and RB69pol fixes 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 PMID:22571765

  3. 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.

  4. 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.

  5. 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.

  6. Double probing of human spermatozoa for persistent histones, surplus cytoplasm, apoptosis and DNA fragmentation.

    PubMed

    Sati, Leyla; Ovari, Laszlo; Bennett, David; Simon, Stephen D; Demir, Ramazan; Huszar, Gabor

    2008-04-01

    Individual spermatozoa were assessed with pairs of probes for persistent histones and cytoplasmic retention, persistent histones and DNA fragmentation, and persistent histones and apoptotic markers. The individual spermatozoa were treated sequentially with combinations of probes for these cytoplasmic and nuclear biochemical markers. Sperm fields were recorded with computer-assisted imaging, and staining patterns with the two probes in the same spermatozoa were examined and scored as light, intermediate or dark (mature to arrested-maturity spermatozoa). The effects of arrested sperm maturation were similar with respect to the cytoplasmic and nuclear characteristics of spermatozoa in 84% of cells, indicating that cytoplasmic and nuclear attributes of arrested sperm maturation are related. However, there were moderate (intermediate-dark or intermediate-light patterns, 14.5% of cells) or major (light-dark patterns, 1.6% of cells) discrepancies in the intensity of the double staining patterns. Thus, testing with single maturity markers may not be fully reliable. These findings are important with respect to: (i) arrested sperm maturation; (ii) potential efficacy of antioxidant and similar therapeutic strategies in subfertile men, as spermatozoa with infrastructure defects due to mismaturation or maturation arrest are unlikely to respond to interventions; and (iii) detection of adverse male environmental exposures.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. Enzyme-amplified electrochemical hybridization assay based on PNA, LNA and DNA probe-modified micro-magnetic beads.

    PubMed

    Laschi, Serena; Palchetti, Ilaria; Marrazza, Giovanna; Mascini, Marco

    2009-09-01

    In the present study, we investigated the properties of PNA and LNA capture probes in the development of an electrochemical hybridization assay. Streptavidin-coated paramagnetic micro-beads were used as a solid phase to immobilize biotinylated DNA, PNA and LNA capture probes, respectively. The target sequence was then recognized via hybridization with the capture probe. After labeling the biotinylated hybrid with a streptavidin-enzyme conjugate, the electrochemical detection of the enzymatic product was performed onto the surface of a disposable electrode. The assay was applied to the analytical detection of biotinylated DNA as well as RNA sequences. Detection limits, calculated considering the slope of the linear portion of the calibration curve in the range 0-2 nM were found to be 152, 118 and 91 pM, coupled with a reproducibility of the analysis equal to 5, 9 and 6%, calculated as RSD%, for DNA, PNA and LNA probes respectively, using the DNA target. In the case of the RNA target, the detection limits were found to be 51, 60 and 78 pM for DNA, PNA and LNA probes respectively.

  13. DNA and RNA "traffic lights": synthetic wavelength-shifting fluorescent probes based on nucleic acid base substitutes for molecular imaging.

    PubMed

    Holzhauser, Carolin; Wagenknecht, Hans-Achim

    2013-08-02

    The DNA base substitute approach by the (S)-3-amino-1,2-propanediol linker allows placing two fluorophores in a precise way inside a given DNA framework. The double helical architecture around the fluorophores, especially the DNA-induced twist, is crucial for the desired photophysical interactions. Excitonic, excimer, and energy transfer interactions yield fluorescent DNA and RNA probes with dual emission color readout. Especially, our DNA and RNA "traffic light" that combines the green emission of TO with the red emission of TR represents an important tool for molecular imaging and can be applied as aptasensors and as probes to monitor the siRNA delivery into cells. The concept can be extended to the synthetically easier to access postsynthetic 2'-modifications and the NIR range. Thereby, the pool of tailor-made fluorescent nucleic acid conjugates can be extended.

  14. Chemiluminescence-imaging detection of DNA on a solid-phase membrane by using a peroxidase-labeled macromolecular probe.

    PubMed

    Azam, Md Golam; Yamasuji, Mutsumi; Krawczyk, Tomasz; Shibata, Takayuki; Kabashima, Tsutomu; Kai, Masaaki

    2015-07-01

    We have developed a novel method for sensitive chemiluminescence (CL)-imaging detection of DNA by using a macromolecular probe synthesized by attaching multiple molecules of horseradish peroxidase (HRP) and biotin in dextran backbone. The probe formed a macromolecular assembly by binding to streptavidin which specifically recognized biotinylated complementary DNA, which was hybridized to a target DNA on a solid-phase membrane. This methodology was applied to CL-imaging detection of a synthetic telomere DNA (TTAGGG)10 and human telomere DNA by using the CL probe comprising of dextranT2000 (MW=ca. 2000kDa) bonded to approximately 42 molecules of HRP and 210 molecules of biotin. The human telomere DNA in a small number of buccal mucous cells (ca. 70 cell numbers) of cheek tissue was quantitatively determined by the proposed CL detection method that afforded approximately 10 times higher sensitivity than that of the conventional CL method using commercially available HRP-avidin probe. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. 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.

  16. 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.

  17. Probing DNA in nanopores via tunneling: from sequencing to ``quantum'' analogies

    NASA Astrophysics Data System (ADS)

    di Ventra, Massimiliano

    2012-02-01

    Fast and low-cost DNA sequencing methods would revolutionize medicine: a person could have his/her full genome sequenced so that drugs could be tailored to his/her specific illnesses; doctors could know in advance patients' likelihood to develop a given ailment; cures to major diseases could be found faster [1]. However, this goal of ``personalized medicine'' is hampered today by the high cost and slow speed of DNA sequencing methods. In this talk, I will discuss the sequencing protocol we suggest which requires the measurement of the distributions of transverse currents during the translocation of single-stranded DNA into nanopores [2-5]. I will support our conclusions with a combination of molecular dynamics simulations coupled to quantum mechanical calculations of electrical current in experimentally realizable systems [2-5]. I will also discuss recent experiments that support these theoretical predictions. In addition, I will show how this relatively unexplored area of research at the interface between solids, liquids, and biomolecules at the nanometer length scale is a fertile ground to study quantum phenomena that have a classical counterpart, such as ionic quasi-particles, ionic ``quantized'' conductance [6,7] and Coulomb blockade [8]. Work supported in part by NIH. [4pt] [1] M. Zwolak, M. Di Ventra, Physical Approaches to DNA Sequencing and Detection, Rev. Mod. Phys. 80, 141 (2008).[0pt] [2] M. Zwolak and M. Di Ventra, Electronic signature of DNA nucleotides via transverse transport, Nano Lett. 5, 421 (2005).[0pt] [3] J. Lagerqvist, M. Zwolak, and M. Di Ventra, Fast DNA sequencing via transverse electronic transport, Nano Lett. 6, 779 (2006).[0pt] [4] J. Lagerqvist, M. Zwolak, and M. Di Ventra, Influence of the environment and probes on rapid DNA sequencing via transverse electronic transport, Biophys. J. 93, 2384 (2007).[0pt] [5] M. Krems, M. Zwolak, Y.V. Pershin, and M. Di Ventra, Effect of noise on DNA sequencing via transverse electronic transport

  18. 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.

  19. 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.

  20. 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.

  1. Direct fluorescence in situ hybridization on human metaphase chromosomes using quantum dot-platinum labeled DNA probes

    SciTech Connect

    Hwang, Gyoyeon; Lee, Hansol; Lee, Jiyeon

    2015-11-13

    The telomere shortening in chromosomes implies the senescence, apoptosis, or oncogenic transformation of cells. Since detecting telomeres in aging and diseases like cancer, is important, the direct detection of telomeres has been a very useful biomarker. We propose a telomere detection method using a newly synthesized quantum dot (QD) based probe with oligonucleotide conjugation and direct fluorescence in situ hybridization (FISH). QD-oligonucleotides were prepared with metal coordination bonding based on platinum-guanine binding reported in our previous work. The QD-oligonucleotide conjugation method has an advantage where any sequence containing guanine at the end can be easily bound to the starting QD-Pt conjugate. A synthesized telomeric oligonucleotide was bound to the QD-Pt conjugate successfully and this probe hybridized specifically on the telomere of fabricated MV-4-11 and MOLT-4 chromosomes. Additionally, the QD-telomeric oligonucleotide probe successfully detected the telomeres on the CGH metaphase slide. Due to the excellent photostability and high quantum yield of QDs, the QD-oligonucleotide probe has high fluorescence intensity when compared to the organic dye-oligonucleotide probe. Our QD-oligonucleotide probe, conjugation method of this QD probe, and hybridization protocol with the chromosomes can be a useful tool for chromosome painting and FISH. - Highlights: • We prepared a probe linked between QD and telomeric oligonucleotide with platinum-guanine bonding. • Telomeres were detected by our new telomere probes successfully in three different human metaphase chromosomes. • QDPt-DNA probe has high fluorescence intensity in comparison with organic dye-DNA probe.

  2. 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.

  3. PhenDV, a turn-off fluorescent quadruplex DNA probe for improving the sensitivity of drug screening assays.

    PubMed

    Beauvineau, Claire; Guetta, Corinne; Teulade-Fichou, Marie-Paule; Mahuteau-Betzer, Florence

    2017-08-30

    We report a new turn-off fluorescent probe, PhenDV, for the identification of high affinity quadruplex (G4) DNA ligands. This push-pull fluorophore displays a high fluorescence quantum yield in water (ΦF = 0.21) and is a selective and strong quadruplex DNA binder. We describe its use as a fluorescent indicator for the G4 Fluorescent Intercalator Displacement (FID) assay as its fluorescence is strongly quenched when bound to G4 DNA and fully restored when displaced by ligand. This probe improves the sensitivity of the G4-FID assay, as the read out relies on increased fluorescence instead of quenching observed with classical on/off probes.

