WorldWideScience

Sample records for hybridization fish probes

  1. Marine Fish Hybridization

    KAUST Repository

    He, Song

    2017-04-01

    Natural hybridization is reproduction (without artificial influence) between two or more species/populations which are distinguishable from each other by heritable characters. Natural hybridizations among marine fishes were highly underappreciated due to limited research effort; it seems that this phenomenon occurs more often than is commonly recognized. As hybridization plays an important role in biodiversity processes in the marine environment, detecting hybridization events and investigating hybridization is important to understand and protect biodiversity. The first chapter sets the framework for this disseration study. The Cohesion Species Concept was selected as the working definition of a species for this study as it can handle marine fish hybridization events. The concept does not require restrictive species boundaries. A general history and background of natural hybridization in marine fishes is reviewed during in chapter as well. Four marine fish hybridization cases were examed and documented in Chapters 2 to 5. In each case study, at least one diagnostic nuclear marker, screened from among ~14 candidate markers, was found to discriminate the putative hybridizing parent species. To further investigate genetic evidence to support the hybrid status for each hybrid offspring in each case, haploweb analysis on diagnostic markers (nuclear and/or mitochondrial) and the DAPC/PCA analysis on microsatellite data were used. By combining the genetic evidences, morphological traits, and ecological observations together, the potential reasons that triggered each hybridization events and the potential genetic/ecology effects could be discussed. In the last chapter, sequences from 82 pairs of hybridizing parents species (for which COI barcoding sequences were available either on GenBank or in our lab) were collected. By comparing the COI fragment p-distance between each hybridizing parent species, some general questions about marine fish hybridization were discussed: Is

  2. Temperature controlling system based on FISH probe hybridization facility%基于FISH探针杂交设备的温度控制系统

    Institute of Scientific and Technical Information of China (English)

    李天庆; 张化; 张婉尧

    2015-01-01

    以玻片为基础的FISH,它的变性杂交过程完全实现自动化.FISH探针杂交仪内置一个ARM7微处理器,以控制不同温度变化,满足变性、杂交和固定温度等不同模式,可预置用按键编程进去的程序(显示屏上能指导编程),方便客户调用,提高工作效率.温度控制系统是FISH探针杂交设备的核心部分,决定了杂交仪的可靠性、稳定性及精准性.%By using FISH based on slide,automation in the degeneration process of hybrid can be achieved.Installed an internal ARM7 microprocessor,the FISH probe hybridization facility can control temperature,meet different modes such as denaturation,hybridization,fixed temperature,and so on.Procedure input by means of push-button can be preset(there are guidance on display screen)to make things easy for user,as a result,work efficiency can be improved.The temperature controlling system,which is the core of FISH probe hybridization facility and ensure its reliability,stability and accuracy.

  3. Fluorescence in situ hybridization (FISH using non-commercial probes in the diagnosis of clinically suspected microdeletion syndromes

    Directory of Open Access Journals (Sweden)

    Ashutosh Halder

    2013-01-01

    Interpretation & conclusion: We conclude that FISH should not be the method of choice for clinically suspected microdeletion syndromes. We propose to follow strict clinical criteria for FISH testing or preferably to follow better methods (genotype first approach. Whole genome screening may be used as first line of test and FISH may be used for confirmation of screening result, screening of family members and prenatal diagnosis.

  4. International, collaborative assessment of limitations of chromosome-specific probes (CSP) and fluorescent in situ hybridization (FISH): Analysis of expected detections in 73,000 prenatal cases

    Energy Technology Data Exchange (ETDEWEB)

    Evans, M.I.; Henry, G.P.; Miller, W.A. [Wayne State Univ., Detroit, MI (United States)] [and others

    1994-09-01

    FISH and CSP have been proposed to reduce karyotyping need. The purpose of this study was to assess the potential efficacy of CSP-FISH using currently available probes (13, 18, 21, X, & Y) in large, prenatal diagnostic centers. Results (1990-1993) from 7 centers in 4 countries were divided by those expected to be detectable by currently available probes, and those which would be missed assuming 10% probe efficacy. 72,994 karyotypes included 699 trisomy 21`s, 352 trisomy 18`s, 136 trisomy 13`s, 358 sex chromosome aneuploidies, 70 triploidies, and 855 others (translocations, inversions, deletions, markers). Of 2,613 abnormalities, 1,745 would be detectable (66.8%). [Detroit 55.7%, Stockholm 68.3%, Boston 52.6%, Denver 61.3%, Muenster 77.0%, London 84.5%, Philadelphia 69.4%]. Centers with high proportions of referrals for ultrasound anomalies had the highest CSP-FISH positives secondary to increased T 18 & 13. We conclude: (1) 73,000 karyotypes show relatively consistent incidences of the common trisomies, sex chromosome abnormalities, and other chromosome abnormalities among the centers. (2) The proportion expected detectable by FISH-CSP technology varies from 52.6% to 84.5%, averaging 66.8%. (3) 1/3 of the karyotypic abnormalities would be missed, and therefore, replacement of complete karyotyping with FISH would have unacceptably high false-negative rates for routine evaluation. (4) FISH-CSP, while useful when positive for anomalies, is not sufficient when negative to obviate the need for a complete karyotype.

  5. Distant hybridization leads to different ploidy fishes

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Distant hybridization makes it possible to transfer the genome of one species to another, which results in changes in phenotypes and genotypes of the progenies. This study shows that distant hybridization or the combination of this method with gynogenesis or androgenesis lead to different ploidy fishes with genetic variation, including fertile tetraploid hybrids, sterile triploid hybrids, fertile diploid hybrids, fertile diploid gynogenetic fish, and their derived progenies. The formations of the different ploidy fishes depend on the genetic relationship between the parents. In this study, several types of distant hybridization, including red crucian carp (Carassius auratus red var.) (2n=100, abbreviated as RCC) (♀)×common carp (Cyprinus carpio L.) (2n=100, abbreviated as CC) (♂), and RCC (2n=100) (♀)×blunt snout bream (Megalobrama amblycephala) (2n=48, abbreviated as BSB) (♂) are described. In the distant hybridization of RCC (♀)×CC (♂), bisexual fertile F3–F18 allotetraploid hybrids (4n=200, abbreviated as 4nAT) were formed. The diploid hybrid eggs and diploid sperm generated by the females and males of 4nAT developed into diploid gynogenetic hybrids and diploid androgenetic hybrids, respectively, by gynogenesis and androgenesis, without treatment for doubling the chromosome. Improved tetraploid hybrids and improved diploid fishes with genetic variation were derived from the gynogenetic hybrid line. The improved diploid fishes included the high-body RCC and high-body goldfish. The formation of the tetraploid hybrids was related to the occurrence of unreduced gametes generated from the diploid hybrids, which involved in premeiotic endoreduplication, endomitosis, or fusion of germ cells. The sterile triploid hybrids (3n=150) were produced on a large scale by crossing the males of tetraploid hybrids with females of diploid fish (2n=100). In another distant hybridization of RCC (♀)×BSB (♂), different ploidy fishes were obtained, including

  6. Distant hybridization leads to different ploidy fishes.

    Science.gov (United States)

    Liu, ShaoJun

    2010-04-01

    Distant hybridization makes it possible to transfer the genome of one species to another, which results in changes in phenotypes and genotypes of the progenies. This study shows that distant hybridization or the combination of this method with gynogenesis or androgenesis lead to different ploidy fishes with genetic variation, including fertile tetraploid hybrids, sterile triploid hybrids, fertile diploid hybrids, fertile diploid gynogenetic fish, and their derived progenies. The formations of the different ploidy fishes depend on the genetic relationship between the parents. In this study, several types of distant hybridization, including red crucian carp (Carassius auratus red var.) (2n=100, abbreviated as RCC) (female) x common carp (Cyprinus carpio L.) (2n=100, abbreviated as CC) (male), and RCC (2n=100) (female) x blunt snout bream (Megalobrama amblycephala) (2n=48, abbreviated as BSB) (male) are described. In the distant hybridization of RCC (female) x CC (male), bisexual fertile F(3)-F(18) allotetraploid hybrids (4n=200, abbreviated as 4nAT) were formed. The diploid hybrid eggs and diploid sperm generated by the females and males of 4nAT developed into diploid gynogenetic hybrids and diploid androgenetic hybrids, respectively, by gynogenesis and androgenesis, without treatment for doubling the chromosome. Improved tetraploid hybrids and improved diploid fishes with genetic variation were derived from the gynogenetic hybrid line. The improved diploid fishes included the high-body RCC and high-body goldfish. The formation of the tetraploid hybrids was related to the occurrence of unreduced gametes generated from the diploid hybrids, which involved in premeiotic endoreduplication, endomitosis, or fusion of germ cells. The sterile triploid hybrids (3n=150) were produced on a large scale by crossing the males of tetraploid hybrids with females of diploid fish (2n=100). In another distant hybridization of RCC (female) x BSB (male), different ploidy fishes were

  7. Fluorescence In Situ Hybridization Probe Preparation.

    Science.gov (United States)

    Tolomeo, Doron; Stanyon, Roscoe R; Rocchi, Mariano

    2017-01-01

    The public human genome sequencing project utilized a hierarchical approach. A large number of BAC/PAC clones, with an insert size approximate from 50 kb to 300 kb, were identified and finely mapped with respect to the Sequence Tagged Site (STS) physical map and with respect to each other. A "golden path" of BACs, covering the entire human genome, was then selected and each clone was fully sequenced. The large number of remaining BACs was not fully sequenced, but the availability of the end sequence (~800-1000 bp) at each end allowed them to be very precisely mapped on the human genome.The search for copy number variations of the human genome used several strategies. One of these approaches took advantage of the fact that fosmid clones, contrary to BAC/PAC clones, have a fixed insert size (~40 kb) (Kidd et al., Nature 453: 56-64, 2008). In this context, the ends of ~7 million fosmid clones were sequenced, and therefore it was possible to precisely map these clones on the human genome.In summary, a large number of genomic clones (GC) are available for FISH experiments. They usually yield bright FISH signals and are extremely precious for molecular cytogenetics, and in particular cancer cytogenetics. The already-labeled probes available commercially are usually based on a combination of such GCs. The present chapter summarizes the protocols for extracting, labeling, and hybridization onto slides of DNA obtained from GC.

  8. Fluorescent hybridization probes for nucleic acid detection.

    Science.gov (United States)

    Guo, Jia; Ju, Jingyue; Turro, Nicholas J

    2012-04-01

    Due to their high sensitivity and selectivity, minimum interference with living biological systems, and ease of design and synthesis, fluorescent hybridization probes have been widely used to detect nucleic acids both in vivo and in vitro. Molecular beacons (MBs) and binary probes (BPs) are two very important hybridization probes that are designed based on well-established photophysical principles. These probes have shown particular applicability in a variety of studies, such as mRNA tracking, single nucleotide polymorphism (SNP) detection, polymerase chain reaction (PCR) monitoring, and microorganism identification. Molecular beacons are hairpin oligonucleotide probes that present distinctive fluorescent signatures in the presence and absence of their target. Binary probes consist of two fluorescently labeled oligonucleotide strands that can hybridize to adjacent regions of their target and generate distinctive fluorescence signals. These probes have been extensively studied and modified for different applications by modulating their structures or using various combinations of fluorophores, excimer-forming molecules, and metal complexes. This review describes the applicability and advantages of various hybridization probes that utilize novel and creative design to enhance their target detection sensitivity and specificity.

  9. Application of locked nucleic acid-based probes in fluorescence in situ hybridization

    DEFF Research Database (Denmark)

    Fontenete, Sílvia; Carvalho, Daniel R; Guimarães, Nuno

    2016-01-01

    Fluorescence in situ hybridization (FISH) employing nucleic acid mimics as probes is becoming an emerging molecular tool in the microbiology area for the detection and visualization of microorganisms. However, the impact that locked nucleic acid (LNA) and 2′-O-methyl (2′-OMe) RNA modifications have...... on the probe that is targeting microorganisms is unknown. In this study, the melting and hybridization efficiency properties of 18 different probes in regards to their use in FISH for the detection of the 16S rRNA of Helicobacter pylori were compared. For the same sequence and target, probe length and the type...

  10. Hybrid seine for full fish community collections

    Science.gov (United States)

    McKenna, James E.; Waldt, Emily M.; Abbett, Ross; David, Anthony; Snyder, James

    2013-01-01

    Seines are simple and effective fish collection gears, but the net mesh size influences how well the catch represents the fish communities. We designed and tested a hybrid seine with a dual-mesh bag (1/4″ and 1/8″) and compared the fish assemblage collected by each mesh. The fine-mesh net retained three times as many fish and collected more species (as many as eight), including representatives of several rare species, than did the coarser mesh. The dual-mesh bag permitted us to compare both sizes and species retained by each layer and to develop species-specific abundance correction factors, which allowed comparison of catches with the coarse-mesh seine used for earlier collections. The results indicate that a hybrid seine with coarse-mesh wings and a fine-mesh bag would enhance future studies of fish communities, especially when small-bodied fishes or early life stages are the research focus.

  11. Polymerase Chain Reaction-based Suppression of Repetitive Sequences in Whole Chromosome Painting Probes for FISH

    Energy Technology Data Exchange (ETDEWEB)

    Dugan, L C; Pattee, M; Williams, J; Eklund, M; Bedford, J S; Christian, A T

    2004-04-21

    We have developed a method to suppress the PCR amplification of repetitive sequences in whole chromosome painting probes by adding Cot-1 DNA to the amplification mixture. The repetitive sequences in the Cot-1 DNA bind to their homologous sequences in the probe library, prevent the binding of primers, and interfere with extension of the probe sequences, greatly decreasing PCR efficiency selectively across these blocked regions. A second labeling reaction is then done and this product is resuspended in FISH hybridization mixture without further addition of blocking DNA. The hybridization produces little if any non-specific binding on any other chromosomes. We have been able to successfully use this procedure with both human and rat chromosome probes. This technique should be applicable in producing probes for CGH, M-FISH and SKY, as well as reducing the presence of repetitive DNA in genomic libraries.

  12. Detection of MDM2/CDK4 amplification in lipomatous soft tissue tumors from formalin-fixed, paraffin-embedded tissue: comparison of multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH).

    Science.gov (United States)

    Creytens, David; van Gorp, Joost; Ferdinande, Liesbeth; Speel, Ernst-Jan; Libbrecht, Louis

    2015-02-01

    In this study, the detection of MDM2 and CDK4 amplification was evaluated in lipomatous soft tissue tumors using multiplex ligation-dependent probe amplification (MLPA), a PCR-based technique, in comparison with fluorescence in situ hybridization (FISH). These 2 techniques were evaluated in a series of 77 formalin-fixed, paraffin-embedded lipomatous tumors (27 benign adipose tumors, 28 atypical lipomatous tumors/well-differentiated liposarcomas, 18 dedifferentiated liposarcomas, and 4 pleomorphic liposarcomas). Using MLPA, with a cut-off ratio of >2, 36/71 samples (22 atypical lipomatous tumors/well-differentiated liposarcomas, and 14 dedifferentiated liposarcomas) showed MDM2 and CDK4 amplification. Using FISH as gold standard, MLPA showed a sensitivity of 90% (36/40) and a specificity of 100% (31/31) in detecting amplification of MDM2 and CDK4 in lipomatous soft tissue tumors. In case of high-level amplification (MDM2-CDK4/CEP12 ratio >5), concordance was 100%. Four cases of atypical lipomatous tumor/well-differentiated liposarcoma (4/26, 15%) with a low MDM2 and CDK4 amplification level (MDM2-CDK4/CEP12 ratio ranging between 2 and 2.5) detected by FISH showed no amplification by MLPA, although gain of MDM2 and CDK4 (ratios ranging between 1.6 and 1.9) was seen with MLPA. No amplification was detected in benign lipomatous tumors and pleomorphic liposarcomas. Furthermore, there was a very high concordance between the ratios obtained by FISH and MLPA. In conclusion, MLPA proves to be an appropriate and straightforward technique for screening MDM2/CDK4 amplification in lipomatous tumors, especially when a correct cut-off value and reference samples are chosen, and could be considered a good alternative to FISH to determine MDM2 and CDK4 amplification in liposarcomas. Moreover, because MLPA, as a multiplex technique, allows simultaneous detection of multiple chromosomal changes of interest, it could be in the future a very reliable and fast molecular analysis on

  13. Role of fluorescence in situ hybridization (FISH) in sequencing the tomato genome

    Science.gov (United States)

    Chromosomes at various stages of the cell cycle can be used for localization of DNA probes via Fluorescence in situ hybridization (FISH). While mitotic metaphase chromosomes are demonstrably too short and compact for this purpose, long pachytene chromosomes are ideal. BACs that hybridize to euchrom...

  14. Application of fluorescence in situ hybridization (FISH) to the ...

    African Journals Online (AJOL)

    Application of fluorescence in situ hybridization (FISH) to the analysis of ... In this study, fluorescent in situ hybridization (FISH) as a culture-independent molecular ... a high percentage and took place in an oily biological system under aerobic ...

  15. Identification of Cannabis sativa L. using the 1-kbTHCA synthase-fluorescence in situ hybridization probe.

    Science.gov (United States)

    Jeangkhwoa, Pattraporn; Bandhaya, Achirapa; Umpunjun, Puangpaka; Chuenboonngarm, Ngarmnij; Panvisavas, Nathinee

    2017-03-01

    This study reports a successful application of fluorescence in situ hybridization (FISH) technique in the identification of Cannabis sativa L. cells recovered from fresh and dried powdered plant materials. Two biotin-16-dUTP-labeled FISH probes were designed from the Cannabis-specific tetrahydrocannabinolic acid synthase (THCAS) gene and the ITS region of the 45S rRNA gene. Specificity of probe-target hybridization was tested against the target and 4 non-target plant species, i.e., Humulus lupulus, Mitragyna speciosa, Papaver sp., and Nicotiana tabacum. The 1-kb THCA synthase hybridization probe gave Cannabis-specific hybridization signals, unlike the 700-bp Cannabis-ITS hybridization probe. Probe-target hybridization was also confirmed against 20 individual Cannabis plant samples. The 1-kb THCA synthase and 700-bp Cannabis-ITS hybridization probes clearly showed 2 hybridization signals per cell with reproducibility. The 1-kb THCA synthase probe did not give any FISH signal when tested against H. lupulus, its closely related member of the Canabaceae family. It was also showed that 1-kb THCA synthase FISH probe can be applied to identify small amount of dried powdered Cannabis material with an addition of rehydration step prior to the experimental process. This study provided an alternative identification method for Cannabis trace. Copyright © 2016. Published by Elsevier B.V.

  16. Optimized Fast-FISH with a-satellite probes: acceleration by microwave activation

    Directory of Open Access Journals (Sweden)

    Durm M.

    1997-01-01

    Full Text Available It has been shown for several DNA probes that the recently introduced Fast-FISH (fluorescence in situ hybridization technique is well suited for quantitative microscopy. For highly repetitive DNA probes the hybridization (renaturation time and the number of subsequent washing steps were reduced considerably by omitting denaturing chemical agents (e.g., formamide. The appropriate hybridization temperature and time allow a clear discrimination between major and minor binding sites by quantitative fluorescence microscopy. The well-defined physical conditions for hybridization permit automatization of the procedure, e.g., by a programmable thermal cycler. Here, we present optimized conditions for a commercially available X-specific a-satellite probe. Highly fluorescent major binding sites were obtained for 74oC hybridization temperature and 60 min hybridization time. They were clearly discriminated from some low fluorescent minor binding sites on metaphase chromosomes as well as in interphase cell nuclei. On average, a total of 3.43 ± 1.59 binding sites were measured in metaphase spreads, and 2.69 ± 1.00 in interphase nuclei. Microwave activation for denaturation and hybridization was tested to accelerate the procedure. The slides with the target material and the hybridization buffer were placed in a standard microwave oven. After denaturation for 20 s at 900 W, hybridization was performed for 4 min at 90 W. The suitability of a microwave oven for Fast-FISH was confirmed by the application to a chromosome 1-specific a-satellite probe. In this case, denaturation was performed at 630 W for 60 s and hybridization at 90 W for 5 min. In all cases, the results were analyzed quantitatively and compared to the results obtained by Fast-FISH. The major binding sites were clearly discriminated by their brightness

  17. TM7 detection in human microbiome: are PCR primers and FISH probes specific enough?

    Science.gov (United States)

    Sizova, Maria V.; Doerfert, Sebastian N.; Gavrish, Ekaterina; Epstein, Slava S.

    2015-01-01

    TM7 appears important and omnipresent because it is repeatedly detected by molecular techniques in diverse environments. Here we report that most of primers and FISH probes thought to be TM7-specific do hybridize with multiple species from oral and vaginal cavity. This calls for re-examination of TM7 distribution and abundance. PMID:25957511

  18. [Optimized protocols for interphase FISH analysis of imprints and sections using split signal probes].

    Science.gov (United States)

    Pelluard-Nehme, F; Dupont, T; Turmo, M; Merlio, J-P; Belaud-Rotureau, M-A

    2007-03-01

    Fluorescent in situ hybridization (FISH) analysis is a molecular technique allowing the detection of recurrent translocations in cancer. Several hybridization protocols were assayed in order to evaluate their performances for interphase FISH analysis of histological sections and imprints using split probes. Adult and foetal lymphoid tissues were selected. Touch imprints of fresh (EF) or frozen (EC) tissues, sections (CF) and isolated nuclei (NI) of formol-fixed paraffin-embedded tissues were performed. The cut-off values of the IGH, IGlambda, BCL-2, BCL-6, CCND1 and MYC DNA FISH split signal probes were calculated for adult reactive lymph nodes on the different histological preparations (EC, CF, CC, NI) and on several tissues for the IGH and BCL-6 probes. In reactive lymph nodes, the cut-off values of the probes were between 3 and 13% and found independent of the preparation type. Conversely, slight but significant variations of the cut-off level were observed when different foetal control tissues were assayed with the same probe set. Finally, this study provided optimized-protocols for FISH analysis of either fresh/frozen imprints or formalin-fixed paraffin-embedded sections using split signal DNA probes.

  19. Automated design of probes for rRNA-targeted fluorescence in situ hybridization reveals the advantages of using dual probes for accurate identification.

    Science.gov (United States)

    Wright, Erik S; Yilmaz, L Safak; Corcoran, Andrew M; Ökten, Hatice E; Noguera, Daniel R

    2014-08-01

    Fluorescence in situ hybridization (FISH) is a common technique for identifying cells in their natural environment and is often used to complement next-generation sequencing approaches as an integral part of the full-cycle rRNA approach. A major challenge in FISH is the design of oligonucleotide probes with high sensitivity and specificity to their target group. The rapidly expanding number of rRNA sequences has increased awareness of the number of potential nontargets for every FISH probe, making the design of new FISH probes challenging using traditional methods. In this study, we conducted a systematic analysis of published probes that revealed that many have insufficient coverage or specificity for their intended target group. Therefore, we developed an improved thermodynamic model of FISH that can be applied at any taxonomic level, used the model to systematically design probes for all recognized genera of bacteria and archaea, and identified potential cross-hybridizations for the selected probes. This analysis resulted in high-specificity probes for 35.6% of the genera when a single probe was used in the absence of competitor probes and for 60.9% when up to two competitor probes were used. Requiring the hybridization of two independent probes for positive identification further increased specificity. In this case, we could design highly specific probe sets for up to 68.5% of the genera without the use of competitor probes and 87.7% when up to two competitor probes were used. The probes designed in this study, as well as tools for designing new probes, are available online (http://DECIPHER.cee.wisc.edu).

  20. Probing Compositional Variation within Hybrid Nanostructures

    Energy Technology Data Exchange (ETDEWEB)

    Yuhas, Benjamin D.; Habas, Susan E.; Fakra, Sirine C.; Mokari, Taleb

    2010-06-22

    We present a detailed analysis of the structural and magnetic properties of solution-grown PtCo-CdS hybrid structures in comparison to similar free-standing PtCo alloy nanoparticles. X-ray absorption spectroscopy is utilized as a sensitive probe for identifying subtle differences in the structure of the hybrid materials. We found that the growth of bimetallic tips on a CdS nanorod substrate leads to a more complex nanoparticle structure composed of a PtCo alloy core and thin CoO shell. The core-shell architecture is an unexpected consequence of the different nanoparticle growth mechanism on the nanorod tip, as compared to free growth in solution. Magnetic measurements indicate that the PtCo-CdS hybrid structures are superparamagnetic despite the presence of a CoO shell. The use of X-ray spectroscopic techniques to detect minute differences in atomic structure and bonding in complex nanosystems makes it possible to better understand and predict catalytic or magnetic properties for nanoscale bimetallic hybrid materials.

  1. Fluorescent in situ hybridization (FISH on the veterinary diagnostic field

    Directory of Open Access Journals (Sweden)

    Ján Dianovský

    2016-09-01

    Full Text Available Proceeding deals with of genomic changes detectable by FISH . The DSD syndrom in Yorkshire terrier 78,XY t (Y-;6p+ was observed by the use of X and Y FISH WCP probes. Following results indicated numerous genomic changes in cancers. Using comparative genomic hybridization numerous chromosomal rearrangements were detected, which indicated the heterogeneity in tumour growth. In Bernese Mountain Dog bitch,8 loses on chromosomes and gains on 18 different of chromosomes were detected. The last study was focused on chromosomal position and nucleotide sequencing of the LCA5L exons. Those were analysed in cattle of BTA1q44, sheep OAR1q43 and of CHI1q44 in the goats.

  2. The Selection and Use of Sorghum (Sorghum propinquum Bacterial Artificial Chromosomes as Cytogenetic FISH Probes for Maize (Zea mays L.

    Directory of Open Access Journals (Sweden)

    Debbie M. Figueroa

    2011-01-01

    Full Text Available The integration of genetic and physical maps of maize is progressing rapidly, but the cytogenetic maps lag behind, with the exception of the pachytene fluorescence in situ hybridization (FISH maps of maize chromosome 9. We sought to produce integrated FISH maps of other maize chromosomes using Core Bin Marker loci. Because these 1 Kb restriction fragment length polymorphism (RFLP probes are below the FISH detection limit, we used BACs from sorghum, a small-genome relative of maize, as surrogate clones for FISH mapping. We sequenced 151 maize RFLP probes and compared in silico BAC selection methods to that of library filter hybridization and found the latter to be the best. BAC library screening, clone verification, and single-clone selection criteria are presented along with an example of transgenomic BAC FISH mapping. This strategy has been used to facilitate the integration of RFLP and FISH maps in other large-genome species.

  3. The selection and use of sorghum (Sorghum propinquum) bacterial artificial chromosomes as cytogenetic FISH probes for maize (Zea mays L.).

    Science.gov (United States)

    Figueroa, Debbie M; Davis, James D; Strobel, Cornelia; Conejo, Maria S; Beckham, Katherine D; Ring, Brian C; Bass, Hank W

    2011-01-01

    The integration of genetic and physical maps of maize is progressing rapidly, but the cytogenetic maps lag behind, with the exception of the pachytene fluorescence in situ hybridization (FISH) maps of maize chromosome 9. We sought to produce integrated FISH maps of other maize chromosomes using Core Bin Marker loci. Because these 1 Kb restriction fragment length polymorphism (RFLP) probes are below the FISH detection limit, we used BACs from sorghum, a small-genome relative of maize, as surrogate clones for FISH mapping. We sequenced 151 maize RFLP probes and compared in silico BAC selection methods to that of library filter hybridization and found the latter to be the best. BAC library screening, clone verification, and single-clone selection criteria are presented along with an example of transgenomic BAC FISH mapping. This strategy has been used to facilitate the integration of RFLP and FISH maps in other large-genome species.

  4. FISH of uncultured amniocytes for prenatal diagnosis: Experience in 24 cases using commercially available probes

    Energy Technology Data Exchange (ETDEWEB)

    Weremowicz, S.; Sandstrom, M.McH.; Walsh, K.A. [Harvard Medical School, Boston, MA (United States)] [and others

    1994-09-01

    Rapid prenatal diagnosis of chromosomal aneuploidies is being requested increasingly by physicians at our institution. We report our experience in providing rapid diagnoses in prenatal samples referred following an abnormal ultrasound examination (n=22) and for confirmation of trisomy 21 prior to selective termination in a twin gestation (n=22). Uncultured amniocytes (46,XY) and cultured lymphocytes (46,SY) were used as control cells and a DXZ1 probe was hybridized separately from the test probes as a control probe. In 23 cases our FISH interpretation was concordant with the cytogenetic analysis. In one case referred to rule out trisomy 21 in which cystic hygroma was detected on ultrasound exam in a 35 y.o. G2 P1, a FISH interpretation of disomy 21 was based on 18% of cells with 1 signal, 65% with 2, 15% with 3, and 2% with 4; the large percentage of 3 signals was also reported. Cytogenetic analysis was 47,XX,+21 in 63 metaphases. Subsequent FISH analysis of metaphases revealed a large number of chromosomes 21 with only one site of hybridization that might have contributed to the discordant interpretation. Whether this result reflects population polymorphism in hybridization of this cosmid remains to be elucidated. Our findings confirm use of FISH as an invaluable adjunct to conventional cytogenetics; however, results must be interpreted cautiously until larger numbers of cases have been analyzed to detect potentially rare events.

  5. Towards Fluorescence In Vivo Hybridization (FIVH) Detection of H. pylori in Gastric Mucosa Using Advanced LNA Probes

    DEFF Research Database (Denmark)

    Fontenete, Sílvia; Leite, Marina; Guimarães, Nuno

    2015-01-01

    acid (LNA)/ 2' O-methyl RNA (2'OMe) probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization......In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo...

  6. A simple protocol for attenuating the auto-fluorescence of cyanobacteria for optimized fluorescence in situ hybridization (FISH) imaging.

    Science.gov (United States)

    Zeller, Perrine; Ploux, Olivier; Méjean, Annick

    2016-03-01

    Cyanobacteria contain pigments, which generate auto-fluorescence that interferes with fluorescence in situ hybridization (FISH) imaging of cyanobacteria. We describe simple chemical treatments using CuSO4 or H2O2 that significantly reduce the auto-fluorescence of Microcystis strains. These protocols were successfully applied in FISH experiments using 16S rRNA specific probes and filamentous cyanobacteria.

  7. Marine genetic swamping: hybrids replace an obligately estuarine fish.

    Science.gov (United States)

    Roberts, David G; Gray, Charles A; West, Ronald J; Ayre, David J

    2010-02-01

    Populations of obligately estuarine taxa are potentially small and isolated and may lack genetic variation and display regional differentiation as a result of drift and inbreeding. Hybridization with a wide-ranging marine congener should introduce genetic variation and reduce the effects of inbreeding depression and genetic drift. However, high levels of hybridization can cause demographic and genetic swamping. In southeastern Australia hybridization occurs between obligately estuarine Black bream (Acanthopagrus butcheri) and migratory marine Yellowfin bream (Acanthopagrus australis). Here, we surveyed genetic variation at eight microsatellite loci and the mitochondrial control region of juvenile fish from five coastal lagoons (including temporal replication in two lagoons) (total n = 970) to determine the frequency and persistence of hybridization, and its likely consequence for the estuarine restricted A. butcheri. Of 688 juvenile fish genotyped 95% were either A. australis (347) or hybrids (309); only 5% (32) were A. butcheri. Most hybrids were later generation hybrids or A. butcheri backcrosses, which are likely multi-generational residents within lagoons. Far greater proportions of hybrid juveniles were found within two lagoons that are generally closed to the ocean (>90% hybrid fish within generally closed lagoons vs. 12-27% in permanently or intermittently open lagoons). In both lagoons, this was consistent across multiple cohorts of fish [79-97% hybrid fish (n = 282)]. Hybridization and introgression represent a major threat to the persistence of A. butcheri and have yet to be investigated for large numbers of estuarine taxa.

  8. Fluoroscence in situ hybridization of chicken intestinal samples with bacterial rRNA targeted oligonucleotide probes

    DEFF Research Database (Denmark)

    Olsen, Katja Nyholm; Francesch, M.; Christensen, Henrik

    2006-01-01

    The objective was to develop a fast and accurate molecular method for the quantification of the intestinal flora in chickens by rRNA fluorescence in situ hybridization (FISH). Seven weeks old conventionally reared Lohmann hens were used to set up the method. To sample ileal intestinal content......, the distal part from Meckels diverticulum to the ileo-caecal junction was removed. Fixation was performed in ethanol and phosphate buffered saline. After washing by centrifugation, the sample was resuspended in pre-heated hybridization buffer with oligonucleotide probe labelled with Cy3 (10ng/µl). The cells...... were hybridized for 24-72h, centrifuged, washed with pre-heated hybridization buffer, centrifuged and resuspended in Millipore quality water before filtration onto a 0.22 µm black polycarbonate filter. The probes used in this study were, LGC354A, LGC354B, LGC354C, Strc493, Bacto1080, Sal3, Chis150, EUB...

  9. Telomere analysis of platyhelminths and acanthocephalans by FISH and Southern hybridization.

    Science.gov (United States)

    Bombarová, Marta; Vítková, Magda; Spakulová, Marta; Koubková, Bozena

    2009-11-01

    We examined the composition of telomeres in chromosomes of parasitic worms, representatives of the flatworm groups Monogenea and Cestoda (Platyhelminthes), and thorny-headed worms (Syndermata: Acanthocephala) by fluorescence in situ hybridization (FISH) with different telomeric repeat probes. Our results show that the (TTAGGG)n sequence, supposed to be the ancestral telomeric repeat motif of Metazoa, is conserved in Monogenea (Paradiplozoon homoion) and Cestoda (Caryophyllaeus laticeps, Caryophyllaeides fennica, and Nippotaenia mogurndae) but not in Acanthocephala (Pomphorhynchus laevis and Pomphorhynchus tereticollis). In the Pomphorhynchus species, no hybridization signals were obtained with the "nematode" (TTAGGC)n, "arthropod" (TTAGG)n, and bdelloid (TGTGGG)n telomeric probes using FISH with their chromosomes and Southern hybridization with P. laevis DNA. Therefore, we suggest that parasitic Acanthocephala have evolved yet unknown telomeric repeat motifs or different mechanisms of telomere maintenance.

  10. APPLICATION OF TWO-COLOR INTERPHASE FISH USING SEX PROBE IN ALLOGENEIC STEM CELL TRANSPLANTATION

    Institute of Scientific and Technical Information of China (English)

    曾慧兰; 李建勇; 朱康儿; 薛永权; 李杨秋; 刘晓力; 过宇

    2002-01-01

    Objective: To evaluate the significance of two-color interphase fluorescence in situ hybridization (FISH) using X and Y centromere probe in the engraftment estimation and minimal residual disease (MRD) monitoring after allogeneic stem cell transplantation (alloSCT). Methods: Samples from 12 cases patients in different periods after alloSCT were detected by interphase FISH. Results: All of the 12 patients were proved to obtain engraftment 22(35 days after alloSCT. While traditional karyotype showed as 100%XX or 100%XY invariably, FISH showed different percentages of donor original sex chromosome. Conclusion: Two-color interphase FISH is a more sensitive and simple test for engraftment evaluation and MRD monitoring post SCT, though, it can not entirely replace traditional karyotype analysis and gene detection by RT-PCR.

  11. Studies of the Ecophysiology of Single Cells in Microbial Communities by (Quantitative) Microautoradiography and Fluorescence In Situ Hybridization (MAR-FISH)

    DEFF Research Database (Denmark)

    Nierychlo, Marta; Nielsen, Jeppe Lund; Nielsen, Per Halkjær

    2015-01-01

    Microautoradiography (MAR) in combination with fluorescence in situ hybridization (FISH) is a powerful method of obtaining information about the ecophysiology of probe-defined single cells in mixed microbial communities. The incorporation of radiolabelled substrates can be quantified by automated...

  12. Validation of break-apart and fusion MYC probes using a digital fluorescence in situ hybridization capture and imaging system

    Directory of Open Access Journals (Sweden)

    Michael Liew

    2016-01-01

    Full Text Available Introduction: Detection of MYC translocations using fluorescence in situ hybridization (FISH is important in the evaluation of lymphomas, in particular, Burkitt lymphoma and diffuse large B-cell lymphoma. Our aim was to validate a digital FISH capture and imaging system for the detection of MYC 8q24 translocations using LSI-MYC (a break-apart probe and MYC 8;14 translocation using IGH-MYC (a fusion probe. Materials and Methods: LSI-MYC probe was evaluated using tissue sections from 35 patients. IGH-MYC probe was evaluated using tissue sections from forty patients. Sections were processed for FISH and analyzed using traditional methods. FISH slides were then analyzed using the GenASIs capture and analysis system. Results: Results for LSI-MYC had a high degree of correlation between traditional method of FISH analysis and digital FISH analysis. Results for IGH-MYC had a 100% concordance between traditional method of FISH analysis and digital FISH analysis. Conclusion: Annotated whole slide images of H and E and FISH sections can be digitally aligned, so that areas of tumor within a section can be matched and evaluated with a greater degree of accuracy. Images can be archived permanently, providing a means for examining the results retrospectively. Digital FISH imaging of the MYC translocations provides a better diagnostic tool compared to traditional methods for evaluating lymphomas.

  13. Validation of break-apart and fusion MYC probes using a digital fluorescence in situ hybridization capture and imaging system

    Science.gov (United States)

    Liew, Michael; Rowe, Leslie; Clement, Parker W.; Miles, Rodney R.; Salama, Mohamed E.

    2016-01-01

    Introduction: Detection of MYC translocations using fluorescence in situ hybridization (FISH) is important in the evaluation of lymphomas, in particular, Burkitt lymphoma and diffuse large B-cell lymphoma. Our aim was to validate a digital FISH capture and imaging system for the detection of MYC 8q24 translocations using LSI-MYC (a break-apart probe) and MYC 8;14 translocation using IGH-MYC (a fusion probe). Materials and Methods: LSI-MYC probe was evaluated using tissue sections from 35 patients. IGH-MYC probe was evaluated using tissue sections from forty patients. Sections were processed for FISH and analyzed using traditional methods. FISH slides were then analyzed using the GenASIs capture and analysis system. Results: Results for LSI-MYC had a high degree of correlation between traditional method of FISH analysis and digital FISH analysis. Results for IGH-MYC had a 100% concordance between traditional method of FISH analysis and digital FISH analysis. Conclusion: Annotated whole slide images of H and E and FISH sections can be digitally aligned, so that areas of tumor within a section can be matched and evaluated with a greater degree of accuracy. Images can be archived permanently, providing a means for examining the results retrospectively. Digital FISH imaging of the MYC translocations provides a better diagnostic tool compared to traditional methods for evaluating lymphomas. PMID:27217970

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

    Science.gov (United States)

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

    2014-01-01

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

  15. Interphase cytogenetics in pathology: principles, methods, and applications of fluorescence in situ hybridization (FISH).

    Science.gov (United States)

    Werner, M; Wilkens, L; Aubele, M; Nolte, M; Zitzelsberger, H; Komminoth, P

    1997-01-01

    Characteristic chromosome aberrations have been identified in various tumors. Fluorescence in situ hybridization (FISH) using specific probes that are generated by vector cloning or in vitro amplification and labeled with fluorescent dyes allow for the detection of these genetic changes in interphase cells. This technique, that is also referred to as "interphase cytogenetics", can be performed in cytological preparations as well as in sections of routinely formaldehyde-fixed and paraffin-embedded tissue. In cancer research and diagnostics, interphase cytogenetics by FISH is used to detect numerical chromosome changes and structural aberrations, e.g., translocations, deletions, or amplifications. In this technical overview, we explain the principles of the FISH method and provide protocols for FISH in cytological preparations and paraffin sections. Moreover, possible applications of FISH are discussed.

  16. Construction of a repeat-free dual color fluorescent in situ hybridization probe for ROS1 gene in non-small cell lung cancer diagnosis

    Institute of Scientific and Technical Information of China (English)

    程弘夏

    2014-01-01

    Objective To establish a repeat-free ROS1 gene fluorescence in situ hybridization(FISH)probe,and to compare its efficacy with those of commercial FISH probes in non-small cell lung cancer.Methods The probe was constructed by combining human Cot-1 DNA genome into double-stranded sequence,and then digested by duples specific nuclease to establish a repeat-free sequene.The final repeat-free ROS1 FISH probe was labeled by red and green fluoresceins.Results Compared

  17. itFISH: Enhanced Staining by Iterative Fluorescent In Situ Hybridization.

    Science.gov (United States)

    Row, Richard H; Martin, Benjamin L

    2017-03-20

    Fluorescent in situ hybridization (FISH) is an important tool for zebrafish research, particularly when observing the expression of two different genes in the same embryo. Peroxidase-catalyzed deposition of tyramide-conjugated dyes is a widely used and cost-effective approach to performing FISH. A major limitation of the technique is that it does not work well for weakly expressed genes. Here we present a method adapted from planarian research for use in zebrafish that provides a dramatic enhancement of weak staining. By iterating the antibody staining and development steps, a strong signal can be obtained from probes that were previously too weak to detect.

  18. Phylogeny and FISH probe analysis of the “Candidatus Competibacter”-lineage in wastewater treatment systems

    DEFF Research Database (Denmark)

    Nittami, Tadashi; McIlroy, Simon Jon; Kanai, Eri

    . This study aimed at resolving the phylogeny and reassessing FISH probes for coverage of the diversity of the lineage in EBPR systems. Analysis of the level of 16S rRNA gene sequence diversity of lineage members (> 89%) suggests that it is more appropriately viewed as a family rather than a single genus......Our understanding of the microbial ecology of enhanced biological phosphorus removal (EBPR) wastewater treatment systems has been greatly advanced through the application of molecular methods such as fluorescence in situ hybridization (FISH). Considerable attention has been directed...... the gammaproteobacterial “Candidatus Competibacter”-lineage. The group is currently delineated by 8 FISH probe defined phylotypes, although further undescribed phylogenetic diversity beyond what is covered by these probes is evident. Where studied, marked differences in physiology between members are observed, including...

  19. LNA probes substantially improve the detection of bacterial endosymbionts in whole mount of insects by fluorescent in-situ hybridization

    Directory of Open Access Journals (Sweden)

    Priya Natarajan

    2012-05-01

    Full Text Available Abstract Background Detection of unculturable bacteria and their localization in the host, by fluorescent in-situ hybridization (FISH, is a powerful technique in the study of host-bacteria interaction. FISH probes are designed to target the 16 s rRNA region of the bacteria to be detected. LNA probes have recently been used in FISH studies and proven to be more efficient. To date no report has employed LNA probes for FISH detection of bacterial endosymbiont in the whole mount tissues. Further, though speculated, bacteriocytes have not been reported from males of Bemisia tabaci. Results In this study, we compared the efficiency in detecting bacteria by fluorescent DNA oligonucleotides versus modified probes containing Locked Nucleic Acid (LNA substitution in their structure. We used the insect Bemisia tabaci as the experimental material since it carried simultaneous infection by two bacteria: one a primary endosymbiont, Portiera (and present in more numbers while the other a secondary endosymbiont Arsenophonus (and present in less numbers. Thus a variation in the abundance of bacteria was expected. While detecting both the bacteria, we found a significant increase in the signal whenever LNA probes were used. However, the difference was more pronounced in detecting the secondary endosymbiont, wherein DNA probes gave weak signals when compared to LNA probes. Also, signal to noise ratio for LNA probes was higher than DNA probes. We found that LNA considerably improved sensitivity of FISH, as compared to the commonly used DNA oligonucleotide probe. Conclusion By employing LNA probes we could detect endosymbiotic bacteria in males, which have never been reported previously. We were able to detect bacteriocytes containing Portiera and Arsenophonus in the males of B. tabaci. Thus, employing LNA probes at optimized conditions will help to significantly improve detection of bacteria at the lowest concentration and may give a comprehensible depiction

  20. Ancient hybridization and genomic stabilization in a swordtail fish.

    Science.gov (United States)

    Schumer, Molly; Cui, Rongfeng; Powell, Daniel L; Rosenthal, Gil G; Andolfatto, Peter

    2016-06-01

    A rapidly increasing body of work is revealing that the genomes of distinct species often exhibit hybrid ancestry, presumably due to postspeciation hybridization between closely related species. Despite the growing number of documented cases, we still know relatively little about how genomes evolve and stabilize following hybridization, and to what extent hybridization is functionally relevant. Here, we examine the case of Xiphophorus nezahualcoyotl, a teleost fish whose genome exhibits significant hybrid ancestry. We show that hybridization was relatively ancient and is unlikely to be ongoing. Strikingly, the genome of X. nezahualcoyotl has largely stabilized following hybridization, distinguishing it from examples such as human-Neanderthal hybridization. Hybridization-derived regions are remarkably distinct from other regions of the genome, tending to be enriched in genomic regions with reduced constraint. These results suggest that selection has played a role in removing hybrid ancestry from certain functionally important regions. Combined with findings in other systems, our results raise many questions about the process of genomic stabilization and the role of selection in shaping patterns of hybrid ancestry in the genome. © 2016 John Wiley & Sons Ltd.

  1. Parasitic fauna in hybrid tambacu from fish farms

    Directory of Open Access Journals (Sweden)

    Ronilson Macedo Silva

    2013-08-01

    Full Text Available The objective of this work was to evaluate the parasitic fauna of hybrid tambacu (Colossoma macropomum x Piaractus mesopotamicus from fish farms and the host-parasite relationship. A hundred and fourteen fish were collected from four fish farms in Macapá, in the state of Amapá, Brazil, 80.7% of which were infected by: Ichthyophthirius multifiliis (Ciliophora; Piscinoodinium pillulare (Dinoflagellida; Anacanthorus spatulatus, Notozothecium janauachensis, and Mymarothecium viatorum (Monogenoidea; Neoechinorhynchus buttnerae (Acanthocephala; Cucullanus colossomi (Nematoda; Perulernaea gamitanae (Lernaeidae; and Proteocephalidae larvae (Cestoda. A total of 8,136,252 parasites were collected from the examined fish. This is the first record of N. buttnerae, C. colossomi, N. janauachensis, M. viatorum, and Proteocephalidae for hybrid tambacu in Brazil. Ichthyophthirius multifiliis was the most prevalent parasite, whereas endohelminths were the less. A positive correlation was observed between number of I. multifiliis and total length and weight of fish, as well as between number of P. gamitanae and total length. The infection by I. multifiliis had association with the parasitism by Monogenoidea. Low water quality contributes to high parasitism of hybrid tambacu by ectoparasites, which, however, does not influence the relative condition factor of fish.

  2. Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

    DEFF Research Database (Denmark)

    Ronander, Elena; Bengtsson, Dominique C; Joergensen, Louise;

    2012-01-01

    and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA...... fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE(1). Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human...... erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System(2) (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription...

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

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Gyoyeon [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Biological Chemistry, Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Deajeon (Korea, Republic of); Lee, Hansol [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Lee, Jiyeon, E-mail: jylee@kist.re.kr [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Biological Chemistry, Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Deajeon (Korea, Republic of)

    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.

  4. Prediction of melting temperatures in fluorescence in situ hybridization (FISH) procedures using thermodynamic models.

    Science.gov (United States)

    Fontenete, Sílvia; Guimarães, Nuno; Wengel, Jesper; Azevedo, Nuno Filipe

    2016-01-01

    The thermodynamics and kinetics of DNA hybridization, i.e. the process of self-assembly of one, two or more complementary nucleic acid strands, has been studied for many years. The appearance of the nearest-neighbor model led to several theoretical and experimental papers on DNA thermodynamics that provide reasonably accurate thermodynamic information on nucleic acid duplexes and allow estimation of the melting temperature. Because there are no thermodynamic models specifically developed to predict the hybridization temperature of a probe used in a fluorescence in situ hybridization (FISH) procedure, the melting temperature is used as a reference, together with corrections for certain compounds that are used during FISH. However, the quantitative relation between melting and experimental FISH temperatures is poorly described. In this review, various models used to predict the melting temperature for rRNA targets, for DNA oligonucleotides and for nucleic acid mimics (chemically modified oligonucleotides), will be addressed in detail, together with a critical assessment of how this information should be used in FISH.

  5. RNA probes, transcribed from synthetic DNA, for in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Brysch, W.; Hagendorff, G.; Schlingensiepen, K.H.

    1988-03-25

    Single stranded cRNA probes are ideal for in-situ-hybridization. Synthetic oligodesoxy-ribonucleotides on the other hand allow one to chose nucleotide sequences independently of restriction sites and availability of cloned templates. To combine the advantages of these two methods, the authors used an oligonucleotide, containing a T7-RNA-polymerase promotor sequence and a starting sequence of 6 bases as a template for an in-vitro-transcription reaction with T7-RNA-polymerase. A second oligonucleotide, complementary to basepairs 1-101 was also synthesized and the two strands heated to 95/sup 0/ for 3 min, then kept at 65/sup 0/C for one hour in 80 mM Tris, 12mM MgCl, 4 mM Spermidine, 0,04% Triton and finally cooled on ice. The resulting double stranded DNA was used as a template to transcribe /sup 35/S-labelled cRNA, using DNA, T7-Polymerase, /sup 35/S-UTP, ATP, GTP and CTP and RNasin (Promega). No difference could be observed comparing the resulting hybridization pattern with that obtained by using a plasmid derived cRNA probe of rat brain sodium channel II. Moreover the hybridization signal was clearly distinct from the background labelling resulting from hybridization with a sense control probe of the same specific activity.

  6. The chromosomal constitution of fish hybrid lineage revealed by 5S rDNA FISH.

    Science.gov (United States)

    Zhang, Chun; Ye, Lihai; Chen, Yiyi; Xiao, Jun; Wu, Yanhong; Tao, Min; Xiao, Yamei; Liu, Shaojun

    2015-12-03

    The establishment of the bisexual fertile fish hybrid lineage including the allodiploid and allotetraploid hybrids, from interspecific hybridization of red crucian carp (Carassius auratus red var. 2n = 100, 2n = AA) (♀) × common carp (Cyprinus carpio L. 2n = 100, 2n = BB) (♂), provided a good platform to investigate genetic relationship between the parents and their hybrid progenies. The chromosomal inheritance of diploid and allotetraploid hybrid progenies in successive generations, was studied by applying 5S rDNA fluorescence in situ hybridization. Signals of 5S rDNA distinguished the chromosomal constitution of common carp (B-genome) from red crucian carp (A-genome), in which two strong signals were observed on the first submetacentric chromosome, while no major signal was found in common carp. After fish hybridization, one strong signal of 5S rDNA was detected in the same locus on the chromosome of diploid hybrids. As expected, two strong signals were observed in 4nF3 tetraploid hybrids offspring and it is worth mentioning that two strong signals were detected in a separating bivalent of a primary spermatocyte in 4nF3. Furthermore, the mitosis of heterozygous chromosomes was shown normal and stable with blastular tissue histological studies. We revealed that 5S rDNA signal can be applied to discern A-genome from B-genome, and that 5S rDNA bearing chromosomes can be stably passed down in successive generations. Our work provided a significant method in fish breeding and this is important for studies in fish evolutionary biology.

  7. Detection of integrated herpesvirus genomes by fluorescence in situ hybridization (FISH).

    Science.gov (United States)

    Kaufer, Benedikt B

    2013-01-01

    Fluorescence in situ hybridization (FISH) is widely used to visualize nucleotide sequences in interphase cells or on metaphase chromosomes using specific probes that are complementary to the respective targets. Besides its broad application in cytogenetics and cancer research, FISH facilitates the localization of virus genomes in infected cells. Some herpesviruses, including human herpesvirus 6 (HHV-6) and Marek's disease virus (MDV), have been shown to integrate their genetic material into host chromosomes, which allows transmission of HHV-6 via the germ line and is required for efficient MDV-induced tumor formation. We describe here the detection by FISH of integrated herpesvirus genomes in metaphase chromosomes and interphase nuclei of herpesvirus-infected cells.

  8. Minimally invasive prostate cancer detection test using FISH probes

    Directory of Open Access Journals (Sweden)

    Tinawi-Aljundi R

    2016-07-01

    Full Text Available Rima Tinawi-Aljundi,1 Shannon T Knuth,2 Michael Gildea,2 Joshua Khal,2 Jason Hafron,1 Kenneth Kernen,1 Robert Di Loreto,1 Joan Aurich-Costa2 1Pathology and Research Department, Michigan Institute of Urology, St Clair Shores, MI, USA; 2Research and Development, Cellay, Inc., Cambridge, MA, USA Purpose: The ability to test for and detect prostate cancer with minimal invasiveness has the potential to reduce unnecessary prostate biopsies. This study was conducted as part of a clinical investigation for the development of an OligoFISH® probe panel for more accurate detection of prostate cancer.Materials and methods: One hundred eligible male patients undergoing transrectal ultrasound biopsies were enrolled in the study. After undergoing digital rectal examination with pressure, voided urine was collected in sufficient volume to prepare at least two slides using ThinPrep. Probe panels were tested on the slides, and 500 cells were scored when possible. From the 100 patients recruited, 85 had more than 300 cells scored and were included in the clinical performance calculations.Results: Chromosomes Y, 7, 10, 20, 6, 8, 16, and 18 were polysomic in most prostate carcinoma cases. Of these eight chromosomes, chromosomes 7, 16, 18, and 20 were identified as having the highest clinical performance as a fluorescence in situ hybridization test and used to manufacture the fluorescence in situ hybridization probe panels. The OligoFISH® probes performed with 100% analytical specificity. When the OligoFISH® probes were compared with the biopsy results for each individual, the test results highly correlated with positive and negative prostate biopsy pathology findings, supporting their high specificity and accuracy. Probes for chromosomes 7, 16, 18, and 20 showed in the receiver operator characteristics analysis an area under the curve of 0.83, with an accuracy of 81% in predicting the biopsy result.Conclusion: This investigation demonstrates the ease of use

  9. Towards Fluorescence In Vivo Hybridization (FIVH Detection of H. pylori in Gastric Mucosa Using Advanced LNA Probes.

    Directory of Open Access Journals (Sweden)

    Sílvia Fontenete

    Full Text Available In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo FISH-based method (FIVH that directly detects and allows the visualization of the bacterium within the human body would significantly reduce the time of analysis, allowing the diagnosis to be performed during endoscopy. In a previous study we designed and synthesized a phosphorothioate locked nucleic acid (LNA/ 2' O-methyl RNA (2'OMe probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH. In conclusion, this methodology is a promising approach that might be used in vivo in the future in combination with a confocal laser endomicroscope for H. pylori visualization.

  10. Towards Fluorescence In Vivo Hybridization (FIVH) Detection of H. pylori in Gastric Mucosa Using Advanced LNA Probes

    Science.gov (United States)

    Fontenete, Sílvia; Leite, Marina; Guimarães, Nuno; Madureira, Pedro; Ferreira, Rui Manuel; Figueiredo, Céu; Wengel, Jesper; Azevedo, Nuno Filipe

    2015-01-01

    In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo FISH-based method (FIVH) that directly detects and allows the visualization of the bacterium within the human body would significantly reduce the time of analysis, allowing the diagnosis to be performed during endoscopy. In a previous study we designed and synthesized a phosphorothioate locked nucleic acid (LNA)/ 2’ O-methyl RNA (2’OMe) probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH). In conclusion, this methodology is a promising approach that might be used in vivo in the future in combination with a confocal laser endomicroscope for H. pylori visualization. PMID:25915865

  11. Risk stratification of plasma cell neoplasm: insights from plasma cell-specific cytoplasmic immunoglobulin fluorescence in situ hybridization (cIg FISH) vs. conventional FISH.

    Science.gov (United States)

    Dong, Henry; Yang, Hai-Su; Jagannath, Sundar; Stephenson, Christine F; Brenholz, Pauline; Mazumder, Amitabha; Chari, Ajai

    2012-10-01

    We directly compared the results of routine fluorescence in situ hybridization (FISH) and plasma cell-specific cytoplasmic immunoglobulin (cIg) FISH from 75 paired samples for myeloma risk stratification. CIg FISH improves test specificity and sensitivity and tends to eliminate borderline results. It proves that most plasma cells (PCs) consistently carry the abnormality in myelomas with an IGH translocation, whereas routine FISH detects these cells only at variably low levels. Routine cytogenetic analysis of plasma cell neoplasms (PCNs) has a low sensitivity. Conventional fluorescence in situ hybridization (FISH) is not plasma cell (PC) specific and results are diluted by other cells in the sample. Although PC-specific FISH testing has been recommended for multiple myeloma (MM) risk stratification, eg, by combining cytoplasmic immunoglobulin (cIg) staining with FISH, the benefits of cIg FISH have never been directly demonstrated in a controlled study. Seventy-five samples from patients with PCNs were analyzed by concomitant conventional FISH and cIg FISH with probes for t(4;14), t(11;14), t(14;16), -13, 17p-, and +3. The results were compared for their reliability, specificity, and consistency. Apart from marginally improving detection threshold in samples with low PC burden, cIg FISH identified more abnormal cases (50 vs. 47 cases) and more chromosome abnormalities (113 vs. 103 events) than did conventional FISH. It differentiated del(13q) in myelodysplasia from MM. Remarkably, cIg FISH consistently identified a high percentage of abnormal PCs in all cases. It detected IGH translocation in 78% to 100% of PCs in all but 2 positive cases, whereas conventional FISH detected 0% to 46% in these cases (median, 91% vs. 9%). The abnormal cells found in patients with 17p- were 19% to 96% by cIg FISH vs. 0% to 13% by conventional FISH (median, 54% vs. 9%). Cases with insufficient PCs for cIg FISH had only normal conventional FISH results. CIg FISH improves reliability of

  12. Semi-automated scoring of triple-probe FISH in human sperm using confocal microscopy.

    Science.gov (United States)

    Branch, Francesca; Nguyen, GiaLinh; Porter, Nicholas; Young, Heather A; Martenies, Sheena E; McCray, Nathan; Deloid, Glen; Popratiloff, Anastas; Perry, Melissa J

    2017-07-05

    Structural and numerical sperm chromosomal aberrations result from abnormal meiosis and are directly linked to infertility. Any live births that arise from aneuploid conceptuses can result in syndromes such as Kleinfelter, Turners, XYY and Edwards. Multi-probe fluorescence in situ hybridization (FISH) is commonly used to study sperm aneuploidy, however manual FISH scoring in sperm samples is labor-intensive and introduces errors. Automated scoring methods are continuously evolving. One challenging aspect for optimizing automated sperm FISH scoring has been the overlap in excitation and emission of the fluorescent probes used to enumerate the chromosomes of interest. Our objective was to demonstrate the feasibility of combining confocal microscopy and spectral imaging with high-throughput methods for accurately measuring sperm aneuploidy. Our approach used confocal microscopy to analyze numerical chromosomal abnormalities in human sperm using enhanced slide preparation and rigorous semi-automated scoring methods. FISH for chromosomes X, Y, and 18 was conducted to determine sex chromosome disomy in sperm nuclei. Application of online spectral linear unmixing was used for effective separation of four fluorochromes while decreasing data acquisition time. Semi-automated image processing, segmentation, classification, and scoring were performed on 10 slides using custom image processing and analysis software and results were compared with manual methods. No significant differences in disomy frequencies were seen between the semi automated and manual methods. Samples treated with pepsin were observed to have reduced background autofluorescence and more uniform distribution of cells. These results demonstrate that semi-automated methods using spectral imaging on a confocal platform are a feasible approach for analyzing numerical chromosomal aberrations in sperm, and are comparable to manual methods. © 2017 International Society for Advancement of Cytometry. © 2017

  13. HER2 in situ hybridization in gastric and gastroesophageal adenocarcinoma: comparison of automated dual ISH to FISH.

    Science.gov (United States)

    Grin, Andrea; Brezden-Masley, Christine; Bauer, Sharon; Streutker, Catherine J

    2013-12-01

    Patients with gastric and gastroesophageal junction (GEJ) adenocarcinomas that are HER2 positive by immunohistochemistry (IHC) or in situ hybridization show a significant survival benefit with trastuzumab therapy. In situ hybridization is traditionally done by fluorescence in situ hybridization (FISH), despite some limitations. An alternative is the dual in situ hybridization (Dual ISH) technique that is fully automated and uses differentially labeled CEP17 and HER2 probes that can be read by light microscopy on 1 slide. The aim of this study was to assess the utility of Dual ISH in gastric/GEJ cancer and to compare the results with those obtained by IHC and FISH. Cases of gastric/GEJ adenocarcinoma were analyzed by IHC, FISH, and Dual ISH and the correlation between methods calculated. Results for 50 patients were available. There was a 98% (49/50) concordance rate between Dual ISH and FISH. One discrepant case was nonamplified by FISH but showed focal amplification by Dual ISH. Discrepancy was attributed to tumor heterogeneity, which was a frequent finding (78% of HER2-positive cases). There was excellent correlation between Dual ISH and FISH for assessment of HER2 amplification. Dual ISH was rapid, easy to interpret, and maintained cell morphology, which was valuable in identifying tumor heterogeneity.

  14. Identification of triclosan-degrading bacteria using stable isotope probing, fluorescence in situ hybridization and microautoradiography

    DEFF Research Database (Denmark)

    Lolas, Ihab Bishara Lolas; Chen, Xijuan; Bester, Kai

    2012-01-01

    Triclosan is considered a ubiquitous pollutant and can be detected in a wide range of environmental samples. Triclosan removal by wastewater treatment plants has been largely attributed to biodegradation processes; however, very little is known about the micro-organisms involved. In this study, DNA......-based stable isotope probing (DNA-SIP) combined with microautoradiography-fluorescence in situ hybridization (MAR-FISH) was applied to identify active triclosan degraders in an enrichment culture inoculated with activated sludge. Clone library sequences of 16S rRNA genes derived from the heavy DNA fractions...... of enrichment culture incubated with 13C-labelled triclosan showed a predominant enrichment of a single bacterial clade most closely related to the betaproteobacterial genus Methylobacillus. To verify that members of the genus Methylobacillus were actively utilizing triclosan, a specific probe targeting...

  15. Discrimination of bacteriophage infected cells using locked nucleic acid fluorescent in situ hybridization (LNA-FISH).

    Science.gov (United States)

    Vilas Boas, Diana; Almeida, Carina; Sillankorva, Sanna; Nicolau, Ana; Azeredo, Joana; Azevedo, Nuno F

    2016-01-01

    Bacteriophage-host interaction studies in biofilm structures are still challenging due to the technical limitations of traditional methods. The aim of this study was to provide a direct fluorescence in situ hybridization (FISH) method based on locked nucleic acid (LNA) probes, which targets the phage replication phase, allowing the study of population dynamics during infection. Bacteriophages specific for two biofilm-forming bacteria, Pseudomonas aeruginosa and Acinetobacter, were selected. Four LNA probes were designed and optimized for phage-specific detection and for bacterial counterstaining. To validate the method, LNA-FISH counts were compared with the traditional plaque forming unit (PFU) technique. To visualize the progression of phage infection within a biofilm, colony-biofilms were formed and infected with bacteriophages. A good correlation (r = 0.707) was observed between LNA-FISH and PFU techniques. In biofilm structures, LNA-FISH provided a good discrimination of the infected cells and also allowed the assessment of the spatial distribution of infected and non-infected populations.

  16. Study of the Artificial Fish Swarm Algorithm for Hybrid Clustering

    Directory of Open Access Journals (Sweden)

    Hongwei Zhao

    2015-06-01

    Full Text Available The basic Artificial Fish Swarm (AFS Algorithm is a new type of an heuristic swarm intelligence algorithm, but it is difficult to optimize to get high precision due to the randomness of the artificial fish behavior, which belongs to the intelligence algorithm. This paper presents an extended AFS algorithm, namely the Cooperative Artificial Fish Swarm (CAFS, which significantly improves the original AFS in solving complex optimization problems. K-medoids clustering algorithm is being used to classify data, but the approach is sensitive to the initial selection of the centers with low quality of the divided cluster. A novel hybrid clustering method based on the CAFS and K-medoids could be used for solving clustering problems. In this work, first, CAFS algorithm is used for optimizing six widely-used benchmark functions, coming up with comparative results produced by AFS and CAFS, then Particle Swarm Optimization (PSO is studied. Second, the hybrid algorithm with K-medoids and CAFS algorithms is used for data clustering on several benchmark data sets. The performance of the hybrid algorithm based on K-medoids and CAFS is compared with AFS and CAFS algorithms on a clustering problem. The simulation results show that the proposed CAFS outperforms the other two algorithms in terms of accuracy and robustness.

  17. Identification and characterization of marker chromosomes, de novo rearrangements and microdeletions in 100 cases with fluorescence in situ hybridization (FISH)

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, S.M.; Liu, Y.; Papenhausen, P.R. [Roche Biomedical Labs., Research Triangle Park, NC (United States)

    1994-09-01

    Results of molecular cytogenetic analysis are presented for 100 cases in which fluorescence in situ hybridization (FISH) was used as an adjunct to standard cytogenetics. Commercially available centromeric, telomeric, chromosome painting and unique sequence probes were used. Cases were from a 12-month period (June 1993-May 1994) and included examples of sex chromosome abnormalities (8), duplications (5), de novo translocations (6), satellited (12) and non-satellited (7) markers, and microdeletion syndromes (62). Satellited marker chromosomes were evaluated using a combination of DAPI/Distamycin A staining, hybridization with a classical satellite probe for chromosome 15 and hybridization with alpha-satellite probes for chromosomes 13, 14, 21 and 22. Markers positive for the chromosome 15 probe were further evaluated using unique sequence probes for the Prader-Willi/Angelman region. Microdeletion analysis was performed for Prader-Willi/Angelman (49) and DiGeorge/VCF (13) syndromes. Seven cases evaluated for Prader-Willi/Angelman syndrome demonstrated evidence of a deletion within this region. Uniparental disomy analysis was available in cases where a deletion was not detected by FISH, yet follow-up was clinically indicated. Two cases evaluated for DiGeorge/VCF syndrome demonstrated molecular evidence of a deletion. Included in our analysis is an example of familial DiGeorge syndrome.

  18. Ancient hybridization fuels rapid cichlid fish adaptive radiations

    Science.gov (United States)

    Meier, Joana I.; Marques, David A.; Mwaiko, Salome; Wagner, Catherine E.; Excoffier, Laurent; Seehausen, Ole

    2017-01-01

    Understanding why some evolutionary lineages generate exceptionally high species diversity is an important goal in evolutionary biology. Haplochromine cichlid fishes of Africa's Lake Victoria region encompass >700 diverse species that all evolved in the last 150,000 years. How this ‘Lake Victoria Region Superflock' could evolve on such rapid timescales is an enduring question. Here, we demonstrate that hybridization between two divergent lineages facilitated this process by providing genetic variation that subsequently became recombined and sorted into many new species. Notably, the hybridization event generated exceptional allelic variation at an opsin gene known to be involved in adaptation and speciation. More generally, differentiation between new species is accentuated around variants that were fixed differences between the parental lineages, and that now appear in many new combinations in the radiation species. We conclude that hybridization between divergent lineages, when coincident with ecological opportunity, may facilitate rapid and extensive adaptive radiation. PMID:28186104

  19. A novel fluorescent probe: europium complex hybridized T7 phage.

    Science.gov (United States)

    Liu, Chin-Mei; Jin, Qiaoling; Sutton, April; Chen, Liaohai

    2005-01-01

    We report on the creation of a novel fluorescent probe of europium-complex hybridized T7 phage. It was made by filling a ligand-displayed T7 ghost phage with a fluorescent europium complex particle. The structure of the hybridized phage, which contains a fluorescent inorganic core surrounded by a ligand-displayed capsid shell, was confirmed by electron microscope, energy-dispersive X-ray analysis (EDX), bioassays, and fluorescence spectrometer. More importantly, as a benefit of the phage display technology, the hybridized phage has the capability to integrate an affinity reagent against virtually any target molecules. The approach provides an original method to fluorescently "tag" a bioligand and/or to "biofunctionalize" a fluorophore particle. By using other types of materials such as radioactive or magnetic particles to fill the ghost phage, we envision that the hybridized phages represent a new class of fluorescent, magnetic, or radioprobes for imaging and bioassays and could be used both in vitro and in vivo.

  20. Detection of a complex translocation using fluorescent in situ hybridization (FISH)

    Energy Technology Data Exchange (ETDEWEB)

    Rosen, B.A. [Brandeis Univ., Waltham, MA (United States); Abuelo, D.N. [Rhode Island Hospital, Providence, RI (United States); Mark, H.F. [Brown Univ. School of Medicine, Providence, RI (United States)

    1994-09-01

    The use of fluorescent in situ hybridization (FISH) allowed the detection of a complex 3-way translocation in a patient with multiple congenital malformations and mental retardation. The patient was a 10-year-old girl with mental retardation, seizures, repaired cleft palate, esotropia, epicanthal folds, broad nasal bridge, upward slanting palpebral fissures, single transverse palmar crease, brachydactyly, hypoplastic nails, ectrodactyly between the third and fourth right toes, and hypoplasia of the left third toe. Chromosome analysis performed at birth was reported as normal. We performed high resolution banding analysis which revealed an apparently balanced translocation between chromosomes 2 and 9. However, because of her multiple abnormalities, further studies were ordered. Fluorescent in situ hybridization (FISH) using chromosome painting probes revealed a karyotype of 46,XX,t(2;8;9) (2pter{yields}q31::8q21.2{yields}8qter; 8pter{yields}q21.2::2q31{yields}q34::9q34{yields}qter; 9pter{yields}q34::2q34{yields}qter). The 3-way translocation appears to be de novo, as neither parent is a translocation carrier. This case illustrates the importance of using FISH to further investigate cases of apparently balanced translocations in the presence of phenotypic abnormalities and/or mental retardation.

  1. Detection of Helicobacter pylori in raw bovine milk by fluorescence in situ hybridization (FISH).

    Science.gov (United States)

    Angelidis, Apostolos S; Tirodimos, Ilias; Bobos, Mattheos; Kalamaki, Mary S; Papageorgiou, Demetrios K; Arvanitidou, Malamatenia

    2011-12-02

    The transmission pathways of Helicobacter pylori in humans have not been fully elucidated. Research in the last decade has proposed that foodborne transmission, among others, may be a plausible route of human infection. Owing to the organism's fastidious growth characteristics and its ability to convert to viable, yet unculturable states upon exposure to stress conditions, the detection of H. pylori in foods via culture-dependent methods has been proven to be laborious, difficult and in most cases unsuccessful. Hence, nucleic acid-based methods have been proposed as alternative methods but, to date, only PCR-based methods have been reported in the literature. In the current study, fluorescence in situ hybridization (FISH) was used for the detection of H. pylori in raw, bulk-tank bovine milk. After repeated milk centrifugation and washing steps, the bacterial flora of raw milk was subjected to fixation and permeabilization and H. pylori detection was conducted by FISH after hybridization with an H. pylori-specific 16S rRNA-directed fluorescent oligonucleotide probe. Using this protocol, H. pylori was detected in four out of the twenty (20%) raw milk samples examined. The data presented in this manuscript indicate that FISH can serve as an alternative molecular method for screening raw bovine milk for the presence of H. pylori.

  2. Comparison of viable cell counts and fluorescence in situ hybridization using specific rRNA-based probes for the quantification of human fecal bacteria

    NARCIS (Netherlands)

    Harmsen, HJM; Gibson, GR; Elfferich, P; Raangs, GC; Wildeboer-Veloo, ACM; Argaiz, A; Roberfroid, MB; Welling, GW

    2000-01-01

    Conventional cultivation and fluorescence in situ hybridization (FISH) using 16S rRNA-based probes were compared for the enumeration of human colonic bacteria. Groups of common intestinal anaerobic bacteria were enumerated in slurries prepared From fecal samples of three healthy volunteers. To intro

  3. Chromogenic in situ hybridization: a multicenter study comparing silver in situ hybridization with FISH.

    Science.gov (United States)

    Bartlett, J M S; Campbell, Fiona M; Ibrahim, Merdol; Wencyk, Peter; Ellis, Ian; Kay, Elaine; Connolly, Yvonne; O'Grady, Anthony; Di Palma, Silvana; Starczynski, Jane; Morgan, John M; Jasani, Bharat; Miller, Keith

    2009-10-01

    Our purposes were to perform a robust assessment of a new HER2 chromogenic in situ hybridization test and report on concordance of silver in situ hybridization (SISH) data with fluorescence in situ hybridization (FISH) data and on intraobserver and interlaboratory scoring consistency. HER2 results were scored from 45 breast cancers in 7 laboratories using the Ventana (Tucson, AZ) INFORM HER-2 SISH assay and in 1 central laboratory using a standard FISH assay. Overall, 94.8% of cases were successfully analyzed by SISH across the 6 participating laboratories that reported data. Concordance for diagnosis of HER2 amplification by SISH compared with FISH was high (96.0% overall). Intraobserver variability (8.0%) and intersite variability (12.66%) of absolute HER2/chromosome 17 ratios appear to be tightly controlled across all 6 participating laboratories. The Ventana INFORM HER-2 SISH assay is robust and reproducible, shows good concordance with a standard FISH assay, and complies with requirements in national guidelines for performance of diagnostic tests.

  4. Photoswitchable non-fluorescent thermochromic dye-nanoparticle hybrid probes

    Science.gov (United States)

    Harrington, Walter N.; Haji, Mwafaq R.; Galanzha, Ekaterina I.; Nedosekin, Dmitry A.; Nima, Zeid A.; Watanabe, Fumiya; Ghosh, Anindya; Biris, Alexandru S.; Zharov, Vladimir P.

    2016-11-01

    Photoswitchable fluorescent proteins with controllable light-dark states and spectral shifts in emission in response to light have led to breakthroughs in the study of cell biology. Nevertheless, conventional photoswitching is not applicable for weakly fluorescent proteins and requires UV light with low depth penetration in bio-tissue. Here we introduce a novel concept of photoswitchable hybrid probes consisting of thermochromic dye and absorbing nanoparticles, in which temperature-sensitive light-dark states and spectral shifts in absorption can be switched through controllable photothermal heating of doped nanoparticles. The proof-of-concept is demonstrated through the use of two different types of temperature-sensitive dyes doped with magnetic nanoparticles and reversibly photoswitched by a near-infrared laser. Photoacoustic imaging revealed the high contrast of these probes, which is sufficient for their visualization in cells and deep tissue. Our results suggest that these new photoswitchable multicolour probes can be used for multimodal cellular diagnostics and potentially for magnetic and photothermal therapy.

  5. Detection of iron-depositing Pedomicrobium species in native biofilms from the Odertal National Park by a new, specific FISH probe.

    Science.gov (United States)

    Braun, Burga; Richert, Inga; Szewzyk, Ulrich

    2009-10-01

    Iron-depositing bacteria play an important role in technical water systems (water wells, distribution systems) due to their intense deposition of iron oxides and resulting clogging effects. Pedomicrobium is known as iron- and manganese-oxidizing and accumulating bacterium. The ability to detect and quantify members of this species in biofilm communities is therefore desirable. In this study the fluorescence in situ hybridization (FISH) method was used to detect Pedomicrobium in iron and manganese incrusted biofilms. Based on comparative sequence analysis, we designed and evaluated a specific oligonucleotide probe (Pedo 1250) complementary to the hypervariable region 8 of the 16S rRNA gene for Pedomicrobium. Probe specificities were tested against 3 different strains of Pedomicrobium and Sphingobium yanoikuyae as non-target organism. Using optimized conditions the probe hybridized with all tested strains of Pedomicrobium with an efficiency of 80%. The non-target organism showed no hybridization signals. The new FISH probe was applied successfully for the in situ detection of Pedomicrobium in different native, iron-depositing biofilms. The hybridization results of native bioflims using probe Pedo_1250 agreed with the results of the morphological structure of Pedomicrobium bioflims based on scanning electron microscopy.

  6. Flow cytometric sorting of fecal bacteria after in situ hybridization with polynucleotide probes.

    Science.gov (United States)

    Bruder, Lena M; Dörkes, Marcel; Fuchs, Bernhard M; Ludwig, Wolfgang; Liebl, Wolfgang

    2016-10-01

    The gut microbiome represents a key contributor to human physiology, metabolism, immune function, and nutrition. Elucidating the composition and genetics of the gut microbiota under various conditions is essential to understand how microbes function individually and as a community. Metagenomic analyses are increasingly used to study intestinal microbiota. However, for certain scientific questions it is sufficient to examine taxon-specific submetagenomes, covering selected bacterial genera in a targeted manner. Here we established a new variant of fluorescence in situ hybridization (FISH) combined with fluorescence-activated cell sorting (FACS), providing access to the genomes of specific taxa belonging to the complex community of the intestinal microbiota. In contrast to standard oligonucleotide probes, the RNA polynucleotide probe used here, which targets domain III of the 23S rRNA gene, extends the resolution power in environmental samples by increasing signal intensity. Furthermore, cells hybridized with the polynucleotide probe are not subjected to harsh pretreatments, and their genetic information remains intact. The protocol described here was tested on genus-specifically labeled cells in various samples, including complex fecal samples from different laboratory mouse types that harbor diverse intestinal microbiota. Specifically, as an example for the protocol described here, RNA polynucleotide probes could be used to label Enterococcus cells for subsequent sorting by flow cytometry. To detect and quantify enterococci in fecal samples prior to enrichment, taxon-specific PCR and qPCR detection systems have been developed. The accessibility of the genomes from taxon-specifically sorted cells for subsequent molecular analyses was demonstrated by amplification of functional genes. Copyright © 2016 Elsevier GmbH. All rights reserved.

  7. Fluorescent in situ hybridization (FISH) assessment of chromosome copy number in sperm

    Energy Technology Data Exchange (ETDEWEB)

    Sheu, M.; Sigman, M.; Mark, H.F.L. [Brown Univ. School of Medicine, Providence, RI (United States)

    1994-09-01

    Approximately 15% of all recognized pregnancies end in spontaneous abortions. The overall frequency of chromosome abnormalities in spontaneous abortions is approximately 50%. Thus aneuploidy is a significant cause of fetal wastage. In addition, structural and numerical abnormalities of chromosomes can also lead to birth defects, developmental delay, mental retardation and infertility. Conventional cytogenetic analysis via GTG- and other banding techniques is a powerful tool in the elucidation of the nature of chromosomal abnormalities. Fluorescent in situ hybridization (FISH) enables detection of numerical chromosomal abnormalities, especially trisomies, in intact cells. Using FISH and commercially available biotin-labeled probes, we have initiated a prospective study to assess specific chromosome copy number of preparations of unstained smears from men referred for a male infertility evaluation as well as smears from normal control males chosen randomly from the sample of sperm donors. A total of approximately 19,000 sperm nuclei have been examined thus far. Of those suitable for analysis, 7382 (38.75%) were normal possessing one copy of chromosome 8, 155 (0.81%) were disomic, and 15 (0.079%) had more than two copies of chromosome 8. Comparisons with data available in the literature will be discussed. Work is ongoing to increase the efficiency of hybridization using both reported and previously untried pretreatment and fixation protocols. We have also initiated studies using multicolor FISH with various chromosome enumeration probes. The assay described here is a potentially powerful tool for detecting rare events such as spontaneous germ cell aneuploidy, aneuploidy detected in semen from men with carcinoma in situ of the testis and aneuploidy induced by potential environmental genotoxicants. It can also be utilized for segregation analysis and for correlating chromosome copy number with germ cell morphology.

  8. A New Type of Hybrid Fish-like Microrobot

    Institute of Scientific and Technical Information of China (English)

    Wei Zhang; Shu-Xiang Guo; Kinji Asaka

    2006-01-01

    In order to develop a new type of fish-like microrobot with swimming, walking, and floating motions, in our past research, we developed a hybrid microrobot actuated by ionic conducting polymer film (ICPF) actuators. But the microrobot had some problems in walking and floating motions. In this paper, we propose a concept of hybrid microrobot (see Fig. 1). The microrobot is actuated by a pair of caudal fins, a base with legs and an array of artificial swim bladders. We have developed a prototype of the base with legs and one artificial swim bladder, respectively, and carried out experiments for evaluating their characteristics. Experimental results show the base with legs can realize walking speed of 6 mm/s and rotating speed of 7.1 degrees/s respectively, and the prototype of the artificial swim bladder has a maximum floatage of 2.6 mN. The experimental results also indicate that the microrobot has some advantages, such as walking motion with 2 degrees of freedom, the walking ability on rough surface (sand paper), the controllable floatage, etc. This kind of fish-like microrobot is expected for industrial and medical applications.

  9. Introgressive hybridization as a promoter of genome reshuffling in natural homoploid fish hybrids (Cyprinidae, Leuciscinae).

    Science.gov (United States)

    Pereira, C S A; Aboim, M A; Ráb, P; Collares-Pereira, M J

    2014-03-01

    Understanding the mechanisms underlying diversification and speciation by introgressive hybridization is currently one of the major challenges in evolutionary biology. Here, the analysis of hybridization between two pairs of Iberian Leuciscinae provided new data on independent hybrid zones involving Achondrostoma oligolepis (AOL) and Pseudochondrostoma duriense (PDU), and confirmed the occurrence of hybrids between AOL and Pseudochondrostoma polylepis (PPO). A multilevel survey combining morphological, genetic and cytogenomic markers on a vast population screening successfully sorted the selected fishes as admixed. Results were similar in both AOL × PDU and AOL × PPO systems. Overall, hybrid morphotypes, cytogenomic data and genetic profiling indicated preferential backcrossing and suggested AOL as a major genomic contributor. Moreover, results implied AOL as more permissive to introgression than PDU or PPO. Although PDU- and PPO-like individuals appeared more resilient to genome modifications, AOL appeared to be more involved and affected by the ongoing hybridization events, as chromosomal translocations were only found in AOL-like individuals. All hybrids analysed evidenced extensive ribosomal DNA (rDNA) polymorphism that was not found in parental species, but usually seen falling within the range of possible parental combinations. Yet, transgressive phenotypes that cannot be explained by normal recombination, including more rDNA clusters than expected or the occurrence of syntenic rDNAs, were also detected. Present results proved rapid genomic evolution providing the genetic novelty for species to persist. In addition, although the ultimate consequences of such apparently extensive and recurrent events remain unknown, modern genome-wide methodologies are of great promise towards answering questions concerning the causes, dynamics and impacts of hybridization.

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

    Science.gov (United States)

    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.

  11. Molecular cytogenetic analysis of the Appenine endemic cyprinid fish Squalius lucumonis and three other Italian leuciscines using chromosome banding and FISH with rDNA probes.

    Science.gov (United States)

    Rossi, Anna Rita; Milana, Valentina; Hett, Anne Kathrin; Tancioni, Lorenzo

    2012-12-01

    Karyotype and other chromosomal characteristics of the Appenine endemic cyprinid fish, Toscana stream chub Squalius lucumonis, were analysed using conventional banding and FISH with 45S and 5S rDNA probes. The diploid chromosome number (2n = 50) and karyotype characteristics including pericentromeric heterochromatic blocks and GC-rich CMA(3)-positive sites corresponding to both positive Ag-NORs and 45S rDNA loci on the short arms of a single medium-sized submetacentric chromosome pair were consistent with those found in most European leuciscine cyprinids. On other hand, 5S rDNA FISH in the Toscana stream chub and three other Italian leuciscines, S. squalus, Rutilus rubilio and Telestes muticellus, revealed a species-specific hybridization pattern, i.e. signals on four (S. lucumonis), three (S. squalus and R. rubilio) and two (T. muticellus) chromosome pairs. Whereas all the species shared the 5S rDNA loci on the largest subtelocentric chromosome pair, a "leuciscine" cytotaxonomic marker, S. lucumonis showed both classes of rDNA loci tandem aligned on the short arms of chromosome pair No. 12. The present findings suggest that the observed high variability of 5S rDNA loci provides a powerful tool for investigation of karyotype differentiation in karyologically conservative leuciscine fishes.

  12. Dramatically improved RNA in situ hybridization signals using LNA-modified probes

    DEFF Research Database (Denmark)

    Thomsen, Rune; Nielsen, Peter Stein; Jensen, Torben Heick

    2005-01-01

    In situ detection of RNA by hybridization with complementary probes is a powerful technique. Probe design is a critical parameter in successful target detection. We have evaluated the efficiency of fluorescent DNA oligonucleotides modified to contain locked nucleic acid (LNA) residues. This incre......In situ detection of RNA by hybridization with complementary probes is a powerful technique. Probe design is a critical parameter in successful target detection. We have evaluated the efficiency of fluorescent DNA oligonucleotides modified to contain locked nucleic acid (LNA) residues....... This increases the thermal stability of hybrids formed with RNA. The LNA-based probes detect specific RNAs in fixed yeast cells with an efficiency far better than conventional DNA oligonucleotide probes of the same sequence. Using this probe design, we were also able to detect poly(A)+ RNA accumulation within...

  13. Metaphase FISH on a Chip: Miniaturized Microfluidic Device for Fluorescence in situ Hybridization

    DEFF Research Database (Denmark)

    Vedarethinam, Indumathi; Shah, Pranjul Jaykumar; Dimaki, Maria;

    2010-01-01

    Fluorescence in situ Hybridization (FISH) is a major cytogenetic technique for clinical genetic diagnosis of both inherited and acquired chromosomal abnormalities. Although FISH techniques have evolved and are often used together with other cytogenetic methods like CGH, PRINS and PNA-FISH, the pr......Fluorescence in situ Hybridization (FISH) is a major cytogenetic technique for clinical genetic diagnosis of both inherited and acquired chromosomal abnormalities. Although FISH techniques have evolved and are often used together with other cytogenetic methods like CGH, PRINS and PNA...

  14. Application of rRNA probes and fluorescence in situ hybridization for rapid detection of the toxic dinoflagellate Alexandrium minutum

    Science.gov (United States)

    Tang, Xianghai; Yu, Rencheng; Zhou, Mingjiang; Yu, Zhigang

    2012-03-01

    The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade ("Global clade") of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade ("Pacific clade"). The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1, M-PC-3), or just nucleolus (M-PC-2). Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs.

  15. Application of rRNA probes and fluorescence in situ hybridization for rapid detection of the toxic dinoflagellate Alexandrium minutum

    Institute of Scientific and Technical Information of China (English)

    TANG Xianghai; YU Rencheng; ZHOU Mingjiang; YU Zhigang

    2012-01-01

    The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs).This species consists of many strains that differ in their ability to produce toxins but have similar morphology,making identification difficult.In this study,species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A.minutum from two phylogenetic clades.We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes.Three ribotype-specific probes,M-GC-1,M-PC-2,and M-PC-3,were designed.The former is specific for the GC clade (“Global clade”) of A.minutum,the majority of which have been found non-toxic,and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade (“Pacific clade”).The specificity of these three probes was confirmed by FISH.All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions.However,the accessibility of rRNA molecules in ribosomes varied among the probe binding positions.Thus,there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1,M-PC-3),or just nucleolus (M-PC-2).Our results provide a methodological basis for studying the biogeography and population dynamics of A.minutum,and providing an early warning of toxic HABs.

  16. Real-time assays with molecular beacons and other fluorescent nucleic acid hybridization probes.

    Science.gov (United States)

    Marras, Salvatore A E; Tyagi, Sanjay; Kramer, Fred Russell

    2006-01-01

    A number of formats for nucleic acid hybridization have been developed to identify DNA and RNA sequences that are involved in cellular processes and that aid in the diagnosis of genetic and infectious diseases. The introduction of hybridization probes with interactive fluorophore pairs has enabled the development of homogeneous hybridization assays for the direct identification of nucleic acids. A change in the fluorescence of these probes indicates the presence of a target nucleic acid, and there is no need to separate unbound probes from hybridized probes. The advantages of homogeneous hybridization assays are their speed and simplicity. In addition, homogeneous assays can be combined with nucleic acid amplification, enabling the detection of rare target nucleic acids. These assays can be followed in real time, providing quantitative determination of target nucleic acids over a broad range of concentrations.

  17. Characterization of B chromosomes in Lilium hybrids through GISH and FISH

    NARCIS (Netherlands)

    Xie, S.L.; Marasek-Ciolakowska, A.; Ramanna, M.S.; Arens, P.F.P.; Visser, R.G.F.; Tuyl, van J.M.

    2014-01-01

    Supernumerary (B) chromosomes and small aberrant chromosomes were detected in Lilium hybrids and characterized through genomic in situ hybridization (GISH) and florescence in situ hybridization (FISH). Two small, supernumerary or B chromosomes were detected as extra chromosomes in a tetraploid plant

  18. Estimate of true incomplete exchanges using fluorescence in situ hybridization with telomere probes

    Science.gov (United States)

    Wu, H.; George, K.; Yang, T. C.

    1998-01-01

    PURPOSE: To study the frequency of true incomplete exchanges in radiation-induced chromosome aberrations. MATERIALS AND METHODS: Human lymphocytes were exposed to 2 Gy and 5 Gy of gamma-rays. Chromosome aberrations were studied using the fluorescence in situ hybridization (FISH) technique with whole chromosome-specific probes, together with human telomere probes. Chromosomes 2 and 4 were chosen in the present study. RESULTS: The percentage of incomplete exchanges was 27% when telomere signals were not considered. After excluding false incomplete exchanges identified by the telomere signals, the percentage of incomplete exchanges decreased to 11%. Since telomere signals appear on about 82% of the telomeres, the percentage of true incomplete exchanges should be even lower and was estimated to be 3%. This percentage was similar for chromosomes 2 and 4 and for doses of both 2 Gy and 5 Gy. CONCLUSIONS: The percentage of true incomplete exchanges is significantly lower in gamma-irradiated human lymphocytes than the frequencies reported in the literature.

  19. Variability of ribosomal DNA sites in Festuca pratensis, Lolium perenne, and their intergeneric hybrids, revealed by FISH and GISH.

    Science.gov (United States)

    Ksiazczyk, T; Taciak, M; Zwierzykowski, Z

    2010-01-01

    This study focuses on the variability of chromosomal location and number of ribosomal DNA (rDNA) sites in some diploid and autotetraploid Festuca pratensis and Lolium perenne cultivars, as well as on identification of rDNA-bearing chromosomes in their triploid and tetraploid F. pratensis × L. perenne hybrids. The rDNA loci were mapped using fluorescence in situ hybridization (FISH) with 5S and 25S rDNA probes, and the origin of parental genomes was verified by genomic in situ hybridization (GISH) with L. perenne genomic DNA as a probe, and F. pratensis genomic DNA as a block. FISH detected variation in the number and chromosomal location of both 5S and 45S rDNA sites. In F. pratensis mostly additional signals of 5S rDNA loci occurred, as compared with standard F. pratensis karyotypes. Losses of 45S rDNA loci were more frequent in L. perenne cultivars and intergeneric hybrids. Comparison of the F. pratensis and L. perenne genomes approved a higher number of rDNA sites as well as variation in chromosomal rDNA location in L. perenne. A greater instability of F. pratensis-genome-like and L. perenne-genome-like chromosomes in tetraploid hybrids was revealed, indicating gains and losses of rDNA loci, respectively. Our data indicate that the rDNA loci physically mapped on chromosomes 2 and 3 in F. pratensis and on chromosome 3 in L. perenne are useful markers for these chromosomes in intergeneric Festuca × Lolium hybrids.

  20. Interactive fluorophore and quencher pairs for labeling fluorescent nucleic acid hybridization probes.

    Science.gov (United States)

    Marras, Salvatore A E

    2008-03-01

    The use of fluorescent nucleic acid hybridization probes that generate a fluorescence signal only when they bind to their target enables real-time monitoring of nucleic acid amplification assays. Real-time nucleic acid amplification assays markedly improves the ability to obtain qualitative and quantitative results. Furthermore, these assays can be carried out in sealed tubes, eliminating carryover contamination. Fluorescent nucleic acid hybridization probes are available in a wide range of different fluorophore and quencher pairs. Multiple hybridization probes, each designed for the detection of a different nucleic acid sequence and each labeled with a differently colored fluorophore, can be added to the same nucleic acid amplification reaction, enabling the development of high-throughput multiplex assays. In order to develop robust, highly sensitive and specific real-time nucleic acid amplification assays it is important to carefully select the fluorophore and quencher labels of hybridization probes. Selection criteria are based on the type of hybridization probe used in the assay, the number of targets to be detected, and the type of apparatus available to perform the assay. This article provides an overview of different aspects of choosing appropriate labels for the different types of fluorescent hybridization probes used with different types of spectrofluorometric thermal cyclers currently available.

  1. Surface density dependence of PCR amplicon hybridization on PNA/DNA probe layers

    DEFF Research Database (Denmark)

    Yao, Danfeng; Kim, Junyoung; Yu, Fang

    2005-01-01

    at an intermediate sodium concentration (approximately 100 mM). These effects were mainly ascribed to the electrostatic cross talk among the hybridized DNA molecules and the secondary structure of PCR amplicons. For the negatively charged DNA probes, the hybridization reaction was subjected additionally to the DNA...

  2. Mitochondrial Genome Variation after Hybridization and Differences in the First and Second Generation Hybrids of Bream Fishes.

    Directory of Open Access Journals (Sweden)

    Wei-Zhuo Zhang

    Full Text Available Hybridization plays an important role in fish breeding. Bream fishes contribute a lot to aquaculture in China due to their economically valuable characteristics and the present study included five bream species, Megalobrama amblycephala, Megalobrama skolkovii, Megalobrama pellegrini, Megalobrama terminalis and Parabramis pekinensis. As maternal inheritance of mitochondrial genome (mitogenome involves species specific regulation, we aimed to investigate in which way the inheritance of mitogenome is affected by hybridization in these fish species. With complete mitogenomes of 7 hybrid groups of bream species being firstly reported in the present study, a comparative analysis of 17 mitogenomes was conducted, including representatives of these 5 bream species, 6 first generation hybrids and 6 second generation hybrids. The results showed that these 17 mitogenomes shared the same gene arrangement, and had similar gene size and base composition. According to the phylogenetic analyses, all mitogenomes of the hybrids were consistent with a maternal inheritance. However, a certain number of variable sites were detected in all F1 hybrid groups compared to their female parents, especially in the group of M. terminalis (♀ × M. amblycephala (♂ (MT×MA, with a total of 86 variable sites between MT×MA and its female parent. Among the mitogenomes genes, the protein-coding gene nd5 displayed the highest variability. The number of variation sites was found to be related to phylogenetic relationship of the parents: the closer they are, the lower amount of variation sites their hybrids have. The second generation hybrids showed less mitogenome variation than that of first generation hybrids. The non-synonymous and synonymous substitution rates (dN/dS were calculated between all the hybrids with their own female parents and the results indicated that most PCGs were under negative selection.

  3. Dramatically improved RNA in situ hybridization signals using LNA-modified probes

    DEFF Research Database (Denmark)

    Thomsen, Rune; Nielsen, Peter Stein; Jensen, Torben Heick

    2005-01-01

    In situ detection of RNA by hybridization with complementary probes is a powerful technique. Probe design is a critical parameter in successful target detection. We have evaluated the efficiency of fluorescent DNA oligonucleotides modified to contain locked nucleic acid (LNA) residues. This incre...... the nucleus/ nucleolus of wild-type cells. LNA-based probes should be readily applicable to a diverse array of cells and tissue samples....

  4. Interphase FISH detection of BCL2 rearrangement in follicular lymphoma using breakpoint-flanking probes

    NARCIS (Netherlands)

    Vaandrager, J W; Schuuring, E; Raap, T; Philippo, K; Kleiverda, K; Kluin, P

    2000-01-01

    Rearrangement of the BCL2 gene is an important parameter for the differential diagnosis of non-Hodgkin lymphomas. Although a relatively large proportion of breakpoints is clustered, many are missed by standard PCR. A FISH assay is therefore desired. Up to now, a lack of probes flanking the BCL2 gene

  5. Effect of graded fingerlings on hybrid catfish food fish size distribution

    Science.gov (United States)

    It is not unusual to have both 0.5 lb and 5 lb fish harvested from a single-batch hybrid catfish production pond at the end of the growing season. When that happens, farmers may be docked for fish that are either larger or smaller than the processor’s preferred size range. This study was conducted t...