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Effect of a Dual Charge on the DNA-Conjugated Redox Probe on DNA Sensing by Short Hairpin Beacons Tethered to Gold Electrodes.

    PubMed

    Kékedy-Nagy, László; Shipovskov, Stepan; Ferapontova, Elena E

    2016-08-16

    Charges of redox species can critically affect both the interfacial state of DNA and electrochemistry of DNA-conjugated redox labels and, as a result, the electroanalytical performance of those systems. Here, we show that the kinetics of electron transfer (ET) between the gold electrode and methylene blue (MB) label conjugated to a double-stranded (ds) DNA tethered to gold strongly depend on the charge of the MB molecule, and that affects the performance of genosensors exploiting MB-labeled hairpin DNA beacons. Positively charged MB binds to dsDNA via electrostatic and intercalative/groove binding, and this binding allows the DNA-mediated electrochemistry of MB intercalated into the duplex and, as a result, a complex mode of the electrochemical signal change upon hairpin hybridization to the target DNA, dominated by the "on-off" signal change mode at nanomolar levels of the analyzed DNA. When MB bears an additional carboxylic group, the negative charge provided by this group prevents intimate interactions between MB and DNA, and then the ET in duplexes is limited by the diffusion of the MB-conjugated dsDNA (the phenomenon first shown in Farjami , E. ; Clima , L. ; Gothelf , K. ; Ferapontova , E. E. Anal. Chem. 2011 , 83 , 1594 ) providing the robust "off-on" nanomolar DNA sensing. Those results can be extended to other intercalating redox probes and are of strategic importance for design and development of electrochemical hybridization sensors exploiting DNA nanoswitchable architectures.

  15. 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.

  16. 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.

  17. Cyanotryptophans as Novel Fluorescent Probes for Studying Protein Conformational Changes and DNA-Protein Interaction.

    PubMed

    Talukder, Poulami; Chen, Shengxi; Roy, Basab; Yakovchuk, Petro; Spiering, Michelle M; Alam, Mohammad P; Madathil, Manikandadas M; Bhattacharya, Chandrabali; Benkovic, Stephen J; Hecht, Sidney M

    2015-12-29

    Described herein are the syntheses and photophysical characterization of three novel cyanotryptophans, and their efficient incorporation into proteins as fluorescent probes. Photophysical characteristics indicated that each was significantly brighter and red-shifted in fluorescence emission relative to tryptophan. Each analogue was used to activate a suppressor tRNA transcript and was incorporated with good efficiency into two different positions (Trp22 and Trp74) of Escherichia coli dihydrofolate reductase (ecDHFR). The Trp analogues could be monitored selectively in the presence of multiple native Trp residues in DHFR. 6-CNTrp (A) formed an efficient Förster resonance energy transfer (FRET) pair with l-(7-hydroxycoumarin-4-yl)ethylglycine (HCO, D) at position 17. Further, 6-CNTrp (A) was incorporated into two DNA binding proteins, including the Klenow fragment of DNA polymerase I and an RNA recognition motif (RRM2) of heterogeneous nuclear ribonucleoprotein L-like (hnRNP LL). Using these proteins, we demonstrated the use of FRET involving A as a fluorescence donor and benzo[g]quinazoline-2,4-(1H,3H)-dione 2'-deoxyriboside (Tf) or 4-aminobenzo[g]quinazoline-2-one 2'-deoxyriboside (Cf) as fluorescent acceptors to study the binding interaction of the Klenow fragment with duplex DNA oligomers (labeled with Tf), or the domain-specific association between hnRNP LL and the BCL2 i-motif DNA (labeled with Cf). Thus, the non-natural amino acid could be used as a FRET partner for studying protein-nucleic acid interactions. Together, these findings demonstrate the potential utility of 6-CNTrp (A) as a fluorescence donor for the study of protein conformational events.

  18. Foldamers with Hybrid Biological and Synthetic Sequences as Selective DNA Fluorescent Probes

    PubMed Central

    Wang, Wei; Wan, Wei; Stachiw, Andrew; Li, Alexander D.Q.

    2008-01-01

    Foldable polymers with alternating single strand deoxyribonucleic acid (ssDNA) and planar fluorescent organic chromophores can self-organize into folded nanostructures and hence are hybrid foldamers with biological sequences and synthetic properties. The biological sequence provides highly specific molecular recognition properties while the physical properties of synthetic chromophores offer sensitive fluorescence detection. In this paper, we describe that rational designed hybrid foldamers exhibit potential in the detection of polynucleotides. Under strictly controlled laboratory conditions, fluorescence measurements indicate that configuration change due to binding of polynucleotides with one or two mismatched bases can be readily distinguished. These results shed light on the design and construction of nanostructured foldamers with actuator and sensory properties, which may find important applications as biological probes. PMID:16086577

  19. 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.

  20. Rapid method for testing susceptibility of Mycobacterium tuberculosis by using DNA probes.

    PubMed Central

    Martin-Casabona, N; Xairó Mimó, D; González, T; Rossello, J; Arcalis, L

    1997-01-01

    The increasing number of multidrug-resistant Mycobacterium tuberculosis strains has stimulated the interest of investigators in finding a rapid method for susceptibility testing. We used commercially available rRNA DNA-bioluminescence-labelled probes (Accu-Probe, Gen Probe, Inc. San Diego, Calif.) for this purpose. The study was performed in three chronological steps. (i) We studied the correlation between the photometric light units (PLUs) given by the hybridization method, the numbers of CFU per milliliter, and turbidity as nephelometric units for six different inocula of an M. tuberculosis strain over 14 days. A good correlation (c > 0.9; P < 0.05) was found from the third day for all concentrations used. (ii) Over a period of 14 days we studied the evolution of the PLUs for 20 strains growing in medium with 0.2 microl of isoniazid (H) per ml and 18 strains in medium with 1 microl of rifampin (R) per ml to standardize the method. Susceptible and resistant strains were used according to the reference proportions method in Middlebrook 7H10, and the MICs were determined in solid and liquid media. The final inoculum of a 10(-2) dilution from a McFarland no. 1 standard and reading at 3 and 5 days provided the best results. A quotient was established to find a cutoff point between resistant and susceptible strains. (iii) We used the standardized parameters in 117 tests with H and R. On day 3, the sensitivity, specificity, positive predictive value, and negative predictive value for detecting resistant strains were 86.8, 100, 100, and 90.1%, respectively, and on day 5 they were 96.2, 100, 100, and 94%, respectively. We concluded that the method is readily available, is easy to perform, and could be useful for screening resistant M. tuberculosis strains. PMID:9316900

  1. Direct estimation of biofilm density on different pipe material coupons using a specific DNA-probe.

    PubMed

    Chang, Young C; Le Puil, Michael; Biggerstaff, John; Randall, Andrew A; Schulte, Alfons; Taylor, James S

    2003-10-01

    A variety of approaches to quantify biomass in biofilms without disruption due to detachment have been developed over the years. One basic approach is the combination of advanced microscopy with molecular staining. However, many stains (e.g. 4',6-diamino-2-phenylindole, acridine orange or live-dead stains) can be non-specific when corrosion products, precipitates, and pipe material are present. In addition, some pipe materials cause high background when using epifluorescent microscopy. The new refinement discussed in this presentation used fluorescence spectroscopy to obtain the spectra from four common distribution system pipe materials: PVC, 'concrete' lined cast iron, cast iron, and galvanized steel. The emission maximum for all four materials was between 500 and 550 nm, but emissions radically decreased around 575-600 nm. A molecular probe, BO-PRO-3 (Molecular Probes, Inc., Eugene, OR, USA) was identified which has an emission intensity maximum at 599 nm (red), with emission intensity 200 times greater when it is bound to DNA. The BO-PRO-3 has greatly reduced non-specific staining and background problems. In the preliminary experiment, using diluted waste water, a significant exponential relationship was found between stained surface area/total area ratio and fixed biofilm inventory measurements from scraping heterotrophic plate counts (SHPC) on R2A medium. In addition, the biofilm inventory on different pipe material coupons from pilot distribution systems was also correlated to the stained surface area fraction and SHPC.

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. Detection of Pneumocystis carinii in induced sputa from immunocompromised patients using a repetitive DNA probe.

    PubMed

    Graves, D C; Chary-Reddy, S; Becker-Hapak, M

    1997-02-01

    A hybridization assay for the detection of Pneumocystis carinii was developed using a repetitive DNA fragment of P.c. hominis. The assay was specific as different micro-organisms typically found in the respiratory tract, normal human lung DNA (A 549 cell line) and normal rat lung DNA did not react with the repetitive probe. In a slot blot (SB) hybridization assay, the repetitive probe was able to detect as few as 100 P.c. hominis organisms with no false-positives. The results of the SB hybridization assay were compared with an immunofluorescence (IFA) assay for the detection of P.c. hominis in 84 induced sputum (IS) samples obtained from 52 human immunodeficiency virus (HIV)-seropositive patients, 22 HIV-seronegative patients and 10 healthy individuals. Samples from 24 patients clinically diagnosed with P. carinii pneumonia (PCP) were positive for P.c. hominis by both assays. In addition, the SB assay detected P.c. hominis in 14 patients (10 HIV-positive and four HIV-negative) who were negative by IFA. All 14 samples showed a positive PCR signal for the P.c. hominis dihydrofolate reductase gene, further confirming the presence of P.c. hominis in these specimens. Twelve of these patients had a clinical course highly suggestive of PCP and were either on P. carinii prophylaxis or P. carinii chemotherapy. The other two samples were from HIV-positive patients who had respiratory illness due to causes other than P.c. hominis (disseminated histoplasmosis and fatal Bordetella pneumonia). Detection of P.c. hominis in these samples suggests that these patients may have subclinical colonization by P.c. hominis. Furthermore, P.c. hominis was detected in all 12 sequential IS samples from six AIDS patients who had primary episodes of PCR using the SB assay, while P.c. hominis was detected only in eight samples by IFA (66.6%). All six patients developed recurrent PCP within 6 months from the time the assays were performed, further illustrating the potential of the SB

  7. 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

  8. 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...