  6. Hybridization probe for femtomolar quantification of selected nucleic acid sequences on a disposable electrode.

    Science.gov (United States)

    Jenkins, Daniel M; Chami, Bilal; Kreuzer, Matthias; Presting, Gernot; Alvarez, Anne M; Liaw, Bor Yann

    2006-04-01

    Mixed monolayers of electroactive hybridization probes on gold surfaces of a disposable electrode were investigated as a technology for simple, sensitive, selective, and rapid gene identification. Hybridization to the ferrocene-labeled hairpin probes reproducibly diminished cyclic redox currents, presumably due to a displacement of the label from the electrode. Observed peak current densities were roughly 1000x greater than those observed in previous studies, such that results could easily be interpreted without the use of algorithms to correct for background polarization currents. Probes were sensitive to hybridization with a number of oligonucleotide sequences with varying homology, but target oligonucleotides could be distinguished from competing nontarget sequences based on unique "melting" profiles from the probe. Detection limits were demonstrated down to nearly 100 fM, which may be low enough to identify certain genetic conditions or infections without amplification. This technology has rich potential for use in field devices for gene identification as well as in gene microarrays.

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

    Science.gov (United States)

    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.

  8. Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): A Method for Bacterial Small RNA Detection

    Science.gov (United States)

    2012-01-10

    Friedrich, U. & Lenke, J. Improved Enumeration of Lactic Acid Bacteria in Mesophilic Dairy Starter Cultures by Using Multiplex Quantitative Real...messenger RNA using locked nucleic acid probes. Anal. Biochem. 390, 109-114 (2009). 13. Waters, L. & Storz, G. Regulatory RNAs in bacteria . Cell. 136, 615...Video Article Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection Kelly

  9. The Formation of the Polyploid Hybrids From Different Subfamily Fish Crossings and Its Evolutionary Significance

    OpenAIRE

    Liu, Shaojun; Qin, Qinbo; Xiao, Jun; Lu, Wenting; Shen, Jiamin; Li, Wei; Liu, Jifang; Duan, Wei; Zhang, Chun; Tao, De Min; Zhao, Rurong; Yan, Jinpeng; Liu, Yun

    2007-01-01

    This study provides genetic evidences at the chromosome, DNA content, DNA fragment and sequence, and morphological levels to support the successful establishment of the polyploid hybrids of red crucian carp × blunt snout bream, which belonged to a different subfamily of fish (Cyprininae subfamily and Cultrinae subfamily) in the catalog. We successfully obtained the sterile triploid hybrids and bisexual fertile tetraploid hybrids of red crucian carp (RCC) (♀) × blunt snout bream (BSB) (♂) as w...

  10. Fluorescence in situ hybridizations (FISH) for the localization of viruses and endosymbiotic bacteria in plant and insect tissues.

    Science.gov (United States)

    Kliot, Adi; Kontsedalov, Svetlana; Lebedev, Galina; Brumin, Marina; Cathrin, Pakkianathan Britto; Marubayashi, Julio Massaharu; Skaljac, Marisa; Belausov, Eduard; Czosnek, Henryk; Ghanim, Murad

    2014-02-24

    Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells and can be further modified to visualize other components in the cell such as infection with viruses and bacteria. Spatial localization and visualization of viruses and bacteria during the infection process is an essential step that complements expression profiling experiments such as microarrays and RNAseq in response to different stimuli. Understanding the spatiotemporal infections with these agents complements biological experiments aimed at understanding their interaction with cellular components. Several techniques for visualizing viruses and bacteria such as reporter gene systems or immunohistochemical methods are time-consuming, and some are limited to work with model organisms and involve complex methodologies. FISH that targets RNA or DNA species in the cell is a relatively easy and fast method for studying spatiotemporal localization of genes and for diagnostic purposes. This method can be robust and relatively easy to implement when the protocols employ short hybridizing, commercially-purchased probes, which are not expensive. This is particularly robust when sample preparation, fixation, hybridization, and microscopic visualization do not involve complex steps. Here we describe a protocol for localization of bacteria and viruses in insect and plant tissues. The method is based on simple preparation, fixation, and hybridization of insect whole mounts and dissected organs or hand-made plant sections, with 20 base pairs short DNA probes conjugated to fluorescent dyes on their 5' or 3' ends. This protocol has been successfully applied to a number of insect and plant tissues, and can be used to analyze expression of mRNAs or other RNA or DNA species in the cell.

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

    Science.gov (United States)

    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.

  12. Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleicacid probes and tyramide signal amplification

    DEFF Research Database (Denmark)

    Silahtaroglu, Asli N.; Nolting, Dorrit; Andersen, Lars Dyrskjøt

    2007-01-01

    RNAs in frozen tissue sections using fluorescence in situ hybridization (FISH). The method combines the unique miRNA recognition properties of locked nucleic acid (LNA)-modified oligonucleotide probes with FISH using the tyramide signal amplification (TSA) technology. Although both approaches have previously...... been shown to increase detection sensitivity in FISH, combining these techniques into one protocol significantly decreases the time needed for miRNA detection in cryosections, while simultaneously retaining high detection sensitivity. Starting with fixation of the tissue sections, this miRNA FISH...... protocol can be completed within approximately 6 h and allows miRNA detection in a wide variety of animal tissue cryosections as well as in human tumor biopsies at high cellular resolution....

  13. Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice.

    Science.gov (United States)

    Pathak, Rupak; Koturbash, Igor; Hauer-Jensen, Martin

    2017-01-11

    Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

  14. Fluorescent in-situ hybridization (FISH for BCR/ABL in chronic myeloid leukemia after bone marrow transplantation

    Directory of Open Access Journals (Sweden)

    Maria de Lourdes Lopes Ferrari Chauffaille

    2001-01-01

    Full Text Available CONTEXT: Identification of Philadelphia chromosome or BCR/ABL gene rearrangement in chronic myeloid leukemia is important at diagnosis as well as after treatment. OBJECTIVE: To compare the results of karyotyping using fluorescent in-situ hybridization (FISH upon diagnosis and 1 year after bone marrow transplantation in 12 patients. TYPE OF STUDY: Diagnostic test and residual disease detection. SETTING: Hematology and Hemotherapy Department, Federal University of São Paulo/Escola Paulista de Medicina, São Paulo, Brazil. SAMPLE: 12 patients with chronic myeloid leukemia at diagnosis and 1 year after bone marrow transplantation. DIAGNOSTIC TEST: Karyotyping was done in the usual way and the BCR/ABL gene-specific probe was used for FISH. MAIN MEASUREMENTS: Disease at diagnosis and residual. RESULTS: At diagnosis, 10 patients presented t(9;22(q34.1;q11 as well as positive FISH. Two cases did not have metaphases but FISH was positive. After bone marrow transplantation, 8 patients presented normal karyotype, 1 had persistence of identifiable Philadelphia chromosome and 3 had no metaphases. Two cases showed complete chimera and 2 had donor and host cells simultaneously. FISH was possible in all cases after bone marrow transplantation and confirmed the persistence of identifiable Philadelphia chromosome clone in one patient, and identified another that did not present metaphases for analysis. Cases that showed mixed chimera in karyotype were negative for BCR/ABL by FISH. CONCLUSION: The applicability of FISH is clear, particularly for residual disease detection. Classical and molecular cytogenetics are complementary methods.

  15. Probing hybrid modified gravity by stellar motion around Galactic Centre

    CERN Document Server

    Borka, D; Jovanović, P; Jovanović, V Borka

    2015-01-01

    We consider possible signatures for the so called {\\it hybrid gravity} within the Galactic Central Parsec. This modified theory of gravity consists of a superposition of the metric Einstein-Hilbert Lagrangian with an $f(R)$ term constructed {\\it \\`{a} la Palatini } and can be easily reduced to an equivalent scalar-tensor theory. The present analysis is based on the S2 star orbital precession around the massive compact dark object at the Galactic Centre where the simulated orbits in hybrid modified gravity are compared with astronomical observations. These simulations result with strong constraints on the range of hybrid gravity interaction parameter $\\phi_0$ and show that its most probable value, in the case of S2 star, is around -0.0009 to -0.0002. At the same time, we are also able to obtain reliable constrains on the effective mass parameter $m_{\\phi}$ of hybrid modified gravity. Its most probable value, in the case of S2 star, is around -0.0034 to -0.0025. Furthermore, the hybrid gravity potential induces...

  16. The formation of the polyploid hybrids from different subfamily fish crossings and its evolutionary significance.

    Science.gov (United States)

    Liu, Shaojun; Qin, Qinbo; Xiao, Jun; Lu, Wenting; Shen, Jiamin; Li, Wei; Liu, Jifang; Duan, Wei; Zhang, Chun; Tao, Min; Zhao, Rurong; Yan, Jinpeng; Liu, Yun

    2007-06-01

    This study provides genetic evidences at the chromosome, DNA content, DNA fragment and sequence, and morphological levels to support the successful establishment of the polyploid hybrids of red crucian carp x blunt snout bream, which belonged to a different subfamily of fish (Cyprininae subfamily and Cultrinae subfamily) in the catalog. We successfully obtained the sterile triploid hybrids and bisexual fertile tetraploid hybrids of red crucian carp (RCC) (female symbol) x blunt snout bream (BSB) (male symbol) as well as their pentaploid hybrids. The triploid hybrids possessed 124 chromosomes with two sets from RCC and one set from BSB; the tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from BSB. The females of tetraploid hybrids produced unreduced tetraploid eggs that were fertilized with the haploid sperm of BSB to generate pentaploid hybrids with 172 chromosomes with three sets from BSB and two sets from RCC. The ploidy levels of triploid, tetraploid, and pentaploid hybrids were confirmed by counting chromosomal number, forming chromosomal karyotype, and measuring DNA content and erythrocyte nuclear volume. The similar and different DNA fragments were PCR amplified and sequenced in triploid, tetraploid hybrids, and their parents, indicating their molecular genetic relationship and genetic markers. In addition, this study also presents results about the phenotypes and feeding habits of polyploid hybrids and discusses the formation mechanism of the polyploid hybrids. It is the first report on the formation of the triploid, tetraploid, and pentaploid hybrids by crossing parents with a different chromosome number in vertebrates. The formation of the polyploid hybrids is potentially interesting in both evolution and fish genetic breeding.

  17. Brightness through Local Constraint-LNA-Enhanced FIT Hybridization Probes for In Vivo Ribonucleotide Particle Tracking

    DEFF Research Database (Denmark)

    Hövelmann, Felix; Gaspar, Imre; Loibl, Simon

    2014-01-01

    Imaging the dynamics of RNA in living cells is usually performed by means of transgenic approaches that require modification of RNA targets and cells. Fluorogenic hybridization probes would also allow the analysis of wild-type organisms. We developed nuclease-resistant DNA forced intercalation (FIT......) probes that combine the high enhancement of fluorescence upon hybridization with the high brightness required to allow tracking of individual ribonucleotide particles (RNPs). In our design, a single thiazole orange (TO) intercalator dye is linked as a nucleobase surrogate and an adjacent locked nucleic...

  18. Fiber-based hybrid probe for non-invasive cerebral monitoring in neonatology

    Science.gov (United States)

    Rehberger, Matthias; Giovannella, Martina; Pagliazzi, Marco; Weigel, Udo; Durduran, Turgut; Contini, Davide; Spinelli, Lorenzo; Pifferi, Antonio; Torricelli, Alessandro; Schmitt, Robert

    2015-07-01

    Improved cerebral monitoring systems are needed to prevent preterm infants from long-term cognitive and motor restrictions. Combining advanced near-infrared diffuse spectroscopy measurement technologies, time-resolved spectroscopy (TRS) and diffuse correlation spectroscopy (DCS) will introduce novel indicators of cerebral oxygen metabolism and blood flow for neonatology. For non-invasive sensing a fiber-optical probe is used to send and receive light from the infant head. In this study we introduce a new fiber-based hybrid probe that is designed for volume production. The probe supports TRS and DCS measurements in a cross geometry, thus both technologies gain information on the same region inside the tissue. The probe is highly miniaturized to perform cerebral measurements on heads of extreme preterm infants down to head diameters of 6cm. Considerations concerning probe production focus on a reproducible accuracy in shape and precise optical alignment. In this way deviations in measurement data within a series of probes should be minimized. In addition to that, requirements for clinical use like robustness and hygiene are considered. An additional soft-touching sleeve made of FDA compatible silicone allows for a flexible attachment with respect to the individual anatomy of each patient. We present the technical concept of the hybrid probe and corresponding manufacturing methods. A prototype of the probe is shown and tested on tissue phantoms as well as in vivo to verify its operational reliability.

  19. Current and historical hybridization with differential introgression among three species of cyprinid fishes (genus Cyprinella).

    Science.gov (United States)

    Broughton, Richard E; Vedala, Krishna C; Crowl, Tessa M; Ritterhouse, Lauren L

    2011-05-01

    Hybridization is common among freshwater fishes, particular among the Cyprinidae. We used two mitochondrial genes and one nuclear gene to characterize hybridization among two species pairs of Cyprinella in southwestern North America. Genalogical patterns revealed that C. lutrensis and C. venusta are currently hybridizing in several localities producing apparent F(1), F(2) and backcross generations, yet there was no evidence for introgression outside of local hybrid zones. Alternatively, mitochondrial haplotypes from C. lutrensis appear to have introgressed into a C. lepida population in the Nueces River completely replacing the native C. lepida haplotype. There was no evidence of introgression of nuclear DNA and there does not appear to be ongoing hybridization. The population of C. lepida from the nearby Frio River exhibits no evidence of hybridization with C. lutrensis. Thus, contact between C. lutrensis and C. venusta results in the formation of localized hybrid swarms, while contact between C. lutrensis and C. lepida has resulted in complete mitochondrial introgression in the Nueces River or no apparent hybridization in the Frio River. The three different outcomes of contact between these species illustrate the variable nature of interspecific reproductive interactions and provide an excellent system in which to better understand the factors influencing hybridization among freshwater fishes.

  20. Phenylethynylpyrene excimer forming hybridization probes for fluorescence SNP detection

    DEFF Research Database (Denmark)

    Prokhorenko, Igor A.; Astakhova, Irina V.; Momynaliev, Kuvat T.

    2009-01-01

    Excimer formation is a unique feature of some fluorescent dyes (e.g., pyrene) which can be used for probing the proximity of biomolecules. Pyrene excimer fluorescence has previously been used for homogeneous detection of single nucleotide polymorphism (SNP) on DNA. 1-Phenylethynylpyrene (1-1-PEPy...

  1. In situ hybridization with labeled probes: assessment of african Swine Fever virus in formalin-fixed paraffin-embedded tissues.

    Science.gov (United States)

    Ballester, Maria; Rodríguez, Fernando

    2015-01-01

    In situ hybridization (ISH) has become a very valuable molecular diagnostic tool to detect specific DNA or RNA sequences in biological samples through the use of complementary DNA- or RNA-labeled probes. Here, we describe an optimized in situ hybridization protocol to detect African swine fever virus (ASFV) DNA in formalin-fixed, paraffin-embedded tissues using digoxigenin-labeled probes.

  2. Hybridization-based biosensor containing hairpin probes and use thereof

    Science.gov (United States)

    Miller, Benjamin L.; Strohsahl, Christopher M.

    2010-10-12

    A sensor chip that includes: a fluorescence quenching surface; a nucleic acid probe that contains first and second ends with the first end bound to the fluorescence quenching surface, and is characterized by being able to self-anneal into a hairpin conformation; and a first fluorophore bound to the second end of the first nucleic acid molecule. When the first nucleic acid molecule is in the hairpin conformation, the fluorescence quenching surface substantially quenches fluorescent emissions by the first fluorophore; and when the first nucleic acid molecule is in a non-hairpin conformation, fluorescent emissions by the fluorophore are substantially free of quenching by the fluorescence quenching surface. Various nucleic acid probes, methods of making the sensor chip, biological sensor devices that contain the sensor chip, and their methods of use are also disclosed.

  3. Comparison Between Fluorescent In Situ Hybridization (FISH) and ...

    African Journals Online (AJOL)

    ... were collected, for culture method and bacteria characterized by biochemical ... for 24 hours and later preserved in 70% alcohol before in situ hybridization test. ... were observed in 47 to 75% while the bacterium was isolated on culture in 7 ...

  4. Edge turbulence measurement in Heliotron J using a combination of hybrid probe system and fast cameras

    Energy Technology Data Exchange (ETDEWEB)

    Nishino, N., E-mail: nishino@hiroshima-u.ac.jp [Graduate School of Engineering, Hiroshima University, Higashi-Hiroshima (Japan); Zang, L. [Kyoto University, Gokasho, Uji, Kyoto (Japan); Takeuchi, M. [JAEA, Naka, Ibaraki (Japan); Mizuuchi, T.; Ohshima, S. [Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); Kasajima, K.; Sha, M. [Kyoto University, Gokasho, Uji, Kyoto (Japan); Mukai, K. [NIFS, Toki, Gifu (Japan); Lee, H.Y. [Kyoto University, Gokasho, Uji, Kyoto (Japan); Nagasaki, K.; Okada, H.; Minami, T.; Kobayashi, S.; Yamamoto, S.; Konoshima, S.; Nakamura, Y.; Sano, F. [Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan)

    2013-07-15

    The hybrid probe system (a combination of Langmuir probes and magnetic probes), fast camera and gas puffing system were installed at the same toroidal section to study edge plasma turbulence/fluctuation in Heliotron J, especially blob (intermittent filament). Fast camera views the location of the probe head, so that the probe system yields the time evolution of the turbulence/fluctuation while the camera images the spatial profile. Gas puff at the same toroidal section was used to control the plasma density and simultaneous gas puff imaging technique. Using this combined system the filamentary structure associated with magnetic fluctuation was found in Heliotron J at the first time. The other kind of fluctuation was also observed at another experiment. This combination measurement enables us to distinguish MHD activity and electro-static activity.

  5. Probing Structure and Composition of Nickel/Titanium Carbide Hybrid Interfaces at the Atomic Scale (Preprint)

    Science.gov (United States)

    2010-01-01

    The transition in structure and composition across the titanium carbide /nickel hybrid interface has been determined at near atomic resolution by...coupling high-resolution transmission electron microscopy with three-dimensional atom probe tomography. The titanium carbide phase adopts a rocksalt-type

  6. Detection of cyclin D1 mRNA by hybridization sensitive NIC-oligonucleotide probe.

    Science.gov (United States)

    Kovaliov, Marina; Segal, Meirav; Kafri, Pinhas; Yavin, Eylon; Shav-Tal, Yaron; Fischer, Bilha

    2014-05-01

    A large group of fluorescent hybridization probes, includes intercalating dyes for example thiazole orange (TO). Usually TO is coupled to nucleic acids post-synthetically which severely limits its use. Here, we have developed a phosphoramidite monomer, 10, and prepared a 2'-OMe-RNA probe, labeled with 5-(trans-N-hexen-1-yl-)-TO-2'-deoxy-uridine nucleoside, dU(TO), (Nucleoside bearing an Inter-Calating moiety, NIC), for selective mRNA detection. We investigated a series of 15-mer 2'-OMe-RNA probes, targeting the cyclin D1 mRNA, containing one or several dU(TO) at various positions. dU(TO)-2'-OMe-RNA exhibited up to 7-fold enhancement of TO emission intensity upon hybridization with the complementary RNA versus that of the oligomer alone. This NIC-probe was applied for the specific detection of a very small amount of a breast cancer marker, cyclin D1 mRNA, in total RNA extract from cancerous cells (250 ng/μl). Furthermore, this NIC-probe was found to be superior to our related NIF (Nucleoside with Intrinsic Fluorescence)-probe which could detect cyclin D1 mRNA target only at high concentrations (1840 ng/μl). Additionally, dU(T) can be used as a monomer in solid-phase oligonucleotide synthesis, thus avoiding the need for post-synthetic modification of oligonucleotide probes. Hence, we propose dU(TO) oligonucleotides, as hybridization probes for the detection of specific RNA in homogeneous solutions and for the diagnosis of breast cancer.

  7. Homoeologous chromosome pairing in the distant hybrid Alstroemeria aurea x A. inodora and the genome composition of its backcross derivatives determined by fluorescence in situ hybridization with species-specific probes.

    Science.gov (United States)

    Kamstra, S A; Ramanna, M S; de Jeu, M J; Kuipers, A G; Jacobsen, E

    1999-01-01

    A distant hybrid between two diploid species (2n = 2x = 16), Alstroemeria aurea and A. inodora, was investigated for homoeologous chromosome pairing, crossability with A. inodora and chromosome transmission to its BC1 offspring. Fluorescence in situ hybridization (FISH) with two species-specific probes, A001-I (A. aurea specific) and D32-13 (A. inodora specific), was used to analyse chromosome pairing in the hybrid and the genome constitution of its BC1 progeny plants. High frequencies of associated chromosomes were observed in both genotypes of the F1 hybrid, A1P2-2 and A1P4. In the former, both univalents and bivalents were found at metaphase I, whereas the latter plant also showed tri- and quadrivalents. Based on the hybridization sites of DNA probes on the chromosomes of both parental species, it was established that hybrid A1P4 contains a reciprocal translocation between the short arm of chromosome 1 and the long arm of chromosome 8 of A. inodora. Despite regular homoeologous chromosome pairing in 30% of the pollen mother cells, both hybrids were highly sterile. They were backcrossed reciprocally with one of the parental species, A. inodora. Two days after pollination, embryo rescue was applied and, eventually, six BC1 progeny plants were obtained. Among these, two were aneuploids (2n = 2x + 1 = 17) and four were triploids (2n = 3x = 24). The aneuploid plants had originated when the interspecific hybrid was used as a female parent, indicating that n eggs were functional in the hybrid. In addition, 2n gametes were also functional in the hybrid, resulting in the four triploid BC1 plants. Of these four plants, three had received 2n pollen grains from the hybrid and one a 2n egg. Using FISH, homoeologous crossing over between the chromosomes of the two parental species in the hybrid was clearly detected in all BC1 plants. The relevance of these results for the process of introgression and the origin of n and 2n gametes are discussed.

  8. DNA breakage detection-fluorescence in situ hybridization (DBD-FISH in buccal cells

    Directory of Open Access Journals (Sweden)

    E. I. Cortés-Gutiérrez

    2012-12-01

    Full Text Available DNA breakage detection-fluorescence in situ hybridization (DBD-FISH is a recently developed technique that allows cell-by-cell detection and quantification of DNA breakage in the whole genome or within specific DNA sequences. The present investigation was conducted to adapt the methodology of DBD-FISH to the visualization and evaluation of DNA damage in buccal epithelial cells. DBD-FISH revealed that DNA damage increased significantly according to H2O2 concentration (r2=0.91. In conclusion, the DBD-FISH technique is easy to apply in buccal cells and provides prompt results that are easy to interpret. Future studies are needed to investigate the potential applicability of a buccal cell DBD-FISH model to human biomonitoring and nutritional work.

  9. DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) in buccal cells.

    Science.gov (United States)

    Cortés-Gutiérrez, E I; Dávila-Rodríguez, M I; Fernández, J L; López-Fernández, C; Gosálvez, J

    2012-12-28

    DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) is a recently developed technique that allows cell-by-cell detection and quantification of DNA breakage in the whole genome or within specific DNA sequences. The present investigation was conducted to adapt the methodology of DBD-FISH to the visualization and evaluation of DNA damage in buccal epithelial cells. DBD-FISH revealed that DNA damage increased significantly according to H2O2 concentration (r2=0.91). In conclusion, the DBD-FISH technique is easy to apply in buccal cells and provides prompt results that are easy to interpret. Future studies are needed to investigate the potential applicability of a buccal cell DBD-FISH model to human biomonitoring and nutritional work.

  10. Characterization of the Arachis (Leguminosae) D genome using fluorescence in situ hybridization (FISH) chromosome markers and total genome DNA hybridization

    OpenAIRE

    Germán Robledo; Guillermo Seijo

    2008-01-01

    Chromosome markers were developed for Arachis glandulifera using fluorescence in situ hybridization (FISH) of the 5S and 45S rRNA genes and heterochromatic 4'-6-diamidino-2-phenylindole (DAPI) positive bands. We used chromosome landmarks identified by these markers to construct the first Arachis species ideogram in which all the homologous chromosomes were precisely identified. The comparison of this ideogram with those published for other Arachis species revealed very poor homeologies with a...

  11. Metaphase FISH on a Chip: Miniaturized Microfluidic Device for Fluorescence in situ Hybridization

    Directory of Open Access Journals (Sweden)

    Niels Tommerup

    2010-11-01

    Full Text Available Fluorescence in situ Hybridization (FISH is a major cytogenetic technique for clinical genetic diagnosis of both inherited and acquired chromosomal abnormalities. Although FISH techniques have evolved and are often used together with other cytogenetic methods like CGH, PRINS and PNA-FISH, the process continues to be a manual, labour intensive, expensive and time consuming technique, often taking over 3–5 days, even in dedicated labs. We have developed a novel microFISH device to perform metaphase FISH on a chip which overcomes many shortcomings of the current laboratory protocols. This work also introduces a novel splashing device for preparing metaphase spreads on a microscope glass slide, followed by a rapid adhesive tape-based bonding protocol leading to rapid fabrication of the microFISH device. The microFISH device allows for an optimized metaphase FISH protocol on a chip with over a 20-fold reduction in the reagent volume. This is the first demonstration of metaphase FISH on a microfluidic device and offers a possibility of automation and significant cost reduction of many routine diagnostic tests of genetic anomalies.

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

    Energy Technology Data Exchange (ETDEWEB)

    Harris, C.; Nunez, M. [Univ. of Wisconsin, WI (United States); Giraldez, R. [ONCOR, Inc., Gaithersburg, MD (United States)

    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.

  13. Validation of interphase fluorescence in situ hybridization (iFISH for multiple myeloma using CD138 positive cells

    Directory of Open Access Journals (Sweden)

    Renata Kiyomi Kishimoto

    2016-06-01

    Full Text Available ABSTRACT BACKGROUND: Multiple myeloma is a plasma cell neoplasm with acquired genetic abnormalities of clinical and prognostic importance. Multiple myeloma differs from other hematologic malignancies due to a high fraction of low proliferating malignant plasma cells and the paucity of plasma cells in bone marrow aspiration samples, making cytogenetic analysis a challenge. An abnormal karyotype is found in only one-third of patients with multiple myeloma and interphase fluorescence in situ hybridization is the most useful test for studying the chromosomal abnormalities present in almost 90% of cases. However, it is necessary to study the genetic abnormalities in plasma cells after their identification or selection by morphology, immunophenotyping or sorting. Other challenges are the selection of the most informative FISH panel and determining cut-off levels for FISH probes. This study reports the validation of interphase fluorescence in situ hybridization using CD138 positive cells, according to proposed guidelines published by the European Myeloma Network (EMN in 2012. METHOD: Bone marrow samples from patients with multiple myeloma were used to standardize a panel of five probes [1q amplification, 13q14 deletion, 17p deletion, t(4;14, and t(14;16] in CD138+ cells purified by magnetic cell sorting. RESULTS: This test was validated with a low turnaround time and good reproducibility. Five of six samples showed genetic abnormalities. Monosomy/deletion 13 plus t(4;14 were found in two cases. CONCLUSION: This technique together with magnetic cell sorting is effective and can be used in the routine laboratory practice. In addition, magnetic cell sorting provides a pure plasma cell population that allows other molecular and genomic studies.

  14. Inconsistent phylogeographic pattern between a sperm dependent fish and its host: in situ hybridization vs dispersal.

    Science.gov (United States)

    Vergilino, Roland; Leung, Christelle; Angers, Bernard

    2016-09-06

    Co-dispersal of sperm-dependent hybrids and their sexual relatives is expected to result in consistent spatial patterns between assemblages of hybrids and genetic structure of parental species. However, local hybridization events may blur this signal as assemblages could be organized under different connectivity constraints. This study aims at testing the hypothesis of local hybridization events by comparing the assemblage of hybrid fish Chrosomus eos-neogaeus to the genetic diversity of one of its parental species, Chrosomus eos. An extensive survey performed on a total of 132 sites located in two regions of Southern Quebec (West-Qc and East-Qc) revealed a distinct organization of hybrid lineages. One of the six hybrid lineages detected in West-Qc is widespread throughout this region resulting in a low α-diversity (1.38) and β-diversity (4.35). On the other hand, 36 hybrid lineages were detected in East-Qc and displayed narrow geographic distributions leading to a high α-diversity (2.30) and β-diversity (15.68). In addition, the C. eos multilocus haplotype of several of these hybrids is assigned to their respective sympatric C. eos population. Finally, contrasting with hybrids, the paternal species C. eos displayed a higher ρST in West-Qc (0.2300) than in East-Qc (0.0734). The unusually high diversity of hybrid lineages in East-Qc as well as the spatial organization and the close genetic relationship with C. eos sympatric populations support the hypothesis that multiple hybridization events occurred in situ. These findings coupled to the near absence of the maternal species Chrosomus neogeaus suggest that the decline of this species could be the trigger event at the origin of the high rates of spontaneous hybridization in this region.

  15. Behavioural isolation may facilitate homoploid hybrid speciation in cichlid fish

    NARCIS (Netherlands)

    Selz, O. M.; Thommen, R.; Maan, M. E.; Seehausen, O.

    Hybrid speciation is constrained by the homogenizing effects of gene flow from the parental species. In the absence of post-mating isolation due to structural changes in the genome, or temporal or spatial premating isolation, another form of reproductive isolation would be needed for homoploid

  16. Water quality in hybrid catfish ponds after partial fish harvest

    Science.gov (United States)

    Intensification of United States catfish aquaculture involves hybrid catfish ('channel catfish Ictalurus punctatus x ' blue catfish I. furcatus) grown in ponds with abundant aeration and high feeding rates. High feeding rates cause water quality deterioration because most of the nitrogen, phosphorus...

  17. Behavioural isolation may facilitate homoploid hybrid speciation in cichlid fish

    NARCIS (Netherlands)

    Selz, O. M.; Thommen, R.; Maan, M. E.; Seehausen, O.

    2014-01-01

    Hybrid speciation is constrained by the homogenizing effects of gene flow from the parental species. In the absence of post-mating isolation due to structural changes in the genome, or temporal or spatial premating isolation, another form of reproductive isolation would be needed for homoploid hybri

  18. [Interphase cytogenetics of the breast tumors with fluorescence in situ hybridization (FISH) on cytologic preparation--its practice and clinical applications].

    Science.gov (United States)

    Shibuya, M; Osamura, R Y

    1996-05-01

    Fluorescence in situ hybridization (FISH) study with the chromosome specific probes is performed in the interphase nuclei of the routinely processed cytologic preparation of the breast tumors. Numerical aberrations on the chromosomes 1, 3, 11 or 17 were detected in more than 80% of the malignant tumors, but not in the benign tumors. Marked heterogeneity of the polysomies is noted in the malignant tumor cells. A few malignant cases revealed monosomy of chromosome 17. No apparent correlation between the numerical abnormalities and the histological features in malignant tumors is identified. These results suggest that the interphase cytogenetics with FISH for the breast tumors may be useful for differential diagnosis of malignancy. The practice and the clinical applications of the FISH study are discussed.

  19. FISH Detection of PML-RARA Fusion in ins(15;17 Acute Promyelocytic Leukaemia Depends on Probe Size

    Directory of Open Access Journals (Sweden)

    Lynda J. Campbell

    2013-01-01

    Full Text Available The diagnosis of acute promyelocytic leukaemia (APL is usually confirmed by cytogenetics showing the characteristic t(15;17, but a minority of patients have a masked PML/RARA fusion. We report ten patients with APL and no evidence of the t(15;17, in whom the insertion of RARA into PML could not be demonstrated by initial FISH studies using a standard dual fusion probe but was readily identified using smaller probes. Given the need for rapid diagnosis of APL, it is important to be aware of the false negative rate for large PML/RARA FISH probes in the setting of masked rearrangements.

  20. Comparison of high resolution chromosome banding and fluorescence in situ hybridization (FISH) for the laboratory evaluation of Prader-Willi syndrome and Angelman syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Delach, J.A.; Rosengren, S.S.; Kaplan, L.; Greenstein, R.M.; Cassidy, S.B.; Benn, P.A.

    1994-08-01

    The development of probes containing segments of DNA from chromosome region 15q11-q13 provides the opportunity to confirm the diagnosis of Prader-Willi syndrome (PWS) and Angelman syndrome (AS) by fluorescence in situ hybridization (FISH). We have evaluated FISH studies and high resolution chromosome banding studies in 14 patients referred to confirm or rule out AS. In four patients (three from the PWS category and 1 from the AS group) chromosome analysis suggested that a deletion was present but FISH failed to confirm the finding. In one AS group patient, FISH identified a deletion not detectable by high resolution banding. Review of the clinical findings in the discrepant cases suggested that FISH results were correct and high resolution findings were erroneous. Studies with a chromosome 15 alpha satellite probe (D15Z) on both normal and abnormal individuals suggested that incorrect interpretation of chromosome banding may occasionally be attributable to alpha satellite polymorphism but other variation of 15q11-q13 chromosome bands also contributes to misinterpretation. We conclude that patients who have been reported to have a cytogenetic deletion of 15q11-q13 and who have clinical findings inconsistent with PWS and AS should be re-evaluated by molecular genetic techniques. 31 refs., 3 figs., 2 tabs.

  1. mRNA-targeted Fluorescent in Situ Hybridization (FISH) of Gram-negative Bacteria Without Template Amplification or Tyramide Signal Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Coleman, James R.; Culley, David E.; Chrisler, William B.; Brockman, Fred J.

    2007-12-01

    Technologies are needed to study gene expression at the level of individual cells within a population or microbial community. Fluorescent in situ hybridization (FISH) supplies high-resolution spatial information and has been widely applied to study microbial communities at the rRNA level. While mRNA-targeted FISH has been popular for studying gene expression in eukaryotic cells, very little success has been achieved with prokaryotes. At present, detection of specific mRNAs in individual prokaryotic cells requires the use of in situ-RT-PCR or tyramide signal amplification (TSA). In this study we used DNA oligonucleotide probes labeled with a single near-infrared dye in FISH assays to detect multicopy plasmid-based and endogenous mRNA molecules in Escherichia coli and Shewanella oneidensis MR-1. We took advantage of the fact there is much less background signal produced by biological materials and support matrices in the near-infrared spectrum and thus long camera exposure times could be used. In addition, we demonstrate that a combination of probes targeting both rRNA and mRNA could be successfully employed within the same FISH assay. These results, as well as ongoing R&D improvements in NIR and infrared dyes, indicate the FISH approach we demonstrated could be applied in certain environmental settings to monitor gene expression in mixed populations.

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

    Directory of Open Access Journals (Sweden)

    Yuji Miyahara

    2013-02-01

    Full Text Available 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. Label-free potentiometry for detecting DNA hybridization using peptide nucleic acid and DNA probes.

    Science.gov (United States)

    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.

  4. Clinical Application of Fluorescence in Situ Hybridization (FISH) to Detect HER-2 Gene in Breast Cancer

    Institute of Scientific and Technical Information of China (English)

    Maurie Buehler; Ellie Guardino; Jung Sik Park; Eun Jeong Jang

    2014-01-01

    Objective: To investigate the clinical application of the detection of HER-2 gene by lfuorescence in situ hybridization (FISH) in breast cancer and the correlation between HER-2 gene ampliifcation and clinicopathology of breast cancer. Methods:Parafifn-embedded breast inifltrating ductal carcinoma from 48 patients were detected by FISH and immunohistochemistry (IHC) respectively for comparing the results of two methods. Results: HER-2 protein expressions were classified into three groups (3+/2+/1+ or 0) and the positive rates of HER-2 gene ampliifcation by FISH were 77.8%, 57.1% and 10.5%, respectively. Of the 29 cases with positive axillary lymph node, 12 were with HER-2 gene ampliifcation (P0.05). Conclusion:The false positive and negative rates are higher in HER-2 protein expression by IHC. Compared with IHC, FISH, being more effective and precise, can be applied extensively in clinic. HER-2 gene ampliifcation is concerned with axillary nodes metastases.

  5. Nitrifying bacterial communities in an aquaculture wastewater treatment system using fluorescence in situ hybridization (FISH), 16S rRNA gene cloning, and phylogenetic analysis.

    Science.gov (United States)

    Paungfoo, Chanyarat; Prasertsan, Poonsuk; Burrell, Paul C; Intrasungkha, Nugul; Blackall, Linda L

    2007-07-01

    Aquaculture, especially shrimp farming, has played a major role in the growth of Thailand's economy in recent years, as well as in many South East Asian countries. However, the nutrient discharges from these activities have caused adverse impacts on the quality of the receiving waterways. In particular nitrogenous compounds, which may accumulate in aquaculture ponds, can be toxic to aquatic animals and cause environmental problems such as eutrophication. The mineralization process is well known, but certain aspects of the microbial ecology of nitrifiers, the microorganisms that convert ammonia to nitrate, are poorly understood. A previously reported enrichment of nitrifying bacteria (ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) from a shrimp farm inoculated in a sequencing batch reactor (SBR) was studied by molecular methods. The initial identification and partial quantification of the nitrifying bacteria (AOB and NOB) were carried out by fluorescence in situ hybridization (FISH) using previously published 16S rRNA-targeting oligonucleotide probes. The two dominant bacterial groups detected by FISH were from the Cytophaga-Flavobacterium-Bacteroides and Proteobacteria (beta subdivision) phyla. Published FISH probes for Nitrobacter and Nitrospira did not hybridize to any of the bacterial cells. Therefore it is likely that new communities of NOBs, differing from previously reported ones, exist in the enrichments. Molecular genetic techniques (cloning, sequencing, and phylogenetic analysis) targeting the 16S rRNA genes from the nitrifying enrichments were performed to identify putative AOBs and NOBs.

  6. Fluorescent in situ hybridization analysis of open lactic acid fermentation of kitchen refuse using rRNA-targeted oligonucleotide probes.

    Science.gov (United States)

    Sakai, Kenji; Mori, Masatsugu; Fujii, Akira; Iwami, Yuko; Chukeatirote, Ekachai; Shirai, Yoshihito

    2004-01-01

    Reproducible amounts of lactic acid accumulate in minced kitchen refuse under open conditions with intermittent pH neutralization [Sakai et al., Food Sci. Technol. Res., 6, 140 (2000)]. Here, we showed that such pH-controlled open fermentation of kitchen refuse reproducibly resulted a selective proliferation of a major lactic acid bacterial (LAB) species. In one experiment, the predominant microorganisms isolated during the early phase (6 h) were Gammaproteobacteria. In contrast, those that predominated during the late phase (48 h) were always Lactobacillus plantarum in three independent experiments. To further quantify the microbial community within open lactic acid fermentation, we performed fluorescent in situ hybridization (FISH) analysis targeting 16S (23S) rRNA. We designed two new group-specific DNA probes: LAC722(L) was active for most LAB including the genera Lactobacillus, Pediococcus, Leuconostoc and Weisella, whereas Lplan477 was specific for L. plantarum and its related species. We then optimized sample preparation using lysozyme and hybridization conditions including temperature, as well as the formamide concentration and the salt concentration in the washing buffer. We succeeded in quantification of microorganisms in semi-solid, complex biological materials such as minced kitchen refuse by taking color microphotographs in modified RGB balance on pre-coated slides. FISH analysis of the fermentation of kitchen refuse indicated that control of the pH swing leads to domination by the LAB population in minced kitchen refuse under open conditions. We also confirmed that L. plantarum, which generates lactic acid in high quantities but with low optical activity, became the dominant microorganism in kitchen refuse during the late phase of open fermentation.

  7. Ocean warming, a rapid distributional shift, and the hybridization of a coastal fish species.

    Science.gov (United States)

    Potts, Warren M; Henriques, Romina; Santos, Carmen V; Munnik, Kate; Ansorge, Isabelle; Dufois, Francois; Booth, Anthony J; Kirchner, Carola; Sauer, Warwick H H; Shaw, Paul W

    2014-09-01

    Despite increasing awareness of large-scale climate-driven distribution shifts in the marine environment, no study has linked rapid ocean warming to a shift in distribution and consequent hybridization of a marine fish species. This study describes rapid warming (0.8 °C per decade) in the coastal waters of the Angola-Benguela Frontal Zone over the last three decades and a concomitant shift by a temperature sensitive coastal fish species (Argyrosomus coronus) southward from Angola into Namibia. In this context, rapid shifts in distribution across Economic Exclusive Zones will complicate the management of fishes, particularly when there is a lack of congruence in the fisheries policy between nations. Evidence for recent hybridization between A. coronus and a congener, A. inodorus, indicate that the rapid shift in distribution of A. coronus has placed adults of the two species in contact during their spawning events. Ocean warming may therefore revert established species isolation mechanisms and alter the evolutionary history of fishes. While the consequences of the hybridization on the production of the resource remain unclear, this will most likely introduce additional layers of complexity to their management.

  8. Simultaneous 16S and 18S rRNA fluorescence in situ hybridization (FISH) on LR White sections demonstrated in Vestimentifera (Siboglinidae) tubeworms.

    Science.gov (United States)

    Schimak, Mario P; Toenshoff, Elena R; Bright, Monika

    2012-02-01

    Traditional morphological identification of invertebrate marine species is limited in early life history stages for many taxa. In this study, we demonstrate, by example of Vestimentiferan tubeworms (Siboglinidae, Polychaeta), that the simultaneous fluorescence in situ hybridization (FISH) of both eukaryotic host and bacterial symbiont cells is possible on a single semi-thin (1 μm) section. This allows the identification of host specimens to species level as well as offering visualization of bacteria distributed within the host tissue. Previously published 18S rRNA host-specific oligonucleotide probes for Riftia pachyptila, Tevnia jerichonana and a newly designed Oasisia alvinae probe, as well as a 16S rRNA probe targeting symbionts found in all host species, were applied. A number of standard fixation and hybridization parameters were tested and optimized for the best possible signal intensity and cellular resolution. Ethanol conserved samples embedded in LR White low viscosity resin yielded the best results with regard to both signal intensity and resolution. We show that extended storage times of specimens does not affect the quality of signals attained by FISH and use our protocol to identify morphologically unidentifiable tubeworm individuals from a small data set, conforming to previous findings in succession studies of the Siboglinidae family.

  9. Assessment of chromosomal abnormalities in sperm of infertile men using sperm karyotyping and multicolour fluorescence in situ hybridization (FISH)

    Energy Technology Data Exchange (ETDEWEB)

    Moosani, N.; Martin, R.H. [Alberta Children`s Hospital and Univ. of Calgary (Canada)

    1994-09-01

    Individuals with male factor infertility resulting from idiopathic oligo-, astheno- or teratozoospermia are frequently offered IVF in an attempt to increase their chances of having a child. A concern remains whether these infertile males have an elevated risk of transmitting chromosomal abnormalities to their offspring. Sperm chromosomal complements from these men were assayed using the human sperm/hamster oocyte fusion system and fluorescence in situ hybridization (FISH) on sperm nuclei. For each of 5 infertile patients, 100 sperm karyotypes were analyzed and multicolour FISH analysis was performed on a minimum of 10,000 sperm nuclei for each chromosome-specific DNA probe for chromosomes 1 (pUC1.77), 12 (D12Z3), X (XC) and Y (DYZ3). As a group, the infertile patients showed increased frequencies of both numerical ({chi}{sup 2}=17.26, {proportional_to} <0.001) and total abnormalities ({chi}{sup 2}=7.78, {proportional_to} <0.01) relative to control donors when assessed by sperm karyotypes. Analysis of sperm nuclei by FISH indicated a significant increase in the frequency of disomy for chromosome 1 in three of the five patients as compared to control donors ({chi}{sup 2}>8.35, {proportional_to} <0.005). In addition, the frequency of XY disomy was significantly higher in four of the five patients studied by FISH ({chi}{sup 2}>10.58, {proportional_to}<0.005), suggesting that mis-segregation caused by the failure of the XY bivalent to pair may play a role in idiopathic male infertility.

  10. Identification of cDNAs by direct hybridization using cosmid probes

    Energy Technology Data Exchange (ETDEWEB)

    Lennon, G.G.; Lieuallen, K.

    1993-12-01

    The goal of this effort is to quickly obtain as many chromosome-specific cDNAs with as much map and sequence detail as possible. Many techniques have been proposed to isolate and identify genes within defined genomic regions; the technique discussed here is direct hybridization of a relatively complex genomic probe, an entire cosmid clone, to cDNA libraries. This method continues to be a straightforward technique with a fair number of successes.

  11. Automation of ALK gene rearrangement testing with fluorescence in situ hybridization (FISH): a feasibility study.

    Science.gov (United States)

    Zwaenepoel, Karen; Merkle, Dennis; Cabillic, Florian; Berg, Erica; Belaud-Rotureau, Marc-Antoine; Grazioli, Vittorio; Herelle, Olga; Hummel, Michael; Le Calve, Michele; Lenze, Dido; Mende, Stefanie; Pauwels, Patrick; Quilichini, Benoit; Repetti, Elena

    2015-02-01

    In the past several years we have observed a significant increase in our understanding of molecular mechanisms that drive lung cancer. Specifically in the non-small cell lung cancer sub-types, ALK gene rearrangements represent a sub-group of tumors that are targetable by the tyrosine kinase inhibitor Crizotinib, resulting in significant reductions in tumor burden. Phase II and III clinical trials were performed using an ALK break-apart FISH probe kit, making FISH the gold standard for identifying ALK rearrangements in patients. FISH is often considered a labor and cost intensive molecular technique, and in this study we aimed to demonstrate feasibility for automation of ALK FISH testing, to improve laboratory workflow and ease of testing. This involved automation of the pre-treatment steps of the ALK assay using various protocols on the VP 2000 instrument, and facilitating automated scanning of the fluorescent FISH specimens for simplified enumeration on various backend scanning and analysis systems. The results indicated that ALK FISH can be automated. Significantly, both the Ikoniscope and BioView system of automated FISH scanning and analysis systems provided a robust analysis algorithm to define ALK rearrangements. In addition, the BioView system facilitated consultation of difficult cases via the internet.

  12. Probe hybridization array typing: a binary typing method for Escherichia coli.

    Science.gov (United States)

    Srinivasan, U; Zhang, L; France, A M; Ghosh, D; Shalaby, W; Xie, J; Marrs, C F; Foxman, B

    2007-01-01

    The ability to distinguish between Escherichia coli strains is critical for outbreak investigations. Binary typing, based on the presence or absence of genetic material, provides a high-throughput alternative to gel- and PCR-based typing techniques that generate complex banding patterns and lack uniform interpretation criteria. We developed, validated, and determined the discriminatory power of an E. coli binary typing method, probe hybridization array typing (PHAT). In PHAT, the absence or presence of genetic material is identified by using DNA hybridization to produce a reproducible and portable fingerprint for each genome. PHAT probes were generated from genome subtractive hybridization experiments. We PHAT typed the ECOR collection of strains from a variety of geographical locations, and 33 rectal E. coli strains selected from college-aged women with urinary tract infection. In the set of 33 human rectal strains, the discriminatory power of PHAT (98%) equaled that of multilocus sequence typing (MLST) and pulsed-field gel electrophoresis. However, for ECOR strains, which include nonhuman strains, the current set of PHAT probes was less discriminating than MLST, ribotyping, and enterobacterial repetitive intergenic consensus sequence PCR (80% versus 97, 92, and 97%, respectively). When we limited the analysis to ECOR strains of B2 and D lineage, which are associated with human infection, current PHAT probes were highly discriminatory (94%). PHAT can be applied in a high-throughput format (i.e., "library on a slide"), the discriminatory ability can be varied based on the probe set, and PHAT is readily adapted to other bacterial species with high variation in genetic content.

  13. Cloned polynucleotide and synthetic oligonucleotide probes used in colony hybridization are equally efficient in the identification of enterotoxigenic Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Sommerfelt, H.; Kalland, K.H.; Raj, P.; Moseley, S.L.; Bhan, M.K.; Bjorvatn, B.

    1988-11-01

    Restriction endonuclease-generated polynucleotide and synthetically produced oligonucleotide gene probes used in colony hybridization assays proved to be efficient for the detection and differentiation of enterotoxigenic Escherichia coli. To compare their relative efficiencies, these two sets of probes were radiolabeled with /sup 32/P and were applied to 74 strains of E. coli with known enterotoxin profiles and to 156 previously unexamined E. coli isolates. The enterotoxigenic bacteria Vibrio cholerae O1, Vibrio cholerae non-O1 (NAG), Yersinia enterocolitica, and E. coli harboring the plasmid vectors of the polynucleotide gene probes were examined for further evaluation of probe specificity. The two classes of probes showed a perfect concordance in their specific detection and differentiation of enterotoxigenic E. coli. In the analysis of six strains, the signal strength on autoradiography after hybridization with oligonucleotides was weaker than that obtained after hybridization with polynucleotide probes. The probes did not hybridize with DNA from V. cholerae O1, V. cholerae non-O1 (NAG), or Y. enterocolitica. The strains of E. coli harboring the plasmid vectors of the polynucleotide gene probes were, likewise, negative in the hybridization assays.

  14. Fluorescence in situ hybridization (FISH screening for the 22q11.2 deletion in patients with clinical features of velocardiofacial syndrome but without cardiac anomalies

    Directory of Open Access Journals (Sweden)

    Paula Sandrin-Garcia

    2007-01-01

    Full Text Available The velocardiofacial syndrome (VCFS, a condition associated with 22q11.2 deletions, is characterized by a typical facies, palatal anomalies, learning disabilities, behavioral disturbances and cardiac defects. We investigated the frequency of these chromosomal deletions in 16 individuals with VCFS features who presented no cardiac anomalies, one of the main characteristics of VCFS. Fluorescent in situ hybridization (FISH with the N25 (D22S75; 22q11.2 probe revealed deletions in ten individuals (62%. Therefore, even in the absence of cardiac anomalies testing for the 22q11.2 microdeletions in individuals showing other clinical features of this syndrome is recommended.

  15. Design and application of a fish-shaped lateral line probe for flow measurement

    Science.gov (United States)

    Tuhtan, J. A.; Fuentes-Pérez, J. F.; Strokina, N.; Toming, G.; Musall, M.; Noack, M.; Kämäräinen, J. K.; Kruusmaa, M.

    2016-04-01

    We introduce the lateral line probe (LLP) as a measurement device for natural flows. Hydraulic surveys in rivers and hydraulic structures are currently based on time-averaged velocity measurements using propellers or acoustic Doppler devices. The long-term goal is thus to develop a sensor system, which includes spatial gradients of the flow field along a fish-shaped sensor body. Interpreting the biological relevance of a collection of point velocity measurements is complicated by the fact that fish and other aquatic vertebrates experience the flow field through highly dynamic fluid-body interactions. To collect body-centric flow data, a bioinspired fish-shaped probe is equipped with a lateral line pressure sensing array, which can be applied both in the laboratory and in the field. Our objective is to introduce a new type of measurement device for body-centric data and compare its output to estimates of conventional point-based technologies. We first provide the calibration workflow for laboratory investigations. We then provide a review of two velocity estimation workflows, independent of calibration. Such workflows are required as existing field investigations consist of measurements in environments where calibration is not feasible. The mean difference for uncalibrated LLP velocity estimates from 0 to 50 cm/s under in a closed flow tunnel and open channel flume was within 4 cm/s when compared to conventional measurement techniques. Finally, spatial flow maps in a scale vertical slot fishway are compared for the LLP, direct measurements, and 3D numerical models where it was found that the LLP provided a slight overestimation of the current velocity in the jet and underestimated the velocity in the recirculation zone.

  16. Prediction of melting temperatures in fluorescence in situ hybridization (FISH) procedures using thermodynamic models

    DEFF Research Database (Denmark)

    Fontenete, Sílvia; Guimarães, Nuno; Wengel, Jesper

    2016-01-01

    Abstract The thermodynamics and kinetics of DNA hybridization, i.e. the process of self-assembly of one, two or more complementary nucleic acid strands, has been studied for many years. The appearance of the nearest-neighbor model led to several theoretical and experimental papers on DNA thermody......Abstract The thermodynamics and kinetics of DNA hybridization, i.e. the process of self-assembly of one, two or more complementary nucleic acid strands, has been studied for many years. The appearance of the nearest-neighbor model led to several theoretical and experimental papers on DNA...... thermodynamics that provide reasonably accurate thermodynamic information on nucleic acid duplexes and allow estimation of the melting temperature. Because there are no thermodynamic models specifically developed to predict the hybridization temperature of a probe used in a fluorescence in situ hybridization...

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

    Directory of Open Access Journals (Sweden)

    Ahsan Munir

    2017-05-01

    Full Text Available 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. Application of hybridization control probe to increase accuracy on ligation detection or minisequencing diagnostic microarrays

    Directory of Open Access Journals (Sweden)

    Hultman Jenni

    2009-12-01

    Full Text Available Abstract Background Nucleic acid detection based on ligation reaction or single nucleotide extension of ssDNA probes followed by tag microarray hybridization provides an accurate and sensitive detection tool for various diagnostic purposes. Since microarray quality is crucial for reliable detection, these methods can benefit from correcting for microarray artefacts using specifically adapted techniques. Findings Here we demonstrate the application of a per-spot hybridization control oligonucleotide probe and a novel way of computing normalization for tag array data. The method takes into account the absolute value of the detection probe signal and the variability in the control probe signal to significantly alleviate problems caused by artefacts and noise on low quality microarrays. Conclusions Diagnostic microarray platforms require experimental and computational tools to enable efficient correction of array artefacts. The techniques presented here improve the signal to noise ratio and help in determining true positives with better statistical significance and in allowing the use of arrays with poor quality that would otherwise be discarded.

  19. Uprobe 2008: an online resource for universal overgo hybridization-based probe retrieval and design.

    Science.gov (United States)

    Sullivan, Robert T; Morehouse, Caroline B; Thomas, James W

    2008-07-01

    Cross-species sequence comparisons are a prominent method for analyzing genomic DNA and an ever increasing number of species are being selected for whole-genome sequencing. Targeted comparative genomic sequencing is a complementary approach to whole-genome shotgun sequencing and can produce high-quality sequence assemblies of orthologous chromosomal regions of interest from multiple species. Genomic libraries necessary to support targeted mapping and sequencing projects are available for more than 90 vertebrates. An essential step for utilizing these and other genomic libraries for targeted mapping and sequencing is the development of the hybridization-based probes, which are necessary to screen a genomic library of interest. The Uprobe website (http://uprobe.genetics.emory.edu) provides a public online resource for identifying or designing 'universal' overgo-hybridization probes from conserved sequences that can be used to efficiently screen one or more genomic libraries from a designated group of species. Currently, Uprobe provides the ability to search or design probes for use in broad groups of species, including mammals and reptiles, as well as more specific clades, including marsupials, carnivores, rodents and nonhuman primates. In addition, Uprobe has the capability to design custom probes from multiple-species sequence alignments provided by the user, thus providing a general tool for targeted comparative physical mapping.

  20. Hybridization-Based Detection of Helicobacter pylori at Human Body Temperature Using Advanced Locked Nucleic Acid (LNA) Probes

    DEFF Research Database (Denmark)

    Fontenete, Sílvia; Guimarães, Nuno; Leite, Marina

    2013-01-01

    the possibility of developing a variant of fluorescence in situ hybridization (FISH), named fluorescence in vivo hybridization (FIVH), for the detection of Helicobacter pylori. Using oligonucleotide variations comprising locked nucleic acids (LNA) and 2'-O-methyl RNAs (2'OMe) with two types of backbone linkages...

  1. Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleicacid probes and tyramide signal amplification

    DEFF Research Database (Denmark)

    Silahtaroglu, Asli N.; Nolting, Dorrit; Andersen, Lars Dyrskjøt;

    2007-01-01

    The ability to determine spatial and temporal microRNA (miRNA) accumulation at the tissue, cell and subcellular levels is essential for understanding the biological roles of miRNAs and miRNA-associated gene regulatory networks. This protocol describes a method for fast and effective detection of mi......RNAs in frozen tissue sections using fluorescence in situ hybridization (FISH). The method combines the unique miRNA recognition properties of locked nucleic acid (LNA)-modified oligonucleotide probes with FISH using the tyramide signal amplification (TSA) technology. Although both approaches have previously...... protocol can be completed within approximately 6 h and allows miRNA detection in a wide variety of animal tissue cryosections as well as in human tumor biopsies at high cellular resolution....

  2. Characterization of the Arachis (Leguminosae D genome using fluorescence in situ hybridization (FISH chromosome markers and total genome DNA hybridization

    Directory of Open Access Journals (Sweden)

    Germán Robledo

    2008-01-01

    Full Text Available Chromosome markers were developed for Arachis glandulifera using fluorescence in situ hybridization (FISH of the 5S and 45S rRNA genes and heterochromatic 4'-6-diamidino-2-phenylindole (DAPI positive bands. We used chromosome landmarks identified by these markers to construct the first Arachis species ideogram in which all the homologous chromosomes were precisely identified. The comparison of this ideogram with those published for other Arachis species revealed very poor homeologies with all A and B genome taxa, supporting the special genome constitution (D genome of A. glandulifera. Genomic affinities were further investigated by dot blot hybridization of biotinylated A. glandulifera total DNA to DNA from several Arachis species, the results indicating that the D genome is positioned between the A and B genomes.

  3. Phylogeny-function analysis of (meta)genomic libraries: screening for expression of ribosomal RNA genes by large-insert library fluorescent in situ hybridization (LIL-FISH).

    Science.gov (United States)

    Leveau, Johan H J; Gerards, Saskia; de Boer, Wietse; van Veen, Johannes A

    2004-09-01

    We assessed the utility of fluorescent in situ hybridization (FISH) in the screening of clone libraries of (meta)genomic or environmental DNA for the presence and expression of bacterial ribosomal RNA (rRNA) genes. To establish proof-of-principle, we constructed a fosmid-based library in Escherichia coli of large-sized genomic DNA fragments of the mycophagous soil bacterium Collimonas fungivorans, and hybridized 768 library clones with the Collimonas-specific fluorescent probe CTE998-1015. Critical to the success of this approach (which we refer to as large-insert library FISH or LIL-FISH) was the ability to induce fosmid copy number, the exponential growth status of library clones in the FISH assay and the use of a simple pooling strategy to reduce the number of hybridizations. Twelve out of 768 E. coli clones were suspected to harbour and express Collimonas 16S rRNA genes based on their hybridization to CTE998-1015. This was confirmed by the finding that all 12 clones were also identified in an independent polymerase chain reaction-based screening of the same 768 clones using a primer set for the specific detection of Collimonas 16S ribosomal DNA (rDNA). Fosmids isolated from these clones were grouped by restriction analysis into two distinct contigs, confirming that C. fungivorans harbours at least two 16S rRNA genes. For one contig, representing 1-2% of the genome, the nucleotide sequence was determined, providing us with a narrow but informative view of Collimonas genome structure and content.

  4. Design of Hybrid Solar and Wind Energy Harvester for Fishing Boat

    Science.gov (United States)

    Banjarnahor, D. A.; Hanifan, M.; Budi, E. M.

    2017-07-01

    In southern beach of West Java, Indonesia, there are many villagers live as fishermen. They use small boats for fishing, in one to three days. Therefore, they need a fish preservation system. Fortunately, the area has high potential of solar and wind energy. This paper presents the design of a hybrid solar and wind energy harvester to power a refrigerator in the fishing boat. The refrigerator should keep the fish in 2 - 4 °C. The energy needed is 720 Wh daily. In the area, the daily average wind velocity is 4.27 m/s and the sun irradiation is 672 W/m2. The design combined two 100W solar panels and a 300W wind turbine. The testing showed that the solar panels can harvest 815 - 817 Wh of energy, while the wind turbine can harvest 43 - 62 Wh of energy daily. Therefore, the system can fulfil the energy requirement in fishing boat, although the solar panels were more dominant. To install the wind turbine on the fishing-boat, a computational design had been conducted. The boat hydrostatic dimension was measured to determine its stability condition. To reach a stable equilibrium condition, the wind turbine should be installed no more than 1.7 m of height.

  5. Detection of VHSV IVb within the gonads of Great Lakes fish using in situ hybridization.

    Science.gov (United States)

    Al-Hussinee, L; Lumsden, J S

    2011-05-24

    Viral haemorrhagic septicaemia virus (VHSV) genotype IVb was recently detected as the cause of numerous mortality events in Great Lakes fish. In situ hybridization was used to examine the gonads from 13 fish, including freshwater drum Aplodinotus grunniens and muskellunge Esox masquinongy that were infected naturally, as well as rainbow trout Oncorhynchus mykiss and fathead minnows Pimphales promelas, which were experimentally infected. Although the ovaries and testes of fish infected by VHSV IVb had few lesions, viral RNA was present in the ovaries of the rainbow trout and fathead minnow and was abundant in the gonads of muskellunge and in the ovaries of freshwater drum. Viral RNA was present mainly surrounding yolk vacuoles/granules or adjacent to the germinal vesicle, with lesser amounts found within the germinal vesicle, in the mesovarium and/or tunica albuginea and blood vessels of the ovary. Viral RNA was also found in and surrounding primary and secondary spermatocytes of the muskellunge.

  6. Progress on FISH Probes of Sludge Bulking Caused by Filamentous Bacteria%丝状细菌污泥膨胀的FISH探针研究进展

    Institute of Scientific and Technical Information of China (English)

    王萍; 余志晟; 齐嵘; 张洪勋

    2012-01-01

    Excessive growth of filamentous bacteria can cause a mass of bubbles and sludge bulking in activated sludge wastewater treatment plants (WWTPs), and result in that sludge can't be deposited drastically in the secondary clarifier and the biomass float out of the clarifier, which may impact negatively on performance and operation of WWTPs. The reported seven types of filamentous bacteria causing sludge bulking and their relative ecological physiology characteristics were summarized, and the probes detecting existing potential filamentous bacteria and their related hybridization conditions were listed in this paper. At present, fluorescence in situ hybridization (FISH) has been widely used in identification, extracellular enzymes, cell surface characteristics, and ecological physiology of filamentous bacteria in active sludge. However, few studies focus on it in China. The design of specific probe, and the quantitative FISH based on the probe is proposed as an important research direction in the future. Tab 7, Ref 57%在活性污泥法处理污水的工艺中,丝状细菌的过度繁殖常引起大量的泡沫并引发污泥膨胀.该现象的发生导致二沉池的污泥不能有效地沉淀,并大量流出,影响了污水处理厂的正常操作本文综述了引起污泥膨胀发生的7大类潜在丝状细菌及其相关生理生态学特性;列举了国内外现有的潜在丝状细菌的FISH探针及其相关的杂交条件.目前,在活性污泥丝状细菌的分类鉴定、胞外酶、细胞表面特性和相关生态生理学特性方面,荧光原位杂交(Fluorescence in situ hybridization,FISH)技术均起到重要的作用,而国内相关的研究很少.设计特异性的FISH探针,并以此进行定量荧光原位杂交,将是国内污泥膨胀问题未来研究的重点方向之一.

  7. Intrinsically Labeled Fluorescent Oligonucleotide Probes on Quantum Dots for Transduction of Nucleic Acid Hybridization.

    Science.gov (United States)

    Shahmuradyan, Anna; Krull, Ulrich J

    2016-03-15

    Quantum dots (QDs) have been widely used in chemical and biosensing due to their unique photoelectrical properties and are well suited as donors in fluorescence resonance energy transfer (FRET). Selective hybridization interactions of oligonucleotides on QDs have been determined by FRET. Typically, the QD-FRET constructs have made use of labeled targets or have implemented labeled sandwich format assays to introduce dyes in proximity to the QDs for the FRET process. The intention of this new work is to explore a method to incorporate the acceptor dye into the probe molecule. Thiazole orange (TO) derivatives are fluorescent intercalating dyes that have been used for detection of double-stranded nucleic acids. One such dye system has been reported in which single-stranded oligonucleotide probes were doubly labeled with adjacent thiazole orange derivatives. In the absence of the fully complementary (FC) oligonucleotide target, the dyes form an H-aggregate, which results in quenching of fluorescence emission due to excitonic interactions between the dyes. The hybridization of the FC target to the probe provides for dissociation of the aggregate as the dyes intercalate into the double stranded duplex, resulting in increased fluorescence. This work reports investigation of the dependence of the ratiometric signal on the type of linkage used to conjugate the dyes to the probe, the location of the dye along the length of the probe, and the distance between adjacent dye molecules. The limit of detection for 34mer and 90mer targets was found to be identical and was 10 nM (2 pmol), similar to analogous QD-FRET using labeled oligonucleotide target. The detection system could discriminate a one base pair mismatch (1BPM) target and was functional without substantial compromise of the signal in 75% serum. The 1BPM was found to reduce background signal, indicating that the structure of the mismatch affected the environment of the intercalating dyes.

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

    Science.gov (United States)

    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. A populational survey of 45S rDNA polymorphism in the Jefferson salamander Ambystoma jeffersonianum revealed by fluorescence in situ hybridization (FISH)

    Institute of Scientific and Technical Information of China (English)

    Ke BI; James P.BOGART; Jinzhong FU

    2009-01-01

    The chromosomal localization of 45S ribosomal RNA genes in Ambystoma jeffersonianum was determined by fluorescence in situ hybridization with 18S rDNA fragment as a probe (FISH-rDNA). Our results revealed the presence of rDNA polymorphism among A.jeffersonianum populations in terms of number, location and FISH signal intensity on the chromosomes. Nine rDNA cytotypes were found in ten geographically isolated populations and most of them contained derivative rDNA sites. Our preliminary study provides strong indication of karyotypic diversification of A.jeffersonianum that is demonstrated by intraspecific variation of 45S rDNA cytotypes. rDNA cytotype polymorphism has been described in many other caudate amphibians. We predict that habitat isolation, low dispersal ability and decline of effective population size could facilitate the fixation and accumulation of variable rDNA cytotypes during their chromosome evolution [Current Zoology 55(2):145-149,2009].

  10. A populational survey of 45S rDNA polymorphism in the Jefferson salamander Ambystoma jeffersonianum revealed by fluorescence in situ hybridization (FISH

    Directory of Open Access Journals (Sweden)

    Jinzhong FU

    2009-04-01

    Full Text Available The chromosomal localization of 45S ribosomal RNA genes in Ambystoma jeffersonianum was determined by fluorescence in situ hybridization with 18S rDNA fragment as a probe (FISH-rDNA. Our results revealed the presence of rDNA polymorphism among A.jeffersonianum populations in terms of number, location and FISH signal intensity on the chromosomes. Nine rDNA cytotypes were found in ten geographically isolated populations and most of them contained derivative rDNA sites. Our preliminary study provides strong indication of karyotypic diversification of A.jeffersonianum that is demonstrated by intraspecific variation of 45S rDNA cytotypes. rDNA cytotype polymorphism has been described in many other caudate amphibians. We predict that habitat isolation, low dispersal ability and decline of effective population size could facilitate the fixation and accumulation of variable rDNA cytotypes during their chromosome evolution.

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

    Science.gov (United States)

    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.

  12. Carrier-phonon interactions in hybrid halide perovskites probed with ultrafast anisotropy studies

    Science.gov (United States)

    Rivett, Jasmine P. H.; Richter, Johannes M.; Price, Michael B.; Credgington, Dan; Deschler, Felix

    2016-09-01

    Hybrid halide perovskites are at the frontier of optoelectronic research due to their excellent semiconductor properties and solution processability. For this reason, much attention has recently been focused on understanding photoexcited charge-carrier generation and recombination in these materials. Conversely, very few studies have so far been devoted to understanding carrier-carrier and carrier-phonon scattering mechanisms in these materials. This is surprising given that carrier scattering mechanisms fundamentally limit charge-carrier motilities and therefore the performance of photovoltaic devices. We apply linear polarization selective transient absorption measurements to polycrystalline CH3NH3PbBr3 hybrid halide perovskite films as an effective way of studying the scattering processes in these materials. Comparison of the photo induced bleach signals obtained when the linear polarizations of the pump and probe are aligned either parallel or perpendicular to one another, reveal a significant difference in spectral intensity and shape within the first few hundred femtoseconds after photoexcitation.

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

    Science.gov (United States)

    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.

  14. [Stain hybridization method with pRepHind probe for the diagnosis of Plasmodium falciparum].

    Science.gov (United States)

    Moleón Borodowsky, I

    1992-01-01

    A study was conducted on the parasitemia detection level and the specificity of the pRepHind DNA probe for diagnosing Plasmodium falciparum by the stain hybridization method. The parasitemia detection level was studied by using dilutions of a P. falciparum in vitro culture, adjusted by direct microscopic examination to 1; 0.1; 0.01; 0.001; 0.0001 and 0.00001% of parasited red cells. Specificity was increased by using DNA extractions from P. Yoelii, P. berghei and human leucocytes. The results showed that the method was able to detect 0.0001% of parasitemia starting from DNA extractions of 100 L infected red cells. The pRepHind probe only detected specifically DNA from P. falciparum. It is concluded that the method is suitable for being used in the diagnosis of infection due to P. falciparum.

  15. Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification.

    Science.gov (United States)

    Silahtaroglu, Asli N; Nolting, Dorrit; Dyrskjøt, Lars; Berezikov, Eugene; Møller, Morten; Tommerup, Niels; Kauppinen, Sakari

    2007-01-01

    The ability to determine spatial and temporal microRNA (miRNA) accumulation at the tissue, cell and subcellular levels is essential for understanding the biological roles of miRNAs and miRNA-associated gene regulatory networks. This protocol describes a method for fast and effective detection of miRNAs in frozen tissue sections using fluorescence in situ hybridization (FISH). The method combines the unique miRNA recognition properties of locked nucleic acid (LNA)-modified oligonucleotide probes with FISH using the tyramide signal amplification (TSA) technology. Although both approaches have previously been shown to increase detection sensitivity in FISH, combining these techniques into one protocol significantly decreases the time needed for miRNA detection in cryosections, while simultaneously retaining high detection sensitivity. Starting with fixation of the tissue sections, this miRNA FISH protocol can be completed within approximately 6 h and allows miRNA detection in a wide variety of animal tissue cryosections as well as in human tumor biopsies at high cellular resolution.

  16. The complete mitochondrial genome of Channa argus, Channa maculata and hybrid snakehead fish [Channa maculata (♀) × Channa argus (♂)].

    Science.gov (United States)

    Zhu, Shu-Ren; Ma, Ke-Yi; Xing, Zhi-Jun; Xie, Nan; Wang, Yu-Xi; Wang, Qun; Li, Jia-Le

    2013-06-01

    We sequenced and characterized the complete mitochondrial genome of Channa argus, Channa maculata and their hybrid [C. maculata (♀) and C. argus (♂)]. All the three mitochondrial genomes contained the typical complement of 13 protein-coding genes, 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs) and 1 control region. The entire mitochondrial DNA (mtDNA) molecule of C. maculata was 16,559 bp long while the complete mtDNA molecule of C. argus and hybrid snakehead fish was 16,558 bp long. This is the first report on the complete mitogenome sequence of C. maculata and hybrid snakehead fish.

  17. Effect of unlabeled helper probes on detection of an RNA target by bead-based sandwich hybridization

    DEFF Research Database (Denmark)

    Barken, K.B.; Cabig-Ciminska, M.; Holmgren, A.;

    2004-01-01

    Unlabeled helper oligonucleotides assisting a bead-based sandwich hybridization assay were tested for the optimal placement of the capture and detection probes. The target used was a full-length in vitro synthesized mRNA molecule. Helper probes complementary to regions adjacent to the binding sit....... Using an electrical chip linked to the detection probe for the detection of p-ominophenol, which is produced by alkaline phosphatase, a detection limit of 2 x 10(-13) M mRNA molecules was reached without the use of a nucleic acid amplification step.......Unlabeled helper oligonucleotides assisting a bead-based sandwich hybridization assay were tested for the optimal placement of the capture and detection probes. The target used was a full-length in vitro synthesized mRNA molecule. Helper probes complementary to regions adjacent to the binding site...

  18. Detection of airborne Legionella while showering using liquid impingement and fluorescent in situ hybridization (FISH).

    Science.gov (United States)

    Deloge-Abarkan, Magali; Ha, Thi-Lan; Robine, Enric; Zmirou-Navier, Denis; Mathieu, Laurence

    2007-01-01

    Aerosols of water contaminated with Legionella bacteria constitute the only mode of exposure for humans. However, the prevention strategy against this pathogenic bacteria risk is managed through the survey of water contamination. No relationship linked the Legionella bacteria water concentration and their airborne abundance. Therefore, new approaches in the field of the metrological aspects of Legionella bioaerosols are required. This study was aimed at testing the main principles for bioaerosol collection (solid impaction, liquid impingement and filtration) and the in situ hybridization (FISH) method, both in laboratory and field assays, with the intention of applying such methodologies for airborne Legionella bacteria detection while showering. An aerosolization chamber was developed to generate controlled and reproducible L. pneumophila aerosols. This tool allowed the identification of the liquid impingement method as the most appropriate one for collecting airborne Legionella bacteria. The culturable fraction of airborne L. pneumophila recovered with the liquid impingement principle was 4 and 700 times higher compared to the impaction and filtration techniques, respectively. Moreover, the concentrations of airborne L. pneumophila in the impinger fluid were on average 7.0 x 10(5) FISH-cells m(-3) air with the fluorescent in situ hybridization (FISH) method versus 9.0 x 10(4) CFU m(-3) air with the culture method. These results, recorded under well-controlled conditions, were confirmed during the field experiments performed on aerosols generated by hot water showers in health institutions. This new approach may provide a more accurate characterization of aerobiocontamination by Legionella bacteria.

  19. Segregation of Species-Specific Male Attractiveness in F2 Hybrid Lake Malawi Cichlid Fish

    Directory of Open Access Journals (Sweden)

    Ola Svensson

    2011-01-01

    Full Text Available Among the huge radiations of haplochromine cichlid fish in Lakes Malawi and Victoria, closely related species are often reproductively isolated via female mate choice although viable fertile hybrids can be produced when females are confined only with heterospecific males. We generated F2 hybrid males from a cross between a pair of closely related sympatric cichlid fish from Lake Malawi. Laboratory mate choice experiments using microsatellite paternity analysis demonstrated that F2 hybrid males differed significantly in their attractiveness to females of the two parental species, indicating heritable variation in traits involved in mate choice that may contribute to reproductive isolation between these species. We found no significant correlation between male mating success and any measurement of male colour pattern. A simple quantitative genetic model of reproductive isolation suggests that there may be as few as two chromosomal regions controlling species-specific attractiveness. We propose that adaptive radiation of Lake Malawi cichlids could be facilitated by the presence of genes with major effects on mate choice and reproductive isolation.

  20. Water quality and plankton communities in hybrid catfish (female channel catfish, Ictalurus punctatus x male blue catfish, I. furcatus) ponds after partial fish harvest

    Science.gov (United States)

    Twelve, 0.4-ha ponds were stocked with 10,000 hybrid catfish fingerlings in March 2015. Six ponds were partially harvested in August to remove fish larger than ~ 0.57 kg. All remaining fish were removed in October and November. Partial harvest of faster-growing fish removed ~26% of fish initially st...

  1. Preliminary test of an imaging probe for nuclear medicine using hybrid pixel detectors

    CERN Document Server

    Bertolucci, Ennio; Mettivier, G; Montesi, M C; Russo, P

    2002-01-01

    We are investigating the feasibility of an intraoperative imaging probe for lymphoscintigraphy with Tc-99m tracer, for sentinel node radioguided surgery, using the Medipix series of hybrid detectors coupled to a collimator. These detectors are pixelated semiconductor detectors bump-bonded to the Medipix1 photon counting read-out chip (64x64 pixel, 170 mu m pitch) or to the Medipix2 chip (256x256 pixel, 55 mu m pitch), developed by the European Medipix collaboration. The pixel detector we plan to use in the final version of the probe is a semi-insulating GaAs detector or a 1-2 mm thick CdZnTe detector. For the preliminary tests presented here, we used 300-mu m thick silicon detectors, hybridized via bump-bonding to the Medipix1 chip. We used a tungsten parallel-hole collimator (7 mm thick, matrix array of 64x64 100 mu m circular holes with 170 mu m pitch), and a 22, 60 and 122 keV point-like (1 mm diameter) radioactive sources, placed at various distances from the detector. These tests were conducted in order ...

  2. Highly Sensitive Fluorescent-labeled Probes and Glass Slide Hybridization for the Detection of Plant RNA Viruses and a Viroid

    Institute of Scientific and Technical Information of China (English)

    Zhiyou DU; Bo JIN; Wenhong LIU; Liang CHEN; Jishuang CHEN

    2007-01-01

    In this study, a modified method of the conventional RNA dot-blot hybridization was established, by replacing 32P labels with CY5 labels and replacing nylon membranes with positive-charged glass slides, for detecting plant RNA viruses and a viroid. The modified RNA dot-blot hybridization method was named glass slide hybridization. The optimum efficiency of RNA binding onto the surfaces of activated glass slide was achieved using aminosilane-coated glass slide as a solid matrix and 5×saline sodium citrate (SSC) as a spotting solution. Using a CY5-labeled DNA probe prepared through PCR amplification, the optimized glass slide hybridization could detect as little as 1.71 pg of tobacco mosaic virus (TMV) RNA.The sensitivity of the modified method was four times that of dot-blot hybridization on nylon membrane with a 32P-labeled probe. The absence of false positive within the genus Potyvirus [potato virus A, potato virus Y (PVY) and zucchini yellow mosaic virus] showed that this method was highly specific. Furthermore,potato spindle tuber viroid (PSTVd) was also detected specifically. A test of 40 field potato samples showed that this method was equivalent to the conventional dot-blot hybridization for detecting PVY and PSTVd. To our knowledge, this is the first report of using dot-blot hybridization on glass slides with fluorescent-labeled probes for detecting plant RNA viruses and a viroid.

  3. Optical response of a quantum dot-metal nanoparticle hybrid interacting with a weak probe field.

    Science.gov (United States)

    Kosionis, Spyridon G; Terzis, Andreas F; Sadeghi, Seyed M; Paspalakis, Emmanuel

    2013-01-30

    We study optical effects in a hybrid system composed of a semiconductor quantum dot and a spherical metal nanoparticle that interacts with a weak probe electromagnetic field. We use modified nonlinear density matrix equations for the description of the optical properties of the system and obtain a closed-form expression for the linear susceptibilities of the quantum dot, the metal nanoparticle, and the total system. We then investigate the dependence of the susceptibility on the interparticle distance as well as on the material parameters of the hybrid system. We find that the susceptibility of the quantum dot exhibits optical transparency for specific frequencies. In addition, we show that there is a range of frequencies of the applied field for which the susceptibility of the semiconductor quantum dot leads to gain. This suggests that in such a hybrid system quantum coherence can reverse the course of energy transfer, allowing flow of energy from the metallic nanoparticle to the quantum dot. We also explore the susceptibility of the metal nanoparticle and show that it is strongly influenced by the presence of the quantum dot.

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

    Science.gov (United States)

    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.

  5. Identification of intracellular bacteria in adenoid and tonsil tissue specimens: the efficiency of culture versus fluorescent in situ hybridization (FISH).

    Science.gov (United States)

    Stępińska, M; Olszewska-Sosińska, O; Lau-Dworak, M; Zielnik-Jurkiewicz, B; Trafny, E A

    2014-01-01

    Monocyte/macrophage cells from human nasopharyngeal lymphoid tissue can be a source of bacteria responsible for human chronic and recurrent upper respiratory tract infection. Detection and characterization of pathogens surviving intracellularly could be a key element in bacteriological diagnosis of the infections as well as in the study on interactions between bacteria and their host. The present study was undertaken to assess the possibility of isolation of viable bacteria from the cells expressing monocyte/macrophage marker CD14 in nasopharyngeal lymphoid tissue. Overall, 74 adenotonsillectomy specimens (adenoids and tonsils) from 37 children with adenoid hypertrophy and recurrent infections as well as 15 specimens from nine children with adenoid hypertrophy, which do not suffer from upper respiratory tract infections (the control group), were studied. The suitability of immunomagnetic separation for extraction of CD14(+) cells from lymphoid tissue and for further isolation of the intracellular pathogens has been shown. The coexistence of living pathogens including Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pyogenes with the bacteria representing normal nasopharyngeal microbiota inside CD14(+) cells was demonstrated. Twenty-four strains of these pathogens from 32.4 % of the lysates of CD14(+) cells were isolated. Concurrently, the fluorescent in situ hybridization (FISH) with a universal EUB388, and the species-specific probes demonstrated twice more often the persistence of these bacterial species in the lysates of CD14(+) cells than conventional culture. Although the FISH technique appears to be more sensitive than traditional culture in the intracellular bacteria identification, the doubts on whether the bacteria are alive, and therefore, pathogenic would still exist without the strain cultivation.

  6. Comparison of immunohistochemistry (IHC and fluorescence in situ hybridization (FISH assessment for Her-2 status in breast cancer

    Directory of Open Access Journals (Sweden)

    Qi Minfang

    2009-11-01

    Full Text Available Abstract Background The concordance rate between IHC and FISH according to clinical performance is still controversial. We report a prospective study to reflect the concordance between IHC and FISH in Guilin city, People's Republic of China. Methods Fifty cases of invasive ductal carcinoma of breast tested by IHC and scored as 0, 1+, 2+ and 3+ by pathologists were further analyzed by FISH using a commercially available double-color probe, and the FISH findings were compared with IHC test results. Results A total concordance of 82.0% was observed with a Kappa coefficient of 0.640 (P Conclusion The IHC can be used firstly to screen the HER-2 status, and FISH can be used as a supplementary role to IHC and 2+ and some negative cases. And only those cases with Her-2 status of IHC 3+ or FISH positive should be treated with Herceptin.

  7. Insertional translocation detected using FISH confirmation of array-comparative genomic hybridization (aCGH) results.

    Science.gov (United States)

    Kang, Sung-Hae L; Shaw, Chad; Ou, Zhishuo; Eng, Patricia A; Cooper, M Lance; Pursley, Amber N; Sahoo, Trilochan; Bacino, Carlos A; Chinault, A Craig; Stankiewicz, Pawel; Patel, Ankita; Lupski, James R; Cheung, Sau Wai

    2010-05-01

    Insertional translocations (ITs) are rare events that require at least three breaks in the chromosomes involved and thus qualify as complex chromosomal rearrangements (CCR). In the current study, we identified 40 ITs from approximately 18,000 clinical cases (1:500) using array-comparative genomic hybridization (aCGH) in conjunction with fluorescence in situ hybridization (FISH) confirmation of the aCGH findings, and parental follow-up studies. Both submicroscopic and microscopically visible IT events were detected. They were divided into three major categories: (1) simple intrachromosomal and interchromosomal IT resulting in pure segmental trisomy, (2) complex IT involving more than one abnormality, (3) deletion inherited from a parent with a balanced IT resulting in pure segmental monosomy. Of the cases in which follow-up parental studies were available, over half showed inheritance from an apparently unaffected parent carrying the same unbalanced rearrangement detected in the propositi, thus decreasing the likelihood that these IT events are clinically relevant. Nevertheless, we identified six cases in which small submicroscopic events were detected involving known disease-associated genes/genomic segments and are likely to be pathogenic. We recommend that copy number gains detected by clinical aCGH analysis should be confirmed using FISH analysis whenever possible in order to determine the physical location of the duplicated segment. We hypothesize that the increased use of aCGH in the clinic will demonstrate that IT occurs more frequently than previously considered but can identify genomic rearrangements with unclear clinical significance.

  8. Fluorescence in situ hybridization (FISH analysis of primary ocular adnexal MALT lymphoma

    Directory of Open Access Journals (Sweden)

    Harada Mine

    2006-10-01

    Full Text Available Abstract Background It remains unknown whether primary ocular adnexal extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma is a homogeneous entity, as there are few reports of the results of cytogenetic or molecular analyses of these tumors. Methods We performed interphase fluorescence in situ hybridization (FISH analysis to detect translocations and aneuploidy in 34 cases of primary ocular adnexal MALT lymphoma, and reviewed the histopathological findings. Correlations between the results of FISH analysis, the histopathological features and the clinical data were also analyzed. Results Among the 34 cases, FISH analysis revealed t(14;18(q32;q21 in one case, trisomy 3 in 21 cases (62%, and trisomy 18 in 16 cases (47%. The cases with trisomy 18 had significantly more prominent lymphoepithelial lesions (LELs and less nodularity in the tumors. In regard to the clinical correlations, tumors with trisomy 18 were observed predominantly in females and younger patients; also, in the majority of the cases, the tumor was of conjunctival origin. All the cases with recurrence showed trisomy 18 in the tumor. Conclusion Primary ocular adnexal MALT lymphoma is a significantly heterogeneous entity. Cases with trisomy 18 may have unique clinicopathological features.

  9. Identification of Dekkera bruxellensis (Brettanomyces) from wine by fluorescence in situ hybridization using peptide nucleic acid probes.

    Science.gov (United States)

    Stender, H; Kurtzman, C; Hyldig-Nielsen, J J; Sørensen, D; Broomer, A; Oliveira, K; Perry-O'Keefe, H; Sage, A; Young, B; Coull, J

    2001-02-01

    A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the species D. bruxellensis and that the new method is able to identify Brettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity.

  10. Comet-FISH with strand-specific probes reveals transcription-coupled repair of 8-oxoGuanine in human cells.

    Science.gov (United States)

    Guo, Jia; Hanawalt, Philip C; Spivak, Graciela

    2013-09-01

    Oxidized bases in DNA have been implicated in cancer, aging and neurodegenerative disease. We have developed an approach combining single-cell gel electrophoresis (comet) with fluorescence in situ hybridization (FISH) that enables the comparative quantification of low, physiologically relevant levels of DNA lesions in the respective strands of defined nucleotide sequences and in the genome overall. We have synthesized single-stranded probes targeting the termini of DNA segments of interest using a polymerase chain reaction-based method. These probes facilitate detection of damage at the single-molecule level, as the lesions are converted to DNA strand breaks by lesion-specific endonucleases or glycosylases. To validate our method, we have documented transcription-coupled repair of cyclobutane pyrimidine dimers in the ataxia telangiectasia-mutated (ATM) gene in human fibroblasts irradiated with 254 nm ultraviolet at 0.1 J/m2, a dose ∼100-fold lower than those typically used. The high specificity and sensitivity of our approach revealed that 7,8-dihydro-8-oxoguanine (8-oxoG) at an incidence of approximately three lesions per megabase is preferentially repaired in the transcribed strand of the ATM gene. We have also demonstrated that the hOGG1, XPA, CSB and UVSSA proteins, as well as actively elongating RNA polymerase II, are required for this process, suggesting cross-talk between DNA repair pathways.

  11. Dominant Microbial Composition and Its Vertical Distribution in Saline Meromictic Lake Kaiike (Japan) as Revealed by Quantitative Oligonucleotide Probe Membrane Hybridization

    OpenAIRE

    Koizumi, Yoshikazu; Kojima, Hisaya; Fukui, Manabu

    2004-01-01

    Vertical distributions of dominant bacterial populations in saline meromictic Lake Kaiike were investigated throughout the water column and sediment by quantitative oligonucleotide probe membrane hybridization. Three oligonucleotide probes specific for the small-subunit (SSU) rRNA of three groups of Chlorobiaceae were newly designed. In addition, three general domain (Bacteria, Archaea, and Eukarya)-specific probes, two δ-Proteobacteria-specific probes, a Chlorobiaceae-specific probe, and a C...

  12. Genotyping of the CCR5 chemokine receptor by isothermal NASBA amplification and differential probe hybridization.

    Science.gov (United States)

    Romano, J W; Tetali, S; Lee, E M; Shurtliff, R N; Wang, X P; Pahwa, S; Kaplan, M H; Ginocchio, C C

    1999-11-01

    The human CCR5 chemokine receptor functions as a coreceptor with CD4 for infection by macrophage-tropic isolates of human immunodeficiency virus type 1 (HIV-1). A mutated CCR5 allele which encodes a protein that does not function as a coreceptor for HIV-1 has been identified. Thus, expression of the wild-type and/or mutation allele is relevant to determining the infectability of patient peripheral blood mononuclear cells (PBMC) and affects disease progression in vivo. We developed a qualitative CCR5 genotyping assay using NASBA, an isothermal nucleic acid amplification technology. The method involves three enzymes and two oligonucleotides and targets the CCR5 mRNA, which is expressed in PBMC at a copy number higher than 2, the number of copies of DNA present encoding the gene. The single oligonucleotide set amplifies both alleles, and genotyping is achieved by separate hybridizations of wild-type- and mutation-specific probes directly to the single-stranded RNA amplification product. Assay sensitivity and specificity were demonstrated with RNAs produced in vitro from plasmid clones bearing the DNA encoding each allele. No detectable cross-reactivity between wild-type and mutation probes was found, and 50 copies of each allele were readily detectable. Analysis of patient samples found that 20% were heterozygous and 1% were homozygous for the CCR5 mutation. Thus, NASBA is a sensitive and specific means of rapidly determining CCR5 genotype and provides several technical advantages over alternative assay systems.

  13. A Sensitive Alternative for MicroRNA In Situ Hybridizations Using Probes of 2'-O-Methyl RNA + LNA

    DEFF Research Database (Denmark)

    Søe, Martin Jensen; Møller, Trine; Dufva, Martin;

    2011-01-01

    The use of short, high-affinity probes consisting of a combination of DNA and locked nucleic acid (LNA) has enabled the specific detection of microRNAs (miRNAs) by in situ hybridization (ISH). However, detection of low–copy number miRNAs is still not always possible. Here the authors show...... that probes consisting of 2'-O-methyl RNAs (2OMe) and LNA at every third base (2:1 ratio), under optimized hybridization conditions, excluding yeast RNA from the hybridization buffer, can provide superior performance in detection of miRNA targets in terms of sensitivity and signal-to-noise ratio compared...... to DNA + LNA probes. Furthermore, they show that hybridizations can be performed in buffers of 4M urea instead of 50% formamide, thereby yielding an equally specific but nontoxic assay. The use of 2OMe + LNA–based probes and the optimized ISH assay enable simple and fast detection of low–copy number mi...

  14. A LightCycler real-time PCR hybridization probe assay for detecting food-borne thermophilic Campylobacter

    DEFF Research Database (Denmark)

    Perelle, S.; Josefsen, Mathilde Hartmann; Hoorfar, Jeffrey

    2004-01-01

    Cycler real-time PCR assay (LC-PCR), which used fluorescent hybridization probes was developed. The test incorporated an internal amplification control co-amplified with the 16S rRNA gene of Campylobacter to monitor potential PCR inhibitors and ensure successful amplifications. The specificity study involving...

  15. Exploring the Hybridization Thermodynamics of Spherical Nucleic Acids to Tailor Probes for Diagnostic and Therapeutic Applications

    Science.gov (United States)

    Randeria, Pratik Shailesh

    Spherical nucleic acids (SNAs), three-dimensional nanoparticle conjugates composed of densely packed and highly oriented oligonucleotides around organic or inorganic nanoparticles, are an emergent class of nanostructures that show promise as single-entity agents for intracellular messenger RNA (mRNA) detection and gene regulation. SNAs exhibit superior biocompatibility and biological properties compared to linear oligonucleotides, enabling them to overcome many of the limitations of linear oligonucleotides for use in biomedical applications. However, the origins of these biologically attractive properties are not well understood. In this dissertation, the chemistry underlying one such property is studied in detail, and the findings are applied towards the rational design of more effective SNAs for diagnostic and therapeutic applications. Chapter 1 introduces the synthesis of SNAs, the unique properties that make them superior to linear nucleic acids for biomedicine, and previously studied applications of these structures. Chapter 2 focuses on quantitatively studying the impact of the chemical structure of the SNA on its ability to hybridize multiple complementary nucleic acids. This chapter lays the groundwork for understanding the factors that govern SNA hybridization thermodynamics and how to tailor SNAs to increase their binding affinity to target mRNA strands. Chapters 3 and 4 capitalize on this knowledge to engineer probes for intracellular mRNA detection and gene regulation applications. Chapter 3 reports the development of an SNA-based probe that can simultaneously report the expression level of two different mRNA transcripts in live cells and differentiate diseased cells from non-diseased cells. Chapter 4 investigates the use of topically-applied SNAs to down-regulate a critical mediator of impaired wound healing in diabetic mice to accelerate wound closure. This study represents the first topical therapeutic application of SNA nanotechnology to treat open

  16. Fluorescence In Situ Hybridization (FISH-Based Karyotyping Reveals Rapid Evolution of Centromeric and Subtelomeric Repeats in Common Bean (Phaseolus vulgaris and Relatives

    Directory of Open Access Journals (Sweden)

    Aiko Iwata-Otsubo

    2016-04-01

    Full Text Available Fluorescence in situ hybridization (FISH-based karyotyping is a powerful cytogenetics tool to study chromosome organization, behavior, and chromosome evolution. Here, we developed a FISH-based karyotyping system using a probe mixture comprised of centromeric and subtelomeric satellite repeats, 5S rDNA, and chromosome-specific BAC clones in common bean, which enables one to unambiguously distinguish all 11 chromosome pairs. Furthermore, we applied the karyotyping system to several wild relatives and landraces of common bean from two distinct gene pools, as well as other related Phaseolus species, to investigate repeat evolution in the genus Phaseolus. Comparison of karyotype maps within common bean indicates that chromosomal distribution of the centromeric and subtelomeric satellite repeats is stable, whereas the copy number of the repeats was variable, indicating rapid amplification/reduction of the repeats in specific genomic regions. In Phaseolus species that diverged approximately 2–4 million yr ago, copy numbers of centromeric repeats were largely reduced or diverged, and chromosomal distributions have changed, suggesting rapid evolution of centromeric repeats. We also detected variation in the distribution pattern of subtelomeric repeats in Phaseolus species. The FISH-based karyotyping system revealed that satellite repeats are actively and rapidly evolving, forming genomic features unique to individual common bean accessions and Phaseolus species.

  17. Trade-off between morphological convergence and opportunistic diet behavior in fish hybrid zone

    Directory of Open Access Journals (Sweden)

    Grey Jonathan

    2009-10-01

    Full Text Available Abstract Background The invasive Chondrostoma nasus nasus has colonized part of the distribution area of the protected endemic species Chondrostoma toxostoma toxostoma. This hybrid zone is a complex system where multiple effects such as inter-species competition, bi-directional introgression, strong environmental pressure and so on are combined. Why do sympatric Chondrostoma fish present a unidirectional change in body shape? Is this the result of inter-species interactions and/or a response to environmental effects or the result of trade-offs? Studies focusing on the understanding of a trade-off between multiple parameters are still rare. Although this has previously been done for Cichlid species flock and for Darwin finches, where mouth or beak morphology were coupled to diet and genetic identification, no similar studies have been done for a fish hybrid zone in a river. We tested the correlation between morphology (body and mouth morphology, diet (stable carbon and nitrogen isotopes and genomic combinations in different allopatric and sympatric populations for a global data set of 1330 specimens. To separate the species interaction effect from the environmental effect in sympatry, we distinguished two data sets: the first one was obtained from a highly regulated part of the river and the second was obtained from specimens coming from the less regulated part. Results The distribution of the hybrid combinations was different in the two part of the sympatric zone, whereas all the specimens presented similar overall changes in body shape and in mouth morphology. Sympatric specimens were also characterized by a larger diet behavior variance than reference populations, characteristic of an opportunistic diet. No correlation was established between the body shape (or mouth deformation and the stable isotope signature. Conclusion The Durance River is an untamed Mediterranean river despite the presence of numerous dams that split the river from

  18. Trade-off between morphological convergence and opportunistic diet behavior in fish hybrid zone.

    Science.gov (United States)

    Corse, Emmanuel; Costedoat, Caroline; Pech, Nicolas; Chappaz, Rémi; Grey, Jonathan; Gilles, André

    2009-10-27

    The invasive Chondrostoma nasus nasus has colonized part of the distribution area of the protected endemic species Chondrostoma toxostoma toxostoma. This hybrid zone is a complex system where multiple effects such as inter-species competition, bi-directional introgression, strong environmental pressure and so on are combined. Why do sympatric Chondrostoma fish present a unidirectional change in body shape? Is this the result of inter-species interactions and/or a response to environmental effects or the result of trade-offs? Studies focusing on the understanding of a trade-off between multiple parameters are still rare. Although this has previously been done for Cichlid species flock and for Darwin finches, where mouth or beak morphology were coupled to diet and genetic identification, no similar studies have been done for a fish hybrid zone in a river. We tested the correlation between morphology (body and mouth morphology), diet (stable carbon and nitrogen isotopes) and genomic combinations in different allopatric and sympatric populations for a global data set of 1330 specimens. To separate the species interaction effect from the environmental effect in sympatry, we distinguished two data sets: the first one was obtained from a highly regulated part of the river and the second was obtained from specimens coming from the less regulated part. The distribution of the hybrid combinations was different in the two part of the sympatric zone, whereas all the specimens presented similar overall changes in body shape and in mouth morphology. Sympatric specimens were also characterized by a larger diet behavior variance than reference populations, characteristic of an opportunistic diet. No correlation was established between the body shape (or mouth deformation) and the stable isotope signature. The Durance River is an untamed Mediterranean river despite the presence of numerous dams that split the river from upstream to downstream. The sympatric effect on morphology and

  19. PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei.

    Science.gov (United States)

    Müller, Patrick; Rößler, Jens; Schwarz-Finsterle, Jutta; Schmitt, Eberhard; Hausmann, Michael

    2016-07-01

    Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes). Gene targets can be specifically labelled with at least about 20 PNA-probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3d-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy.