  9. Comparison of API 50 CH Strips to Whole-Chromosomal DNA Probes for Identification of Lactobacillus Species

    PubMed Central

    Boyd, Melinda A.; Antonio, May A. D.; Hillier, Sharon L.

    2005-01-01

    The API 50 CH identification system was evaluated for the identification of 97 strains of commensal lactobacilli. This system agreed with the species-level identifications for none of the 7 reference strains and only 4 of 90 vaginal isolates identified using whole-chromosomal DNA probes. PMID:16208005

  10. 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...

  11. 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.

  12. 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.

  13. Spectrophotometric determination of DNA using a near infrared probe 1,1'-disulfobutyl-3,3,3',3'-tetramethylindotricarbocyanine

    NASA Astrophysics Data System (ADS)

    Wang, Hong; Li, Wei-Rong; Lu, Yin; Fu, Ni-Na; Zhang, Hua-Shan

    2005-07-01

    A direct spectrophotometric determination of DNA in solution has been developed using a near infrared probe, 1,1'-disulfobutyl-3,3,3',3'-tetramethylindotricarbocyanine (DSTCY). In pH = 7.5 Tris-HCl buffer, the reaction of DSTCY with DNA was complete within 10 min at room temperature in the presence of cetyltrimethylammonium bromide (CTAB), which leaded to a sharp increase of the absorbance at 474 nm. The concentration of DNA can be determined in such wavelength with the linear range of 0.5-8.0 μg ml -1 and the detection limit of 45 ng ml -1.

  14. A liquid chromatography - mass spectrometry method to measure ¹³C-isotope enrichment for DNA stable-isotope probing.

    PubMed

    Auclair, Julie; Lépine, François; Villemur, Richard

    2012-03-01

    DNA stable-isotope probing (DNA-SIP) is a cultivation-independent technique that makes it possible to associate metabolic function and taxonomic identity in a wide range of terrestrial and aquatic environments. In DNA-SIP, DNA is labeled via the assimilation of a labeled growth substrate that is subsequently used to identify microorganisms involved in assimilation of the substrate. However, the labeling time has to be sufficient to obtain labeled DNA but not so long such that cross-feeding of ¹³C-labeled metabolites from the primary consumers to nontarget species can occur. Confirmation that the DNA is isotopically labeled in DNA-SIP assays can be achieved using an isotope ratio mass spectrometer. In this study, we describe the development of a method using liquid chromatography (HPLC) coupled to a quadrupole mass spectrometer (QMS) to measure the ¹³C enrichment of thymine incorporated into DNA in Escherichia coli cultures fed with [¹³C]acetate. The method involved the hydrolysis of DNA extracted from the cultures that released the nucleotides, followed by the separation of the thymine by HPLC on a reverse-phase C₈ column in isocratic elution mode and the detection and quantification of ¹³C-labeled thymine by QMS. To mimic a DNA-SIP assay, a DNA mixture was made using ¹³C-labeled E. coli DNA with DNA extracted from five bacterial species. The HPLC-MS method was able to measure the correct proportion of ¹³C-DNA in the mix. This method can then be used as an alternative to the use of isotope ratio mass spectrometry in DNA-SIP assays.

  15. 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

  16. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. Multiple components in restriction enzyme digests of mammalian (insectivore), avian and reptilian genomic DNA hybridize with murine immunoglobulin VH probes.

    PubMed Central

    Litman, G W; Berger, L; Jahn, C L

    1982-01-01

    High molecular weight genomic DNAs isolated from an insectivore, Tupaia, and a representative reptilian, Caiman, and avian, Gallus, were digested with restriction endonucleases transferred to nitrocellulose and hybridized with nick-translated probes of murine VH genes. The derivations of the probes designated S107V (1) and mu 107V (2,3) have been described previously. Under conditions of reduced stringency, multiple hybridizing components were observed with Tupaia and Caiman; only mu mu 107V exhibited significant hybridization with the separated fragments of Gallus DNA. The nick-translated S107V probe was digested with Fnu4H1 and subinserts corresponding to the 5' and 3' regions both detected multiple hybridizing components in Tupaia and Caiman DNA. A 5' probe lacking the leader sequence identified the same components as the intact 5' probe, suggesting that VH coding regions distant as the reptilians may possess multiple genetic components which exhibit significant homology with murine immunoglobulin in VH regions. Images PMID:6285298

  4. Anion-exchanged nanosolid support of magnetic nanoparticle in combination with PNA probes for DNA sequence analysis

    NASA Astrophysics Data System (ADS)

    Theppaleak, T.; Rutnakornpituk, M.; Wichai, U.; Vilaivan, T.; Rutnakornpituk, B.

    2013-12-01

    Poly((2-diethylamino)ethyl methacrylate) (poly(DEAEMA))-grafted magnetite nanoparticle (MNP) with a positively charged surface was used as an anion exchanger for detection of deoxyribonucleic acid (DNA) sequences by employing peptide nucleic acid (PNA) as a probe. The cationic MNP with a size ranging between 6 and 10 nm in diameter can electrostatically adsorb DNA with a capacity of 171 nmol nucleotide/g MNP. The electrostatically neutral pyrrolidinyl PNA-bearing prolyl-2-aminocyclopentane carboxylic acid backbone (acpcPNA) can be adsorbed by the cationic MNP only when the sequence of the PNA and DNA is complementary, and the presence of adsorbed PNA could be examined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Two DNA sequences, the sequence stimulating Kirsten Rat Sarcoma ( K- ras) gene and the sequence having 5' methylated CpG site, were used in this study. It was found that the particles can be used as nanosolid support to differentiate between complementary and single-base mismatched DNA sequences using both single and two acpcPNA probes. This polymer-grafted MNP might be applicable for use as a magnetically guidable tool for detection of real DNA samples in the future.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. A model of binding on DNA microarrays: understanding the combined effect of probe synthesis failure, cross-hybridization, DNA fragmentation and other experimental details of affymetrix arrays

    PubMed Central

    2012-01-01

    Background DNA microarrays are used both for research and for diagnostics. In research, Affymetrix arrays are commonly used for genome wide association studies, resequencing, and for gene expression analysis. These arrays provide large amounts of data. This data is analyzed using statistical methods that quite often discard a large portion of the information. Most of the information that is lost comes from probes that systematically fail across chips and from batch effects. The aim of this study was to develop a comprehensive model for hybridization that predicts probe intensities for Affymetrix arrays and that could provide a basis for improved microarray analysis and probe development. The first part of the model calculates probe binding affinities to all the possible targets in the hybridization solution using the Langmuir isotherm. In the second part of the model we integrate details that are specific to each experiment and contribute to the differences between hybridization in solution and on the microarray. These details include fragmentation, wash stringency, temperature, salt concentration, and scanner settings. Furthermore, the model fits probe synthesis efficiency and target concentration parameters directly to the data. All the parameters used in the model have a well-established physical origin. Results For the 302 chips that were analyzed the mean correlation between expected and observed probe intensities was 0.701 with a range of 0.88 to 0.55. All available chips were included in the analysis regardless of the data quality. Our results show that batch effects arise from differences in probe synthesis, scanner settings, wash strength, and target fragmentation. We also show that probe synthesis efficiencies for different nucleotides are not uniform. Conclusions To date this is the most complete model for binding on microarrays. This is the first model that includes both probe synthesis efficiency and hybridization kinetics/cross-hybridization. These

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. Supersandwich cytosensor for selective and ultrasensitive detection of cancer cells using aptamer-DNA concatamer-quantum dots probes.

    PubMed

    Liu, Hongying; Xu, Shouming; He, Zhimei; Deng, Anping; Zhu, Jun-Jie

    2013-03-19

    In this work, a signal amplification supersandwich strategy was developed for highly selective and sensitive detection of cancer cells using aptamer-DNA concatamer-quantum dots (QDs) probes. First of all, electrode materials denoted as MWCNTs@PDA@AuNPs were fabricated by multiwall carbon nanotubes (MWCNTs), gold nanoparticles (AuNPs), and polydopamine (PDA) using a layer-by-layer technique. Then, the prepared bases as matrices were applied to bind concanavalin A (Con A), resulting in high stability, bioactivity, and capability for cell capture. Meanwhile, aptamer-DNA concatamer-QDs were designed via DNA hybridization followed by covalent assembling, which incorporated the specific recognition of the aptamer with the signal amplification of the DNA concatamer and QDs. With aptamer-DNA concatamer-QDs as recognizing probes, the model cancer cells (CCRF-CEM cells) were detected using a MWCNTs@PDA@AuNPs modified electrode with trapped Con A by means of fluorescence and electrochemical methods. The proposed supersandwich cytosensor showed high sensitivity with the detection limit of 50 cells mL(-1). More importantly, it could distinguish cancer cells from normal cells, which indicated the promising applications of our method in clinical diagnosis and treatment of cancers.