  20. Salmonellae in fish feces analyzed by in situ hybridization and quantitative polymerase chain reaction.

    Science.gov (United States)

    Sha, Qiong; Forstner, Michael R J; Bonner, Timothy H; Hahn, Dittmar

    2013-09-01

    The potential of fish to transfer salmonellae from heterogeneous aquatic biofilms into feces was assessed in controlled aquarium studies with Suckermouth Catfish Hypostomus plecostomus and with biofilms inoculated with salmonellae. Neither the presence of catfish nor inoculation with salmonellae had detectable effects on the abundance of the microbial community. Densities of the microbial community were about 10(5) cells/mL in the water during a 1-week period, whereas densities of the microbial community increased 10-fold (10(6) to 10(7) cells/mg) in catfish feces during the same period. Salmonellae were detected by both quantitative polymerase chain reaction (qPCR) and situ hybridization in water samples immediately after inoculation, in numbers of about 10(4) cells/mL, representing up to 20% of the cells of the microbial community. Numbers decreased by three orders of magnitude within the first 3 d of the study, which represented only 0.01% of the community, and became undetectable after day 5. In catfish feces, numbers of Salmonella initially increased to up to 6% of the cells of the community but then declined. These results suggest that Salmonella are not biomagnified during gut passage, and thus, fish only provide a means for the translocation of this pathogen.

  1. CONGESTION MANAGEMENT BY OPTIMAL ALLOCATION OF FACTS CONTROLLERS USING HYBRID FISH BEE OPTIMIZATION

    Directory of Open Access Journals (Sweden)

    S. Thangalakshmi

    2014-01-01

    Full Text Available The role of Independent System Operator (ISO in the restructured power industry includes system control, capacity planning, transmission tariff and congestion management; the challenging task being minimizing the congestion. One of the popular techniques used to alleviate congestion is using Flexible AC Transmission Systems (FACTS devices. The power system generally operates near its rated capacity in deregulated market because of intensive usage of transmission grids. So, the major issues that need to be addressed are improving the voltage profile and reducing the power loss in the electrical network. Motivation: The location of FACTS devices can improve the power flow in the line, maintain the bus profile and reduce the losses. However locating the ideal location is a NP problem. This study presents a novel heuristic method to determine the types of FACTS devices and its optimal location in a power system without violating the thermal and voltage limits. Power flow sensitivity index to find the optimal location of UPFC is suggested in this study. A hybrid fish bee swarm optimization is proposed which is based on Artificial Bee Colony (ABC and Fish School Search (FSS methods. This proposed algorithm is tested based on IEEE 30 bus system and line performances are studied.

  2. Diagnosis of a constitutional five-chromosome rearrangement by fluorescent in situ hybridization (FISH)

    Energy Technology Data Exchange (ETDEWEB)

    Tsien, F.; Shapira, E. [Tulane Univ. School of Medicine, New Orleans, LA (United States); Carvalho, T. [Hospital Sarah Kubitschek, Brasilia (Brazil)] [and others

    1994-09-01

    Complex chromosomal rearrangements are structural rearrangements involving at least three chromosomes and three or more chromosome breakpoints. Such karyotypes are often acquired during cancer multi-step development and in chromosome instability syndromes. However, extremely rare constitutional forms have been reported, most of which are incompatible with life. We present a 2-year-old female with de novo complex rearrangement consisting of five chromosomes and nine breakpoints. Clinical evaluation at two years of age revealed a weight of 5 kg, length of 66 cm, and had circumference of 38 cm, all below the 5th percentile, microcephaly, trigonocephaly, epicanthal folds, inguinal hernia, left clubfoot, hypertonicity, and developmental delay. The neurological examination revealed chorea-acanthocytosis and psychomotor delay. Cultured lymphocytes and fibroblasts revealed a karyotype consisting of five derivative chromosomes. The metaphases were further analyzed by FISH using chromosome-specific libraries and telomeric probes in order to delineate the composition of the rearranged chromosomes; FISH results demonstrated a karyotype of: 46,XX,1pter{r_arrow}1q25::1q42.1{r_arrow}1qter, 2pter{r_arrow}q32.3::1q32.3{r_arrow}2q41::2q37.3{r_arrow}2qter, 7qter{r_arrow}7q21.2::6q22.3{r_arrow}6qter::1q31{r_arrow}1q32.3::6p23{r_arrow}6q22.3, 7pter{r_arrow}7q21.1::6p23{r_arrow}6pter, 2q33{r_arrow}2q37, 1::9p21{r_arrow}9qter. This analysis demonstrates the usefulness of FISH in characterizing complex chromosome rearrangements otherwise difficult to correctly interpret using classical cytogenetics alone.

  3. Robust hybridization-based genotyping probes for HPV 6, 11, 16 and 18 obtained via in vitro selection

    Directory of Open Access Journals (Sweden)

    Ivan B. Brukner

    2010-04-01

    Full Text Available This paper describes the technical and analytical performance of a novel set of hybridization probes for the four GARDASIL® vaccine-relevant HPV types (6, 11, 16 and 18. These probes are obtained through in vitro selection from a pool of random oligonucleotides, rather than the traditional “rational design” approach typically used as the initial step in assay development. The type-specific segment of the HPV genome was amplified using a GP5+/6+ PCR protocol and 39 synthetic oligonucleotide templates derived from each of the HPV types, as PCR targets. The robust performance of the 4 selected hybridization probes was demonstrated by monitoring the preservation of the specificity and sensitivity of the typing assay over all 39 HPV types, using a different spectrum of HPV (genome equivalent: 103-109 and human DNA concentrations (10-100 ng as well as temperature and buffer composition variations. To the Authors’ knowledge, this is a unique hybridization-based multiplex typing assay. It performs at ambient temperatures, does not require the strict temperature control of hybridization conditions, and is functional with a number of different non-denaturing buffers, thereby offering downstream compatibility with a variety of detection methods. Studies aimed at demonstrating clinical performance are needed to validate the applicability of this strategy.

  4. Dot-Blot Hybridization for Detection of Five Cucurbit Viruses by Digoxigenin-Labelled cDNA Probes

    Institute of Scientific and Technical Information of China (English)

    MENG Juan; GU Qin-sheng; LIN Shi-ming; PENG Bin; LIU Li-feng; TIAN Yan-ping; LI Li

    2007-01-01

    Dot-blot hybridization was applied in this paper to detect five viruses infecting cucurbitaceous crops,Zuccini yellow mosaic virus(ZYMV),Watermelon mosaic virus(WMV),Cucumber mosaic virus(CMV),Papaya ringspot virus watermelon strain(PRSV-W)and Squash mosaic virus(SqMV),as a good alternative assay in seed health test and epidemiological and transgenic research.Digoxigenin-labelled cDNA probes of the five viruses were synthesized by PCR with the specific primers and applied in dot-blot hybridization to detect five viruses in crude extraction of the infected leaves.And three SqMV probes of different lengths(0.55,1.6,and 2.7 kb,respectively)were designed to investigate the effect of hybridization.The results showed that the sensitivity for detecting the crude extraction of infected leaves by ZYMV,WMV,CMV,PRSV-W,and SqMV was down to 1:160,1:160,1:320,1:160,and 1:320,respectively.Three SqMV probes of different length showed no differences on the sensitivity and specificity.The digoxigenin-labelled probes prepared by PCR could be used for accurate and rapid identification of 5 viruses infecting cucurbitaceous crops with good stabilities,sensitivities,specificity,and reproducibilities.

  5. Trypanosomiasis in hybrid catfish (Clarias macrocephalus x Clarias gariepinus and other freshwater fishes

    Directory of Open Access Journals (Sweden)

    Promkhunthong, W.

    2005-02-01

    Full Text Available The infestation of Trypanosoma sp. (Kinetoplastida in the hybrid catfish and some fresh water fish was studied showing such parasitic infestation was specific only in catfish. The virulence as determined by LD50 for 5 days was 2.28x1010 cell in a fish sample. The parasitic infestation caused hematological changes by the reduction of red as well as white blood cells. The reductions were highly significant as compared to the healthy sample (p<0.05 as noted by the red and white blood cell count which dropped from 2.14±0.48x106 to 1.62±0.27x106 cells/ml and from 1.45±3.76x105 to 2.42±0.78x104 cells/ml blood in the infested samples, respectively. Similar trend was noted for hemoglobin and hematocrit which dropped significantly (p<0.05. The hemoglobin in healthy fish is 7.075±0.929g/100g, which dropped to 6.268±0.697g/100g in the samples with infestation. The percentage of hematocrit in healthy sample is 25.275±3.318%, which dropped to 21.722±3.068% in the samples with infestation. The reverse trend was recognized for serum protein andleukocrit which increased in the samples with Trypanosoma sp. infestation. The density gradient centrifugation technique was employed in the isolation of parasites in which 50% Percoll solution in 0.85% final preparation of saline solution was capable of removing Trypanosoma sp. from the blood. The study of antibody levels in serum showed that the infested hybrid catfish could develop the antibody which reached a peak 14 days after the infestation. Trypanosoma sp. was unable to cause histological changes in the tissues of gill, liver, kidney, spleen, stomach and intestine. Minor inflammations were observed, even the cases that large number of parasites were found in the tissues, blood streams and sinuses. Marked reductions were recorded for mature red blood cells while there were the formation of immature red blood and phagocytotic cells at higher rates as compared to the healthy individual.

  6. Group-specific small-subunit rRNA hybridization probes to characterize filamentous foaming in activated sludge systems.

    Science.gov (United States)

    de los Reyes, F L; Ritter, W; Raskin, L

    1997-03-01

    Foaming in activated sludge systems is characterized by the formation of a thick, chocolate brown-colored scum that floats on the surface of aeration basins and secondary clarifiers. These viscous foams have been associated with the presence of filamentous mycolic acid-containing actinomycetes. To aid in evaluating the microbial representation in foam, we developed and characterized group-, genus-, and species-specific oligonucleotide probes targeting the small subunit rRNA of the Mycobacterium complex, Gordona spp., and Gordona (Nocardia) amarae, respectively. The use of a universal base analog, 5-nitroindole, in oligonucleotide probe design was evaluated by comparing the characteristics of two different versions of the Mycobacterium complex probe. The temperature of dissociation of each probe was determined. Probe specificity studies with a diverse collection of 67 target and nontarget rRNAs demonstrated the specificity of the probes to the target groups. Whole-cell hybridizations with fluorescein- and rhodamine-labeled probes were performed with pure cultures of various members of the Mycobacterium complex as well as with environmental samples from a full-scale activated sludge plant which experienced foaming. Quantitative membrane hybridizations with activated sludge and anaerobic digester foam showed that 15.0 to 18.3% of the total small-subunit rRNAs could be attributed to members of the Mycobacterium complex, of which a vast majority consisted of Gordona rRNA. Several G. amarae strains made up only a very small percentage of the Gordona strains present. We demonstrated that group-specific rRNA probes are useful tools for the in situ monitoring and identification of filamentous bacteria in activated sludge systems.

  7. One-year monitoring of an oligonucleotide fluorescence in situ hybridization probe panel laboratory-developed test for bladder cancer detection

    Directory of Open Access Journals (Sweden)

    Tinawi-Aljundi R

    2015-04-01

    Full Text Available Rima Tinawi-Aljundi,1 Lauren King,2 Shannon T Knuth,2 Michael Gildea,2 Carrie Ng,2 Josh Kahl,2 Jacqueline Dion,2 Chris Young,2 Edward W Schervish,1 J Rene Frontera,1 Jason Hafron,1 Kenneth M Kernen,1 Robert Di Loreto,1 Joan Aurich-Costa21Michigan Institute of Urology, St Claire Shores, MI, USA; 2Cellay, Inc., Cambridge, MA, USA Background: Previously, we had developed and manufactured an oligonucleotide fluorescence in situ hybridization (OligoFISH probe panel based on the most clinically sensitive chromosomes found in a reference set of bladder carcinoma cases. The panel was clinically validated for use as a diagnostic and monitoring assay for bladder cancer, reaching 100% correlation with the results of the UroVysion test. After 1 year of using this probe panel, we present here the comparison of cytology, cystoscopy, and pathology findings to the OligoFISH probe panel results to calculate its clinical performance. Materials and methods: In order to calculate clinical performance, we compared the OligoFISH results to the cytology and cystoscopy/pathology findings for 147 initial diagnoses and 399 recurrence monitorings. Finally, we compared clinical performance to published values for the UroVysion test, including both low- and high-grade tumors. Results: Chromosomes 3, 6, 7, and 20 were highly involved in bladder carcinoma aneuploidy. At the initial diagnosis, we obtained 90.5% (95% confidence interval [CI]: 84.5%–94.7% accuracy, 96.8% sensitivity (95% CI: 91.0%–99.3%, 79.2% specificity (95% CI: 65.9%–87.8%, 89.2% positive predictive value (PPV; 95% CI: 81.5%–94.5%, and 93.3% negative predictive value (NPV; 95% CI: 81.7%–97.3%. When monitoring for recurrence, we obtained 85.2% accuracy (95% CI: 81.3%–88.5%, 82.0% sensitivity (95% CI: 76.0%–87.1%, 88.4% specificity (95% CI: 83.2%–92.5%, 87.7% PPV (95% CI: 82.1%–92.0%, and 83.0% NPV (95% CI: 77.3%–87.8%. When looking at low- and high-grade tumors, the test showed 100

  8. Frequencies of complex chromosome exchange aberrations induced by 238Pu alpha-particles and detected by fluorescence in situ hybridization using single chromosome-specific probes.

    Science.gov (United States)

    Griffin, C S; Marsden, S J; Stevens, D L; Simpson, P; Savage, J R

    1995-04-01

    We undertook an analysis of chromosome-type exchange aberrations induced by alpha-particles using fluorescence in situ hybridization (FISH) with whole chromosome-specific probes for human chromosomes 1 or 4, together with a pan-centromeric probe. Contact-inhibited primary human fibroblasts (in G1) were irradiated with 0.41-1.00 Gy 238Pu alpha-particles and aberrations were analysed at the next mitosis following a single chromosome paint. Exchange and aberration painting patterns were classified according to Savage and Simpson (1994a). Of exchange aberrations, 38-47% were found to be complex derived, i.e. resulting from three or more breaks in two or more chromosomes, and the variation with dose was minimal. The class of complex aberrations most frequently observed were insertions, derived from a minimum of three breaks in two chromosomes. There was also an elevated frequency of rings. The high level of complex aberrations observed after alpha-particle irradiation indicates that, when chromosome domains are traversed by high linear energy transfer alpha-particle tracks, there is an enhanced probability of production of multiple localized double-strand breaks leading to more complicated interactions.

  9. Probing nanoscale chemical segregation and surface properties of antifouling hybrid xerogel films.

    Science.gov (United States)

    Destino, Joel F; Gatley, Caitlyn M; Craft, Andrew K; Detty, Michael R; Bright, Frank V

    2015-03-24

    Over the past decade there has been significant development in hybrid polymer coatings exhibiting tunable surface morphology, surface charge, and chemical segregation-all believed to be key properties in antifouling (AF) coating performance. While a large body of research exists on these materials, there have yet to be studies on all the aforementioned properties in a colocalized manner with nanoscale spatial resolution. Here, we report colocalized atomic force microscopy, scanning Kelvin probe microscopy, and confocal Raman microscopy on a model AF xerogel film composed of 1:9:9 (mol:mol:mol) 3-aminopropyltriethoxysilane (APTES), n-octyltriethoxysilane (C8), and tetraethoxysilane (TEOS) formed on Al2O3. This AF film is found to consist of three regions that are chemically and physically unique in 2D and 3D across multiple length scales: (i) a 1.5 μm thick base layer derived from all three precursors; (ii) 2-4 μm diameter mesa-like features that are enriched in free amine (from APTES), depleted in the other species and that extend 150-400 nm above the base layer; and (iii) 1-2 μm diameter subsurface inclusions within the base layer that are enriched in hydrogen-bonded amine (from APTES) and depleted in the other species.

  10. Evaluating a hybrid three-dimensional metrology system: merging data from optical and touch probe devices

    Science.gov (United States)

    Gerde, Janice R.; Christens-Barry, William A.

    2011-08-01

    In a project to meet requirements for CBP Laboratory analysis of footwear under the Harmonized Tariff Schedule of the United States (HTSUS), a hybrid metrology system comprising both optical and touch probe devices has been assembled. A unique requirement must be met: To identify the interface-typically obscured in samples of concern-of the "external surface area upper" (ESAU) and the sole without physically destroying the sample. The sample outer surface is determined by discrete point cloud coordinates obtained using laser scanner optical measurements. Measurements from the optically inaccessible insole region are obtained using a coordinate measuring machine (CMM). That surface similarly is defined by point cloud data. Mathematically, the individual CMM and scanner data sets are transformed into a single, common reference frame. Custom software then fits a polynomial surface to the insole data and extends it to intersect the mesh fitted to the outer surface point cloud. This line of intersection defines the required ESAU boundary, thus permitting further fractional area calculations to determine the percentage of materials present. With a draft method in place, and first-level method validation underway, we examine the transformation of the two dissimilar data sets into the single, common reference frame. We also will consider the six previously-identified potential error factors versus the method process. This paper reports our on-going work and discusses our findings to date.

  11. Karyotype of Zea luxurians and Z. mays subsp. mays using FISH/DAPI, and analysis of meiotic behavior of hybrids.

    Science.gov (United States)

    González, Graciela E; Poggio, Lidia

    2011-01-01

    The karyotypes of Zea luxurians and a race of maize from northwestern Argentina are described and compared using 4′,6-diamidino-2-phenylindole (DAPI) banding and fluorescent in situ hybridization (FISH) to localize the 180 bp knobs. The meiotic behavior of the F₁ artificial hybrids Z. luxurians × maize is also analyzed to determine the genomic relationships between both species. Neocentromere activity at knobs in the meiosis of the hybrids is particularly discussed. The meiotic behavior and the high pollen sterility of the hybrid revealed genetical and (or) chromosomal divergences, leading to postzygotic reproductive isolation among their parents. Here, we propose that maize shows lower genomic affinity to Z. luxurians than to other species of the genus with 2n = 20.

  12. Multiplex fluorescence in situ hybridization (M-FISH) and confocal laser scanning microscopy (CLSM) to analyze multispecies oral biofilms.

    Science.gov (United States)

    Karygianni, Lamprini; Hellwig, Elmar; Al-Ahmad, Ali

    2014-01-01

    Multiplex fluorescence in situ hybridization (M-FISH) constitutes a favorable microbiological method for the analysis of spatial distribution of highly variable phenotypes found in multispecies oral biofilms. The combined use of confocal laser scanning microscopy (CLSM) produces high-resolution three-dimensional (3D) images of individual bacteria in their natural environment. Here, we describe the application of M-FISH on early (Streptococcus spp., Actinomyces naeslundii) and late colonizers (Fusobacterium nucleatum, Veillonella spp.) of in situ-formed oral biofilms, the acquisition of CLSM images, as well as the qualitative and quantitative analysis of these digitally obtained and processed images.

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

    Science.gov (United States)

    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. Quantification of syntrophic fatty acid-beta-oxidizing bacteria in a mesophilic biogas reactor by oligonucleotide probe hybridization

    DEFF Research Database (Denmark)

    Hansen, K.W.; Ahring, Birgitte Kiær; Raskin, L.

    1999-01-01

    -beta-oxidizing syntrophic bacteria, were developed and characterized. The probes were designed to be specific at the family, genus, and species levels and were characterized by temperature of-dissociation and specificity studies, To demonstrate the usefulness of the probes for the detection and quantification of saturated......Small-subunit rRNA sequences were obtained for two saturated fatty acid-beta-oxidizing syntrophic bacteria, Syntrophomonas sapovorans and Syntrophomonas wolfei LYE, and sequence analysis confirmed their classification as members of the family Syntrophomonadaceae. S, wolfei LYE was closely related...... fatty acid-beta-oxidizing syntrophic bacteria in methanogenic environments, the microbial community structure of a sample from a full-scale biogas plant was determined. Hybridization results with probes for syntrophic bacteria-and methanogens were compared to specific methanogenic activities...

  15. Development of a fluorescent in situ hybridization (FISH) technique for visualizing CGMMV in plant tissues.

    Science.gov (United States)

    Shargil, D; Zemach, H; Belausov, E; Lachman, O; Kamenetsky, R; Dombrovsky, A

    2015-10-01

    Cucumber green mottle mosaic virus (CGMMV), which belongs to the genus Tobamovirus, is a major pathogen of cucurbit crops grown indoors and in open fields. Currently, immunology (e.g., ELISA) and molecular amplification techniques (e.g., RT-PCR) are employed extensively for virus detection in plant tissues and commercial seed lots diagnostics. In this study, a fluorescent in situ hybridization (FISH) technique, using oligonucleotides whose 5'-terminals were labeled with red cyanine 3 (Cy3) or green fluorescein isothiocyanate (FITC), was developed for the visualization of the pathogen in situ. This simple and reliable method allows detection and localization of CGMMV in the vegetative and reproductive tissues of cucumber and melon. When this technique was applied in male flowers, anther tissues were found to be infected; whereas the pollen grains were found to be virus-free. These results have meaningful epidemiological implications for the management of CGMMV, particularly with regard to virus transfer via seed and the role of insects as CGMMV vectors.

  16. When biogeographical provinces collide: Hybridization of reef fishes at the crossroads of marine biogeographical provinces in the Arabian Sea

    KAUST Repository

    DiBattista, Joseph

    2015-04-01

    Aim: Suture zones are areas where closely related species from different biogeographical regions come into contact and interbreed. This concept originated from the study of terrestrial ecosystems but it remains unclear whether a similar phenomenon occurs in the marine environment. Here we investigate a potential suture zone from a previously unknown hybrid hotspot at the Socotra Archipelago (Yemen), located in the Arabian Sea, where fauna from the Red Sea, Gulf of Aden, Arabian Sea, western Indian Ocean and greater Indo-Polynesian Province intersect. Location: Red Sea, Gulf of Aden, Arabian Sea and Indian Ocean. Methods: Putative hybrid reef fish were identified based on intermediate coloration and morphology. Underwater observations and collections were conducted to determine: (1) whether parent species form heterospecific social groups or breeding pairs; (2) the sex and reproductive status of morphologically intermediate individuals; and (3) whether parent species were forming mixed species associations owing to a dearth of conspecific partners. To support hybrid status, morphologically intermediate and parental individuals were genotyped using mitochondrial DNA cytochrome c oxidase subunit I (COI), nuclear recombination-activating gene 2 (RAG2) and the nuclear TMO-4C4 (TMO) gene. Results: We observed putative hybrids involving 14 species from four reef fish families at Socotra. Most cases involved a parental species with a restricted distribution (e.g. Red Sea or Arabian Sea) and a broadly distributed Indo-Pacific species. In most cases, at least one of the parent species was rare at Socotra. Hybrid gene flow was largely unidirectional, and although introgression was rare, we found evidence that some butterflyfish and surgeonfish hybrids were fertile and formed breeding groups with parental species. Main conclusions: The rate of hybrid discovery at Socotra is much greater than that recorded elsewhere in the marine environment and involved both allopatric and

  17. Experimental characterization of the lower hybrid wave field on the first pass using a magnetic probe array

    Science.gov (United States)

    Shinya, T.; Baek, S. G.; Wallace, G. M.; Parker, R. R.; Shiraiwa, S.; Takase, Y.

    2016-10-01

    Experimental characterization of the lower hybrid (LH) wave propagation from the launcher to the core plasma is important to validate an antenna spectrum model and to identify parasitic wave-edge plasma interactions occurring in front of the launcher. On Alcator C-Mod, the wave frequency spectrum and dominant parallel wavenumber are characterized with two probe arrays installed near the edge plasma. The first one is mounted on a radially movable structure that is about 108 deg toroidally away from the launcher. A phasing scan experiment at moderate density suggests a resonance-cone propagation of the launched slow LH wave with a finite spectral width. As plasma density is raised, the measured power decreases, correlated with the observed loss of efficiency. Recently, the second probe array with an increased number of probes has been installed on a limiter that is 54 deg. toroidally away from the launcher, which is expected to be dominantly sensitive to the wave-field directly leaving the launcher. An initial measurement shows that the probe array detects a coherent wave field. A full-wave model to evaluate the wave electric-field pattern in front of the probe array is under development. If available, further experimental and modeling results will be presented. Supported by USDoE Award(s) DE-FC02-99ER54512 and Japan/U.S. Cooperation in Fusion Research and Development.

  18. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Science.gov (United States)

    2010-04-01

    ... detection and enumeration of FISH signals in interphase nuclei of formalin-fixed, paraffin-embedded human.... This device is intended for in vitro diagnostic use with FISH assays as an aid in the...

  19. Quantification of syntrophic fatty acid-beta-oxidizing bacteria in a mesophilic biogas reactor by oligonucleotide probe hybridization

    DEFF Research Database (Denmark)

    Hansen, K.W.; Ahring, Birgitte Kiær; Raskin, L.

    1999-01-01

    Small-subunit rRNA sequences were obtained for two saturated fatty acid-beta-oxidizing syntrophic bacteria, Syntrophomonas sapovorans and Syntrophomonas wolfei LYE, and sequence analysis confirmed their classification as members of the family Syntrophomonadaceae. S, wolfei LYE was closely related...... to S. wolfei subsp. wolfei, but S. sapovorans did not cluster with the other members of the genus Syntrophomonas, Five oligonucleotide probes targeting the small-subunit rRNA of different groups within the family Syntrophomonadaceae, which contains all currently known saturated fatty acid...... fatty acid-beta-oxidizing syntrophic bacteria in methanogenic environments, the microbial community structure of a sample from a full-scale biogas plant was determined. Hybridization results with probes for syntrophic bacteria-and methanogens were compared to specific methanogenic activities...

  20. Fluorescence in situ hybridization for the tissue detection of bacterial pathogens associated with porcine infections

    DEFF Research Database (Denmark)

    Jensen, Henrik Elvang; Jensen, Louise Kruse; Barington, Kristiane

    2015-01-01

    sequences within intact cells. FISH allows direct histological localization of the bacteria in the tissue and thereby a correlation between the infection and the histopathological changes present. This chapter presents protocols for FISH identification of bacterial pathogens in fixed deparaffinized tissue......Fluorescence in situ hybridization (FISH) is an efficient technique for the identification of specific bacteria in tissue of both experimental and spontaneous infections. The method detects specific sequences of nucleic acids by hybridization of fluorescently labeled probes to complementary target...

  1. Fluorescence In Situ Hybridization for the Tissue Detection of Bacterial Pathogens Associated with Porcine Infections

    DEFF Research Database (Denmark)

    Elvang Jensen, Henrik; Jensen, Louise Kruse; Barington, Kristiane

    2015-01-01

    sequences within intact cells. FISH allows direct histological localization of the bacteria in the tissue and thereby a correlation between the infection and the histopathological changes present. This chapter presents protocols for FISH identification of bacterial pathogens in fixed deparaffinized tissue......Fluorescence in situ hybridization (FISH) is an efficient technique for the identification of specific bacteria in tissue of both experimental and spontaneous infections. The method detects specific sequences of nucleic acids by hybridization of fluorescently labeled probes to complementary target...

  2. Probing diversity in freshwater fishes from Mexico and Guatemala with DNA barcodes.

    Science.gov (United States)

    Valdez-Moreno, M; Ivanova, N V; Elías-Gutiérrez, M; Contreras-Balderas, S; Hebert, P D N

    2009-02-01

    The freshwater fish fauna of Mexico and Guatemala is exceptionally diverse with >600 species, many endemic. In this study, patterns of sequence divergence were analysed in representatives of this fauna using cytochrome c oxidase subunit 1 (COI) DNA barcodes for 61 species in 36 genera. The average divergence among conspecific individuals was 0.45%, while congeneric taxa showed 5.1% divergence. Three species of Poblana, each occupying a different crater lake in the arid regions of Central Mexico, have had a controversial taxonomic history but are usually regarded as endemics to a single lake. They possess identical COI barcodes, suggesting a very recent history of isolation. Representatives of the Cichlidae, a complex and poorly understood family, were well discriminated by barcodes. Many species of Characidae seem to be young, with low divergence values (clusters were apparent in the Bramocharax-Astyanax complex. The symbranchid, Opisthernon aenigmaticum, has been regarded as a single species ranging from Guatemala to Mexico, but it includes two deeply divergent barcode lineages, one a possible new endemic species. Aside from these special cases, the results confirm that DNA barcodes will be highly effective in discriminating freshwater fishes from Central America and that a comprehensive analysis will provide new important insights for understanding diversity of this fauna.

  3. Fishing

    Institute of Scientific and Technical Information of China (English)

    姜群山

    2002-01-01

    @@ Last Saturday my cousin (表兄) came to my home. We were very happy to see each other. We decided that the next day we went to fish. We got up very early that day. When we left home,the moon could still be seen in the sky.

  4. Dominant microbial composition and its vertical distribution in saline meromictic Lake Kaiike (Japan) as revealed by quantitative oligonucleotide probe membrane hybridization.

    Science.gov (United States)

    Koizumi, Yoshikazu; Kojima, Hisaya; Fukui, Manabu

    2004-08-01

    Vertical distributions of dominant bacterial populations in saline meromictic Lake Kaiike were investigated throughout the water column and sediment by quantitative oligonucleotide probe membrane hybridization. Three oligonucleotide probes specific for the small-subunit (SSU) rRNA of three groups of Chlorobiaceae were newly designed. In addition, three general domain (Bacteria, Archaea, and Eukarya)-specific probes, two delta-Proteobacteria-specific probes, a Chlorobiaceae-specific probe, and a Chloroflexi-specific probe were used after optimization of their washing conditions. The abundance of the sum of SSU rRNAs hybridizing with probes specific for three groups of Chlorobiaceae relative to total SSU rRNA peaked in the chemocline, accounting for up to 68%. The abundance of the delta-proteobacterial SSU rRNA relative to total SSU rRNA rapidly increased just below the chemocline up to 29% in anoxic water and peaked at the 2- to 3-cm sediment depth at ca. 34%. The abundance of SSU rRNAs hybridizing with the probe specific for the phylum Chloroflexi relative to total SSU rRNA was highest (31 to 54%) in the top of the sediment but then steeply declined with depth and became stable at 11 to 19%, indicating the robust coexistence of sulfate-reducing bacteria and Chloroflexi in the top of the sediment. Any SSU rRNA of Chloroflexi in the water column was under the detection limit. The summation of the signals of group-specific probes used in this study accounted for up to 89% of total SSU rRNA, suggesting that the DGGE-oligonucleotide probe hybridization approach, in contrast to conventional culture-dependent approaches, was very effective in covering dominant populations.

  5. Progress in molecular diagnosis of Charcot-Marie-Tooth-disease type 1 (CMT 1, HMSN I) and hereditary neuropathy with liability to pressure palsies (HNPP) by fluorescence in situ hybridization (FISH)-detection of a potential genetic mosaicism

    Energy Technology Data Exchange (ETDEWEB)

    Bathke, K.; Liehr. T.; Ekici, A. [Institute for Human Genetics, Erlange (Germany)] [and others

    1994-09-01

    We tested 20 CMT 1 patients characterized according to the criteria of the European CMT consortium by Southern hybridization of MspI restricted genomic DNA with probes pVAW409R1, pVAW412Hec and pEW401HE. In 11 of the 20 CMT 1 cases (55%), we observed a duplication in 17q11.2; one patient had a dinucleotide insertion in exon 6 of the PO-gene (5%). One HNPP case had a typical 17p11.2 deletion. Analysis of CA-repeats was performed with primers RM11GT and Mfd41; SSCP-analysis of the PO, PMP22 and Cx32-genes is in progress. FISH was carried out with probe pVAW409R1. 125 interphase nuclei were analyzed for each proband by counting the signals per nucleus. Normal cells show a characteristic distribution of signals: 1 signal in 5.9% of nuclei, 2 in 86.3% and 3 in 7.8%. A duplication is indicated by a shift to 3 signals in more than approximately 60% and 2 in less than 25% of the nuclei. In contrast, the 17p11.2 deletion of the HNPP patient shifts to 82.4% of nuclei with a single hybridization signal versus 14.4% with 2 signals. We detected one case with significantly abnormal distribution of interphase nuclei hybridization signals compared to cultures of normal cells and to those with 17p11.2 duplication or deletion: 3.2% nuclei revealed 1 signal, 48.0% two signals and 48.8% 3 signals, indicating a pathogenic but moderate dosis increase compared to the throughout duplicated cases. FISH with probe pVAW409R1 is a versatile tool to detect the HNPP deletion both in interphase nuclei and in metaphase chromosomes. In CMT 1 disease interphase nuclei are required for FISH analysis due to the small duplication of 1.5 Mbp. In contrast to Southern techniques, FISH is able to detect genetic mosaicism.

  6. Human disturbance causes the formation of a hybrid swarm between two naturally sympatric fish species.

    Science.gov (United States)

    Hasselman, Daniel J; Argo, Emily E; McBride, Meghan C; Bentzen, Paul; Schultz, Thomas F; Perez-Umphrey, Anna A; Palkovacs, Eric P

    2014-03-01

    Most evidence for hybrid swarm formation stemming from anthropogenic habitat disturbance comes from the breakdown of reproductive isolation between incipient species, or introgression between allopatric species following secondary contact. Human impacts on hybridization between divergent species that naturally occur in sympatry have received considerably less attention. Theory predicts that reinforcement should act to preserve reproductive isolation under such circumstances, potentially making reproductive barriers resistant to human habitat alteration. Using 15 microsatellites, we examined hybridization between sympatric populations of alewife (Alosa pseudoharengus) and blueback herring (A. aestivalis) to test whether the frequency of hybridization and pattern of introgression have been impacted by the construction of a dam that isolated formerly anadromous populations of both species in a landlocked freshwater reservoir. The frequency of hybridization and pattern of introgression differed markedly between anadromous and landlocked populations. The rangewide frequency of hybridization among anadromous populations was generally 0-8%, whereas all landlocked individuals were hybrids. Although neutral introgression was observed among anadromous hybrids, directional introgression leading to increased prevalence of alewife genotypes was detected among landlocked hybrids. We demonstrate that habitat alteration can lead to hybrid swarm formation between divergent species that naturally occur sympatrically, and provide empirical evidence that reinforcement does not always sustain reproductive isolation under such circumstances.

  7. The Zebra Fish IBD Model Assessed By Novel Probe Based TagMan Assays

    DEFF Research Database (Denmark)

    Kania, Per Walter; Buchmann, Kurt; Haarder, Simon

    2015-01-01

    models it is known that oxazolone and TNBS can induce conditions mimicking ulcerative colitis (Th2 like response) and Crohn's disease (Th1/Th17 like response), respectively. Zebra fish (body weight 0.5 g) were divided into 4 groups (each with 2 replicates) and instilled rectally with H2O, ethanol, TNBS......, itself up-regulates genes (T-bet, INF, IL-17A, TGF) primarily from the Th1/Th17 response but also IL-10 of the Th2 response. The gene expression pattern of the oxazolone and TNBS instilled groups were partly similar pointing primarily towards a Th2 response. TNF was the only Th1response specific gene...

  8. Formation of a hybrid plasmonic waveguide mode probed by dispersion measurement

    Energy Technology Data Exchange (ETDEWEB)

    Saito, H. [Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan); Quantum Nanoelectronics Research Center, Tokyo Institute of Technology, Oh-Okayama, Meguro-ku, Tokyo 152-8551 (Japan); Kurata, H., E-mail: kurata@eels.kuicr.kyoto-u.ac.jp [Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2015-04-07

    Hybrid waveguides, i.e., dielectric waveguides combined with plasmonic waveguides, have great potential for concomitantly exhibiting subwavelength confinement and long range propagation, enabling a highly integrated photonic circuit. We report the characterization of hybrid waveguide modes excited in Si/SiO{sub 2}/Al films, by dispersion measurement using angle-resolved electron energy-loss spectroscopy. This experiment directly verifies the formation of the hybrid waveguide mode with a strongly localized electromagnetic field in a 6-nm-thick SiO{sub 2} layer. The results clearly describe the characteristic behavior of the hybrid waveguide mode, which depends on the effective index of the constituent dielectric waveguide and the surface plasmon-polariton modes.

  9. Bimodal Imaging Probes for Combined PET and OI: Recent Developments and Future Directions for Hybrid Agent Development

    Directory of Open Access Journals (Sweden)

    Uwe Seibold

    2014-01-01

    Full Text Available Molecular imaging—and especially positron emission tomography (PET—has gained increasing importance for diagnosis of various diseases and thus experiences an increasing dissemination. Therefore, there is also a growing demand for highly affine PET tracers specifically accumulating and visualizing target structures in the human body. Beyond the development of agents suitable for PET alone, recent tendencies aim at the synthesis of bimodal imaging probes applicable in PET as well as optical imaging (OI, as this combination of modalities can provide clinical advantages. PET, due to the high tissue penetration of the γ-radiation emitted by PET nuclides, allows a quantitative imaging able to identify and visualize tumors and metastases in the whole body. OI on the contrary visualizes photons exhibiting only a limited tissue penetration but enables the identification of tumor margins and infected lymph nodes during surgery without bearing a radiation burden for the surgeon. Thus, there is an emerging interest in bimodal agents for PET and OI in order to exploit the potential of both imaging techniques for the imaging and treatment of tumor diseases. This short review summarizes the available hybrid probes developed for dual PET and OI and discusses future directions for hybrid agent development.

  10. On the Translation of Hybridity in the Chinese Version of Saving Fish from Drowning in the Light of Bakhtin’s Theory of Dialogue

    Institute of Scientific and Technical Information of China (English)

    HU Zheng-yan; WANG Ji-ping

    2015-01-01

    In translation, hybridity often poses a big challenge to the translator, especially when the hybrid text is a novel. The au⁃thor of this paper hopes to contribute to the study of the translation of hybridity in English novels by drawing on Bakhtin ’s theory of dialogue. By analyzing the translation of linguistic hybridity in Saving Fish from Drowning, the author of this paper finds that the translation by Caiju is not good enough and tries to provide a new direction by virtue of which translators can choose right transla⁃tion strategies.

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

    Science.gov (United States)

    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.

  12. Detection of Helicobacter Pylori Genome with an Optical Biosensor Based on Hybridization of Urease Gene with a Gold Nanoparticles-Labeled Probe

    Science.gov (United States)

    Shahrashoob, M.; Mohsenifar, A.; Tabatabaei, M.; Rahmani-Cherati, T.; Mobaraki, M.; Mota, A.; Shojaei, T. R.

    2016-05-01

    A novel optics-based nanobiosensor for sensitive determination of the Helicobacter pylori genome using a gold nanoparticles (AuNPs)-labeled probe is reported. Two specific thiol-modified capture and signal probes were designed based on a single-stranded complementary DNA (cDNA) region of the urease gene. The capture probe was immobilized on AuNPs, which were previously immobilized on an APTES-activated glass, and the signal probe was conjugated to different AuNPs as well. The presence of the cDNA in the reaction mixture led to the hybridization of the AuNPs-labeled capture probe and the signal probe with the cDNA, and consequently the optical density of the reaction mixture (AuNPs) was reduced proportionally to the cDNA concentration. The limit of detection was measured at 0.5 nM.

  13. Fish predation on a Daphnia hybrid species complex: A factor explaining species coexistence?

    NARCIS (Netherlands)

    Spaak, P.; Hoekstra, J.F.

    1997-01-01

    Recent studies on the life histories of Daphnia hybrids and their parental species have revealed that hybrids can combine an intermediate size with a relatively high reproductive rate, which might explain their success in many European lakes. Based on this information, we formulated the temporal

  14. Computationally Probing the Performance of Hybrid, Heterogeneous, and Homogeneous Iridium-Based Catalysts for Water Oxidation

    Energy Technology Data Exchange (ETDEWEB)

    García-Melchor, Max [SUNCAT Center for Interface Science and Catalysis, Department of Chemical Engineering, Stanford University, Stanford CA (United States); Vilella, Laia [Institute of Chemical Research of Catalonia (ICIQ), The Barcelona Institute of Science and Technology (BIST),Tarragona (Spain); Departament de Quimica, Universitat Autonoma de Barcelona, Barcelona (Spain); López, Núria [Institute of Chemical Research of Catalonia (ICIQ), The Barcelona Institute of Science and Technology (BIST), Tarragona (Spain); Vojvodic, Aleksandra [SUNCAT Center for Interface Science and Catalysis, SLAC National Accelerator Laboratory, Menlo Park CA (United States)

    2016-04-29

    An attractive strategy to improve the performance of water oxidation catalysts would be to anchor a homogeneous molecular catalyst on a heterogeneous solid surface to create a hybrid catalyst. The idea of this combined system is to take advantage of the individual properties of each of the two catalyst components. We use Density Functional Theory to determine the stability and activity of a model hybrid water oxidation catalyst consisting of a dimeric Ir complex attached on the IrO2(110) surface through two oxygen atoms. We find that homogeneous catalysts can be bound to its matrix oxide without losing significant activity. Hence, designing hybrid systems that benefit from both the high tunability of activity of homogeneous catalysts and the stability of heterogeneous systems seems feasible.

  15. Fully automated fluorescent in situ hybridization (FISH staining and digital analysis of HER2 in breast cancer: a validation study.

    Directory of Open Access Journals (Sweden)

    Elise M J van der Logt

    Full Text Available HER2 assessment is routinely used to select patients with invasive breast cancer that might benefit from HER2-targeted therapy. The aim of this study was to validate a fully automated in situ hybridization (ISH procedure that combines the automated Leica HER2 fluorescent ISH system for Bond with supervised automated analysis with the Visia imaging D-Sight digital imaging platform. HER2 assessment was performed on 328 formalin-fixed/paraffin-embedded invasive breast cancer tumors on tissue microarrays (TMA and 100 (50 selected IHC 2+ and 50 random IHC scores full-sized slides of resections/biopsies obtained for diagnostic purposes previously. For digital analysis slides were pre-screened at 20x and 100x magnification for all fluorescent signals and supervised-automated scoring was performed on at least two pictures (in total at least 20 nuclei were counted with the D-Sight HER2 FISH analysis module by two observers independently. Results were compared to data obtained previously with the manual Abbott FISH test. The overall agreement with Abbott FISH data among TMA samples and 50 selected IHC 2+ cases was 98.8% (κ = 0.94 and 93.8% (κ = 0.88, respectively. The results of 50 additionally tested unselected IHC cases were concordant with previously obtained IHC and/or FISH data. The combination of the Leica FISH system with the D-Sight digital imaging platform is a feasible method for HER2 assessment in routine clinical practice for patients with invasive breast cancer.

  16. Identification of human rotavirus serotype by hybridization to polymerase chain reaction-generated probes derived from a hyperdivergent region of the gene encoding outer capsid protein VP7

    Energy Technology Data Exchange (ETDEWEB)

    Flores, J.; Sears, J.; Schael, I.P.; White, L.; Garcia, D.; Lanata, C.; Kapikian, A.Z. (National Institutes of Health, Bethesda, MD (USA))

    1990-08-01

    We have synthesized {sup 32}P-labeled hybridization probes from a hyperdivergent region (nucleotides 51 to 392) of the rotavirus gene encoding the VP7 glycoprotein by using the polymerase chain reaction method. Both RNA (after an initial reverse transcription step) and cloned cDNA from human rotavirus serotypes 1 through 4 could be used as templates to amplify this region. High-stringency hybridization of each of the four probes to rotavirus RNAs dotted on nylon membranes allowed the specific detection of corresponding sequences and thus permitted identification of the serotype of the strains dotted. The procedure was useful when applied to rotaviruses isolated from field studies.

  17. In situ hybridization of cytokine mRNA using alkaline phosphatase-labelled oligodeoxynucleotide probes

    DEFF Research Database (Denmark)

    Clausen, Bettina Hjelm; Fenger, Christina; Finsen, B.

    2013-01-01

    In situ hybridization is a powerful tool for visualizing cellular gene expression in morphologically preserved brain tissue giving precise information on the regional expression of specific mRNA sequences in cells of diverse phenotype. Here, we describe a sensitive, simple, and robust method using...

  18. Action spectrum for melanoma induction in hybrid fish of the genus Xiphophorus

    Energy Technology Data Exchange (ETDEWEB)

    Setlow, R.B.

    1997-03-01

    Cutaneous malignant melanoma (CMM) is a complicated disease that is dependent on a number of factors that influence its incidence in ways that are quantitatively uncertain. The incidence of CMM increases with proximity to the Equator -- an observation in line with the conclusion that sun exposure is the most important etiologic agent. However, the latitude effect does not implicate UVB because the intensities of all spectral regions increase toward the Equator. An understanding of the useful public health measures to lower the incidence of CMM would benefit greatly if the spectral region of sunlight implicated in melanoma incidence were known. Such knowledge requires animal models to evaluate the incidence as a function of wavelength. There are marsupial models, a transgenic mouse model, and a fish model. To date, only the fish model has been employed to obtain an action spectrum. The paper describes a fish model, implications of the fish spectrum, and epidemiological data.

  19. Next-generation repeat-free FISH probes for DNA amplification in glioblastoma in vivo: Improving patient selection to MDM2-targeted inhibitors.

    Science.gov (United States)

    Brunelli, Matteo; Eccher, Albino; Cima, Luca; Trippini, Tobia; Pedron, Serena; Chilosi, Marco; Barbareschi, Mattia; Scarpa, Aldo; Pinna, Giampietro; Cabrini, Giulio; Pilotto, Sara; Carbognin, Luisa; Bria, Emilio; Tortora, Giampaolo; Fioravanzo, Adele; Schiavo, Nicola; Meglio, Mario; Sava, Teodoro; Belli, Laura; Martignoni, Guido; Ghimenton, Claudio

    2017-01-01

    A next-generation FISH probe mapping to the MDM2 locus-specific region has recently been designed. The level of MDM2 gene amplification (high versus low) may allow selection of patients for cancer treatment with MDM2 inhibitors and may predict their responsiveness. We investigated the spectrum of MDM2 gene alterations using the new probes in vivo after visualizing single neoplastic cells in situ from a series of glioblastomas. Signals from next-generation repeat-free FISH interphase probes were identified in tissue microarrays that included 3 spots for each of the 48 cases. The murine double minutes (MDM2)-specific DNA probe and the satellite enumeration probe for chromosome 12 were used. Three cases (6%) showed more than 25 signals (high gene amplification), and 7 (15%) showed 3-10 signals (gains); among these, 4 cases (8%) had an equal number of MDM2 and centromeric signals on chromosome 12 (polyploidy). Genomic heterogeneity was observed only in 3 cases with low gene amplification. In our series, 6% of glioblastomas exhibited high MDM2 amplification (in vivo) with a pattern related to the known double minutes/chromothripsis phenomenon (in situ), and only cases with low amplification showed genomic heterogeneity. We concluded that the rate of MDM2 gene amplification can be a useful predictive biomarker to improve patient selection.

  20. Hybrid intracerebral probe with integrated bare LED chips for optogenetic studies.

    Science.gov (United States)

    Ayub, Suleman; Gentet, Luc J; Fiáth, Richárd; Schwaerzle, Michael; Borel, Mélodie; David, François; Barthó, Péter; Ulbert, István; Paul, Oliver; Ruther, Patrick

    2017-09-01

    This article reports on the development, i.e., the design, fabrication, and validation of an implantable optical neural probes designed for in vivo experiments relying on optogenetics. The probes comprise an array of ten bare light-emitting diode (LED) chips emitting at a wavelength of 460 nm and integrated along a flexible polyimide-based substrate stiffened using a micromachined ladder-like silicon structure. The resulting mechanical stiffness of the slender, 250-μm-wide, 65-μm-thick, and 5- and 8-mm-long probe shank facilitates its implantation into neural tissue. The LEDs are encapsulated by a fluropolymer coating protecting the implant against the physiological conditions in the brain. The electrical interface to the external control unit is provided by 10-μm-thick, highly flexible polyimide cables making the probes suitable for both acute and chronic in vivo experiments. Optical and electrical properties of the probes are reported, as well as their in vivo validation in acute optogenetic studies in transgenic mice. The depth-dependent optical stimulation of both excitatory and inhibitory neurons is demonstrated by altering the brain activity in the cortex and the thalamus. Local network responses elicited by 20-ms-long light pulses of different optical power (20 μW and 1 mW), as well as local modulation of single unit neuronal activity to 1-s-long light pulses with low optical intensity (17 μW) are presented. The ability to modulate neural activity makes these devices suitable for a broad variety of optogenetic experiments.

  1. In vitro formation and thermal transition of novel hybrid fibrils from type I fish scale collagen and type I porcine collagen

    OpenAIRE

    Song Chen, Toshiyuki Ikoma, Nobuhiro Ogawa, Satoshi Migita, Hisatoshi Kobayashi and Nobutaka Hanagata

    2010-01-01

    Novel type I collagen hybrid fibrils were fabricated by neutralizing a mixture of type I fish scale collagen solution and type I porcine collagen solution with a phosphate buffer saline at 28 °C. Their structure was discussed in terms of the volume ratio of fish/porcine collagen solution. Scanning electron and atomic force micrographs showed that the diameter of collagen fibrils derived from the collagen mixture was larger than those derived from each collagen, and all resultant fibrils exhib...

  2. Using NV centers to probe magnetization dynamics in normal metal/magnetic insulator hybrid system at the nanoscale

    Science.gov (United States)

    Zhang, Huiliang; Ku, Mark J. H.; Han, Minyong; Casola, Francesco; van der Sar, Toeno; Yacoby, Amir; Walsworth, Ronald L.

    2016-05-01

    Understanding magnetization dynamics induced by electric current is of great interest for both fundamental and practical reasons. Great endeavor has been dedicated to spin-orbit torques (SOT) in metallic structures, while quantitative study of analogous phenomena in magnetic insulators remains challenging where transport measurements are not feasible. Recently we have developed techniques using nitrogen vacancy (NV) centers in diamond to probe few-nanometre-scale correlated-electron magnetic excitations (i.e., spin waves). Here we demonstrate how this powerful tool can be implemented to study magnetization dynamics inside ferromagnetic insulator, Yttrium iron garnet (YIG) with spin injection from electrical current through normal metal (Platinum in our case). Particularly our work will focus on NV magnetic detection, imaging, and spectroscopy of coherent auto-oscillations in Pt/YIG microdisc. Magnetic fluctuations and local temperature measurements, both with nearby NV centers, will also be interesting topics relevant to SOT physics in Pt/YIG hybrid system.

  3. Analysis of messenger RNA expression by in situ hybridization using RNA probes synthesized via in vitro transcription

    Science.gov (United States)

    Carter, Bradley S.; Fletcher, Jonathan S.; Thompson, Robert C.

    2010-01-01

    The analysis of the spatial patterning of mRNA expression is critically important for assigning functional and physiological significance to a given gene product. Given the tens of thousands of mRNAs in the mammalian genome, a full assessment of individual gene functions would ideally be overlaid upon knowledge of the specific cell types expressing each mRNA. In situ hybridization approaches represent a molecular biological/histological method that can reveal cellular patterns of mRNA expression. Here, we present detailed procedures for the detection of specific mRNAs using radioactive RNA probes in tissue sections followed by autoradiographic detection. These methods allow for the specific and sensitive detection of spatial patterns of mRNA expression, thereby linking mRNA expression with cell type and function. Radioactive detection methods also facilitate semi-quantitative analyses of changes in mRNA gene expression. PMID:20699122

  4. 全染色体涂染探针FISH技术在AIH产前诊断唐氏综合征的应用%Application of FISH technique with whole chromosome probe for prenatal diagnosis of Downs syndrome after AIH

    Institute of Scientific and Technical Information of China (English)

    刘新雄; 梁荣伟; 符可鹏; 陈哲; 孙雷; 翁勋锦

    2012-01-01

    Objective; To explore the application value of fluorescence in situ hybridization ( FISH) by using self - designed human whole chromosome 21 special DNA probe for prenatal diagnosis of Downs syndrome after artificial insemination by husband (AIH) . Methods; FISH of uncultured amniotic fluid cells abstracted from pregnant women of 16 - 26 gestational weeks after AIH treatment was performed with self - designed human whole chromosome 21 special DNA probe, routine cell culture and chromosomal karyotype analysis were conducted at the same time, and the results of the two methods were compared. Results; The result of FISH was obtained within 24 hours, one child with trisomy 21 and one child with triple X syndrome were found. The coincidence rate of self - designed human whole chromosome 21 special DNA probe for detection of chromosome 21 in uncultured amniotic fluid cells was as high as 99.42% , no abnormal FISH result was found in the patients whose chromosomal karyotype was 47, XXX. The detection results were identical with the results of chromosomal karyotype analysis and follow - up. Conclusion; FISH technique with self - designed human whole chromosome 21 special DNA probe has the advantages of speediness and accuracy, which can quicken the diagnostic time, FISH technique has good application value for the high risk pregnant women after successful AIH in the prenatal diagnosis of Downs syndrome.%目的:探讨应用自制的人21号全染色体特异DNA涂染探针FISH技术在AIH产前诊断唐氏综合征的应用价值.方法:对经AIH治疗成功受孕的妊娠16 ~26周孕妇抽取的未培养羊水细胞采用已制备的人21号全染色体特异DNA涂染探针进行荧光原位杂交,同时进行常规细胞培养及染色体核型分析,并比较两种检测方法的结果.结果:自制探针FISH检测均于24h内出结果,检测出患儿2例,其中1例为标准21三体,1例为X三体.自制的人21号全染色体特异DNA涂染探针

  5. QUANTITATIVE FLUORESCENCE IN-SITU HYBRIDIZATION OF BIFIDOBACTERIUM SPP WITH GENUS-SPECIFIC 16S RIBOSOMAL-RNA-TARGETED PROBES AND ITS APPLICATION IN FECAL SAMPLES

    NARCIS (Netherlands)

    LANGENDIJK, PS; SCHUT, F; JANSEN, GJ; RAANGS, GC; KAMPHUIS, GR; WILKINSON, MHF; WELLING, GW

    1995-01-01

    Three 16S rRNA hybridization probes were developed and tested for genus-specific detection of Bifidobacterium species in the human fecal flora. Variable regions V2, V4, and VS of the 16S rRNA contained sequences unique to this genus and proved applicable as target sites for oligodeoxynucleotide prob

  6. Fuel consumption analysis of diesel-electric hybrid system of fishing boat%渔船柴电混合动力系统油耗分析

    Institute of Scientific and Technical Information of China (English)

    曹建军; 李胜勇

    2016-01-01

    为了探索海洋渔船推进系统的节油减排,以一艘安装有柴电混合动力系统,成功实现节能减排的金枪鱼延绳钓船为例,通过剖析柴电混合动力系统的结构,柴油机理论负荷特性,结合渔船实际使用情况,分析其节能原因。结果显示,渔船柴电混合动力系统在使用过程中,可根据工况需要,随意切换常规动力与电力推进系统;通过选择合理的主机工况,提高柴油机输出效率,降低柴油机油耗率;综合各种工况,能节油15%以上。研究表明,现阶段在金枪鱼延绳钓渔船上使用混合动力技术是可行的,能起到节能减排的作用。在核心技术不变的情况下,运用一些创新技术手段,如采用多机多桨拆分系统,赋予系统更高的智能调配作用,可以把混合动力系统推广到拖网渔船和围网渔船上。%In order to explore the energy conservation performance of ocean fishing boat propulsion systems,in this paper,a tuna longline vessel that has realized energy-saving and emission-reducing was taken as an example to analyze the reasons behind,through probing into the structure of its diesel-electric hybrid system, engine load characteristics and the actual using conditions of the vessel. The results showed that the diesel-electric hybrid system of the vessel in use can easily switch between the conventional power and the electricity propulsion systems in accordance with the actual working conditions; by selecting the reasonable working conditions of the main engine,the output efficiency of the diesel engine could thus be improved and the diesel fuel consumption rate decreased,and considering the various working conditions,the system could save energy by more than 15%.The research showed that,at present,using hybrid power technology in tuna longline vessels is feasible and can play a role in energy conservation. With the core technology remaining the same and by adopting some

  7. Fluorescent in situ hybridization as an adjunct to conventional cytogenetics.

    Science.gov (United States)

    Mark, H F

    1994-01-01

    Fluorescent in situ hybridization (FISH) is a molecular cytogenetic technique that exploits the availability of recombinant deoxyribonucleic acid (DNA) technology. In metaphase FISH, a specific nucleic acid sequence (probe) is bound to the homologous segment on a metaphase chromosome in a fixed preparation on a glass slide. The presence of a region-specific DNA sequence in a nondividing cell can be detected using interphase FISH. Interphase cytogenetics via FISH can be performed on fixed cells harvested during a routine culture, on tissue sections and on many cytologic specimens. Specific examples of clinical and research applications are discussed to illustrate the utility of FISH in the detection of constitutional and acquired chromosomal abnormalities.

  8. Probing the structure and function of biopolymer-carbon nanotube hybrids with molecular dynamics

    Science.gov (United States)

    Johnson, Robert R.

    2009-12-01

    Nanoscience deals with the characterization and manipulation of matter on the atomic/molecular size scale in order to deepen our understanding of condensed matter and develop revolutionary technology. Meeting the demands of the rapidly advancing nanotechnological frontier requires novel, multifunctional nanoscale materials. Among the most promising nanomaterials to fulfill this need are biopolymer-carbon nanotube hybrids (Bio-CNT). Bio-CNT consists of a single-walled carbon nanotube (CNT) coated with a self-assembled layer of biopolymers such as DNA or protein. Experiments have demonstrated that these nanomaterials possess a wide range of technologically useful properties with applications in nanoelectronics, medicine, homeland security, environmental safety and microbiology. However, a fundamental understanding of the self-assembly mechanics, structure and energetics of Bio-CNT is lacking. The objective of this thesis is to address this deficiency through molecular dynamics (MD) simulation, which provides an atomic-scale window into the behavior of this unique nanomaterial. MD shows that Bio-CNT composed of single-stranded DNA (ssDNA) self-assembles via the formation of high affinity contacts between DNA bases and the CNT sidewall. Calculation of the base-CNT binding free energy by thermodynamic integration reveals that these contacts result from the attractive pi--pi stacking interaction. Binding affinities follow the trend G > A > T > C. MD reveals that long ssDNA sequences are driven into a helical wrapping about CNT with a sub-10 nm pitch by electrostatic and torsional interactions in the backbone. A large-scale replica exchange molecular dynamics simulation reveals that ssDNA-CNT hybrids are disordered. At room temperature, ssDNA can reside in several low-energy conformations that contain a sequence-specific arrangement of bases detached from CNT surface. MD demonstrates that protein-CNT hybrids composed of the Coxsackie-adenovirus receptor are biologically

  9. A nucleic acid probe labeled with desmethyl thiazole orange: a new type of hybridization-sensitive fluorescent oligonucleotide for live-cell RNA imaging.

    Science.gov (United States)

    Okamoto, Akimitsu; Sugizaki, Kaori; Yuki, Mizue; Yanagisawa, Hiroyuki; Ikeda, Shuji; Sueoka, Takuma; Hayashi, Gosuke; Wang, Dan Ohtan

    2013-01-14

    A new fluorescent nucleotide with desmethyl thiazole orange dyes, D'(505), has been developed for expansion of the function of fluorescent probes for live-cell RNA imaging. The nucleoside unit of D'(505) for DNA autosynthesis was soluble in organic solvents, which made the preparation of nucleoside units and the reactions in the cycles of DNA synthesis more efficient. The dyes of D'(505)-containing oligodeoxynucleotide were protonated below pH 7 and the oligodeoxynucleotide exhibited hybridization-sensitive fluorescence emission through the control of excitonic interactions of the dyes of D'(505). The simplified procedure and effective hybridization-sensitive fluorescence emission produced multicolored hybridization-sensitive fluorescent probes, which were useful for live-cell RNA imaging. The acceptor-bleaching method gave us information on RNA in a specific cell among many living cells.

  10. Gill monogeneans of Nile tilapia (Oreochromis niloticus) and red hybrid tilapia (Oreochromis spp.) from the wild and fish farms in Perak, Malaysia: infection dynamics and spatial distribution.

    Science.gov (United States)

    Lim, Shen-Yin; Ooi, Ai-Lin; Wong, Wey-Lim

    2016-01-01

    Tilapia is one of the commercially important fish in Malaysia as well as in other parts of the world. An understanding of monogenean infection dynamics in tilapia fish may assist us in searching for some intervention measures in reducing the loss of fish caused by parasitic diseases. The present study aimed (1) to compare infection level of monogeneans between the wild and cultured Oreochromis niloticus, and between the cultured O. niloticus and cultured red hybrid tilapia, and (2) to examine the spatial distribution of monogenean species over the gills of the different host species. From a total of 75 fish specimens, six species of monogeneans from two genera: Cichlidogyrus (C. halli, C. mbirizei, C. sclerosus, C. thurstonae, C. tilapiae) and Scutogyrus (S. longicornis) were identified. Data showed that the infection level of cultured O. niloticus was higher than that of the wild O. niloticus, however, the former was lower than that of the cultured red hybrid tilapia. Higher species richness of monogeneans was observed in the cultured red hybrid tilapia as compared to the others. Results for spatial distribution showed that the monogeneans have no preference on the left or right sides of the gills. However, C. halli, C. mbirizei, and C. tilapiae showed preferences on specific gill arches in the cultured O. niloticus and red hybrid tilapia. In general, the gill arch IV harboured the least number of monogeneans. The susceptibility of monogenean infection between the different types of tilapia is discussed.

  11. Phylogeny-function analysis of (meta)genomic libraries: screening for expression of ribosomal RNA genes by large-insert library fluorescent in situ hybridization (LIL-FISH)

    NARCIS (Netherlands)

    Leveau, J.H.J.; Gerards, S.; De Boer, W.; Van Veen, J.A.

    2004-01-01

    We assessed the utility of fluorescent in situ hybridization (FISH) in the screening of clone libraries of (meta)genomic or environmental DNA for the presence and expression of bacterial ribosomal RNA (rRNA) genes. To establish proof-of-principle, we constructed a fosmid-based library in Escherichia

  12. Distribution characteristics of ammonia-oxidizing bacteria in the Typha latifolia constructed wetlands using fluorescent in situ hybridization (FISH).

    Science.gov (United States)

    Yan, Li; Inamori, Ryuhei; Gui, Ping; Xu, Kai-qin; Kong, Hai-nan; Matsumura, Masatoshi; Inamori, Yuhei

    2005-01-01

    A molecular biology method, fluorescent in situ hybridization (FISH), in which the pre-treatment was improved in allusion to the media of the constructed wetlands (CW), e.g. the soil and the grit, was used to investigate the vertical distribution characteristics of ammonia-oxidizing bacteria (AOB) quantity and the relation with oxidation-reduction potential (ORP) in the Typha latifolia constructed wetlands under three different loadings in summer from May to September. Results showed that the quantity of the AOB decreased in the Typha latifolia CW with the increase of vertical depth. However, the AOB quantity was 2-4 times the quantity of the control in the root area. Additionally, ORP in the rhizosphere was found to be higher than other areas, which showed that Typha latifolia CW was in an aerobic state in summer when using simulated non-point sewage at the rural area of Taihu Lake in China and small town combined sewage.

  13. Hybrid striped bass feeds based on fish oil, beef tallow, and eicosapentaenoic acid/docosahexaenoic acid supplements: Insight regarding fish oil sparing and demand for -3 long-chain polyunsaturated fatty acids.

    Science.gov (United States)

    Bowzer, J; Jackson, C; Trushenski, J

    2016-03-01

    Previous research suggests that saturated (SFA) and monounsaturated fatty acid (MUFA) rich lipids, including beef tallow, can make utilization or diet-to-tissue transfer of long-chain polyunsaturated fatty acids (LC-PUFA) more efficient. We hypothesized that using beef tallow as an alternative to fish oil may effectively reduce the LC-PUFA demand of hybrid striped bass × and allow for greater fish oil sparing. Accordingly, we evaluated growth performance and tissue fatty acid profiles of juvenile fish (23.7 ± 0.3 g) fed diets containing menhaden fish oil (considered an ideal source of LC-PUFA for this taxon), beef tallow (BEEF ONLY), or beef tallow amended with purified sources of eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) to achieve levels corresponding to 50 or 100% of those observed in the FISH ONLY feed. Diets were randomly assigned to quadruplicate tanks of fish ( = 4; 10 fish/tank), and fish were fed assigned diets to apparent satiation once daily for 10 wk. Survival (98-100%) was equivalent among treatments, but weight gain (117-180%), specific growth rate (1.1-1.5% BW/d), feed intake (1.4-1.8% BW/d), thermal growth coefficient (0.50-0.70), and feed conversion ratio (FCR; 1.1-1.4, DM basis) varied. Except for FCR, no differences were observed between the FISH ONLY and BEEF ONLY treatments, but performance was generally numerically superior among fish fed the diets containing beef tallow supplemented with DHA at the 100% or both EPA and DHA at the 50% or 100% level. Tissue fatty acid composition was significantly distorted in favor among fish fed the beef tallow-based feeds; however, profile distortion was most overt in peripheral tissues. Results suggest that beef tallow may be used as a primary lipid source in practical diets for hybrid striped bass, but performance may be improved by supplementation with LC-PUFA, particularly DHA. Furthermore, our results suggest that -3 LC-PUFA requirements reported for hybrid striped bass may not be

  14. In vitro formation and thermal transition of novel hybrid fibrils from type I fish scale collagen and type I porcine collagen

    Energy Technology Data Exchange (ETDEWEB)

    Chen Song; Ogawa, Nobuhiro; Migita, Satoshi; Kobayashi, Hisatoshi [Biomaterials Center, National Institute for Materials Science, 1-2-1 Sengen, Tsukuba, Ibaraki 305-0047 (Japan); Ikoma, Toshiyuki [Department of Metallurgy and Ceramics Science, Tokyo Institute of Technology, O-okayama 2-12-1, Meguro-ku, Tokyo 152-8550 (Japan); Hanagata, Nobutaka, E-mail: HANAGATA.Nobutaka@nims.go.j [Nanotechnology Innovation Center, National Institute for Materials Science, 1-2-1 Sengen, Tsukuba, Ibaraki 305-0047 (Japan)

    2010-06-15

    Novel type I collagen hybrid fibrils were fabricated by neutralizing a mixture of type I fish scale collagen solution and type I porcine collagen solution with a phosphate buffer saline at 28 {sup 0}C. Their structure was discussed in terms of the volume ratio of fish/porcine collagen solution. Scanning electron and atomic force micrographs showed that the diameter of collagen fibrils derived from the collagen mixture was larger than those derived from each collagen, and all resultant fibrils exhibited a typical D-periodic unit of {approx}67 nm, irrespective of volume ratio of both collagens. Differential scanning calorimetry revealed only one endothermic peak for the fibrils derived from collagen mixture or from each collagen solution, indicating that the resultant collagen fibrils were hybrids of type I fish scale collagen and type I porcine collagen.

  15. In vitro formation and thermal transition of novel hybrid fibrils from type I fish scale collagen and type I porcine collagen

    Science.gov (United States)

    Chen, Song; Ikoma, Toshiyuki; Ogawa, Nobuhiro; Migita, Satoshi; Kobayashi, Hisatoshi; Hanagata, Nobutaka

    2010-06-01

    Novel type I collagen hybrid fibrils were fabricated by neutralizing a mixture of type I fish scale collagen solution and type I porcine collagen solution with a phosphate buffer saline at 28 °C. Their structure was discussed in terms of the volume ratio of fish/porcine collagen solution. Scanning electron and atomic force micrographs showed that the diameter of collagen fibrils derived from the collagen mixture was larger than those derived from each collagen, and all resultant fibrils exhibited a typical D-periodic unit of ~67 nm, irrespective of volume ratio of both collagens. Differential scanning calorimetry revealed only one endothermic peak for the fibrils derived from collagen mixture or from each collagen solution, indicating that the resultant collagen fibrils were hybrids of type I fish scale collagen and type I porcine collagen.

  16. In vitro formation and thermal transition of novel hybrid fibrils from type I fish scale collagen and type I porcine collagen

    Directory of Open Access Journals (Sweden)

    Song Chen, Toshiyuki Ikoma, Nobuhiro Ogawa, Satoshi Migita, Hisatoshi Kobayashi and Nobutaka Hanagata

    2010-01-01

    Full Text Available Novel type I collagen hybrid fibrils were fabricated by neutralizing a mixture of type I fish scale collagen solution and type I porcine collagen solution with a phosphate buffer saline at 28 °C. Their structure was discussed in terms of the volume ratio of fish/porcine collagen solution. Scanning electron and atomic force micrographs showed that the diameter of collagen fibrils derived from the collagen mixture was larger than those derived from each collagen, and all resultant fibrils exhibited a typical D-periodic unit of ~67 nm, irrespective of volume ratio of both collagens. Differential scanning calorimetry revealed only one endothermic peak for the fibrils derived from collagen mixture or from each collagen solution, indicating that the resultant collagen fibrils were hybrids of type I fish scale collagen and type I porcine collagen.

  17. Fusobacterium necrophorum determined as abortifacient in sheep by laser capture microdissection and fluorescence in situ hybridization

    DEFF Research Database (Denmark)

    Boye, Mette; Aalbæk, Bent; Agerholm, Jørgen S.

    2006-01-01

    at late pregnancy by a technique that combines laser capture microdissection (LCM) and fluorescent in situ hybridization (LCM-FISH). Cultural bacteriological examination had failed to identify an infectious agent but by histological examination, large colonies of bacteria associated with tissue......Fluorescent in situ hybridization (FISH) has been extensively used for identification of individual microbial cells within their natural environment. The present work describes the identification of Fusobacterium necrophorum in formalin-fixed tissue samples from three sets of ovine twins aborted......RNA-targeting oligonucleotide probe specific for F. necrophorum was used in a FISH assay. In situ hybridization showed a high density of F. necrophorum in all examined tissue sections. Simultaneous probing with a general bacterial probe EUB338 and the specific probe for F. necrophorum showed that no other bacteria could...

  18. Feasibility of using fluorescence in situ hybridization (FISH) to detect early gene changes in sputum cells from uranium miners

    Energy Technology Data Exchange (ETDEWEB)

    Neft, R.E.; Rogers, J.L.; Belinsky, S.A. [and others

    1995-12-01

    Epidemiological studies have shown that combined exposure to radon progeny and tobacco smoke produce a greater than additive or synergistic increase in lung cancer risk. Lung cancer results from multiple genetic changes over a long period of time. An early change that occurs in lung cancer is trisomy 7 which is found in 50% of non-small cell lung cancer and in the far margins of resected lung tumors. The 80% mortality associated with lung cancer is in part related to the high proportion of patients who present with an advanced, unresectable tumor. Therefore, early detection of patients at risk for tumor development is critical to improve treatment of this disease. Currently, it is difficult to detect lung cancer early while it is still amendable by surgery. Saccomanno, G. has shown that premalignant cytologic changes in sputum cells collected from uranium miners can be detected by a skilled, highly trained cytopathologist. A more objective alternative for identifying premalignant cells in sputum may be to determine whether an early genetic change such as trisomy 7 is present in these cells. Fluorescence in situ hybridization (FISH) can be used to identify cells with trisomy 7. The results of this investigation indicate that FISH may prove to be an accurate, efficient method to test at-risk individuals for genetic alterations in bronchial epithelial cells from sputum.

  19. Improving Probe Immobilization for Label-Free Capacitive Detection of DNA Hybridization on Microfabricated Gold Electrodes

    Directory of Open Access Journals (Sweden)

    Sandro Carrara

    2008-02-01

    Full Text Available Alternative approaches to labeled optical detection for DNA arrays are actively investigated for low-cost point-of-care applications. In this domain, label-free capacitive detection is one of the most intensely studied techniques. It is based on the idea to detect the Helmholtz ion layer displacements when molecular recognition occurs at the electrodes/solution interface. The sensing layer is usually prepared by using thiols terminated DNA single-strength oligonucleotide probes on top of the sensor electrodes. However, published data shows evident time drift, which greatly complicates signal conditioning and processing and ultimately increases the uncertainty in DNA recognition sensing. The aim of this work is to show that newly developed ethylene-glycol functionalized alkanethiols greatly reduce time drift, thereby significantly improving capacitance based label-free detection of DNA.

  20. Phenotypic consequences of a mosaic marker chromosome identified by fluorescence in situ hybridization (FISH) as being derived from chromosome 16

    Energy Technology Data Exchange (ETDEWEB)

    Ray, J.H.; Zhou, X.; Pletcher, B.A. [Cornell Univ. Medical College, Manhasset, NY (United States)] [and others

    1994-09-01

    De novo marker chromosomes are detected in 1 in 2500 amniotic fluid samples and are associated with a 10-15% risk for phenotypic abnormality. FISH can be utilized as a research tool to identify the origins of marker chromosomes. The phenotypic consequences of a marker chromosome derived from the short arm of chromosome 16 are described. A 26-year-old woman underwent amniocentesis at 28 weeks gestation because of a prenatally diagnosed tetralogy of Fallot. Follow-up ultrasounds also showed ventriculomegaly and cleft lip and palate. 32 of 45 cells had the karyotype 47,XY,+mar; the remaining cells were 46,XY. The de novo marker chromosome was C-band positive and non-satellited and failed to stain with distamycin A/DAPI. At birth the ultrasound findings were confirmed and dysmorphic features and cryptorchidism were noted. Although a newborn blood sample contained only normal cells, mosaicism was confirmed in 2 skin biopsies. FISH using whole-chromosome painting and alpha-satellite DNA probes showed that the marker chromosome had originated from chromosome 16. As proximal 16q is distamycin A/DAPI positive, the marker is apparently derived from proximal 16p. At 15 months of age, this child is hypotonic, globally delayed and is gavage-fed. His physical examination is significant for microbrachycephaly, a round face, sparse scalp hair, ocular hypertelorism, exotropia, a flat, wide nasal bridge and tip, mild micrognathia, and tapered fingers with lymphedema of hands and feet. Inguinal hernias have been repaired. His features are consistent with those described for patients trisomic for most or all of the short arm of chromosome 16. Marker chromosomes derived from the short arm of chromosome 16 appear to have phenotypic consequences. As the origin of more marker chromosomes are identified using FISH, their karyotype/phenotype correlations will become more apparent, which will permit more accurate genetic counseling.

  1. New DAPI and FISH findings on egg maturation processes in related hybridogenetic and parthenogenetic Bacillus hybrids (Insecta, Phasmatodea).

    Science.gov (United States)

    Marescalchi, O; Scali, V

    2001-10-01

    Bacillus stick insects have proved adequate for studying a wide array of reproductive modes: sexual, parthenogenetic, hybridogenetic, androgenetic. Hybridogenetic strains (B. rossius-grandii) were thought to discard the paternal "grandii" haploset during first meiotic division and keep the "rossius" hemiclone, whereas the clonal B. whitei (=rossius/grandii) would maintain its hybrid structure by fusing back two nonsister nuclei-each derived from previously segregated heterospecific complements-by the end of the 2(nd) meiotic division. New investigations on laid eggs and ovariole squashes, either DAPI stained or FISH labeled, revealed that in hybridogens the "grandii" set is excluded from the germ line prior to meiosis and that a DNA extra-synthesis should occur to produce hemiclonal eggs after two cytologically normal meiotic divisions. On the other hand, in B. whitei eggs no genome segregation appears to occur and an intrameiotic DNA extra-synthesis must take place to produce 2n tetrachromatidic oocytes I; these divide twice and give unreduced clonal eggs. The new findings bring hybridogenetic oogenesis of Bacillus to be coincident with that of the known hemiclonal organisms and point to an independent onset of B. whitei from hemiclonal strains. In addition, B. whitei gains a closer resemblance to B. lynceorum owing to the sharing of a cytologically identical egg maturation mechanism, of the same maternal ancestor and of peculiar chromosomal features. It is here suggested that B. lynceorum originated from the incorporation of an "atticus" genome into a B. whitei egg, according to a pathway of repeated hybridization often occurred with other polyploid hybrids.

  2. Differentiation of Moraxella nonliquefaciens, M. lacunata, and M. bovis by using multilocus enzyme electrophoresis and hybridization with pilin-specific DNA probes.

    Science.gov (United States)

    Tønjum, T; Caugant, D A; Bøvre, K

    1992-12-01

    Genetic relationships among strains of Moraxella nonliquefaciens, M. lacunata, and M. bovis were studied by using multilocus enzyme electrophoresis and DNA-DNA hybridization. The 74 isolates analyzed for electrophoretic variation at 12 enzyme loci were assigned to 59 multilocus genotypes. The multilocus genotypes were grouped in four major clusters, one representing strains of M. nonliquefaciens, two representing strains of M. lacunata, and one comprising strains of M. bovis and the single strain of M. equi analyzed. DNA-DNA hybridization with total genomic probes also revealed four major distinctive entities that corresponded to those identified by multilocus enzyme electrophoresis. The two distinct clusters recognized among the M. lacunata strains apparently corresponded to the species previously designated M. lacunata and M. liquefaciens. Distinction of the four entities was improved by hybridization with polymerase chain reaction products of nonconserved parts of pilin genes as DNA probes. With these polymerase chain reaction probes, new isolates of M. nonliquefaciens, M. lacunata, M. liquefaciens, and M. bovis can be identified easily by hybridization.

  3. Unravelling the Bacterial Vaginosis-Associated Biofilm: A Multiplex Gardnerella vaginalis and Atopobium vaginae Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes.

    Directory of Open Access Journals (Sweden)

    Liselotte Hardy

    Full Text Available Bacterial vaginosis (BV, a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in vaginal biofilms using Peptide Nucleic Acid (PNA probes targeting these bacteria. For this purpose, we developed three new PNA probes for A. vaginae. The most specific A. vaginae probe, AtoITM1, was selected and then used in an assay with two existing probes, Gard162 and BacUni-1, to evaluate multiplex FISH on clinical samples. Using quantitative polymerase chain reaction (qPCR as the gold standard, we demonstrated a sensitivity of 66.7% (95% confidence interval: 54.5% - 77.1% and a specificity of 89.4% (95% confidence interval: 76.1% - 96% of the new AtoITM1 probe. FISH enabled us to show the presence of a polymicrobial biofilm in bacterial vaginosis, in which Atopobium vaginae is part of a Gardnerella vaginalis-dominated biofilm. We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis.

  4. Unravelling the Bacterial Vaginosis-Associated Biofilm: A Multiplex Gardnerella vaginalis and Atopobium vaginae Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes.

    Science.gov (United States)

    Hardy, Liselotte; Jespers, Vicky; Dahchour, Nassira; Mwambarangwe, Lambert; Musengamana, Viateur; Vaneechoutte, Mario; Crucitti, Tania

    2015-01-01

    Bacterial vaginosis (BV), a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in vaginal biofilms using Peptide Nucleic Acid (PNA) probes targeting these bacteria. For this purpose, we developed three new PNA probes for A. vaginae. The most specific A. vaginae probe, AtoITM1, was selected and then used in an assay with two existing probes, Gard162 and BacUni-1, to evaluate multiplex FISH on clinical samples. Using quantitative polymerase chain reaction (qPCR) as the gold standard, we demonstrated a sensitivity of 66.7% (95% confidence interval: 54.5% - 77.1%) and a specificity of 89.4% (95% confidence interval: 76.1% - 96%) of the new AtoITM1 probe. FISH enabled us to show the presence of a polymicrobial biofilm in bacterial vaginosis, in which Atopobium vaginae is part of a Gardnerella vaginalis-dominated biofilm. We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis.

  5. Probing particle acceleration in lower hybrid turbulence via synthetic diagnostics produced by PIC simulations

    Science.gov (United States)

    Cruz, F.; Fonseca, R. A.; Silva, L. O.; Rigby, A.; Gregori, G.; Bamford, R. A.; Bingham, R.; Koenig, M.

    2016-10-01

    Efficient particle acceleration in astrophysical shocks can only be achieved in the presence of initial high energy particles. A candidate mechanism to provide an initial seed of energetic particles is lower hybrid turbulence (LHT). This type of turbulence is commonly excited in regions where space and astrophysical plasmas interact with large obstacles. Due to the nature of LH waves, energy can be resonantly transferred from ions (travelling perpendicular to the magnetic field) to electrons (travelling parallel to it) and the consequent motion of the latter in turbulent shock electromagnetic fields is believed to be responsible for the observed x-ray fluxes from non-thermal electrons produced in astrophysical shocks. Here we present PIC simulations of plasma flows colliding with magnetized obstacles showing the formation of a bow shock and the consequent development of LHT. The plasma and obstacle parameters are chosen in order to reproduce the results obtained in a recent experiment conducted at the LULI laser facility at Ecole Polytechnique (France) to study accelerated electrons via LHT. The wave and particle spectra are studied and used to produce synthetic diagnostics that show good qualitative agreement with experimental results. Work supported by the European Research Council (Accelerates ERC-2010-AdG 267841).

  6. In situ hybridization of bat chromosomes with human (TTAGGGn probe, after previous digestion with Alu I

    Directory of Open Access Journals (Sweden)

    Karina de Cassia Faria

    2002-01-01

    Full Text Available The purpose of this work was to verify the ability of the enzyme Alu I to cleave and/or remove satellite DNA sequences from heterochromatic regions in chromosomes of bats, by identifying the occurrence of modifications in the pattern of fluorescence in situ hybridization with telomeric DNA. The localization and fluorescence intensity of the telomeric DNA sites of the Alu-digested and undigested chromosomes of species Eumops glaucinus, Carollia perspicillata, and Platyrrhinus lineatus were analyzed. Telomeric sequences were detected at the termini of chromosomes of all three species, although, in C. perspicillata, the signals were very faint or absent in most chromosomes. This finding was interpreted as being due to a reduced number of copies of the telomeric repeat, resulting from extensive telomeric association and/or rearrangements undergone by the chromosomes of Carollia. Fluorescent signals were also observed in centromeric and pericentromeric regions in several two-arm chromosomes of E. glaucinus and C. perspicillata. In E. glaucinus and P. lineatus, some interstitial and terminal telomeric sites were observed to be in association with regions of constitutive heterochromatin and ribosomal DNA (NORs. After digestion, these telomeric sites showed a significant decrease in signal intensity, indicating that enzyme Alu I cleaves and/or removes part of the satellite DNA present in these regions. These results suggest that the telomeric sequence is a component of the heterochromatin, and that the C-band- positive regions of bat chromosomes have a different DNA composition.

  7. The application of fluorescence in situ hybridization (FISH technique for studying the microbial communities in intestinal tissues of white shrimp (Penaeus vannamei

    Directory of Open Access Journals (Sweden)

    Supamattaya, K.

    2005-02-01

    Full Text Available Fluorescence in situ hybridization technique is very useful for the evaluation of microbial communities in various environments. It is possible to apply this technique to study the intestinal microflora in white shrimp (Penaeus vannamei. Different fixatives and storage temperature were tested in this technique. It was found that fixation with 10% buffered formalin for 12 hours and changed to 70% ethanol shown positive results when compared to the fixation with Davidson's fixative or RF fixative. The best signaling was obtainedfrom the samples which were stored in -20ºC. By using the DNA probe targeted to the Eubacteria domain (EUB338 probe, 5′-GCT GCC TCC CGT AGG AGT-3′ labeled with fluorescein as a hybridizing probe, it was found that most intestinal microflora were aggregated with the intestinal contents, or dispersed in the lumen. There was not evidence of the attachment of the microflora with the intestinal epithelium in this study.

  8. ROS1基因非重复序列双色荧光原位杂交探针建立及在非小细胞肺癌组织检测中的应用%Construction of a repeat-free dual color fluorescent in situ hybridization probe for ROS1 gene in non-small cell lung cancer diagnosis

    Institute of Scientific and Technical Information of China (English)

    程弘夏; 叶伦; 薛力泉

    2014-01-01

    目的 建立ROS1基因的非重复序列荧光原位杂交(FISH)探针,比较其与商品化普通探针在非小细胞肺癌检测方面的差异.方法 探针制备的过程中加入Human Cot-1 DNA,与基因组中的重复序列复性结合成双链,用双链特异性酶特异性的切除重复序列.用红色和绿色荧光素分别标记BAC克隆片段化的PCR产物,最终混合得到ROS1基因不含重复序列的双色探针.将制备的非重复序列FISH探针用于2009至2013年收集的经基因测序检测确定的53例ROS1基因重排的非小细胞肺癌标本,并与传统探针测定结果比较.结果 与商品化的重复序列探针比较,非重复序列FISH探针背景清晰,仅在目的区域存在明亮的荧光杂交信号,噪音明显低于商品化探针.两种探针间的杂交率差异有统计学意义(P<0.05);两组探针之间的准确率差异无统计学意义(P>0.05).结论 经比较显示,非重复序列的ROS1双色FISH探针在对非小细胞肺癌样本检测中明显改善探针的杂交效率和信噪比,有利于提高检测的准确度.%Objective To establish a repeat-free ROS1 gene fluorescence in situ hybridization (FISH) probe,and to compare its efficacy with those of commercial FISH probes in non-small cell lung cancer.Methods The probe was constructed by combining human Cot-1 DNA genome into double-stranded sequence,and then digested by duples specific nuclease to establish a repeat-free sequence.The final repeat-free ROS1 FISH probe was labeled by red and green fluoresceins.Results Compared with the commercialized probe,repeat-free FISH probe exhibited excellent efficiency and low signal to noise ratio (SNR) in samples.There was statistical significance in the difference between the hybridization rate of these two probes(P<0.05),but there was no difference between the accuracy rate (P>0.05).Conclusion The repeat-free ROS1 FISH probe significantly improves the probe hybridization efficiency and SNR in nonsmall

  9. Localization of cannabinoid CB1 receptor mRNA using ribonucleotide probes: methods for double- and single-label in situ hybridization.

    Science.gov (United States)

    Hohmann, Andrea G

    2006-01-01

    This chapter presents a reliable, detailed method for performing double-label in situ hybridization (ISH) that has been validated for use in studies identifying the co-localization of cannabinoid CB1 receptor mRNA with other distinct species of mRNAs. This method permits simultaneous detection of two different species of mRNA within the same tissue section. Double-label ISH may be accomplished by hybridizing tissue sections with a combination of radiolabeled and digoxigenin-labeled RNA probes that are complementary to their target mRNAs. Single-label ISH may be accomplished by following the procedures described for use with radioisotopic probes (here [35S]-labeled) only. Silver grains derived from conventional emulsion autoradiography are used to detect the radiolabeled cRNA probe. An alkaline phosphatase-dependent chromogen reaction product is used to detect the nonisotopic (here, digoxigenin-labeled) cRNA probe. Necessary controls that are required to document the specificity of the labeling of the digoxigenin and radiolabeled probes are described. The methods detailed herein may be employed to detect even low levels of a target mRNA. These methods may be utilized to study co-localization and coregulation of expression of a particular gene within identified neurons in multiple systems.

  10. Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.

    Science.gov (United States)

    Tse, Jenny; Wang, Yuanyuan; Zengeya, Thomas; Rozners, Eriks; Tan-Wilson, Anna

    2015-02-01

    We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Technological exploration of BAC-FISH on mitotic chromosomes of maize

    Institute of Scientific and Technical Information of China (English)

    Yongsheng TAO; Zuxin ZHANG; Yonglin CHEN; Lijia LI; Yonglian ZHENG

    2008-01-01

    The rice BAC-DNA was used as probes and fluorescence in situ hybridization (FISH) was applied to the interphase and metaphase mitotic chromosomes of maize. To optimize the BAC-FISH technique, we respect-ively assayed the effect of several factors, including maize or rice genomic Cot DNA used as blocking reagent of DNA, washing temperatures and FAD concentration in the washing buffer and in the hybrid solution. The results show that Cot DNA of maize genome blocked the repet-itive sequence of the rice BAC-DNA when the Cot value was below 50. Meanwhile, it was necessary to adjust the Cot value according to the different probes and their ratios. Decreasing the concentration of FAD in the hybridization mixtures, adjusting the washing rate after hybridization, and most especially, blocking the rice-specific repetitive sequences of BAC-DNA could improve the positive signals of BAC-FISH.

  12. COmbined Binary RAtio fluorescence in situ hybridiziation (COBRA-FISH): development and applications.

    Science.gov (United States)

    Raap, A K; Tanke, H J

    2006-01-01

    The ability to probe for the location of DNA sequences in morphologically preserved chromosomes and nuclei by fluorescence in situ hybridization (FISH) provided for cytogenetics a quantum leap forward in resolution and ease of detection of chromosomal aberrations. COBRA-FISH, an acronym for COmbined Binary RAtio-FISH is a multicolor FISH methodology, which enables recognition of all human chromosome arms on the basis of color, thus greatly facilitating cytogenetic analysis. It also permits gene and viral integration site mapping in the context of chromosome arm painting. Here we review the principle, practice and applications of COBRA-FISH.

  13. Molecular cytogenetics of pituitary adenomas, assessed by FISH technique.

    Science.gov (United States)

    Kontogeorgos, George

    2004-01-01

    Fluorescent in situ hybridization (FISH) represents a moden molecular pathology technique, alternative to conventional cytogenetics (karyotyping). In addition to metaphase spreads, it can be applied directly to interphase nuclei. The latter makes the FISH technique powerful for pathologists for it integrates molecular genetics and classic cytogenetics and brings them together to a single framework for morphologic evaluation. Interphase FISH can be applied to imprints from fresh tissue or to paraffin sections after proteinase K digestion. Centromeric, telomeric and locus DNA-sequence specific probes can be used to identify aneuploidy or gene mutations. Several protocols combine molecular cytogenetics with classic karyotyping. Other sophisticated, FISH-based protocols have been introduced. Among them, comparative genomic hybridization is very important for it can detect non-balanced chromosomal aberrations of uncultured tumor cells and provide overall genomic information in a single experiment. This review presents the principles and applications of FISH technique for the investigation of the cytogenetic background of pituitary adenomas.

  14. Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16S rRNA-targeted probes by colony hybridization and PCR.

    Science.gov (United States)

    Kaufmann, P; Pfefferkorn, A; Teuber, M; Meile, L

    1997-01-01

    A Bifidobacterium genus-specific target sequence in the V9 variable region of the 16S rRNA has been elaborated and was used to develop a hybridization probe. The specificity of this probe, named lm3 (5'-CGGGTGCTI*CCCACTTTCATG-3'), was used to identify all known type strains and distinguish them from other bacteria. All of the 30 type strains of Bifidobacterium which are available at the German culture collection Deutsche Sammlung von Mikroorganismen und Zellkulturen, 6 commercially available production strains, and 34 closely related relevant strains (as negative controls) were tested. All tested bifidobacteria showed distinct positive signals by colony hybridization, whereas all negative controls showed no distinct dots except Gardnerella vaginalis DSM4944 and Propionibacterium freudenreichii subsp. shermanii DSM4902, which gave slight signals. Furthermore, we established a method for isolation and identification of bifidobacteria from food by using a PCR assay without prior isolation of DNA but breaking the cells with proteinase K. By this method, all Bifidobacterium strains lead to a DNA product of the expected size. We also established a quick assay to quantitatively measure Bifidobacterium counts in food and feces by dilution plating and colony hybridization. We were able to demonstrate that 2.1 x 10(6) to 2.3 x 10(7) colonies/g of sour milk containing bifidobacteria hybridized with the specific nucleotide probe. With these two methods, genus-specific colony hybridization and genus-specific PCR, it is now possible to readily and accurately detect any bifidobacteria in food and fecal samples and to discriminate between them and members of other genera. PMID:9097423

  15. Microsatellite instability typing in serum and tissue of patients with colorectal cancer: comparing real time PCR with hybridization probe and high-performance liquid chromatography.

    Science.gov (United States)

    Mokarram, P; Rismanchi, M; Alizadeh Naeeni, M; Mirab Samiee, S; Paryan, M; Alipour, A; Honardar, Z; Kavousipour, S; Naghibalhossaini, F; Mostafavi-Pour, Z; Monabati, A; Hosseni, S V; Shamsdin, S A

    2014-05-01

    Allelic variation of BAT-25 (a 25-repeat quasimonomorphic poly T) and BAT-26 (a 26-repeat quasimonomorphic polyA) loci as two mononucleotide microsatellite markers, were analyzed with high-performance liquid chromatography (HPLC) compared with Real-Time PCR using hybridization probes. BAT-26 and BAT-25 markers were used to determine an appropriate screening technique with high sensitivity and specificity to diagnose microsatellite instability (MSI) status in patients with colorectal cancer (CRC). One of the pathways in colorectal tumor genesis is microsatellite instability (MSI+). MSI is detected in about 15% of all CRCs; 3% are of these are associated with Lynch syndrome and the other 12% are caused by sporadic. Colorectal tumors with MSI have distinctive features compared with microsatellite stable tumors. Due to the high percentage of MSI+ CRC in Iran, screening of this type of CRC is imperative. Two markers were analyzed in tissues and sera of 44 normal volunteers and tumor and matched normal mucosal tissues as well as sera of 44 patients with sporadic CRC. The sensitivity and specificity of BAT-26 with real time PCR method (Hybridization probe) were 100% in comparison with sequencing method as the gold standard, while HPLC had a lower sensitivity and specificity. According to HPLC data, BAT-26 was more sensitive than BAT-25 in identifying MSI tumors. Therefore, MSI typing using the BAT-26 hybridization probe method compared to HPLC could be considered as an accurate method for diagnosing MSI in CRC tumors but not in serum circulating DNAs.

  16. 荧光原位杂交法(FISH)快速检测尿液中金黄色葡萄球菌%Fluorescence in Situ Hybridization Rapidly Detects Staphylococcus Aureus in Urinary Tract Infection Samples

    Institute of Scientific and Technical Information of China (English)

    吴青; 李艳; 汪明; 顾剑; 胡慧霞; 徐万洲; 韦传东; 吴泽刚

    2011-01-01

    目的 利用荧光原位杂交法快速检测尿液中的金黄色葡萄球菌,筛查金黄色葡萄球菌所致的尿路感染.方法 针对金黄色葡萄球菌16sRNA设计荧光标记的核苷酸探针,利用荧光原位杂交技术(FISH)对132例疑似尿路感染患者中段尿标本进行检测;同时进行中段尿培养.结果 荧光原位杂交法检测阳性的有9例,与中段尿培养比较,其敏感度为100.0%,特异度为99.2%,阳性预期值为90.0%,阴性预期值为100.0%.结论 荧光原位杂交能快速检测尿液中的金黄色葡萄球菌,对金黄色葡萄球菌所致的尿路感染进行快速诊断.%Objective The rapid detection of Staphylococcus aureus in urine by fluorescence in situ hybridization (FISH)method. Screening urinary tract infection (UTIs) caused by Staphylococcus aureus. Methods Probes that were specific for Staphylococcus aureus were designed based on the conserved 16 s RNA sequences. Detected a total of 132 urine samples using FISH method. And all of tbese samples tested via traditional culturing techniques. Results 9 of these samples tested positive for a UTI via FISH analysis. Compared to conventional methods of bacterial identification,the FISH method had a sensitivity of 100.0% ,specificity of 99. 2%,positive expected value of 90. 0% and negative expected value of 100. 0%. Conclusion FISH had the potential to become an extremely useful diagnostic tool for UTIs because it has a quick turnaround time and high accuracy.