  17. 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

  18. 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.

  19. Genetic variation detected by use of the M13 "DNA fingerprint" probe in Malus, Prunus, and Rubus (Rosaceae).

    PubMed

    Nybom, H; Rogstad, S H; Schaal, B A

    1990-02-01

    Recently, "DNA fingerprints" have been reported in a wide array of organisms. We used the M13 repeat probe on several genera and species in the angiosperm family Rosaceae. Four apple cultivars could be differentiated when any one of five restriction enzymes was used to analyze minisatellite DNA. Similarly, four individual trees of Prunus serotina (black cherry) exhibited different "fingerprints" with each of four enyzmes. A total of 14 Rubus (blackberries and raspberries) plants representing four species were investigated with two enzymes. Extensive inter-and intraspecific variation was found. However, some closely growing plants had identical "fingerprints", probably due to their being derived through vegetative propagation.

  20. 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.

  1. 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). Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Multicolor chromosome banding (MCB) with YAC/BAC-based probes and region-specific microdissection DNA libraries

    SciTech Connect

    Liehr, T.; Weise, A.; Heller, A.; Starke, H.; Mrasek, K.; Kuechler, A.; Weier, H.-U.G.; Claussen, U.

    2003-06-23

    Multicolor chromosome banding (MCB) allows the delineation of chromosomal regions with a resolution of a few mega base pairs, i.e., slightly below the size of most visible chromosome bands. Based on the hybridization of over lapping region-specific probe libraries, chromosomal subregions are hybridized with probes that fluoresce in distinct wave length intervals, so they can be assigned predefined pseudo-colors during the digital imaging and visualization process. The present study demonstrates how MCB patterns can be produced by region-specific micro dissection derived (mcd) libraries as well as collections of yeast or bacterial artificial chromosomes (YACs and BACs, respectively). We compared the efficiency of an mcd library based approach with the hybridization of collections of locus-specific probes (LSP) for fluorescent banding of three rather differently sized human chromosomes, i.e., chromosomes 2, 13, and 22. The LSP sets were comprised of 107 probes specific for chromosome 2, 82 probes for chromosome 13, and 31 probes for chromosome 22. The results demonstrated a more homogeneous coverage of chromosomes and thus, more desirable banding patterns using the microdissection library-based MCB. This may be related to the observation that chromosomes are difficult to cover completely with YAC and/or BAC clones as single-color fluorescence in situ hybridization (FISH) experiments showed. Mcd libraries, on the other hand, provide high complexity probes that work well as region specific paints, but do not readily allow positioning of break points on genetic or physical maps as required for the positional cloning of genes. Thus, combinations of mcd libraries and locus-specific large insert DNA probes appear to be the most efficient tools for high-resolution cytogenetic analyses.

  3. 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.

  4. 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.

  5. 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.

  6. Ultrasensitive Detection of MicroRNA in Tumor Cells and Tissues via Continuous Assembly of DNA Probe.

    PubMed

    Liao, Yuhui; Fu, Yu; Wu, Yunxia; Huang, Ru; Zhou, Xiaoming; Xing, Da

    2015-11-09

    Nucleic acids have been engineered to participate in a wide variety of tasks. Among them, the enzyme-free amplification modes, enzyme-free DNA circuits (EFDCs), and hybridization chain reactions (HCRs) have been widely applied in a series of studies of bioanalysis. We demonstrated here an ultrasensitive hairpin probe-based circulation for continuous assemble of DNA probe. This strategy improved the analyte stability-dependent amplification efficiency of EFDC and signal enhancement without being limited by the analyte's initial concentration, and it was used to produce a novel microRNA (miRNA) trace analysis assay with ultrasensitive amplification properties. Through the detection of standard miRNA substances, 1 amol-level sensitivity and satisfactory specificity were achieved. Compared with EFDCs and HCRs, the sensitivity of ultrasensitive hairpin probe-based circulation was higher by 3 or 4 orders of magnitude. Furthermore, the excellent performance of this platform was also demonstrated in the detection of miRNAs in tumor cells. The sensitivities for the detection of miRNAs in HepG2, A549 and MCF-7 tumor cells were 10, 10, and 100 cells, respectively. In addition, a high detection rate of 83% was achieved for tumor tissues. Thus, this ultrasensitive hairpin probe-based circulation possesses the potential to be a technological innovation in the field of tumor diagnosis.

  7. 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

  8. 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.

  9. Interaction between tryptophan-Sm(III) complex and DNA with the use of a acridine orange dye fluorophor probe.

    PubMed

    Xiong, Xiao Li; Zhao, Na; Wang, Xing Ming

    2016-02-01

    The interaction of the Trp-Sm(III) complex with herring sperm DNA (hs-DNA) was investigated with the use of acridine orange (AO) dye as a spectral probe for UV-vis spectrophotometry and fluorescence spectroscopy. The results showed that the both the Trp-Sm(III) complex and the AO molecule could intercalate into the double helix of the DNA. The Sm(III)-(Trp)3 complex was stabilized by intercalation into the DNA with binding constants: K(Ө)25°C  = 7.14 × 10(5)  L·mol(-1) and K(Ө) 37°C  = 5.28 × 10(4)  L·mol(-1), and it could displace the AO dye from the AO-DNA complex in a competitive reaction. Computation of the thermodynamic functions demonstrates that Δr Hm (Ө) is the primary driving power of the interaction between the Sm(III)(Trp)3 complex and the DNA. The results from Scatchard and viscometry methods suggested that the interaction mode between the Sm(III)(Trp)3 complex and the hs-DNA is groove binding and weak intercalation binding. Copyright © 2015 John Wiley & Sons, Ltd.

  10. 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.

  11. 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.

  12. Rapid quantification of hepatitis B virus DNA by real-time PCR using efficient TaqMan probe and extraction of virus DNA.

    PubMed

    Lu, Yan-Qin; Han, Jin-Xiang; Qi, Peng; Xu, Wei; Zu, Yan-Hui; Zhu, Bo

    2006-12-07

    To rapidly quantify hepatitis B virus (HBV) DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA. Three standards were prepared by cloning PCR products which targeted S, C and X region of HBV genome into pGEM-T vector respectively. A pair of primers and matched TaqMan probe were selected by comparing the copy number and the Ct values of HBV serum samples derived from the three different standard curves using certain serum DNA. Then the efficiency of six HBV DNA extraction methods including guanidinium isothiocyanate, proteinase K, NaI, NaOH lysis, alkaline lysis and simple boiling was analyzed in sample A, B and C by real-time PCR. Meanwhile, 8 clinical HBV serum samples were quantified. The copy number of the same HBV serum sample originated from the standard curve of S, C and X regions was 5.7 multiply 10(4)/mL, 6.3 multiply 10(2)/mL and 1.6 multiply 10(3)/mL respectively. The relative Ct value was 26.6, 31.8 and 29.5 respectively. Therefore, primers and matched probe from S region were chosen for further optimization of six extraction methods. The copy number of HBV serum samples A, B and C was 3.49 multiply 10(9)/mL, 2.08 multiply 10(6)/mL and 4.40 multiply 10(7)/mL respectively, the relative Ct value was 19.9, 30 and 26.2 in the method of NaOH lysis, which was the efficientest among six methods. Simple boiling showed a slightly lower efficiency than NaOH lysis. Guanidinium isothiocyanate, proteinase K and NaI displayed that the copy number of HBV serum sample A, B and C was around 10(5)/mL, meanwhile the Ct value was about 30. Alkaline failed to quantify the copy number of three HBV serum samples. Standard deviation (SD) and coefficient variation (CV) were very low in all 8 clinical HBV serum samples, showing that quantification of HBV DNA in triplicate was reliable and accurate. Real-time PCR based on optimized primers and TaqMan probe from S region in combination with NaOH lysis is a simple, rapid and accurate method for

  13. 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.

  14. Use of a multi-thermal washer for DNA microarrays simplifies probe design and gives robust genotyping assays

    PubMed Central

    Petersen, Jesper; Poulsen, Lena; Petronis, Sarunas; Birgens, Henrik; Dufva, Martin

    2008-01-01

    DNA microarrays are generally operated at a single condition, which severely limits the freedom of designing probes for allele-specific hybridization assays. Here, we demonstrate a fluidic device for multi-stringency posthybridization washing of microarrays on microscope slides. This device is called a multi-thermal array washer (MTAW), and it has eight individually controlled heating zones, each of which corresponds to the location of a subarray on a slide. Allele-specific oligonucleotide probes for nine mutations in the beta-globin gene were spotted in eight identical subarrays at positions corresponding to the temperature zones of the MTAW. After hybridization with amplified patient material, the slides were mounted in the MTAW, and each subarray was exposed to different temperatures ranging from 22 to 40°C. When processed in the MTAW, probes selected without considering melting temperature resulted in improved genotyping compared with probes selected according to theoretical melting temperature and run under one condition. In conclusion, the MTAW is a versatile tool that can facilitate screening of a large number of probes for genotyping assays and can also enhance the performance of diagnostic arrays. PMID:18063568

  15. 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

  16. Impedimetric genosensor for detection of hepatitis C virus (HCV1) DNA using viral probe on methylene blue doped silica nanoparticles.

    PubMed

    Singhal, Chaitali; Ingle, Aviraj; Chakraborty, Dhritiman; Pn, Anoop Krishna; Pundir, C S; Narang, Jagriti

    2017-05-01

    An impedimetric genosensor was fabricated for detection of hepatitis C virus (HCV) genotype 1 in serum, based on hybridization of the probe with complementary target cDNA from sample. To achieve it, probe DNA complementary to HCVgene was immobilized on the surface of methylene blue (MB) doped silica nanoparticles MB@SiNPs) modified fluorine doped tin oxide (FTO) electrode. The synthesized MB@SiNPs was characterized using scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM) and X-ray diffraction (XRD) pattern. This modified electrode (ssDNA/MB@SiNPs/FTO) served both as a signal amplification platform (due to silica nanoparticles (SiNPs) as well as an electrochemical indicator (due to methylene blue (MB)) for the detection of the HCV DNA in patient serum sample. The genosensor was optimized and evaluated. The sensor showed a dynamic linear range 100-10(6) copies/mL, with a detection limit of 90 copies/mL. The sensor was applied for detection of HCV in sera of hepatitis patient and could be renewed. The half life of the sensor was 4 weeks. The MB@SiNPs/FTO electrode could be used for preparation of other gensensors also. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. 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.

  18. Controlled Translocation of ds/ss hybrid DNA through Solid State Nanopores with Tuning Fork based Force sensing Probe tip (SSN-TFFSP)

    NASA Astrophysics Data System (ADS)

    Kaur, Harpreet; Hyun, Changbae; Huang, Tao; Walsh, Nathan; Nandivada, Santoshi; Rollings, Ryan; Xiao, Min; McNabb, David; Li, Jiali

    2014-03-01

    Using a newly constructed apparatus that integrates a Solid State Nanopore (SSN) and Tuning Fork based Force sensing Probe tip (SSN-TFFSP), we studied ds/ss hybrid single DNA molecules. The ds/ss hybrid DNA is a 48.6 kb double-stranded λ DNA ligated to a 1kb single-stranded DNA. The λ DNA end was ligated to a biotinlated Oligo for attaching the hybrid DNA to a probe tip. The SSN-TFFSP apparatus combines the measurement of ionic current through a solid-state nanopore with a DNA tethered probe tip that is position controlled and sensed by a tuning fork force sensor and a nanopositioning system. The SSN-TFFSP system monitors the process of DNA molecules being captured and trapped by a voltage-biased nanopore, and the process of pulling the trapped DNA out of the nanopore with a controlled speed of 100 μs/base (1nm/ms) or slower. Here we report on the 3 signals measured simultaneously from this apparatus: ionic current through a nanopore, tip position, and tip vibrational amplitude during the process of a ds/ss hybrid DNA tethered to a Probe tip being captured and released by a nanopore This work is supported by a NHGRI (R21HG004776) Grant.

  19. 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.

  20. Use of a Multiplexed CMOS Microarray to Optimize and Compare Oligonucleotide Binding to DNA Probes Synthesized or Immobilized on Individual Electrodes

    PubMed Central

    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. PMID:22163607

  1. 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.

  2. Surface Plasmon Resonator Using High Sensitive Resonance Telecommunication Wavelengths for DNA Sensors of Mycobacterium Tuberculosis with Thiol-Modified Probes

    PubMed Central

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

    2015-01-01

    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. PMID:25609049

  3. 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.

  4. Transcription-Factor-Mediated DNA Looping Probed by High-Resolution, Single-Molecule Imaging in Live E. coli Cells

    PubMed Central

    Hensel, Zach; Xiao, Jie

    2013-01-01

    DNA looping mediated by transcription factors plays critical roles in prokaryotic gene regulation. The “genetic switch” of bacteriophage λ determines whether a prophage stays incorporated in the E. coli chromosome or enters the lytic cycle of phage propagation and cell lysis. Past studies have shown that long-range DNA interactions between the operator sequences OR and OL (separated by 2.3 kb), mediated by the λ repressor CI (accession number P03034), play key roles in regulating the λ switch. In vitro, it was demonstrated that DNA segments harboring the operator sequences formed loops in the presence of CI, but CI-mediated DNA looping has not been directly visualized in vivo, hindering a deep understanding of the corresponding dynamics in realistic cellular environments. We report a high-resolution, single-molecule imaging method to probe CI-mediated DNA looping in live E. coli cells. We labeled two DNA loci with differently colored fluorescent fusion proteins and tracked their separations in real time with ∼40 nm accuracy, enabling the first direct analysis of transcription-factor-mediated DNA looping in live cells. Combining looping measurements with measurements of CI expression levels in different operator mutants, we show quantitatively that DNA looping activates transcription and enhances repression. Further, we estimated the upper bound of the rate of conformational change from the unlooped to the looped state, and discuss how chromosome compaction may impact looping kinetics. Our results provide insights into transcription-factor-mediated DNA looping in a variety of operator and CI mutant backgrounds in vivo, and our methodology can be applied to a broad range of questions regarding chromosome conformations in prokaryotes and higher organisms. PMID:23853547

  5. Transcription-factor-mediated DNA looping probed by high-resolution, single-molecule imaging in live E. coli cells.

    PubMed

    Hensel, Zach; Weng, Xiaoli; Lagda, Arvin Cesar; Xiao, Jie

    2013-01-01

    DNA looping mediated by transcription factors plays critical roles in prokaryotic gene regulation. The "genetic switch" of bacteriophage λ determines whether a prophage stays incorporated in the E. coli chromosome or enters the lytic cycle of phage propagation and cell lysis. Past studies have shown that long-range DNA interactions between the operator sequences O(R) and O(L) (separated by 2.3 kb), mediated by the λ repressor CI (accession number P03034), play key roles in regulating the λ switch. In vitro, it was demonstrated that DNA segments harboring the operator sequences formed loops in the presence of CI, but CI-mediated DNA looping has not been directly visualized in vivo, hindering a deep understanding of the corresponding dynamics in realistic cellular environments. We report a high-resolution, single-molecule imaging method to probe CI-mediated DNA looping in live E. coli cells. We labeled two DNA loci with differently colored fluorescent fusion proteins and tracked their separations in real time with ∼40 nm accuracy, enabling the first direct analysis of transcription-factor-mediated DNA looping in live cells. Combining looping measurements with measurements of CI expression levels in different operator mutants, we show quantitatively that DNA looping activates transcription and enhances repression. Further, we estimated the upper bound of the rate of conformational change from the unlooped to the looped state, and discuss how chromosome compaction may impact looping kinetics. Our results provide insights into transcription-factor-mediated DNA looping in a variety of operator and CI mutant backgrounds in vivo, and our methodology can be applied to a broad range of questions regarding chromosome conformations in prokaryotes and higher organisms.

  6. Kinetic adsorption profile and conformation evolution at the DNA-gold nanoparticle interface probed by dynamic light scattering.

    PubMed

    Wang, Wenjie; Ding, XiaoFan; He, Miao; Wang, Jing; Lou, Xinhui

    2014-10-21

    The kinetic adsorption profile at the DNA-gold nanoparticle (AuNP) interface is probed by following the binding and organization of thiolated linear DNA and aptamers of varying chain lengths (15, 30, 44, and 51 mer) to the surface of AuNPs (13.0 ± 1.0 nm diameter). A systematic investigation utilizing dynamic light scattering has been performed to directly measure the changes in particle size during the course of a typical aging-salting thiolated DNA/AuNP preparation procedure. We discuss the effect of DNA chain length, composition, salt concentration, and secondary structure on the kinetics and conformation at the DNA-AuNP interface. The adsorption kinetics are chain-length dependent, composition independent, and not diffusion rate limited for the conditions we report here. The kinetic data support a mechanism of stepwise adsorption of thiols to the surface of AuNPs and reorganization of the thiols at the interface. Very interestingly, the kinetic increases of the particle sizes are modeled accurately by the pseudo-second-order rate model, suggesting that DNA could possess the statistically well-defined conformational evolution. Together with other experimental evidence, we propose a dynamic inner-layer and outer-tail (DILOT) model to describe the evolution of the DNA conformation after the initial adsorption of a single oligonucleotide layer. According to this model, the length of the tails that extend from the surface of AuNPs, capable for hybridization or molecular recognition, can be conveniently calculated. Considering the wide applications of DNA/AuNPs, the results should have important implications in sensing and DNA-directed nanoparticle assembly.

  7. 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.

  8. 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.

  9. 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.

  10. DNA Probe for Identification of the Take-All Fungus, Gaeumannomyces graminis.

    PubMed

    Henson, J M

    1989-02-01

    A 4.3-kilobase mitochondrial DNA fragment was cloned from Gaeumannomyces graminis var. tritici, the causative agent of take-all disease of wheat. Although this DNA fragment hybridized with all three varieties of G. graminis, it showed little homology with DNA from other fungi and thus should be useful for identification of Gaeumannomyces sp. recovered from infected plants.

  11. 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.

  12. A novel sequence-specific RNA quantification method using nicking endonuclease, dual-labeled fluorescent DNA probe, and conformation-interchangeable oligo-DNA

    PubMed Central

    Hosoda, Kazufumi; Matsuura, Tomoaki; Kita, Hiroshi; Ichihashi, Norikazu; Tsukada, Koji; Urabe, Itaru; Yomo, Tetsuya

    2008-01-01

    We have developed a novel, single-step, isothermal, signal-amplified, and sequence-specific RNA quantification method (L-assay). The L-assay consists of nicking endonuclease, a dual-labeled fluorescent DNA probe (DL-probe), and conformation-interchangeable oligo-DNA (L-DNA). This signal-amplified assay can quantify target RNA concentration in a sequence-specific manner with a coefficient of variation (Cv) of 5% and a lower limit of detection of 0.1 nM. Moreover, this assay allows quantification of target RNA even in the presence of a several thousandfold excess by weight of cellular RNA. In addition, this assay can be used to measure the changes in RNA concentration in real-time and to quantify short RNAs (<30 nucleotides). The L-assay requires only incubation under isothermal conditions, is inexpensive, and is expected to be useful for basic research requiring high-accuracy, easy-to-use RNA quantification, and real-time quantification. PMID:18230763

  13. 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.