  17. Three sympatric karyomorphs in the fish Astyanax fasciatus (Teleostei, Characidae) do not seem to hybridize in natural populations

    Science.gov (United States)

    Ferreira-Neto, Maressa; Artoni, Roberto Ferreira; Vicari, Marcelo Ricardo; Moreira-Filho, Orlando; Camacho, Juan Pedro Martínez; Bakkali, Mohammed; de Oliveira, Claudio; Foresti, Fausto

    2012-01-01

    Abstract Ninety individuals of the characid fish Astyanax fasciatus (Cuvier, 1819) were collected at Água da Madalena stream (Botucatu, São Paulo, Brazil) and analyzed for diploid chromosome number 2n and karyotype composition as well as for the chromosomal location of the 5S and 18S ribosomal DNA (rDNA). Whereas no chromosome differences were associated with sex, three different karyomorphs with diploid chromosome numbers 2n=46, 2n=48 and 2n=50 were found. No intermediate 2n numbers were discovered. The 2n=50 karyomorph showed some differences in 18S rDNA location compared to the two other karyomorphs. Finally, all specimens with the 2n=46 karyomorph showed the presence of a partly heterochromatic macro supernumerary chromosome, which was absent in all individuals with the two other karyomorphs. All these results suggest that indviduals of the three different karyomorphs are not likely to hybridize in the examined populations. Our findings strongly suggest the presence of three separate species (sensu biological species concept) easily diagnosed on the basis of differences in the diploid chromosome numbers and other chromosomal markers. PMID:24260650

  18. Three sympatric karyomorphs in the fish Astyanax fasciatus (Teleostei, Characidae do not seem to hybridize in natural populations

    Directory of Open Access Journals (Sweden)

    Maressa Ferreira-Neto

    2012-01-01

    Full Text Available Ninety individuals of the characid fish Astyanax fasciatus (Cuvier, 1819 were collected at Água da Madalena stream (Botucatu, São Paulo, Brazil and analyzed for diploid chromosome number 2n and karyotype composition as well as for the chromosomal location of the 5S and 18S ribosomal DNA (rDNA. Whereas no chromosome differences were associated with sex, three different karyomorphs with diploid chromosome numbers 2n=46, 2n=48 and 2n=50 were found. No intermediate 2n numbers were discovered. The 2n=50 karyomorph showed some differences in 18S rDNA location compared to the two other karyomorphs. Finally, all specimens with the 2n=46 karyomorph showed the presence of a partly heterochromatic macro supernumerary chromosome, which was absent in all individuals with the two other karyomorphs. All these results suggest that indviduals of the three different karyomorphs are not likely to hybridize in the examined populations. Our findings strongly suggest the presence of three separate species (sensu biological species concept easily diagnosed on the basis of differences in the diploid chromosome numbers and other chromosomal markers.

  19. Supernumerary ring chromosome 20 characterized by fluorescence in situ hybridization

    NARCIS (Netherlands)

    Van Langen, Irene M.; Otter, Mariëlle A.; Aronson, Daniël C.; Overweg-Plandsoen, W.C.G.; Hennekam, Raoul C.M.; Leschot, Nico J.; Hoovers, Jan M.N.

    1996-01-01

    We report on a boy with mild dysmorphic features and developmental delay, in whom karyotyping showed an additional minute ring chromosome in 60% of metaphases. Fluorescence in situ hybridization (FISH) with a centromere specific probe demonstrated that the ring chromosome contained the centromeric r

  20. In situ optical sequencing and structure analysis of a trinucleotide repeat genome region by localization microscopy after specific COMBO-FISH nano-probing

    Science.gov (United States)

    Stuhlmüller, M.; Schwarz-Finsterle, J.; Fey, E.; Lux, J.; Bach, M.; Cremer, C.; Hinderhofer, K.; Hausmann, M.; Hildenbrand, G.

    2015-10-01

    Trinucleotide repeat expansions (like (CGG)n) of chromatin in the genome of cell nuclei can cause neurological disorders such as for example the Fragile-X syndrome. Until now the mechanisms are not clearly understood as to how these expansions develop during cell proliferation. Therefore in situ investigations of chromatin structures on the nanoscale are required to better understand supra-molecular mechanisms on the single cell level. By super-resolution localization microscopy (Spectral Position Determination Microscopy; SPDM) in combination with nano-probing using COMBO-FISH (COMBinatorial Oligonucleotide FISH), novel insights into the nano-architecture of the genome will become possible. The native spatial structure of trinucleotide repeat expansion genome regions was analysed and optical sequencing of repetitive units was performed within 3D-conserved nuclei using SPDM after COMBO-FISH. We analysed a (CGG)n-expansion region inside the 5' untranslated region of the FMR1 gene. The number of CGG repeats for a full mutation causing the Fragile-X syndrome was found and also verified by Southern blot. The FMR1 promotor region was similarly condensed like a centromeric region whereas the arrangement of the probes labelling the expansion region seemed to indicate a loop-like nano-structure. These results for the first time demonstrate that in situ chromatin structure measurements on the nanoscale are feasible. Due to further methodological progress it will become possible to estimate the state of trinucleotide repeat mutations in detail and to determine the associated chromatin strand structural changes on the single cell level. In general, the application of the described approach to any genome region will lead to new insights into genome nano-architecture and open new avenues for understanding mechanisms and their relevance in the development of heredity diseases.

  1. Rapid discrimination of Haemophilus influenzae, H. parainfluenzae, and H. haemolyticus by fluorescence in situ hybridization (FISH and two matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS platforms.

    Directory of Open Access Journals (Sweden)

    Hagen Frickmann

    Full Text Available BACKGROUND: Due to considerable differences in pathogenicity, Haemophilus influenzae, H. parainfluenzae and H. haemolyticus have to be reliably discriminated in routine diagnostics. Retrospective analyses suggest frequent misidentifications of commensal H. haemolyticus as H. influenzae. In a multi-center approach, we assessed the suitability of fluorescence in situ hybridization (FISH and matrix-assisted laser-desorption-ionization time-of-flight mass-spectrometry (MALDI-TOF-MS for the identification of H. influenzae, H. parainfluenzae and H. haemolyticus to species level. METHODOLOGY: A strain collection of 84 Haemophilus spp. comprising 50 H. influenzae, 25 H. parainfluenzae, 7 H. haemolyticus, and 2 H. parahaemolyticus including 77 clinical isolates was analyzed by FISH with newly designed DNA probes, and two different MALDI-TOF-MS systems (Bruker, Shimadzu with and without prior formic acid extraction. PRINCIPAL FINDINGS: Among the 84 Haemophilus strains analyzed, FISH led to 71 correct results (85%, 13 uninterpretable results (15%, and no misidentifications. Shimadzu MALDI-TOF-MS resulted in 59 correct identifications (70%, 19 uninterpretable results (23%, and 6 misidentifications (7%, using colony material applied directly. Bruker MALDI-TOF-MS with prior formic acid extraction led to 74 correct results (88%, 4 uninterpretable results (5% and 6 misidentifications (7%. The Bruker MALDI-TOF-MS misidentifications could be resolved by the addition of a suitable H. haemolyticus reference spectrum to the system's database. In conclusion, no analyzed diagnostic procedure was free of errors. Diagnostic results have to be interpreted carefully and alternative tests should be applied in case of ambiguous test results on isolates from seriously ill patients.

  2. Resolving colocalization of bacteria and metal(loid)s on plant root surfaces by combining fluorescence in situ hybridization (FISH) with multiple-energy micro-focused X-ray fluorescence (ME μXRF).

    Science.gov (United States)

    Honeker, Linnea K; Root, Robert A; Chorover, Jon; Maier, Raina M

    2016-12-01

    Metal(loid)-contamination of the environment due to anthropogenic activities is a global problem. Understanding the fate of contaminants requires elucidation of biotic and abiotic factors that influence metal(loid) speciation from molecular to field scales. Improved methods are needed to assess micro-scale processes, such as those occurring at biogeochemical interfaces between plant tissues, microbial cells, and metal(loid)s. Here we present an advanced method that combines fluorescence in situ hybridization (FISH) with synchrotron-based multiple-energy micro-focused X-ray fluorescence microprobe imaging (ME μXRF) to examine colocalization of bacteria and metal(loid)s on root surfaces of plants used to phytostabilize metalliferous mine tailings. Bacteria were visualized on a small root section using SytoBC nucleic acid stain and FISH probes targeting the domain Bacteria and a specific group (Alphaproteobacteria, Gammaproteobacteria, or Actinobacteria). The same root region was then analyzed for elemental distribution and metal(loid) speciation of As and Fe using ME μXRF. The FISH and ME μXRF images were aligned using ImageJ software to correlate microbiological and geochemical results. Results from quantitative analysis of colocalization show a significantly higher fraction of As colocalized with Fe-oxide plaques on the root surfaces (fraction of overlap 0.49±0.19) than to bacteria (0.072±0.052) (pbacteria that colocalized with metal(loid)s, Actinobacteria, known for their metal tolerance, had a higher correlation with both As and Fe than Alphaproteobacteria or Gammaproteobacteria. This method demonstrates how coupling these micro-techniques can expand our understanding of micro-scale interactions between roots, metal(loid)s and microbes, information that should lead to improved mechanistic models of metal(loid) speciation and fate. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Fluorescence Image Analyzer - FLIMA: software for quantitative analysis of fluorescence in situ hybridization.

    Science.gov (United States)

    Silva, H C M; Martins-Júnior, M M C; Ribeiro, L B; Matoso, D A

    2017-03-30

    The Fluorescence Image Analyzer (FLIMA) software was developed for the quantitative analysis of images generated by fluorescence in situ hybridization (FISH). Currently, the images of FISH are examined without a coefficient that enables a comparison between them. Through GD Graphics Library, the FLIMA software calculates the amount of pixels on image and recognizes each present color. The coefficient generated by the algorithm shows the percentage of marks (probes) hybridized on the chromosomes. This software can be used for any type of image generated by a fluorescence microscope and is able to quantify digoxigenin probes exhibiting a red color, biotin probes exhibiting a green color, and double-FISH probes (digoxigenin and biotin used together), where the white color is displayed.

  4. DNA breakage detection-fish (DBD-FISH): effect of unwinding time.

    Science.gov (United States)

    Vázquez-Gundín, F; Gosálvez, J; de la Torre, J; Fernández, J L

    2000-09-20

    DBD-FISH is a new procedure that allows detection and quantification of DNA breakage in situ within specific DNA target sites. Cells embedded in an agarose matrix on a slide are treated in an alkaline unwinding solution to transform DNA breaks into single-stranded DNA (ssDNA). After removal of proteins, DNA probes are hybridized and detected. DNA breaks increase the ssDNA and relax supercoiling of DNA loops, so more probe hybridizes, thereby increasing the surface area and fluorescence intensity of the FISH signal. The probe selects the chromatin area to be analysed. In order to restrict the extension of unwound ssDNA to a region closer to the origin of the DNA break, human leukocytes were processed for DBD-FISH with a whole genome probe, after a 10 Gy dose of X-rays, for various unwinding times: 5, 2 min and 30s. Two cell populations were detected after 30s, but not with the 5 or 2 min unwinding times. One cell group had small to medium haloes corresponding to the relaxation of DNA supercoiling after DAPI staining, and strong DBD-FISH labelling of induced DNA breaks, whereas the other cell group showed big haloes of DNA loop unfolding and an absence of DBD-FISH labelling. The latter group was similar to cells processed by DBD-FISH without the unwinding step. Thus, they should correspond to cells unaffected by the alkaline unwinding solution, possibly because very brief unwinding times do not allow the diffusion of the alkali into the cells deep within the gel, thus biasing the results. Taking this into account, 2 min seems to be the minimum unwinding time required for an accurate detection of a signal by DBD-FISH.

  5. Fluorescence in situ hybridization of old G-banded and mounted chromosome preparations

    DEFF Research Database (Denmark)

    Gerdes, A M; Pandis, N; Bomme, L;

    1997-01-01

    , that the amount of added probe is increased approximately 2.5 times, and that the amplification of signals is performed twice. The applicability of the method, which allows double painting with two differently labeled probes using two differently fluorescing colors, was tested on 11 cases involving different......An improved method for fluorescence in situ hybridization (FISH) investigation of old, previously G-banded, mounted chromosome preparations with chromosome specific painting probes and centromere-specific probes is described. Before hybridization, the slides are incubated in xylene until...... the coverslips detach spontaneously; any mechanical manipulation will jeopardize the results. The success of chromosome painting is improved by excluding the regular RNase treatment step prior to hybridization. Additional changes compared with standard FISH protocols are that the 2 x SSC step is omitted...

  6. Cellular delivery of quantum dot-bound hybridization probe for detection of intracellular pre-microRNA using chitosan/poly(γ-glutamic acid complex as a carrier.

    Directory of Open Access Journals (Sweden)

    Yao Geng

    Full Text Available A quantum dot (QD-bound hybridization probe was designed for detection of intracellular pre-miRNA using chitosan (CS/poly(γ-glutamic acid (γ-PGA complex as a gene vector. The probe was prepared by assembling thiolated RNA to gold nanoparticle (Au NP via Au-S bond and then binding 3'-end amine of the RNA to the carboxy group capped on quantum dot surface. The QD-RNA-Au NP probe was assembled on the vector by mixing with aqueous γ-PGA solution and then CS solution to construct a gene delivery system for highly effective cellular uptake and delivery. After the probe was released from CS/γ-PGA complex to the cytoplasm by electrostatic repulsion at intracellular pH, it hybridized with pre-miRNA precursor as target. The formed product was then cleaved by RNase III Dicer, leading to the separation of QDs from Au NPs and fluorescence emission of QDs, which could be detected by confocal microscopic imaging to monitor the amount of the intracellular pre-miRNA precursor. The in vitro assays revealed that the QD-RNA-Au NP was a robust, sensitive and selective probe for quantitative detection of target pre-miRNA. Using MDA-MB231 and MCF-7 breast cancer cells as models, the relative amount of pre-miRNA let-7a could be successfully compared. Since the amount of miRNA is related to the progress and prognosis of cancer, this strategy could be expected to hold promising application potential in medical research and clinical diagnostics.

  7. Evaluation of the Yeast Traffic Light PNA FISH probes for identification of Candida species from positive blood cultures.

    Science.gov (United States)

    Hall, Leslie; Le Febre, Kara M; Deml, Sharon M; Wohlfiel, Sherri L; Wengenack, Nancy L

    2012-04-01

    The Yeast Traffic Light PNA FISH kit (YTL) correctly identified Candida spp. in 207/216 (96%) positive blood cultures. Discordant results were seen with known cross-reacting species and cultures containing Candida lambica and Rhodotorula mucilaginosa. The YTL provides rapid, reliable identification of the five common Candida species found in blood cultures.

  8. Discrepancy between fluorescence in situ hybridization and multiplex ligation-dependent probe amplification in orbital recurrence of uveal melanoma 26 years after enucleation.

    Science.gov (United States)

    Russo, Andrea; Rene, Cornelius; Coupland, Sarah E; Sagili, Suresh; Damato, Bertil

    2012-01-01

    Cytogenetic analysis has transformed the management of uveal melanoma in recent years and allows categorization of such tumors into low-grade tumors with a favorable prognosis and high-grade tumors that metastasize with a fatal outcome. The authors report the case of a 73-year-old man who presented with recurrent melanoma in his left socket, 26 years after enucleation for uveal melanoma. Chromosomal analysis by multiplex ligation-dependent probe amplification revealed partial loss of chromosome 3 and gains in chromosomes 6 and 8, which were missed with fluorescence in situ hybridization. The patient developed multiple liver metastases 14 months after orbital exenteration and died 8 months later. To the best of authors' knowledge, this is the first report of late recurrence of uveal melanoma after enucleation, in which multiplex ligation-dependent probe amplification chromosomal analysis has been used. The case also highlights the limitations of fluorescence in situ hybridization and the benefits of multiplex ligation-dependent probe amplification, which is more reliable at predicting survival.

  9. Determination of Nilestriol in Fish by Coupling Direct Insertion Probe With Mass Spectrometer%直接进样质谱法检测鱼肉中尼尔雌醇

    Institute of Scientific and Technical Information of China (English)

    谢增瑞; 庞立新; 赵亚敏

    2004-01-01

    A method for determination of nilestriol in the fish by coupling direct insertion probe with mass spectrometer was established. When the fish be dewatered, nilestriol can be drawed with chloroform and the analysis can be made by coupling direct insertion probe with mass spectrometer as the primary temperature at 100℃ and maintains for 30s, then the temperation rised to 300℃ at the rate of 150℃ · min-1 and kept for 30s. The ratio of signal to noise(S/N) of nilestriol is 14 : 1 as the sample content is lng. The method issimple in operation and sensitive.

  10. Numbers and locations of native bacteria on field-grown wheat roots quantified by fluorescence in situ hybridization (FISH).

    Science.gov (United States)

    Watt, Michelle; Hugenholtz, Philip; White, Rosemary; Vinall, Kerry

    2006-05-01

    Native bacteria, Pseudomonas and filamentous bacteria were quantified and localized on wheat roots grown in the field using fluorescence in situ hybridization (FISH). Seminal roots were sampled through the season from unploughed soil in a conservation farming system. Such soils are spatially heterogeneous, and many roots grow slowly through hard soil with cracks and pores containing dead roots remnant from previous crops. Root and rhizosphere morphology, and contact with soil particles were preserved, and autofluorescence was avoided by observing sections in the far-red with Cy5 and Cy5.5 fluorochromes. Spatial analyses showed that bacteria were embedded in a stable matrix (biofilm) within 11 microm of the root surface (range 2-30 microm) and were clustered on 40% of roots. Half the clusters co-located with axial grooves between epidermal cells, soil particles, cap cells or root hairs; the other half were not associated with visible features. Across all wheat roots, although variable, bacteria averaged 15.4 x 10(5) cells per mm(3) rhizosphere, and of these, Pseudomonas and filaments comprised 10% and 4%, respectively, with minor effects of sample time, and no effect of plant age. Root caps were most heavily colonized by bacteria along roots, and elongation zones least heavily colonized. Pseudomonas varied little with root development and were 17% of bacteria on the elongation zone. Filamentous bacteria were not found on the elongation zone. The most significant factor to rhizosphere populations along a wheat root, however, was contact with dead root remnants, where Pseudomonas were reduced but filaments increased to 57% of bacteria (P < 0.001). This corresponded with analyses of root remnants showing they were heavily colonized by bacteria, with 48% filaments (P < 0.001) and 1.4%Pseudomonas (P = 0.014). Efforts to manage rhizosphere bacteria for sustainable agricultural systems should continue to focus on root cap and mucilage chemistry, and remnant roots as

  11. Fluorescence in situ hybridization(FISH) technique combined with karyotyping analysis in prenatal diagnosis%FISH技术在产前诊断中的应用价值研究

    Institute of Scientific and Technical Information of China (English)

    沈国松; 张甦; 何平亚; 方嵘

    2011-01-01

    目的 使用荧光原位杂交(FISH)技术对常见胎儿染色体数目异常进行快速诊断,结合羊水细胞培养染色体核型分析技术形成产前诊断体系,并对其临床应用价值进行评价.方法 应用诊断最常见染色体病的5种染色体(13、18、21、X和Y)特异性FISH探针对480例未经培养羊水细胞进行产前诊断,并和同时进行的羊水培养染色体核型分析相比较.结果 480例未经培养羊水细胞FISH实验全部获得检测结果,并发现21-三体综合征2例,18-三体综合征1例,克氏综合征1例,特纳综合征 1例,与羊水培养染色体分析结果一致,但报告时间(2至3d)与羊水染色体分析报告时间(2~3周)相比大为缩短.受固有技术限制有6例结构异常未能检出,但应用FISH技术对其中1例与性染色体有关的结构异常进行辅助诊断,获得了成功.结论 FISH技术在常见染色体数目异常产前诊断中应用行之有效,与羊水染色体核型分析技术相结合,将使产前诊断更加高效和安全.%To evaluate the clinical application of the combination of fluorescence in situ hybridization (FISH)technique and karyotyping analysis in prenatal diagnosis.MethodsFISH technology with the centromeric probes of chromo-some 13, 18, 21 ,X and Y was applied in 480 amniotic fluid specimens, the results were compared with those of the G banding karyotypes from standard cytogenetic analysis in cultured amniotic fluid cells.ResultsTotal 480 uncultured amniotic fluid speci-mens were successfully tested by FISH and two Down' s syndrome, one Edwards syndrome, one Klinefelter syndrome and one Turner syndrome were detected. The results obtained by FISH were consistent with those from karyotyping analysis. FISH (2-3 days) was faster than karyotyping(2-3 weeks) to get results. Karyotypes analysis detected 6 cases of chromosome structural abnormalities which were not detected by FISH, however, one of them with a sex chromosome-related structural

  12. Molecular cytogenetics using fluorescence in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Gray, J.W.; Kuo, Wen-Lin; Lucas, J.; Pinkel, D.; Weier, H-U.; Yu, Loh-Chung.

    1990-12-07

    Fluorescence in situ hybridization (FISH) with chromosome-specific probes enables several new areas of cytogenetic investigation by allowing visual determination of the presence and normality of specific genetic sequences in single metaphase or interphase cells. in this approach, termed molecular cytogenetics, the genetic loci to be analyzed are made microscopically visible in single cells using in situ hybridization with nucleic acid probes specific to these loci. To accomplish this, the DNA in the target cells is made single stranded by thermal denaturation and incubated with single-stranded, chemically modified probe under conditions where the probe will anneal only with DNA sequences to which it has high DNA sequence homology. The bound probe is then made visible by treatment with a fluorescent reagent such as fluorescein that binds to the chemical modification carried by the probe. The DNA to which the probe does not bind is made visible by staining with a dye such as propidium iodide that fluoresces at a wavelength different from that of the reagent used for probe visualization. We show in this report that probes are now available that make this technique useful for biological dosimetry, prenatal diagnosis and cancer biology. 31 refs., 3 figs.

  13. 5-bp Classical Satellite DNA Loci from Chromosome-1 Instability in Cervical Neoplasia Detected by DNA Breakage Detection/Fluorescence in Situ Hybridization (DBD-FISH).

    Science.gov (United States)

    Cortés-Gutiérrez, Elva I; Ortíz-Hernández, Brenda L; Dávila-Rodríguez, Martha I; Cerda-Flores, Ricardo M; Fernández, José Luis; López-Fernández, Carmen; Gosálvez, Jaime

    2013-02-19

    We aimed to evaluate the association between the progressive stages of cervical neoplasia and DNA damage in 5-bp classical satellite DNA sequences from chromosome-1 in cervical epithelium and in peripheral blood lymphocytes using DNA breakage detection/fluorescence in situ hybridization (DBD-FISH). A hospital-based unmatched case-control study was conducted in 2011 with a sample of 30 women grouped according to disease stage and selected according to histological diagnosis; 10 with low-grade squamous intraepithelial lesions (LG-SIL), 10 with high-grade SIL (HG-SIL), and 10 with no cervical lesions, from the Unidad Medica de Alta Especialidad of The Mexican Social Security Institute, IMSS, Mexico. Specific chromosome damage levels in 5-bp classical satellite DNA sequences from chromosome-1 were evaluated in cervical epithelium and peripheral blood lymphocytes using the DBD-FISH technique. Whole-genome DNA hybridization was used as a reference for the level of damage. Results of Kruskal-Wallis test showed a significant increase according to neoplastic development in both tissues. The instability of 5-bp classical satellite DNA sequences from chromosome-1 was evidenced using chromosome-orientation FISH. In conclusion, we suggest that the progression to malignant transformation involves an increase in the instability of 5-bp classical satellite DNA sequences from chromosome-1.

  14. Fluorescence in situ hybridization and sequential catalysed reporter deposition (2C-FISH for the flow cytometric sorting of freshwater ultramicrobacteria

    Directory of Open Access Journals (Sweden)

    Stefan M Neuenschwander

    2015-03-01

    Full Text Available Flow cytometric sorting is a powerful tool to physically separate cells within mixed microbial communities. If combined with phylogenetic staining (fluorescence in situ hybridization, FISH it allows to specifically sort defined genotypic microbial populations from complex natural samples. However, the targeted enrichment of freshwater ultramicrobacteria, such as members of the LD12 clade of Alphaproteobacteria (SAR11-IIIb, is still challenging. Current FISH protocols, even in combination with signal amplification by catalysed reporter deposition (CARD, are not sufficiently sensitive for the distinction of these bacteria from background noise by flow cytometry, presumably due to their low ribosome content and small cell sizes. We, therefore, modified a CARD based flow sorting protocol with the aim of increasing its sensitivity to a level sufficient for ultramicrobacteria. This was achieved by a second signal amplification step mediated by horseradish peroxidase labelled antibodies targeted to the fluorophores that were previously deposited by CARD-FISH staining. The protocol was tested on samples from an oligo-mesotrophic lake. Ultramicrobacteria affiliated with LD12 Alphaproteobacteria could be successfully sorted to high purity by flow cytometry. The ratios of median fluorescence signal to background ranged around 20, and hybridization rates determined by flow cytometry were comparable to those obtained by fluorescence microscopy. Potential downstream applications of our modified cell staining approach range from the analysis of microdiversity within 16S rRNA-defined populations to that of functional properties, such as the taxon-specific incorporation rates of organic substrates.

  15. 5-bp Classical Satellite DNA Loci from Chromosome-1 Instability in Cervical Neoplasia Detected by DNA Breakage Detection/Fluorescence in Situ Hybridization (DBD-FISH)

    Science.gov (United States)

    Cortés-Gutiérrez, Elva I.; Ortíz-Hernández, Brenda L.; Dávila-Rodríguez, Martha I.; Cerda-Flores, Ricardo M; Fernández, José Luis; López-Fernández, Carmen; Gosálvez, Jaime

    2013-01-01

    We aimed to evaluate the association between the progressive stages of cervical neoplasia and DNA damage in 5-bp classical satellite DNA sequences from chromosome-1 in cervical epithelium and in peripheral blood lymphocytes using DNA breakage detection/fluorescence in situ hybridization (DBD-FISH). A hospital-based unmatched case-control study was conducted in 2011 with a sample of 30 women grouped according to disease stage and selected according to histological diagnosis; 10 with low-grade squamous intraepithelial lesions (LG-SIL), 10 with high-grade SIL (HG-SIL), and 10 with no cervical lesions, from the Unidad Medica de Alta Especialidad of The Mexican Social Security Institute, IMSS, Mexico. Specific chromosome damage levels in 5-bp classical satellite DNA sequences from chromosome-1 were evaluated in cervical epithelium and peripheral blood lymphocytes using the DBD-FISH technique. Whole-genome DNA hybridization was used as a reference for the level of damage. Results of Kruskal-Wallis test showed a significant increase according to neoplastic development in both tissues. The instability of 5-bp classical satellite DNA sequences from chromosome-1 was evidenced using chromosome-orientation FISH. In conclusion, we suggest that the progression to malignant transformation involves an increase in the instability of 5-bp classical satellite DNA sequences from chromosome-1. PMID:23429197

  16. 5-bp Classical Satellite DNA Loci from Chromosome-1 Instability in Cervical Neoplasia Detected by DNA Breakage Detection/Fluorescence in Situ Hybridization (DBD-FISH

    Directory of Open Access Journals (Sweden)

    Jaime Gosálvez

    2013-02-01

    Full Text Available We aimed to evaluate the association between the progressive stages of cervical neoplasia and DNA damage in 5-bp classical satellite DNA sequences from chromosome-1 in cervical epithelium and in peripheral blood lymphocytes using DNA breakage detection/fluorescence in situ hybridization (DBD-FISH. A hospital-based unmatched case-control study was conducted in 2011 with a sample of 30 women grouped according to disease stage and selected according to histological diagnosis; 10 with low-grade squamous intraepithelial lesions (LG-SIL, 10 with high-grade SIL (HG-SIL, and 10 with no cervical lesions, from the Unidad Medica de Alta Especialidad of The Mexican Social Security Institute, IMSS, Mexico. Specific chromosome damage levels in 5-bp classical satellite DNA sequences from chromosome-1 were evaluated in cervical epithelium and peripheral blood lymphocytes using the DBD-FISH technique. Whole-genome DNA hybridization was used as a reference for the level of damage. Results of Kruskal-Wallis test showed a significant increase according to neoplastic development in both tissues. The instability of 5-bp classical satellite DNA sequences from chromosome-1 was evidenced using chromosome-orientation FISH. In conclusion, we suggest that the progression to malignant transformation involves an increase in the instability of 5-bp classical satellite DNA sequences from chromosome-1.

  17. Asexual Reproduction Does Not Apparently Increase the Rate of Chromosomal Evolution: Karyotype Stability in Diploid and Triploid Clonal Hybrid Fish (Cobitis, Cypriniformes, Teleostei).

    Science.gov (United States)

    Majtánová, Zuzana; Choleva, Lukáš; Symonová, Radka; Ráb, Petr; Kotusz, Jan; Pekárik, Ladislav; Janko, Karel

    2016-01-01

    Interspecific hybridization, polyploidization and transitions from sexuality to asexuality considerably affect organismal genomes. Especially the last mentioned process has been assumed to play a significant role in the initiation of chromosomal rearrangements, causing increased rates of karyotype evolution. We used cytogenetic analysis and molecular dating of cladogenetic events to compare the rate of changes of chromosome morphology and karyotype in asexually and sexually reproducing counterparts in European spined loach fish (Cobitis). We studied metaphases of three sexually reproducing species and their diploid and polyploid hybrid clones of different age of origin. The material includes artificial F1 hybrid strains, representatives of lineage originated in Holocene epoch, and also individuals of an oldest known age to date (roughly 0.37 MYA). Thereafter we applied GISH technique as a marker to differentiate parental chromosomal sets in hybrids. Although the sexual species accumulated remarkable chromosomal rearrangements after their speciation, we observed no differences in chromosome numbers and/or morphology among karyotypes of asexual hybrids. These hybrids possess chromosome sets originating from respective parental species with no cytogenetically detectable recombinations, suggesting their integrity even in a long term. The switch to asexual reproduction thus did not provoke any significant acceleration of the rate of chromosomal evolution in Cobitis. Asexual animals described in other case studies reproduce ameiotically, while Cobitis hybrids described here produce eggs likely through modified meiosis. Therefore, our findings indicate that the effect of asexuality on the rate of chromosomal change may be context-dependent rather than universal and related to particular type of asexual reproduction.

  18. Chromosome-Specific DNA Repeats: Rapid Identification in Silico and Validation Using Fluorescence in Situ Hybridization

    Directory of Open Access Journals (Sweden)

    Heinz-Ulrich G. Weier

    2012-12-01

    Full Text Available Chromosome enumeration in interphase and metaphase cells using fluorescence in situ hybridization (FISH is an established procedure for the rapid and accurate cytogenetic analysis of cell nuclei and polar bodies, the unambiguous gender determination, as well as the definition of tumor-specific signatures. Present bottlenecks in the procedure are a limited number of commercial, non-isotopically labeled probes that can be combined in multiplex FISH assays and the relatively high price and effort to develop additional probes. We describe a streamlined approach for rapid probe definition, synthesis and validation, which is based on the analysis of publicly available DNA sequence information, also known as “database mining”. Examples of probe preparation for the human gonosomes and chromosome 16 as a selected autosome outline the probe selection strategy, define a timeline for expedited probe production and compare this novel selection strategy to more conventional probe cloning protocols.

  19. A water soluble and fast response fluorescent turn-on copper complex probe for H2S detection in zebra fish.

    Science.gov (United States)

    Palanisamy, Sathyadevi; Lee, Lu-Ying; Wang, Yu-Liang; Chen, Yu-Jen; Chen, Chiao-Yun; Wang, Yun-Ming

    2016-01-15

    According to the displacement method, herein we reported a water soluble copper complex [Cu(MaT-cyclen)2] as a fluorescent probe for the detection of H2S. For this, 1-((1-((10-methylanthracen-9-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)-1,4,7,10-tetraazacyclododecane (MaT-cyclen) was synthesized first. To improve its solubility in aqueous media, sodium acetate group was introduced into 8-hydroxy-2-quinoline successfully. MaT-cyclen was chelated with Cu(II) to form [Cu(MaT-cyclen)2] complex, which displayed high sensitivity and selectivity for H2S over the other possible competitive substances on the basis of forming CuS. Meanwhile, [Cu(MaT-cyclen)2] displayed rapid response (zebra fish.

  20. Quantifying filamentous microorganisms in activated sludge before, during, and after an incident of foaming by oligonucleotide probe hybridizations and antibody staining.

    Science.gov (United States)

    Oerther, D B; de los Reyes, F L; de los Reyes, M F; Raskin, L

    2001-10-01

    Quantitative oligonucleotide probe hybridizations, immunostaining, and a simple foaming potential test were used to follow an incident of seasonal filamentous foaming at the Urbana-Champaign Sanitary District, Northeast Wastewater Treatment Plant. A positive correlation was observed between an increase in foaming potential and the appearance of foam on the surfaces of aeration basins and secondary clarifiers. In addition, during the occurrence of foaming, the mass and activity of Gordonia spp. increased as measured by fluorescence in situ hybridization, antibody staining, and quantitative membrane hybridization of RNA extracts. An increase in Gordonia spp. rRNA levels from 0.25 to 1.4% of total rRNA was observed using quantitative membrane hybridizations, whereas during the same period, the fraction of mixed liquor volatile suspended solids attributed to Gordonia spp. increased from 4% to more than 32% of the total mixed liquor volatile suspended solids. These results indicate that both the activity and biomass level of Gordonia spp. in activated sludge increased relative to the activity aid the biomass level of the complete microbial community during a seasonal occurrence of filamentous foaming. Thus, Gordonia spp. may represent a numerically dominant but metabolically limited fraction of the total biomass, and the role of Gordonia spp. in filamentous foaming may be linked more tightly to the physical presence of filamentous microorganisms than to the metabolic activity of the cells.

  1. Magnetic Fe3O4/Ag Hybrid Nanoparticles as Surface-Enhanced Raman Scattering Substrate for Trace Analysis of Furazolidone in Fish Feeds

    Directory of Open Access Journals (Sweden)

    Wansong Yu

    2014-01-01

    Full Text Available Nanoparticles (NPs composed of ferromagnetic and noble metal materials show dual functions of magnetic activity and local surface plasmon response and have great potential as substrates for surface-enhanced Raman scattering (SERS in trace analysis. Easy-to-prepare superparamagnetic Fe3O4/Ag hybrid NPs were synthesized and optimized by adjusting the ratio of silver particles aggregated with APTMS-modified Fe3O4 NPs. The hybrid NPs were assembled under an external magnetic field before being used as substrate for SERS analysis. The SERS spectral features of furazolidone standard solution were clearly identified at concentrations as low as 40 ng mL−1, and furazolidone in fish feeds could be detected at 500 ng g−1. The results indicated that the Fe3O4/Ag hybrid NPs as SERS substrates had a great potential for detection of trace amount of furazolidone and other prohibited or restricted antibiotics in the animal and fish feeds.

  2. Detection of Listeria monocytogenes by direct colony hybridization on hydrophobic grid-membrane filters by using a chromogen-labeled DNA probe.

    Science.gov (United States)

    Peterkin, P I; Idziak, E S; Sharpe, A N

    1991-02-01

    A DNA probe specific for Listeria monocytogenes was isolated from a beta-hemolytic recombinant clone of an L. monocytogenes gene bank. It was labeled with horseradish peroxidase and used in a direct colony hybridization method on hydrophobic grid-membrane filters for the detection of the organism. Following color development of the chromogen, a commercial counter (HGMF Interpreter) was able to detect and count the organisms electronically. The method gave a positive reaction with 70 L. monocytogenes strains, while showing a negative reaction with 10 strains of other Listeria spp. and with 20 organisms of other genera.

  3. Observation of Lower Hybrid Current Drive Improved Confinement with a Graphite Probe at the Last Closed Flux Surface of the HT-7 Tokamak

    Institute of Scientific and Technical Information of China (English)

    徐国盛; 万宝年; 宋梅; 凌必利; 匡光力; 丁伯江

    2002-01-01

    High time resolution measurements of the electrostatic fluctuations, radial electric field Er and turbulence-induced electron flux Гe have been performed across the transition of lower hybrid current drive improved confinement with a graphite Langmuir probe array at the last closed flux surface of the HT-7 tokamak. The decrease of Гe is dominated by the suppression of fluctuation levels, which follows the change of Er. A reversal of the poloidal propagation direction of turbulence demonstrates that the poloidal propagation is dominated by Eт× Bφ drift. The enhancement of poloidal coherence accompanies the fluctuation suppression, which suggests the subtle variation of turbulence features.

  4. Split-probe hybrid femtosecond/picosecond rotational CARS for time-domain measurement of S-branch Raman linewidths within a single laser shot.

    Science.gov (United States)

    Patterson, Brian D; Gao, Yi; Seeger, Thomas; Kliewer, Christopher J

    2013-11-15

    We introduce a multiplex technique for the single-laser-shot determination of S-branch Raman linewidths with high accuracy and precision by implementing hybrid femtosecond (fs)/picosecond (ps) rotational coherent anti-Stokes Raman spectroscopy (CARS) with multiple spatially and temporally separated probe beams derived from a single laser pulse. The probe beams scatter from the rotational coherence driven by the fs pump and Stokes pulses at four different probe pulse delay times spanning 360 ps, thereby mapping collisional coherence dephasing in time for the populated rotational levels. The probe beams scatter at different folded BOXCARS angles, yielding spatially separated CARS signals which are collected simultaneously on the charge coupled device camera. The technique yields a single-shot standard deviation (1σ) of less than 3.5% in the determination of Raman linewidths and the average linewidth values obtained for N(2) are within 1% of those previously reported. The presented technique opens the possibility for correcting CARS spectra for time-varying collisional environments in operando.

  5. speciation through asexuality in fish: postzygotic reproductive isolation may be completed in spite of fertility of hybrids

    Directory of Open Access Journals (Sweden)

    Karel Janko

    2015-12-01

    Altogether, it appears that initiation of hybrid asexuality and the completion of speciation process through formation of postRIMs are interconnected phenomena. Both processes are linked to the genetic divergence of hybridizing taxa: initially, hybridization between little diverged species leads to recombinant and fertile hybrids allowing intensive gene flow. As the hybridizing taxa continue to diverge, clonally reproducing hybrid females and sterile males become dominant and the gene flow ceases. The speciation may therefore be completed through asexuality of hybrids The work was supported by grant no. 13-12580S provided by the Czech Science Foundation (www.gacr.cz. Further support was provided by the Academy of Sciences of the Czech Republic (www.cas.cz by the grant no. RVO 67985904

  6. Fluorescent in-situ hybridization--some of its applications in clinical cytogenetics.

    Science.gov (United States)

    Gole, L A; Bongso, A

    1997-11-01

    Fluorescent in-situ hybridization (FISH) is becoming more and more relevant as an important future tool in prenatal and pre-implantation genetic diagnosis and cancer cytogenetics. This review describes the FISH technique as applied to whole chromosome spreads and interphase cells and discusses its applications in clinical cytogenetics. Information is presented on the various types of probes and the subsequent hybridization and detection procedures. The potential use of this novel FISH technique in the diagnosis of numerical and structural chromosomal aberrations in routine karyotyping for prenatal diagnosis, tumour cytogenetics and pre-implantation genetic diagnosis is outlined.

  7. Determination of alkali-labile phosphoprotein phosphorus from fish plasma using the Tb3+-tiron complex as a fluorescence probe

    Institute of Scientific and Technical Information of China (English)

    LV Xue-fei; ZHAO Yi-bing; ZHOU Qun-fang; JIANG Gui-bin; SONG Mao-yong

    2007-01-01

    A sensitive method based on the fluorescence quenching effect of the Tb3+-Tiron complex is proposed for the determination of alkali-labile phosphoprotein phosphorus (ALP) released from fish plasma. The detection limit was 5.4 ng/ml (S/N=2), and the relative standard deviation of the quenching effect (6 replicates) was 4.6%. The results obtained by the proposed method were in good agreement with those obtained by the colorimetric assay. The advantages of the present method are its relatively simple detection procedure, the lack of toxic organic solvents, and high sensitivity.

  8. Effects of the acute exposition to glyphosate-based herbicide on oxidative stress parameters and antioxidant responses in a hybrid Amazon fish surubim (Pseudoplatystoma sp).

    Science.gov (United States)

    Sinhorin, Valéria Dornelles Gindri; Sinhorin, Adilson Paulo; Teixeira, Jhonnes Marcos dos Santos; Miléski, Kelly Márcia Lazarotto; Hansen, Paula Carine; Moreira, Paula Sueli Andrade; Kawashita, Nair Honda; Baviera, Amanda Martins; Loro, Vania Lúcia

    2014-08-01

    The aim of this study was to investigate the effects of acute glyphosate (active ingredient) exposure on the oxidative stress biomarkers and antioxidant defenses of a hybrid surubim (Pseudoplatystoma sp). The fish were exposed to different herbicide concentrations for 96 h. The thiobarbituric acid-reactive substances (TBARS), protein carbonyls and antioxidant responses were verified. The 15 mg a.pL(-1) of herbicide resulted in the death of 50% of the fish after 96 h. An increase in liver and muscle TBARS levels was observed when fish were exposed to the herbicide. The protein carbonyl content was also increased in the liver (4.5mg a.pL(-1) concentration) and brain (2.25 mg a.pL(-1) concentration). The antioxidant activities decreased in the liver and brain after exposure to herbicide. Levels of ascorbic acid in the liver (2.25 mg a.pL(-1) and 4.5 mg a.pL(-1) concentrations) and brain (2.25 mg a.pL(-1) concentration) were increased post-treatment. Levels of total thiols were increased in the liver and brain (2.25 mg L(-1) and 7.5mg a.pL(-1), respectively). Glyphosate exposure, at the tested concentrations affects surubim health by promoting changes that can affect their survival in natural environment. Some parameters as TBARS and protein carbonyl could be early biomarkers for Roundup exposure in this fish species.

  9. Comparative genomics in chicken and Pekin duck using FISH mapping and microarray analysis

    NARCIS (Netherlands)

    Skinner, M.; Robertson, L.B.; Tempest, H.G.; Langley, E.J.; Ioannou, D.; Fowler, K.E.; Crooijmans, R.P.M.A.

    2009-01-01

    Background: The availability of the complete chicken (Gallus gallus) genome sequence as well as a large number of chicken probes for fluorescent in-situ hybridization (FISH) and microarray resources facilitate comparative genomic studies between chicken and other bird species. In a previous study, w

  10. PMC- FISHs in Hybrids of Tetraploid Cultivated and Diploid Wild Gossypium Species%四倍体栽培棉与二倍体野生棉杂种PMC-FISH研究

    Institute of Scientific and Technical Information of China (English)

    凌键; 王坤波; 邹美娟; 宋国立; 王春英; 彭仁海; 刘方; 黎绍惠; 张香娣; 王玉红

    2009-01-01

    首次将PMC-FISH(花粉母细胞荧光原位杂交)技术应用于四倍体栽培棉与二倍体野生棉杂交所形成的三倍体杂种棉F1中,成功的鉴定了目标染色体中的单价体、双价体、多价体,还发现在非目标染色体上有星状和片段状的杂交信号.在以A组棉基因组DNA作为探针的PMC-FISH中,分别对三倍体杂种棉F1及其母本四倍体栽培棉的减数分裂中期I的染色体构型进行了鉴定.结果显示,四个三倍体杂种棉F1的减数分裂时期染色体的构型分别是:陆地棉(AD)1×雷蒙德氏棉(D5)为1IIIaad + 1IIaa + 4IIad + 7IIdd + 5Ⅰa + 7Ⅰd;海岛棉(AD)2×旱地棉(D4)为1Ⅴaaaad +1IIIadd + 2IIad + 8IIdd + 6Ⅰa + 5Ⅰd;陆地棉(AD)1×澳洲棉(C3)为2IIIadd (adc or acc) + 1IIaa + 9Ⅰa + 4IIcc (dc or dd) + 14Ⅰd (c);陆地棉(AD)1×南岱华棉(C1-n)为:1IIaa + 1IIad (ac)+ 10Ⅰa + 6IIcc (dc or dd) + 13Ⅰd (c).然而,两个四倍体棉染色体的构型都是13IIaa + 13IIdd.上述结果还显示, AD×D的两个杂种组合中,二价体多,单价体少,AD×C的两个杂种组合中,二价体少,单价体多;并且AD×D型杂种中A亚组染色体单体的数目比其在AD×C型杂种中的更少.这说明,与C染色体组相比较,D染色体组与四倍体棉种的亲缘关系更近.同时,我们的结果也证明PMC-FISH技术在分析三倍体杂种棉F1的亲缘关系中起着不可取代的作用.%PMC-FISH (pollen mother cell-fluorescence in situ hybridization) technology was firstly applied to identify probed chromosomes in univalent, bivalent and multivalent, as well as to distinguish star- or fragment-like hybridization signals from non-probed chromosomes in four triploid F1 hybrids produced by cultivated allotetraploid cotton crossed with different diploid cotton species in the genus Gossypium. Using genomic DNA from A genome species as probe, the meiotic chromosome configurations in metaphase Ⅰof four triploid F1 hybrids were identified as 1Ⅲaad + 1Ⅱaa + 4

  11. 荧光原位杂交三探针联合检测在儿童急性B淋巴细胞白血病诊断中的应用%Application of three-probe fluorescence in situ hybridization panel in the diagnosis of pediatric B cell acute lymphoblastic leukemia

    Institute of Scientific and Technical Information of China (English)

    范婧; 李承文; 赵佳炜; 贡金英; 郑迎春; 汝昆

    2014-01-01

    Objective To evaluate the three-probe fluorescence in situ hybridization (FISH) panel in the diagnosis of pediatric B cell acute lymphoblastic leukemia (B-ALL).Methods Three-probe (TEL-AML1,BCR-ABL and MLL) FISH and conventional cytogenetic analysis were performed in 207 children with B-ALL.Results In 207 B-ALL children,the three-probe FISH panel assay showed that 151 cases carried genetic aberrancies with a positive rate of 72.9%,including 44 cases with typical positive signal patterns and 148 cases with atypical signal patterns (among them 41 cases have multiprobe abberancy).The conventional cytogenetic analysis detected 53 cases chromosomal abnormality with a positive rate of 25.6%.Conclusion The detection rate of genetic abnormalities in newly-diagnosed pediatric B-ALL could be significantly improved by using three-probe FISH panel upon conventional cytogenetic analysis.%目的 探讨荧光原位杂交(FISH)三探针联合检测在儿童急性B淋巴细胞白血病(B-ALL)诊断中的应用价值.方法 对207例B-ALL患儿全部行三探针(TEL-AML1、BCR-ABL、MLL) FISH联合检测和常规染色体核型分析.结果 207例B-ALL患儿经FISH三探针联合检测,151例(72.9%)检出异常,包括典型阳性信号44例、非典型信号148例(其中41例为多探针同时检出异常).常规染色体核型分析技术检出染色体异常53例(25.6%).结论 对初诊的儿童B-ALL患者采用三探针联合FISH检测可以有效提高遗传学异常检出率.

  12. Detection of oligonucleotide hybridization on a single microparticle by time-resolved fluorometry: hybridization assays on polymer particles obtained by direct solid phase assembly of the oligonucleotide probes.