  14. 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.

  15. Systematic Spatial Bias in DNA Microarray Hybridization Is Caused by Probe Spot Position-Dependent Variability in Lateral Diffusion

    PubMed Central

    Haider, Susanne; Horn, Matthias; Wagner, Michael; Stocker, Roman; Loy, Alexander

    2011-01-01

    Background The hybridization of nucleic acid targets with surface-immobilized probes is a widely used assay for the parallel detection of multiple targets in medical and biological research. Despite its widespread application, DNA microarray technology still suffers from several biases and lack of reproducibility, stemming in part from an incomplete understanding of the processes governing surface hybridization. In particular, non-random spatial variations within individual microarray hybridizations are often observed, but the mechanisms underpinning this positional bias remain incompletely explained. Methodology/Principal Findings This study identifies and rationalizes a systematic spatial bias in the intensity of surface hybridization, characterized by markedly increased signal intensity of spots located at the boundaries of the spotted areas of the microarray slide. Combining observations from a simplified single-probe block array format with predictions from a mathematical model, the mechanism responsible for this bias is found to be a position-dependent variation in lateral diffusion of target molecules. Numerical simulations reveal a strong influence of microarray well geometry on the spatial bias. Conclusions Reciprocal adjustment of the size of the microarray hybridization chamber to the area of surface-bound probes is a simple and effective measure to minimize or eliminate the diffusion-based bias, resulting in increased uniformity and accuracy of quantitative DNA microarray hybridization. PMID:21858215

  16. 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.

  17. 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. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. 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.

  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. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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

  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. DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals.

    PubMed

    Hura, Greg L; Tsai, Chi-Lin; Claridge, Shelley A; Mendillo, Marc L; Smith, Jessica M; Williams, Gareth J; Mastroianni, Alexander J; Alivisatos, A Paul; Putnam, Christopher D; Kolodner, Richard D; Tainer, John A

    2013-10-22

    DNA metabolism and processing frequently require transient or metastable DNA conformations that are biologically important but challenging to characterize. We use gold nanocrystal labels combined with small angle X-ray scattering to develop, test, and apply a method to follow DNA conformations acting in the Escherichia coli mismatch repair (MMR) system in solution. We developed a neutral PEG linker that allowed gold-labeled DNAs to be flash-cooled and stored without degradation in sample quality. The 1,000-fold increased gold nanocrystal scattering vs. DNA enabled investigations at much lower concentrations than otherwise possible to avoid concentration-dependent tetramerization of the MMR initiation enzyme MutS. We analyzed the correlation scattering functions for the nanocrystals to provide higher resolution interparticle distributions not convoluted by the intraparticle distribution. We determined that mispair-containing DNAs were bent more by MutS than complementary sequence DNA (csDNA), did not promote tetramer formation, and allowed MutS conversion to a sliding clamp conformation that eliminated the DNA bends. Addition of second protein responder MutL did not stabilize the MutS-bent forms of DNA. Thus, DNA distortion is only involved at the earliest mispair recognition steps of MMR: MutL does not trap bent DNA conformations, suggesting migrating MutL or MutS/MutL complexes as a conserved feature of MMR. The results promote a mechanism of mismatch DNA bending followed by straightening in initial MutS and MutL responses in MMR. We demonstrate that small angle X-ray scattering with gold labels is an enabling method to examine protein-induced DNA distortions key to the DNA repair, replication, transcription, and packaging.

  8. DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals

    PubMed Central

    Hura, Greg L.; Tsai, Chi-Lin; Claridge, Shelley A.; Mendillo, Marc L.; Smith, Jessica M.; Williams, Gareth J.; Mastroianni, Alexander J.; Alivisatos, A. Paul; Putnam, Christopher D.; Kolodner, Richard D.; Tainer, John A.

    2013-01-01

    DNA metabolism and processing frequently require transient or metastable DNA conformations that are biologically important but challenging to characterize. We use gold nanocrystal labels combined with small angle X-ray scattering to develop, test, and apply a method to follow DNA conformations acting in the Escherichia coli mismatch repair (MMR) system in solution. We developed a neutral PEG linker that allowed gold-labeled DNAs to be flash-cooled and stored without degradation in sample quality. The 1,000-fold increased gold nanocrystal scattering vs. DNA enabled investigations at much lower concentrations than otherwise possible to avoid concentration-dependent tetramerization of the MMR initiation enzyme MutS. We analyzed the correlation scattering functions for the nanocrystals to provide higher resolution interparticle distributions not convoluted by the intraparticle distribution. We determined that mispair-containing DNAs were bent more by MutS than complementary sequence DNA (csDNA), did not promote tetramer formation, and allowed MutS conversion to a sliding clamp conformation that eliminated the DNA bends. Addition of second protein responder MutL did not stabilize the MutS-bent forms of DNA. Thus, DNA distortion is only involved at the earliest mispair recognition steps of MMR: MutL does not trap bent DNA conformations, suggesting migrating MutL or MutS/MutL complexes as a conserved feature of MMR. The results promote a mechanism of mismatch DNA bending followed by straightening in initial MutS and MutL responses in MMR. We demonstrate that small angle X-ray scattering with gold labels is an enabling method to examine protein-induced DNA distortions key to the DNA repair, replication, transcription, and packaging. PMID:24101514

  9. 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.

  10. 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

  11. 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.

  12. Probing physical properties of a DNA-protein complex using nanofluidic channels.

    PubMed

    Frykholm, Karolin; Alizadehheidari, Mohammadreza; Fritzsche, Joachim; Wigenius, Jens; Modesti, Mauro; Persson, Fredrik; Westerlund, Fredrik

    2014-03-12

    A method to investigate physical properties of a DNA-protein complex in solution is demonstrated. By using tapered nanochannels and lipid passivation the persistence length of a RecA filament formed on double-stranded DNA is determined to 1.15 μm, in agreement with the literature, without attaching protein or DNA to any handles or surfaces. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Tracking heterotrophic and autotrophic carbon cycling by magnetotactic bacteria in freshwater sediments using DNA stable isotope probing

    NASA Astrophysics Data System (ADS)

    Kürşat Coşkun, Ömer; Roud, Sophie; He, Kuang; Petersen, Nikolai; Gilder, Stuart; Orsi, William D.

    2017-04-01

    Magnetotactic bacteria (MTB) are diverse, widespread, motile prokaryotes which biomineralize nanosize magnetic minerals, either magnetite or gregite, under highly conserved genetic control and have magnetotaxis to align their position in aquatic environment according to Earth's magnetic field. They play important roles on some geobiological cycle of important minerals such as iron, sulphur, nitrogen and carbon. Yet, to date, their importance in carbon cycle and carbon source in their natural environment have not been previously studied. In this study, we focused on freshwater benthic carbon cycling of MTB and total bacteria using DNA stable isotope probing (DNA-SIP) technique coupled with quantitative PCR (qPCR). Pond sediments from Unterlippach (Germany) were amended with 13C-labelled sodium bicarbonate and 13C-labelled organic matter, and incubated in the dark over a two week time period. Applying separate qPCR assays specific for total bacteria and MTB, respectively, allowed us to estimate the contribution of MTB to total heterotrophic and autotrophic carbon cycling via DNA-SIP. After one week, there was a slight degree of autotrophic activity which increased markedly after two weeks. Comparing total DNA to the qPCR data revealed that changes in the buoyant density of DNA was due mainly to autotrophic bacterial production. DNA-SIP also identified heterotrophic utilization of 13C-labelled organic matter by MTB after 1 week. The qPCR data also allowed us to estimate uptake rates based on the incubation times for heterotrophic and autotrophic MTB. High-throughput DNA sequencing of 16S rRNA genes showed that most of the MTB involved in carbon cycling were related to the Magnetococcus genus. This study sheds light on the carbon sources for MTB in a natural environment and helps unravel their ecological role in the carbon cycle.

  14. In vitro DNA binding studies of antiretroviral drug nelfinavir using ethidium bromide as fluorescence probe.

    PubMed

    Ramana, M M V; Betkar, Rahul; Nimkar, Amey; Ranade, Prasanna; Mundhe, Balaji; Pardeshi, Sachin

    2015-10-01

    Understanding the interaction of small molecules with DNA has become an active research area at the interface between biology and chemistry. In the present work, we investigated the mode of interaction of nelfinavir (NFV) with herring sperm DNA (hs DNA) under physiological conditions using various biophysical techniques. Analysis of UV-absorption and fluorescence spectra indicates the formation of complex between NFV and hs DNA. According to the fluorescence results, the binding constant (K) between NFV and hs DNA was found to be 3.30 × 10(4)LM(-1). The calculated thermodynamic parameters (ΔH° and ΔS°) suggested that hydrogen bonding plays a major role in binding between them. Phosphate group binding studies revealed that there was no electrostatic interactions occurred between NFV and hs DNA. Circular dichroism (CD) and DNA melting curve were employed to measure the conformational change of hs DNA in the presence of NFV, which verified the minor groove binding mode. These results were further supported by viscosity measurements and competitive displacement assay study using Hoechst 33258. According to the sequence specificity experiments, NFV binds to A-T rich region of hs DNA. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. A H+/Ag+ dual-target responsive label-free light-up probe based on a DNA triplex.

    PubMed

    Xu, Lijun; Guo, Yahui; Wang, Jine; Zhou, Lu; Zhang, Yuanyuan; Hong, Shanni; Wang, Zhili; Zhang, Jianye; Pei, Renjun

    2015-05-01

    We developed a dual-target responsive sensor for label-free light-up fluorescent detection of protons (H(+)) and silver ions (Ag(+)) using an "OR'' logic gate. Berberine, a cost-effective and non-toxic indicator, partially intercalates the formed triplex DNA in the presence of H(+) or Ag(+), generating enhanced fluorescence. The designed Ag(+) probe has high selectivity and desirable sensitivity, which is necessary for practical use. The robust "OR" logic gate is capable of a rapid and reversible response to the H(+) and/or Ag(+) inputs. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. 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.

  17. 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.

  18. Fluorescence melting curve analysis using self-quenching dual-labeled peptide nucleic acid probes for simultaneously identifying multiple DNA sequences.

    PubMed

    Ahn, Jeong Jin; Kim, Youngjoo; Lee, Seung Yong; Hong, Ji Young; Kim, Gi Won; Hwang, Seung Yong

    2015-09-01

    Previous fluorescence melting curve analysis (FMCA) used intercalating dyes, and this method has restricted application. Therefore, FMCA methods such as probe-based FMCA and molecular beacons were studied. However, the usual dual-labeled probes do not possess adequate fluorescence quenching ability and sufficient specificity, and molecular beacons with the necessary stem structures are hard to design. Therefore, we have developed a peptide nucleic acid (PNA)-based FMCA method. PNA oligonucleotide can have a much higher melting temperature (Tm) value than DNA. Therefore, short PNA probes can have adequate Tm values for FMCA, and short probes can have higher specificity and accuracy in FMCA. Moreover, dual-labeled PNA probes have self-quenching ability via single-strand base stacking, which makes PNA more favorable. In addition, this method can facilitate simultaneous identification of multiple DNA templates. In conventional real-time polymerase chain reaction (PCR), one fluorescence channel can identify only one DNA template. However, this method uses two fluorescence channels to detect three types of DNA. Experiments were performed with one to three different DNA sequences mixed in a single tube. This method can be used to identify multiple DNA sequences in a single tube with high specificity and high clarity. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. 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

  20. Probing the Potential Role of Non-B DNA Structures at Yeast Meiosis-Specific DNA Double-Strand Breaks.

    PubMed

    Kshirsagar, Rucha; Khan, Krishnendu; Joshi, Mamata V; Hosur, Ramakrishna V; Muniyappa, K

    2017-05-23

    A plethora of evidence suggests that different types of DNA quadruplexes are widely present in the genome of all organisms. The existence of a growing number of proteins that selectively bind and/or process these structures underscores their biological relevance. Moreover, G-quadruplex DNA has been implicated in the alignment of four sister chromatids by forming parallel guanine quadruplexes during meiosis; however, the underlying mechanism is not well defined. Here we show that a G/C-rich motif associated with a meiosis-specific DNA double-strand break (DSB) in Saccharomyces cerevisiae folds into G-quadruplex, and the C-rich sequence complementary to the G-rich sequence forms an i-motif. The presence of G-quadruplex or i-motif structures upstream of the green fluorescent protein-coding sequence markedly reduces the levels of gfp mRNA expression in S. cerevisiae cells, with a concomitant decrease in green fluorescent protein abundance, and blocks primer extension by DNA polymerase, thereby demonstrating the functional significance of these structures. Surprisingly, although S. cerevisiae Hop1, a component of synaptonemal complex axial/lateral elements, exhibits strong affinity to G-quadruplex DNA, it displays a much weaker affinity for the i-motif structure. However, the Hop1 C-terminal but not the N-terminal domain possesses strong i-motif binding activity, implying that the C-terminal domain has a distinct substrate specificity. Additionally, we found that Hop1 promotes intermolecular pairing between G/C-rich DNA segments associated with a meiosis-specific DSB site. Our results support the idea that the G/C-rich motifs associated with meiosis-specific DSBs fold into intramolecular G-quadruplex and i-motif structures, both in vitro and in vivo, thus revealing an important link between non-B form DNA structures and Hop1 in meiotic chromosome synapsis and recombination. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  1. Synthesis and incorporation of 13C-labeled DNA building blocks to probe structural dynamics of DNA by NMR

    PubMed Central

    Nußbaumer, Felix; Juen, Michael Andreas; Gasser, Catherina; Kremser, Johannes; Müller, Thomas; Tollinger, Martin

    2017-01-01

    Abstract We report the synthesis of atom-specifically 13C-modified building blocks that can be incorporated into DNA via solid phase synthesis to facilitate investigations on structural and dynamic features via NMR spectroscopy. In detail, 6-13C-modified pyrimidine and 8-13C purine DNA phosphoramidites were synthesized and incorporated into a polypurine tract DNA/RNA hybrid duplex to showcase the facile resonance assignment using site-specific labeling. We also addressed micro- to millisecond dynamics in the mini-cTAR DNA. This DNA is involved in the HIV replication cycle and our data points toward an exchange process in the lower stem of the hairpin that is up-regulated in the presence of the HIV-1 nucleocapsid protein 7. As another example, we picked a G-quadruplex that was earlier shown to exist in two folds. Using site-specific 8-13C-2′deoxyguanosine labeling we were able to verify the slow exchange between the two forms on the chemical shift time scale. In a real-time NMR experiment the re-equilibration of the fold distribution after a T-jump could be monitored yielding a rate of 0.012 min−1. Finally, we used 13C-ZZ-exchange spectroscopy to characterize the kinetics between two stacked X-conformers of a Holliday junction mimic. At 25°C, the refolding process was found to occur at a forward rate constant of 3.1 s−1 and with a backward rate constant of 10.6 s−1.

  2. An in vitro selection scheme for oligonucleotide probes to discriminate between closely related DNA sequences

    PubMed Central

    Brukner, Ivan; El-Ramahi, Razan; Gorska-Flipot, Izabella; Krajinovic, Maja; Labuda, Damian

    2007-01-01

    Using an in vitro selection, we have obtained oligonucleotide probes with high discriminatory power against multiple, similar nucleic acid sequences, which is often required in diagnostic applications for simultaneous testing of such sequences. We have tested this approach, referred to as iterative hybridizations, by selecting probes against six 22-nt-long sequence variants representing human papillomavirus, (HPV). We have obtained probes that efficiently discriminate between HPV types that differ by 3–7 nt. The probes were found effective to recognize HPV sequences of the type 6, 11, 16, 18 and a pair of type 31 and 33, either when immobilized on a solid support or in a reverse configuration, as well to discriminate HPV types from the clinical samples. This methodology can be extended to generate diagnostic kits that rely on nucleic acid hybridization between closely related sequences. In this approach, instead of adjusting hybridization conditions to the intended set of probe–target pairs, we ‘adjust’, through in vitro selection, the probes to the conditions we have chosen. Importantly, these conditions have to be ‘relaxed’, allowing the formation of a variety of not fully complementary complexes from which those that efficiently recognize and discriminate intended from non-intended targets can be readily selected. PMID:17426126

  3. BENA435, a new cell-permeant photoactivated green fluorescent DNA probe

    PubMed Central

    Erve, Alexandra; Saoudi, Yasmina; Thirot, Sylvie; Guetta-Landras, Corinne; Florent, Jean-Claude; Nguyen, Chi-Hung; Grierson, David S.; Popov, Andrei V.

    2006-01-01

    N′-(2,8-Dimethoxy-12-methyl-dibenzo [c,h] [1,5] naphthyridin-6-yl)-N,N-dimethyl-propane-1,3-diamine (BENA435) is a new cell-membrane permeant DNA dye with absorption/emission maxima in complex with DNA at 435 and 484 nm. This new reagent is unrelated to known DNA dyes, and shows a distinct preference to bind double-stranded DNA over RNA. Hydrodynamic studies suggest that BENA435 intercalates between the opposite DNA strands. BENA435 fluoresces much stronger when bound to dA/dT rather than dG/dC homopolymers. We evaluated 14 related dibenzonaphthyridine derivatives and found BENA435 to be superior in its in vivo DNA-binding properties. Molecular modelling was used to develop a model of BENA435 intercalation between base pairs of a DNA helix. BENA435 fluorescence in the nuclei of cells increases upon illumination, suggesting photoactivation. BENA435 represents thus the first known cell-permeant photoactivated DNA-binding dye. PMID:16547198

  4. 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

  5. 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

  6. Synthesis and Assessment of DNA/Silver Nanoclusters Probes for Optimal and Selective Detection of Tristeza Virus Mild Strains.

    PubMed

    Shokri, Ehsan; Hosseini, Morteza; Faridbod, Farnoush; Rahaie, Mahdi

    2016-09-01

    Citrus Tristeza virus (CTV) is one of the most destructive pathogens worldwide that exist as a mixture of malicious (Sever) and tolerable (Mild) strains. Mild strains of CTV can be used to immunize healthy plants from more Severe strains damage. Recently, innovative methods based on the fluorescent properties of DNA/silver nanoclusters have been developed for molecular detection purposes. In this study, a simple procedure was followed to create more active DNA/AgNCs probe for accurate and selective detection of Tristeza Mild-RNA. To this end, four distinct DNA emitter scaffolds (C12, Red, Green, Yellow) were tethered to the Mild capture sequence and investigated in various buffers in order to find highly emissive combinations. Then, to achieve specific and reliable results, several chemical additives, including organic solvents, PEG and organo-soluble salts were used to enhance control fluorescence signals and optimize the hybridization solution. The data showed that, under adjusted conditions, the target sensitivity is enhanced by a factor of five and the high discrimination between Mild and Severe RNAs were obtained. The emission ratio of the DNA/AgNCs was dropped in the presence of target RNAs and I0/I intensity linearly ranged from 1.5 × 10(-8) M to 1.8 × 10(-6) M with the detection limit of 4.3 × 10(-9) M.

  7. 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.

  8. 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.

  9. Theoretical and Instrumental Studies of the Competitive Interaction Between Aromatic α-Aminobisphosphonates with DNA Using Binding Probes.

    PubMed

    Gholivand, M B; Peyman, H; Gholivand, Kh; Roshanfekr, H; Taherpour, A A; Yaghobi, R

    2017-07-01

    Fluorescence spectroscopy, UV-visible absorption spectroscopy, circular dichroism (CD) spectroscopy, viscometry, cyclic voltammetry (CV), and differential pulse voltammetry (DPV) were applied to investigate the competitive interaction of DNA with two aromatic α-aminobisphosphonates and neutral red dye (NR, intercalator) and Hoechst (Ho, groove binder) as spectroscopic probes, in a Tris-hydrogen chloride buffer solution (pH 7.4). The principal component analysis (PCA) was applied to determine the number of chemical components presented in complexation equilibrium of DNA with the aromatic α-aminobisphosphonates (B1 and B2). The spectroscopic and voltammetric studies showed that the groove binding mode of interaction is predominant in the solution containing DNA and α-aminobisphosphonates. Furthermore, the results indicated that α-aminobisphosphonate with the lengthy N-alkyl chains had a stronger interaction. The PCA and theoretical quantum mechanical and molecular mechanic methods were also utilized to determine the structure of DNA with the two α-aminobisphosphonates (B1 and B2).

  10. Comparative study of antipneumocystis agents in rats by using a Pneumocystis carinii-specific DNA probe to quantitate infection.

    PubMed

    Liberator, P A; Anderson, J W; Powles, M; Pittarelli, L A; Worley, M; Becker-Hapak, M; Graves, D C; Schmatz, D M

    1992-11-01

    A repetitive genomic DNA clone (B12-2) that specifically hybridizes to Pneumocystis carinii DNA has been identified. No cross-hybridization to genomic DNA prepared from bacteria, other fungi, protozoa, or mammals was observed. Clone B12-2 is multiply represented in the P. carinii genome. By direct hybridization to DNA prepared from the lungs of immunosuppressed rats, the probe can detect the equivalent of fewer than 1,000 P. carinii organisms. A hybridization assay employing clone B12-2 has been developed to quantitate organism load in the rat model for P. carinii. Application of the assay to track the accumulation of organisms during the immunosuppression regimen as well as to monitor the efficacy of two drug therapies used clinically for the treatment of P. carinii pneumonia is described here. The clone B12-2 hybridization assay for the determination of P. carinii organism load possesses several advantageous features and thus should serve to complement conventional staining and immunohistochemical methods.

  11. Application of steered molecular dynamics (SMD) to study DNA drug complexes and probing helical propensity of amino acids

    NASA Astrophysics Data System (ADS)

    Orzechowski, Marek; Cieplak, Piotr

    2005-05-01

    We present the preliminary results of two computer experiments involving the application of an external force to molecular systems. In the first experiment we simulated the process of pulling out a simple intercalator, the 9-aminoacridine molecule, from its complex with a short DNA oligonucleotide in aqueous solution. Removing a drug from the DNA is assumed to be an opposite process to the complex formation. The force and energy profiles suggest that formation of the DNA-9-aminoacridine complex is preferred when the acridine approaches the DNA from the minor groove rather than the major groove side. For a given mode of pulling the intercalation process is also shown to be nucleotide sequence dependent. In another computer experiment we performed a series of molecular dynamics simulations for stretching short, containing 15 amino acids, helical polypeptides in aqueous solution using an external force. The purpose of these simulations is to check whether this type of approach is sensitive enough to probe the sequence dependent helical propensity of short polypeptides.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. MUTATIONS THAT PROBE THE COOPERATIVE ASSEMBLY OF O6-ALKYLGUANINE-DNA ALKYLTRANSFERASE (AGT) COMPLEXES†

    PubMed Central

    Adams, Claire A.; Fried, Michael G.

    2011-01-01

    O6-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O6-alkylguanine and O4-alkylthymine adducts present in DNA that has been exposed to alkylating agents. AGT binds DNA cooperatively and models of cooperative complexes predict that residues 1–7 of one protein molecule and 163–169 of a neighboring protein are closely juxtaposed. To test these models we used directed mutagenesis to substitute triplets of alanine for triplets of native residues across these two sequences. Six of eight designed mutants expressed AGT at detectable levels. All mutant AGTs that were expressed were folded compactly, bound DNA with stoichiometries equivalent to wild-type protein and were able to protect E. coli to varying degrees from the potent alkylating agent MNNG. All mutations attenuated DNA binding cooperativity, but unexpectedly, they also reduced AGT affinity for DNA. This suggests that the protein-protein and protein-DNA interactions of AGT are strongly coupled. When normalized for differences in AGT expression, cells expressing mutants KDC(3–5)-AAA, DCE(4–6)-AAA and KEW(165–167)-AAA were significantly more susceptible to MNNG than wild-type cells. This is the first evidence, to our knowledge, of a role for residues at the protein-protein interface and by implication, cooperative protein-protein interactions, in the cell-protective mechanisms of AGT. PMID:21226457

  17. Combined spectroscopic and molecular docking approach to probing binding interactions between lovastatin and calf thymus DNA.

    PubMed

    Chen, C-B; Chen, J; Wang, J; Zhu, Y-Y; Shi, J-H

    2015-11-01

    The binding interaction of lovastatin with calf thymus DNA (ct-DNA) was studied using UV/Vis absorption spectroscopy, fluorescence emission spectroscopy, circular dichroism (CD), viscosity measurement and molecular docking methods. The experimental results showed that there was an obvious binding interaction of lovastatin with ct-DNA and the binding constant (Kb ) was 5.60 × 10(3) M(-1) at 298 K. In the binding process of lovastatin with ct-DNA, the enthalpy change (ΔH(0)) and entropy change (ΔS(0)) were -24.9 kJ/mol and -12.0 J/mol/K, respectively, indicating that the main binding interaction forces were van der Waal's force and hydrogen bonding. The molecular docking results suggested that lovastatin preferred to bind on the minor groove of different B-DNA fragments and the conformation change of lovastatin in the lovastatin-DNA complex was obviously observed, implying that the flexibility of lovastatin molecule plays an important role in the formation of the stable lovastatin-ct-DNA complex. Copyright © 2015 John Wiley & Sons, Ltd.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. DNA-Linked Enzyme-Coupled Assay for Probing Glucosyltransferase Specificity.

    PubMed

    Sukovich, David J; Modavi, Cyrus; de Raad, Markus; Prince, Robin N; Anderson, J Christopher

    2015-07-17

    Traditional enzyme characterization methods are low-throughput and therefore limit engineering efforts in synthetic biology and biotechnology. Here, we propose a DNA-linked enzyme-coupled assay (DLEnCA) to monitor enzyme reactions in a high-throughput manner. Throughput is improved by removing the need for protein purification and by limiting the need for liquid chromatography mass spectrometry (LCMS) product detection by linking enzymatic function to DNA modification. We demonstrate the DLEnCA methodology using glucosyltransferases as an illustration. The assay utilizes cell free transcription/translation systems to produce enzymes of interest, while UDP-glucose and T4-β-glucosyltransferase are used to modify DNA, which is detected postreaction using qPCR or a similar means of DNA analysis. OleD and two glucosyltransferases from Arabidopsis were used to verify the assay's generality toward glucosyltransferases. We further show DLEnCA's utility by mapping out the substrate specificity for these enzymes.

  6. 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

  7. 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.

  8. Probing the interaction of anthraquinone with DNA by spectroscopy, molecular modeling and cancer cell imaging technique.

    PubMed

    Yang, Lei; Fu, Zheng; Niu, Xiaoqing; Zhang, Guisheng; Cui, Fengling; Zhou, Chunwu

    2015-05-25

    A new anthraquinone derivative, (E)-2-(1-(4,5-dihydroxy-6-(hydroxymethyl)-tetrahydro-2H-pyran-2-yloxyimino)ethyl)-1,4-dihydroxyanthracene-9,10-dione (AODGlc), was synthesized and its binding properties towards DNA were explored under physiological conditions by fluorescence spectroscopy, DNA melting as well as docking techniques. The experimental results revealed that AODGlc could bind to calf thymus DNA (ctDNA) through intercalation between DNA base pairs. The values of thermodynamic parameters at different temperatures including ΔG, ΔH, and ΔS and the molecular modeling study implied that hydrophobic interactions and hydrogen bonds were the main interactions in the AODGlc-ctDNA system. Cervical cancer cells (HepG2 cells) were used in cell viability assay and cell imaging experiment. AODGlc could interact with HepG2 cells and kill HepG2 cells under high concentration with nice curative effect, indicating its potential bioapplication in the future. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  9. 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; Al