    Science.gov (United States)

    Hakala, H; Heinonen, P; Iitiä, A; Lönnberg, H

    1997-01-01

    Oligodeoxyribonucleotides were assembled by conventional phosphoramidite chemistry on uniformly sized (50 microns) porous glycidyl methacrylate/ethylene dimethacrylate (SINTEF) and compact polystyrene (Dynosphere) particles, the aminoalkyl side chains of which were further derivatized with DMTrO-acetyl groups. The linker was completely resistant toward ammonolytic deprotection of the base moieties. The quality of oligonucleotides was assessed by repeating the synthesis on the same particles derivatized with a cleavable ester linker. The ability of the oligonucleotide-coated particles to bind complementary sequences via hybridization was examined by following the attachment of oligonucleotides bearing a photoluminescent europium(III) chelate to the particles. The fluorescence emission was measured directly on a single particle. The effects of the following factors on the kinetics and efficiency of hybridization were studied: number of particles in a given volume of the assay solution, loading of oligonucleotide on the particle, concentration of the target oligonucleotide in solution, length of the hybridizing sequence, presence of noncomplementary sequences, and ionic strength. The fluorescence signal measured on a single particle after hybridization was observed to be proportional to the concentration of the target oligonucleotide in solution over a concentration range of 5 orders of magnitude.

  13. Diagnosis of canine visceral leishmaniasis with radiolabelled probes: comparison of the kDNA PCR-hybridization with three molecular methods in different clinical samples

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, Aline Leandra C.; Ferreira, Sidney A.; Carregal, Virginia M.; Andrade, Antero Silva R., E-mail: antero@cdtn.br [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil). Lab. de Radiobiologia; Melo, Maria N., E-mail: melo@icb.ufmg.br [Departamento de Parasitologia. Instituto de Ciencias Biologicas. Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil)

    2011-07-01

    Leishmania (Leishmania) chagasi is responsible for visceral leishmaniasis (VL) in Brazil and the dog is the main domestic reservoir. Disease control is based on the elimination of infected animals and the use of a sensitive and specific diagnostic test is necessary. The Brazilian VL control program emphasizes serologic surveys, mainly using the enzyme-linked immunosorbent assay (ELISA) and the immunofluorescence antibody test (IFAT), followed by the elimination of the seropositive dogs. However, these techniques present limitations in terms of sensitivity and specificity. The Polymerase Chain Reaction (PCR) associated to hybridization with DNA probes labeled with {sup 32}P has been recognized as a valuable tool for Leishmania identification. In this study, the sensitivity of kDNA PCR hybridization method was compared with three other molecular methods: Internal Transcribed Spacer 1 Nested PCR (ITS-1nPCR), Leishmania nested PCR (LnPCR) and Seminested kDNA PCR (kDNA snPCR). The comparison was performed in different clinical specimens: conjunctival swab, skin, blood and bone marrow. A group of thirty symptomatic dogs, positive in the parasitological and serological tests, was used. When. The techniques targeting kDNA mini-circles (kDNA snPCR and KDNA PCR-hybridization) showed the worst result for blood samples. The KDNA-PCR hybridization showed the best sensitivity for conjunctival swab. By comparing the samples on the basis of positivity obtained by the sum of all methods, the blood showed the worst outcome (71/120).The bone marrow showed the highest positivity (106/120), followed by conjunctival swab (100/120) and skin (89/120). Since the bone marrow samples are unsuitable for routine epidemiological surveys, the conjunctival swab was recommended because it allows high sensitivity, especially when associated with kDNA PCR hybridization method, and is a noninvasive sampling method. (author)

  14. A hydrophobic dye-encapsulated nano-hybrid as an efficient fluorescent probe for living cell imaging.

    Science.gov (United States)

    Chang, Shu; Wu, Xumeng; Li, Yongsheng; Niu, Dechao; Ma, Zhi; Zhao, Wenru; Gu, Jinlou; Dong, Wenjie; Ding, Feng; Zhu, Weihong; Shi, Jianlin

    2012-07-01

    Water-soluble hydrophobic-dye@nano-hybrids (DPN@NHs) with extraordinarily enhanced fluorescent performance were fabricated by encapsulating the hydrophobic dye molecules into the core of the hybrid nanospheres based on the self-assembly of amphiphilic block copolymers followed by shell cross-linking using 3-mercaptopropyltrimethoxy-silane. The DPN@NHs are 50 nm in size, are monodispersed in aqueous solution and have a quantum yield enhanced by 30 times.

  15. Detection of denitrification genes by in situ rolling circle amplification - fluorescence in situ hybridization (in situ RCA-FISH) to link metabolic potential with identity inside bacterial cells

    DEFF Research Database (Denmark)

    Hoshino, Tatsuhiko; Schramm, Andreas

    2010-01-01

    A target-primed in situ rolling circle amplification (in situ RCA) protocol was developed for detection of single-copy genes inside bacterial cells and optimized with Pseudomonas stutzeri, targeting nitrite and nitrous oxide reductase genes (nirS and nosZ). Two padlock probes were designed per gene......). Nevertheless, multiple genes (nirS and nosZ; nirS and the 16S rRNA gene) could be detected simultaneously in P. stutzeri. Environmental application of in situ RCA-FISH was demonstrated on activated sludge by the differential detection of two types of nirS-defined denitrifiers; one of them was identified...

  16. Rapid Genotyping of the Human Renin (REN Gene by the LightCycler® Instrument: Identification of Unexpected Nucleotide Substitutions within the Selected Hybridization Probe Area

    Directory of Open Access Journals (Sweden)

    Line Wee

    2010-01-01

    Full Text Available Preeclampsia is a serious disorder affecting nearly 3% of all in the Western world. It is associated with hypertension and proteinuria, and several lines of evidence suggest that the renin-angiotensin system (RAS may be involved in the development of hypertension at different stages of a preeclamptic pregnancy. In this study, we developed rapid genotyping assays on the LightCycler® instrument to allow the detection of genetic variants in the renin gene (REN that may predispose to preeclampsia. The method is based on real-time PCR and allele-specific hybridization probes, followed by fluorescent melting curve analysis to expose a change in melting temperature (Tm. Ninety-two mother-father-child triads (n=276 from preeclamptic pregnancies were genotyped for three haplotype-tagging single nucleotide polymorphisms (htSNPs in REN. All three htSNPs (rs5705, rs1464816 and rs3795575 were successfully genotyped. Furthermore, two unexpected nucleotide substitutions (rs11571084 and rs61757041 were identified within the selected hybridization probe area of rs1464816 and rs3795575 due to aberrant melting peaks. In conclusion, genotyping on the LightCycler® instrument proved to be rapid and highly reproducible. The ability to uncover additional nucleotide substitutions is particularly important in that it allows the identification of potentially etiological variants that might otherwise be overlooked by other genotyping methods.

  17. The Interaction of the Solar Wind with Solar Probe Plus - 3D Hybrid Simulation. Report 1; The Study for the Distance 4.5Rs

    Science.gov (United States)

    Lipatov, Alexander S.; Sittler, Edward C.; Hartle, Richard E.; Cooper, John F.

    2010-01-01

    Our report devotes a 3D numerical hybrid model of the interaction of the solar wind with the Solar Probe spacecraft. The Solar Probe Plus (SPP) model includes 3 main parts, namely, a non-conducting heat shield, a support system, and cylindrical section or spacecraft bus that contains the particle analysis devices and antenna. One observes an excitation of the low frequency Alfven and whistler type wave directed by the magnetic field with an amplitude of about (0.06-0.6) V/m. The compression waves and the jumps in an electric field with an amplitude of about (0.15-0.7) V/m were also observed. The wave amplitudes are comparable to or greater than previously estimated max wave amplitudes that SPP is expected to measure. The results of our hybrid simulation will be useful for understanding the plasma environment near the SPP spacecraft at the distance 4.5 Rs. Future simulation will take into account the charging of the spacecraft, the charge separation effects, an outgassing from heat shield, a photoionization and an electron impact ionization effects near the spacecraft.

  18. Subtyping of renal cortical neoplasms in fine needle aspiration biopsies using a decision tree based on genomic alterations detected by fluorescence in situ hybridization

    OpenAIRE

    Gowrishankar, Banumathy; Cahill, Lynnette; Arndt, Alexandra E; Al-Ahmadie, Hikmat; Lin, Oscar; Chadalavada, Kalyani; Chaganti, Seeta; Nanjangud, Gouri J; Murty, Vundavalli V; Chaganti, Raju S K; Reuter, Victor E.; Houldsworth, Jane

    2014-01-01

    Objectives To improve the overall accuracy of diagnosis in needle biopsies of renal masses, especially small renal masses (SRMs), using fluorescence in situ hybridization (FISH), and to develop a renal cortical neoplasm classification decision tree based on genomic alterations detected by FISH. Patients and Methods Ex vivo fine needle aspiration biopsies of 122 resected renal cortical neoplasms were subjected to FISH using a series of seven-probe sets to assess gain or loss of 10 chromosomes ...

  19. Positively charged polymer brush-functionalized filter paper for DNA sequence determination following Dot blot hybridization employing a pyrrolidinyl peptide nucleic acid probe.

    Science.gov (United States)

    Laopa, Praethong S; Vilaivan, Tirayut; Hoven, Voravee P

    2013-01-07

    As inspired by the Dot blot analysis, a well known technique in molecular biology and genetics for detecting biomolecules, a new paper-based platform for colorimetric detection of specific DNA sequences employing peptide nucleic acid (PNA) as a probe has been developed. In this particular study, a pyrrolidinyl PNA bearing a conformationally rigid d-prolyl-2-aminocyclopentanecarboxylic acid backbone (acpcPNA) was used as a probe. The filter paper was modified to be positively charged with grafted polymer brushes of quaternized poly(dimethylamino)ethyl methacrylate (QPDMAEMA) prepared by surface-initiated polymerization of 2-(dimethylamino)ethyl methacrylate from the filter paper via ARGET ATRP followed by quaternization with methyl iodide. Following the Dot blot format, a DNA target was first immobilized via electrostatic interactions between the positive charges of the QPDMAEMA brushes and negative charges of the phosphate backbone of DNA. Upon hybridization with the biotinylated pyrrolidinyl peptide nucleic acid (b-PNA) probe, the immobilized DNA can be detected by naked eye observation of the yellow product generated by the enzymatic reaction employing HRP-labeled streptavidin. It has been demonstrated that this newly developed assay was capable of discriminating between complementary and single base mismatch targets at a detection limit of at least 10 fmol. In addition, the QPDMAEMA-grafted filter paper exhibited a superior performance to the commercial membranes, namely Nylon 66 and nitrocellulose.

  20. Hybridization leads to sensory repertoire expansion in a gynogenetic fish, the Amazon molly (poecilia formosa): a test of the hybrid-sensory expansion hypothesis.

    Science.gov (United States)

    Sandkam, Benjamin A; Joy, Jeffrey B; Watson, Corey T; Gonzalez-Bendiksen, Pablo; Gabor, Caitlin R; Breden, Felix

    2013-01-01

    Expansions in sensory systems usually require processes such as gene duplication and divergence, and thus evolve slowly. We evaluate a novel mechanism leading to rapid sensory repertoire expansion: hybrid-sensory expansion (HSE). HSE occurs when two species with differently tuned sensory systems form a hybrid, bringing together alleles from each of the parental species. In one generation, a sensory repertoire is created that is the sum of the variance between parental species. The Amazon molly presents a unique opportunity to test the HSE hypothesis in a "frozen" hybrid. We compared opsin sequences of the Amazon molly, Poecilia formosa, to those of the parental species. Both parental species are homozygous at the RH2-1 locus and each of the four long wavelength sensitive loci, while P. formosa possess two different alleles at these loci; one matching each parental allele. Gene expression analysis showed P. formosa use the expanded opsin repertoire that was the result of HSE. Additionally, behavioral tests revealed P. formosa respond to colored stimuli in a manner similar or intermediate to the parental species P. mexicana and P. latipinna. Together these results strongly support the HSE hypothesis. Hybrid-sensory repertoire expansion is likely important in other hybrid species and in other sensory systems.

  1. Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries.

    Science.gov (United States)

    Murgha, Yusuf; Beliveau, Brian; Semrau, Kassandra; Schwartz, Donald; Wu, Chao-Ting; Gulari, Erdogan; Rouillard, Jean-Marie

    2015-06-01

    Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.

  2. Fluorescence in situ hybridization analysis of formalin fixed paraffin embedded tissues, including tissue microarrays.

    Science.gov (United States)

    Summersgill, Brenda M; Shipley, Janet M

    2010-01-01

    Formalin fixed paraffin embedded (FFPE) material is frequently the most convenient readily available source of diseased tissue, including tumors. Multiple cores of FFPE material are being used increasingly to construct tissue microarrays (TMAs) that enable simultaneous analyses of many archival samples. Fluorescence in situ hybridization (FISH) is an important approach to analyze FFPE material for specific genetic aberrations that may be associated with tumor types or subtypes, cellular morphology, and disease prognosis. Annealing, or hybridization of labeled nucleic acid sequences, or probes, to detect and locate one or more complementary nucleic acid sequences within fixed tissue sections allows the detection of structural (translocation/inversion) and numerical (deletion/gain) aberrations and their localization within tissues. The robust protocols described include probe preparation, hybridization, and detection and take 2-3 days to complete. A protocol is also described for the stripping of probes for repeat FISH in order to maximize the use of scarce tissue resources.

  3. Validation of microsatellite multiplexes for parentage analysis and species discrimination in two hybridizing species of coral reef fish (Plectropomus spp., Serranidae).

    Science.gov (United States)

    Harrison, Hugo B; Feldheim, Kevin A; Jones, Geoffrey P; Ma, Kayan; Mansour, Hicham; Perumal, Sadhasivam; Williamson, David H; Berumen, Michael L

    2014-06-01

    Microsatellites are often considered ideal markers to investigate ecological processes in animal populations. They are regularly used as genetic barcodes to identify species, individuals, and infer familial relationships. However, such applications are highly sensitive the number and diversity of microsatellite markers, which are also prone to error. Here, we propose a novel framework to assess the suitability of microsatellite datasets for parentage analysis and species discrimination in two closely related species of coral reef fish, Plectropomus leopardus and P. maculatus (Serranidae). Coral trout are important fisheries species throughout the Indo-Pacific region and have been shown to hybridize in parts of the Great Barrier Reef, Australia. We first describe the development of 25 microsatellite loci and their integration to three multiplex PCRs that co-amplify in both species. Using simulations, we demonstrate that the complete suite of markers provides appropriate power to discriminate between species, detect hybrid individuals, and resolve parent-offspring relationships in natural populations, with over 99.6% accuracy in parent-offspring assignments. The markers were also tested on seven additional species within the Plectropomus genus with polymorphism in 28-96% of loci. The multiplex PCRs developed here provide a reliable and cost-effective strategy to investigate evolutionary and ecological dynamics and will be broadly applicable in studies of wild populations and aquaculture brood stocks for these closely related fish species.

  4. Validation of microsatellite multiplexes for parentage analysis and species discrimination in two hybridizing species of coral reef fish (Plectropomus spp., Serranidae)

    KAUST Repository

    Harrison, H.B.

    2014-04-24

    Microsatellites are often considered ideal markers to investigate ecological processes in animal populations. They are regularly used as genetic barcodes to identify species, individuals, and infer familial relationships. However, such applications are highly sensitive the number and diversity of microsatellite markers, which are also prone to error. Here, we propose a novel framework to assess the suitability of microsatellite datasets for parentage analysis and species discrimination in two closely related species of coral reef fish, Plectropomus leopardus and P. maculatus (Serranidae). Coral trout are important fisheries species throughout the Indo-Pacific region and have been shown to hybridize in parts of the Great Barrier Reef, Australia. We first describe the development of 25 microsatellite loci and their integration to three multiplex PCRs that co-amplify in both species. Using simulations, we demonstrate that the complete suite of markers provides appropriate power to discriminate between species, detect hybrid individuals, and resolve parent-offspring relationships in natural populations, with over 99.6% accuracy in parent-offspring assignments. The markers were also tested on seven additional species within the Plectropomus genus with polymorphism in 28-96% of loci. The multiplex PCRs developed here provide a reliable and cost-effective strategy to investigate evolutionary and ecological dynamics and will be broadly applicable in studies of wild populations and aquaculture brood stocks for these closely related fish species. 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.

  5. Assessment of phylloplane yeasts on selected Mediterranean plants by FISH with group- and species-specific oligonucleotide probes.

    Science.gov (United States)

    Inácio, João; Ludwig, Wolfgang; Spencer-Martins, Isabel; Fonseca, Alvaro

    2010-01-01

    A previous culture-dependent survey of phylloplane yeasts from selected Mediterranean plants showed that a few species were present in high densities in almost all leaf samples, regardless of the plant type, location or sampling season. However, a few species appeared to be restricted to Cistus albidus leaves, namely Cryptococcus cistialbidi. Here, we describe a culture-independent FISH assay to detect and quantify whole yeast cells in leaf washings. After optimization, the technique was used to check the apparent association between C. albidus leaves and C. cistialbidi and the abundance and ubiquity of other basidiomycetous yeast species such as Erythrobasidium hasegawianum and Sporobolomyces spp. in leaf samples from this and other neighboring plants (Acer monspessulanum and Quercus faginea). No yeast cells were detected in Pistacia lentiscus leaf samples. We were also able to demonstrate that three phylloplane yeasts (C. cistialbidi, E. hasegawianum and Sporobolomyces spp.) appeared to be log-normally distributed among individual C. albidus leaves. The log-normal distribution has important implications for the quantification of phylloplane yeasts based on the washing and plating of bulk leaf samples, which will tend to overestimate the size of the respective populations and become an error source in yeast surveys or related biocontrol studies.

  6. Rapid and reliable diagnosis of murine myeloid leukemia (ML by FISH of peripheral blood smear using probe of PU. 1, a candidate ML tumor suppressor

    Directory of Open Access Journals (Sweden)

    Ban Nobuhiko

    2008-10-01

    Full Text Available Abstract Background Murine myeloid leukemia (ML provides a good animal model to study the mechanisms of radiation-induced leukemia in humans. This disease has been cytogenetically characterized by a partial deletion of chromosome 2 with G-banding. For the rapid diagnosis of ML, this study reports a FISH method using spleen cells and peripheral blood smears from ML mice exposed to gamma rays and neutrons with PU.1, a candidate ML tumor suppressor, as a probe. Results Among mice that were tentatively diagnosed with ML by clinical findings and blood smear examination, 85% carried spleen cells showing the loss of PU.1 although the frequency of these abnormal cells varied among individuals. Mice with very low frequencies of cells showing the loss of one copy of PU.1 (one-PU.1 frequency were later diagnosed pathologically not with ML but with blastic or eosinophilic leukemia. Some neutron-irradiated mice had cells showing translocated PU.1, although no pathological features differentiated these ML mice from ML mice expressing the simple loss of PU.1. The one-PU.1 frequency can be detected from spleen metaphase cells, spleen interphase cells, and blood smears. There was a good correlation between the one-PU.1 frequency in spleen metaphase cells and that in spleen interphase cells (r = 0.96 and between one-PU.1 frequency in spleen interphase cells and that in blood cells (r = 0.83. Conclusion The FISH method was capable of detecting aberration of copy number of the PU.1 gene on murine chromosome 2, and using a peripheral blood smear is more practical and less invasive than conventional pathological diagnosis or the cytogenetic examination of spleen cells.

  7. Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms

    NARCIS (Netherlands)

    Quevedo, Beatrice; Giertsen, Elin; Zijnge, Vincent; Luethi-Schaller, Helga; Guggenheim, Bernhard; Thurnheer, Thomas; Gmuer, Rudolf

    2011-01-01

    Background: The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotroph

  8. Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms

    NARCIS (Netherlands)

    Quevedo, Beatrice; Giertsen, Elin; Zijnge, Vincent; Luethi-Schaller, Helga; Guggenheim, Bernhard; Thurnheer, Thomas; Gmuer, Rudolf

    2011-01-01

    Background: The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotroph

  9. Hybridization chain reaction-based colorimetric aptasensor of adenosine 5'-triphosphate on unmodified gold nanoparticles and two label-free hairpin probes.

    Science.gov (United States)

    Gao, Zhuangqiang; Qiu, Zhenli; Lu, Minghua; Shu, Jian; Tang, Dianping

    2017-03-15

    This work designs a new label-free aptasensor for the colorimetric determination of small molecules (adenosine 5'-triphosphate, ATP) by using visible gold nanoparticles as the signal-generation tags, based on target-triggered hybridization chain reaction (HCR) between two hairpin DNA probes. The assay is carried out referring to the change in the color/absorbance by salt-induced aggregation of gold nanoparticles after the interaction with hairpins, gold nanoparticles and ATP. To construct such an assay system, two hairpin DNA probes with a short single-stranded DNA at the sticky end are utilized for interaction with gold nanoparticles. In the absence of target ATP, the hairpin DNA probes can prevent gold nanoparticles from the salt-induced aggregation through the interaction of the single-stranded DNA at the sticky end with gold nanoparticles. Upon target ATP introduction, the aptamer-based hairpin probe is opened to expose a new sticky end for the strand-displacement reaction with another complementary hairpin, thus resulting in the decreasing single-stranded DNA because of the consumption of hairpins. In this case, gold nanoparticles are uncovered owing to the formation of double-stranded DNA, which causes their aggregation upon addition of the salt, thereby leading to the change in the red-to-blue color. Under the optimal conditions, the HCR-based colorimetric assay presents good visible color or absorbance responses for the determination of target ATP at a concentration as low as 1.0nM. Importantly, the methodology can be further extended to quantitatively or qualitatively monitor other small molecules or biotoxins by changing the sequence of the corresponding aptamer.

  10. Implementation and verification of a four-probe motion error measurement system for a large-scale roll lathe used in hybrid manufacturing

    Science.gov (United States)

    Chen, Yuan-Liu; Niu, Zengyuan; Matsuura, Daiki; Lee, Jung Chul; Shimizu, Yuki; Gao, Wei; Oh, Jeong Seok; Park, Chun Hong

    2017-10-01

    In this paper, a four-probe measurement system is implemented and verified for the carriage slide motion error measurement of a large-scale roll lathe used in hybrid manufacturing where a laser machining probe and a diamond cutting tool are placed on two sides of a roll workpiece for manufacturing. The motion error of the carriage slide of the roll lathe is composed of two straightness motion error components and two parallelism motion error components in the vertical and horizontal planes. Four displacement measurement probes, which are mounted on the carriage slide with respect to four opposing sides of the roll workpiece, are employed for the measurement. Firstly, based on the reversal technique, the four probes are moved by the carriage slide to scan the roll workpiece before and after a 180-degree rotation of the roll workpiece. Taking into consideration the fact that the machining accuracy of the lathe is influenced by not only the carriage slide motion error but also the gravity deformation of the large-scale roll workpiece due to its heavy weight, the vertical motion error is thus characterized relating to the deformed axis of the roll workpiece. The horizontal straightness motion error can also be synchronously obtained based on the reversal technique. In addition, based on an error separation algorithm, the vertical and horizontal parallelism motion error components are identified by scanning the rotating roll workpiece at the start and the end positions of the carriage slide, respectively. The feasibility and reliability of the proposed motion error measurement system are demonstrated by the experimental results and the measurement uncertainty analysis.

  11. Application and analysis of LNG cold energy for hybrid fishing vessel%混合动力渔船LNG冷量的应用分析

    Institute of Scientific and Technical Information of China (English)

    魏鹏威; 张建一; 詹鋆

    2012-01-01

    液化天然气(LNC)是清洁能源,同时又蕴含大量冷量,我国已经提出渔船推广使用LNG作为动力燃料.目前,渔船制冷能耗占全船能耗30%-40%.为了确定混合动力渔船中LNG的应用优势,本文结合实际生产中的50m型围网渔船进行了计算分析.结果显示,在该型渔船上采用柴油/天然气混合动力,替油率80%时每年可节省燃料费用61万元.将LNG冷量用于冷海水系统,每天可制取0℃冷海水10.4t,减少冷海水制冷系统工作时长1.6h,部分冷量用于系统的过冷循环,可使系统COP提高5.2%.分析表明,利用混合动力渔船的LNG冷能可显著节能,降低营运成本.%Liquified Natural Gas (LNG) is a clean energy with a lot of cold energy.Its use as engine fuel for fishing vessel is promoted currently in China.The energy consumption of refrigeration system in fishing vessels has accounted for 30% to 40% in total energy consumption.In order to determine the advantage of LNG applications in hybrid fishing vessel,some calculation and analysis are conducted based on 50 m model purse seine vessel.The results show that 610 thousands yuan can be saved annually if 80% of diesel oil is replaced by LNG on this type of fishing vessels.Meanwhile,10.4 t cold seawater in 0℃ can be produced and 1.6 hour of the system's working time can be reduced each day by use of the LNG cold energy.The COP can be improved by 5.2% when the surplus LNG cold energy is used for subcooled cycle of the refrigeration system.Energy cost can be significantly reduced by use of LNG cold energy in the hybrid fishing vessels based on the analysis.

  12. A new route for local probing of inner interactions within a layered double hydroxide/benzene derivative hybrid material.

    Science.gov (United States)

    Fleutot, S; Dupin, J C; Baraille, I; Forano, C; Renaudin, G; Leroux, F; Gonbeau, D; Martinez, H

    2009-05-14

    This paper presents the preparation and characterization of hybrid hydrotalcite-type layered double hydroxides (Zn1-xAlx(OH)2HBSx.nH2O, with x=0.33) where HBS is the 4-phenol sulfonate, with a detailed analysis of the grafting process of this organic entity onto the host lattice. As a set of the usual techniques (XRD, TG-DT/MS, FTIR and 27Al MAS NMR) was used to characterize the hybrid materials, this work focuses on a joint study by X-ray photoelectron spectroscopy and some quantum-calculation modeling in order to highlight the nature of the interactions between the organic and the mineral sub-systems. For the as-prepared hybrid material, the main results lead to a quasi-vertical orientation of the organic molecules within the mineral sheets via H-bond stabilization. By heating the hybrid material up to 200 degrees C, the structure shrinks with the condensation of the organics; the different theoretical modeling done gives an energy-stable situation when a direct attachment of the HBS sulfonate group sets up with the mineral layers, in agreement with the recorded XPS experimental data.

  13. Probing the local environment of hybrid materials designed from ionic liquids and synthetic clay by Raman spectroscopy

    Science.gov (United States)

    Siqueira, Leonardo J. A.; Constantino, Vera R. L.; Camilo, Fernanda F.; Torresi, Roberto M.; Temperini, Marcia L. A.; Ribeiro, Mauro C. C.; Izumi, Celly M. S.

    2014-03-01

    Hybrid organic-inorganic material containing Laponite clay and ionic liquids forming cations have been prepared and characterized by FT-Raman spectroscopy, X-ray diffraction, and thermal analysis. The effect of varying the length of the alkyl side chain and conformations of cations has been investigated by using different ionic liquids based on piperidinium and imidazolium cations. The structure of the N,N-butyl-methyl-piperidinium cation and the assignment of its vibrational spectrum have been further elucidated by quantum chemistry calculations. The X-ray data indicate that the organic cations are intercalated parallel to the layers of the clay. Comparison of Raman spectra of pure ionic liquids with different anions and the resulting solid hybrid materials in which the organic cations have been intercalated into the clay characterizes the local environment experienced by the cations in the hybrid materials. The Raman spectra of hybrid materials suggest that the local environment of all confined cations, in spite of this diversity in properties, resembles the liquid state of ionic liquids with a relatively disordered structure.

  14. Detection of chromosome aberrations in interphase nuclei using fluorescence in situ hybridization technique.

    OpenAIRE

    1993-01-01

    We report here several experiences of interphase cytogenetics, using fluorescence in situ hybridization (FISH) technique, for the detection of chromosome aberrations. FISH, using alpha satellite specific probes of 18, X, Y chromosomes, was done in interphase nuclei from peripheral blood of patients with Edwards' syndrome, Klinefelter's syndrome and Turner's syndrome with healthy male and female controls, respectively. The distributions of fluorescent signals in 100 interphase nuclei were well...

  15. Fluorescent in situ hybridization for evaluation of Prader-Willi and Angelman syndromes

    Energy Technology Data Exchange (ETDEWEB)

    Wenger, S.L.; Cummins, J.H. [Univ. of Pittsburgh, PA (United States)

    1995-07-17

    We have found fluorescence in situ hybridization (FISH) results more reliavle than high resolution chromosome analysis for the diagnosis of Prader-Willi (PWS) or Angelman syndromes (AS). Specifically, we have found success in the detection of 15q11q13 deletions among 55 cases. Our study suggests that FISH analysis using PWS/AS probes can facilitate diagnostic evaluation of these cases for deletions. 2 refs., 1 tab.

  16. Laser capture microdissection of bacterial cells targeted by fluorescence in situ hybridization

    DEFF Research Database (Denmark)

    Schou, Kirstine Klitgaard; Mølbak, Lars; Jensen, Tim Kåre

    2005-01-01

    . By this method, a potentially pathogenic strain of the genus Brachyspira from formalin-fixed human colonic biopsies were visualized by fluorescence in situ hybridization (FISH) with a 16S rRNA-targeting oligonucleotide probe, followed by laser capture microdissection (LCM) of the targeted cells. Direct 16S r...

  17. Identification of bacterial invasion in necrotizing enterocolitis specimens using fluorescent in situ hybridization

    NARCIS (Netherlands)

    Heida, F H; Harmsen, H J M; Timmer, A; Kooi, E M W; Bos, A F; Hulscher, J B F

    2016-01-01

    OBJECTIVE: Investigation of bacterial invasion into the intestinal wall in necrotizing enterocolitis (NEC) specimens. STUDY DESIGN: We compared 43 surgical NEC specimens with 43 age-matched controls. We used fluorescent in situ hybridization (FISH), a universal bacterial probe together with species-

  18. Specific detection of neuronal cell bodies: in situ hybridization with a biotin-labelled neurofilament cDNA probe.

    NARCIS (Netherlands)

    P. Liesi; J-P. Julien (Jean-Pierre); P. Vilja; F.G. Grosveld (Frank); L. Rechardt

    1986-01-01

    textabstractWe have used a biotinylated, 300-nucleotide cDNA probe which encodes the 68,000 MW neurofilament protein to detect neurofilament-specific mRNA in situ. The neurofilament message specifically demonstrates the neuronal cell bodies, in contrast to the usual antibody staining which detects t

  19. Preparation of graphene quantum dots based core-satellite hybrid spheres and their use as the ratiometric fluorescence probe for visual determination of mercury(II) ions

    Energy Technology Data Exchange (ETDEWEB)

    Hua, Mengjuan [Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China); Wang, Chengquan [School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013 (China); Qian, Jing, E-mail: qianj@ujs.edu.cn [Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China); Wang, Kan; Yang, Zhenting; Liu, Qian; Mao, Hanping [Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China); Wang, Kun, E-mail: wangkun@ujs.edu.cn [Key Laboratory of Modern Agriculture Equipment and Technology, School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang 212013 (China)

    2015-08-12

    We herein proposed a simple and effective strategy for preparing graphene quantum dots (GQDs)-based core-satellite hybrid spheres and further explored the feasibility of using such spheres as the ratiometric fluorescence probe for the visual determination of Hg{sup 2+}. The red-emitting CdTe QDs were firstly entrapped in the silica nanosphere to reduce their toxicity and improve their photo and chemical stabilities, thus providing a built-in correction for environmental effects, while the GQDs possessing good biocompatibility and low toxicity were electrostatic self-assembly on the silica surface acting as reaction sites. Upon exposure to the increasing contents of Hg{sup 2+}, the blue fluorescence of GQDs can be gradually quenched presumably due to facilitating nonradiative electron/hole recombination annihilation. With the embedded CdTe QDs as the internal standard, the variations of the tested solution display continuous fluorescence color changes from blue to red, which can be easily observed by the naked eye without any sophisticated instrumentations and specially equipped laboratories. This sensor exhibits high sensitivity and selectivity toward Hg{sup 2+} in a broad linear range of 10 nM–22 μM with a low detection limit of 3.3 nM (S/N = 3), much lower than the allowable Hg{sup 2+} contents in drinking water set by U.S. Environmental Protection Agency. This prototype ratiometric probe is of good simplicity, low toxicity, excellent stabilities, and thus potentially attractive for Hg{sup 2+} quantification related biological systems. - Highlights: • A facile strategy for preparing GQDs based core-satellite hybrid spheres was reported. • Such spheres can be used as the ratiometric fluorescence probe for Hg{sup 2+} detection. • The Hg{sup 2+} content can be easily distinguished by the naked eye. • The sensor shows high sensitivity and selectivity toward Hg{sup 2+} detection. • The ratiometric probe is of good simplicity, low toxicity, and

  20. Photochemical properties in flag leaves of a super-high-yielding hybrid rice and a traditional hybrid rice (Oryza sativa L.) probed by chlorophyll a fluorescence transient.

    Science.gov (United States)

    Zhang, Meiping; Shan, YongJie; Kochian, Leon; Strasser, Reto J; Chen, GuoXiang

    2015-12-01

    Chlorophyll a fluorescence of flag leaves in a super-high-yielding hybrid rice (Oryza sativa L.) LYPJ, and a traditional hybrid rice SY63 cultivar with lower grain yield, which were grown in the field, were investigated from emergence through senescence of flag leaves. As the flag leaf matured, there was an increasing trend in photosynthetic parameters such as quantum efficiency of primary photochemistry ([Formula: see text] Po) and efficiency of electron transport from PS II to PS I (Ψ Eo). The overall photosynthetic performance index (PIABS) was significantly higher in the high-yielding LYPJ compared to SY63 during the entire reproductive stage of the plant, the same to MDA content. However, [Formula: see text] Po(=F V/F M), an indicator of the primary photochemistry of the flag leaf, did not display significant changes with leaf age and was not significantly different between the two cultivars, suggesting that PIABS is a more sensitive parameter than [Formula: see text] Po (=F V/F M) during leaf age for distinguishing between cultivars differing in yield.

  1. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries.

    OpenAIRE

    1996-01-01

    A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model sys...

  2. Behavior of meiotic chromosomes in Pinus wallichiana, P. strobus and their hybrid and nrDNA localization in pollen mother cells of the hybrid by using FISH.

    Science.gov (United States)

    Deng, Hui-Sheng; Zhang, Da-Ming; Fu, Cheng-Xin; Hong, De-Yuan

    2008-03-01

    The complete process of meiosis was investigated in Pinus wallichiana, P. strobus and their artificial hybrid (F1) using microsporocytes. It is revealed that there were slightly lower chiasma frequency, lower ring bivalent frequency, lower meiotic index and distinctly higher frequency of aberrance (chromosomal bridges, fragments or micronuclei) in pollen mother cells (PMCs) of the hybrid (F1) than those of the parental species, which showed a certain degree of differentiation between homologous chromosomes of the two parents. However, relatively higher frequency of ring bivalents and higher meiotic index in all the three entities indicate the great stability of genomes of parental species, and the differentiation of genomes between the two parents must have been slight. Total nineteen signal loci of 18S rDNA were observed in nine bivalents of the hybrid (F1), among which one bivalent bears two loci, while the others have only one. It is suggested that distinct differentiation at genetic level existed in homologous chromosomes of the two parental species, whereas only slight differentiation at karyotypic and genomic levels take place between the parent species.

  3. Behavior of Meiotic Chromosomes in Pinus wallichiana, P. strobus and Their Hybrid and nrDNA Localization in Pollen Mother Cells of the Hybrid by Using FISH

    Institute of Scientific and Technical Information of China (English)

    Hui-Sheng Deng; Da-Ming Zhang; Cheng-Xin Fu; De-Yuan Hong

    2008-01-01

    The complete process of meiosis was investigated in Pinus wallichiana, P. strobus and their artificial hybrid (F1) using microsporocytes. It is revealed that there were slightly lower chiasma frequency, lower ring bivalent frequency, lower meiotic Index and distinctly higher frequency of aberrance (chromosomal bridges, fragments or micronuclei) in pollen mother cells (PMCs) of the hybrid (F1) than those of the parental species, which showed a certain degree of differentiation between homologous chromosomes of the two parents. However, relatively higher frequency of ring bivalents and higher meiotic index in all the three entities indicate the great stability of genomes of parental species, and the differentiation of genomes between the two parents must have been slight. Total nineteen signal loci of 18S rDNA were observed in nine bivalents of the hybrid (F1), among which one bivalent bears two loci, while the others have only one. It is suggested that distinct differentiation at genetic level existed in homologous chromosomes of the two parental species, whereas only slight differentiation at karyotypic and genomic levels take place between the parent species.

  4. Group-specific small-subunit rRNA hybridization probes to characterize filamentous foaming in activated sludge systems.

    OpenAIRE

    de los Reyes, F L; Ritter, W; Raskin, L.

    1997-01-01

    Foaming in activated sludge systems is characterized by the formation of a thick, chocolate brown-colored scum that floats on the surface of aeration basins and secondary clarifiers. These viscous foams have been associated with the presence of filamentous mycolic acid-containing actinomycetes. To aid in evaluating the microbial representation in foam, we developed and characterized group-, genus-, and species-specific oligonucleotide probes targeting the small subunit rRNA of the Mycobacteri...

  5. 八探针荧光原位杂交联合R显带技术诊断儿童急性淋巴细胞白血病%Application of eight-probe fluorescence in situ hybridization and R-banding karyotype analysis for the diagnosis of acute lymphoblastic leukemia

    Institute of Scientific and Technical Information of China (English)

    赵鼎; 刘帅; 郭振欣; 李瑞

    2016-01-01

    Objective To assess the value of eight-probe fluorescence in situ hybridization (FISH)and R-banding karyotype analysis for the diagnosis of acute lymphoblastic leukemia (ALL).Methods With the eight-probe FISH (using probes for MYC,P16,E2A,CHIC2/D10Z1/D17Z1,TEL/AML1,MLL,BCR/ABL1,and IGH) and R-banding karyotype analysis,237 cases of ALL were analyzed.Results Cytogenetic changes were detected in 135 (56.96%) of all cases,which have involved MYC,P16,E2A,CHIC2/D10Z1 /D17Z1,TEL/AMLl,MLL,BCR/ABL1,and IGH polyploidies.R-banding karyotype analysis has only detected abnormalities in 48 of such cases,in addition with 14 abnormalities missed by the FISH probes,which have given a total positive rate of 26.16%.The detection rate of the two methods has differed significantly (P<0.05).Conclusion Compared with the R-banding karyotype analysis,the eightprobe FISH is more accurate and efficient.Diagnosis of cytogenetic abnormalities for children with ALL using the combined method can provide a basis for evaluation of prognosis as well as personalized therapy.%目的 探讨八探针荧光原位杂交(fluorescence in situ hybridization,FISH)联合R显带染色体核型分析应用于儿童急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)诊断的价值.方法 应用八探针FISH (MYC、P16、E2A、CHIC2 /D10Z1/D17Z1、TEL/AMLl、MLL、BCR/ABL1、IGH的DNA探针)和R显带染色体核型分析技术,对237例ALL患儿进行了联合检测.结果 八探针FISH技术在135例患儿中总共检出了细胞遗传学改变,总体阳性率为56.96%,包括MYC、P16、E2A、CHIC2/D10Z1/D17Z1、TEL/AML1、MLL、BCR/ABL1、IGH等8种细胞遗传学异常.而R显带核型分析对于相对应的细胞遗传学异常仅检出48例,另检出14例八探针FISH未能检出的异常,总阳性率为26.16%.两种方法检出阳性率的差异有统计学意义(P<0.05).结论 八探针FISH技术较R显带染色体核型分析具有准确、高效、省时、省力等优点,可与

  6. Limits on SUSY GUTs and Defects Formation in Hybrid Inflationary Models with Three-Year Wilkinson Microwave Anisotropy Probe (WMAP) Observations

    CERN Document Server

    Fraisse, A A

    2006-01-01

    We confront the predicted effects of hybrid inflationary models on the Cosmic Microwave Background (CMB) with three years of Wilkinson Microwave Anisotropy Probe (WMAP) observations. Using model selection, we compare the ability of a simple flat power-law LCDM model to describe the data to hybrid inflationary models involving global or local cosmic strings, or global textures. We find that it is statistically impossible to distinguish between these models: they all give a similar description of the data, the maximum ratio of the various evidences involved being never higher than e^{0.1 \\pm 0.5}. We then derive the maximum contribution that topological defects can make to the CMB, and place an upper bound on the possible value of cosmic strings tension of G\\mu \\leq 2.1 \\times 10^{-7} (68% CL). Finally, we give the corresponding constraints on the strings and D-strings masses, as well as limits on the D- and F-term coupling constants (\\kappa and \\lambda) and mass scales (M and \\sqrt{\\xi}).

  7. Identification of human DNA in forensic evidence by loop-mediated isothermal amplification combined with a colorimetric gold nanoparticle hybridization probe.

    Science.gov (United States)

    Watthanapanpituck, Khanistha; Kiatpathomchai, Wansika; Chu, Eric; Panvisavas, Nathinee

    2014-11-01

    A DNA test based on loop-mediated isothermal amplification (LAMP) and colorimetric gold nanoparticle (AuNP) hybridization probe to detect the presence of human DNA in forensic evidence was developed. The LAMP primer set targeted eight regions of the human cytochrome b, and its specificity was verified against the DNA of 11 animal species, which included animals closely related to humans, such as chimpanzee and orangutan. By using the AuNP probe, sequence-specific LAMP product could be detected and the test result could be visualized through the change in color. The limit of detection was demonstrated with reproducibility to be as low as 718 fg of genomic DNA, which is equivalent to approximately 100 plasmid DNA copies containing the cytochrome b DNA target region. A simple DNA extraction method for the commonly found forensic biological samples was also devised to streamline the test process. This LAMP-AuNP human DNA test showed to be a robust, specific, and cost-effective tool for the forensic identification of human specimens without requiring sophisticated laboratory instruments.

  8. PCR associated with hybridization with DNA radioactive probes for diagnosis of asymptomatic infection caused by Leishmania Chagasi; PCR associado a hibridizacao com sondas radioativas de DNA para a identificacao de infeccao subclinica causada por Leishmania Chagasi

    Energy Technology Data Exchange (ETDEWEB)

    Andrade, Antero Silva Ribeiro de [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Belo Horizonte, MG (Brazil); Moreno, Elizabeth Castro [Fundacao Nacional de Saude, Belo Horizonte, MG (Brazil). Coordenacao Regional de Minas Gerais; Gomes, Rosangela Fatima; Melo, Maria Norma de; Carneiro, Mariangela [Minas Gerais Univ., Belo Horizonte, MG (Brazil). Dept. de Parasitologia; Fernandes, Octavio [Fundacao Inst. Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, RJ (Brazil). Dept. de Medicina Tropical

    2002-07-01

    Detection systems for diagnosis of leishmaniasis based on PCR are very promising due to their sensitivity and specificity. Secondary detection by specific radioactive DNA probes, able to type the PCR amplified products, increase the specificity and raise about tem-fold the sensitivity of the assay. The aim of this work was evaluate PCR and hybridization as a tool to identify Leishmania (Leishmania) chagasi (the specie that cause the visceral leishmaniasis in Brazil) infection in asymptomatic persons living in a endemic area. Material and Methods: A group of 226 asymptomatic individuals, living in General Carneiro (MG), was selected. Blood samples were harvested and the DNA extracted from the mononucleate cells. PCR was performed using primers addressed to the kinetoplast DNA minicircles. This protocol gives a positive reaction for all Leishmania species. The amplified products were further hybridized with cloned L.chagasi minicircles labeled with {sup 32} P. Results: were identified 111 samples PCR positive, 2 of them hybridization negative and 133 samples hybridization positive, 24 of them PCR negative. The occurrence of samples with hybridization positive and PCR negative was expected since hybridization, with DNA probes labeled with {sup 32} P, increase the sensitivity of the assay. The samples that presented positive PCR and negative hybridization were probably due the presence of other Leishmania species, likely L. (V.) braziliensis (that produce tegumentary leishmaniasis in the region), since L. (L.) chagasi cloned minicircles were used as hybridization probe. We conclude that this procedure is a valuable tool to access subclinical L. (L.) chagasi infections in epidemiological studies. (author)

  9. Signal enhancement for gene detection based on a redox reaction of [Fe(CN)(6)](4-) mediated by ferrocene at the terminal of a peptide nucleic acid as a probe with hybridization-amenable conformational flexibility.

    Science.gov (United States)

    Aoki, Hiroshi; Tao, Hiroaki

    2008-07-01

    Electrochemically enhanced DNA detection was demonstrated by utilizing the couple of a synthesized ferrocene-terminated peptide nucleic acid (PNA) with a cysteine anchor and a sacrificial electron donor [Fe(CN)(6)](4-). DNA detection sensors were prepared by modifying a gold electrode surface with a mixed monolayer of the probe PNA and 11-hydroxy-1-undecanethiol (11-HUT), protecting [Fe(CN)(6)](4-) from any unexpected redox reaction. Before hybridization, the terminal ferrocene moiety of the probe was subject to a redox reaction due to the flexible probe structure and, in the presence of [Fe(CN)(6)](4-), the observed current was amplified based on regeneration of the ferrocene moiety. Hybridization decreased the redox current of the ferrocene. This occurred because hybridization rigidified the probe structure: the ferrocene moiety was then removed from the electrode surface, and the redox reaction of [Fe(CN)(6)](4-) was again prevented. The change in the anodic current before and after hybridization was enhanced 1.75-fold by using the electron donor [Fe(CN)(6)](4-). Sequence-specific detection of the complementary target DNA was also demonstrated.

  10. A scanning probe-based pick-and-place procedure for assembly of integrated quantum optical hybrid devices

    CERN Document Server

    Schell, Andreas W; Schröder, Tim; Wolters, Janik; Aichele, Thomas; Benson, Oliver

    2011-01-01

    Integrated quantum optical hybrid devices consist of fundamental constituents such as single emitters and tailored photonic nanostructures. A reliable fabrication method requires the controlled deposition of active nanoparticles on arbitrary nanostructures with highest precision. Here, we describe an easily adaptable technique that employs picking and placing of nanoparticles with an atomic force microscope combined with a confocal setup. In this way, both the topography and the optical response can be monitored simultaneously before and after the assembly. The technique can be applied to arbitrary particles. Here, we focus on nanodiamonds containing single nitrogen vacancy centers, which are particularly interesting for quantum optical experiments on the single photon and single emitter level.

  11. Apparent digestibility of Asian carp and common carp-derived fish meals in feeds for hybrid striped bass Morone saxatilis female x M. chrysops male and rainbow trout Oncorhynchus mykiss

    Science.gov (United States)

    Apparent digestibility coefficients (ADCs) of nutrients (crude protein, amino acids, crude lipid, fatty acids, and minerals) were determined for fish meals derived from menhaden, Asian carp (combination of silver and bighead carps), and common carp in feeds for hybrid striped bass and rainbow trout....

  12. Fully Automated Fluorescent in situ Hybridization (FISH) Staining and Digital Analysis of HER2 in Breast Cancer : A Validation Study

    NARCIS (Netherlands)

    van der Logt, Elise M. J.; Kuperus, Deborah A. J.; van Setten, Jan W.; van den Heuvel, Marius C.; Boers, James. E.; Schuuring, Ed; Kibbelaar, Robby E.

    2015-01-01

    HER2 assessment is routinely used to select patients with invasive breast cancer that might benefit from HER2-targeted therapy. The aim of this study was to validate a fully automated in situ hybridization (ISH) procedure that combines the automated Leica HER2 fluorescent ISH system for Bond with su

  13. Four dimensional hybrid ultrasound and optoacoustic imaging via passive element optical excitation in a hand-held probe

    Energy Technology Data Exchange (ETDEWEB)

    Fehm, Thomas Felix; Razansky, Daniel, E-mail: dr@tum.de [Institute for Biological and Medical Imaging (IBMI), Helmholtz Zentrum München, Neuherberg (Germany); Faculty of Medicine, Technische Universität München, Munich (Germany); Deán-Ben, Xosé Luís [Institute for Biological and Medical Imaging (IBMI), Helmholtz Zentrum München, Neuherberg (Germany)

    2014-10-27

    Ultrasonography and optoacoustic imaging share powerful advantages related to the natural aptitude for real-time image rendering with high resolution, the hand-held operation, and lack of ionizing radiation. The two methods also possess very different yet highly complementary advantages of the mechanical and optical contrast in living tissues. Nonetheless, efficient integration of these modalities remains challenging owing to the fundamental differences in the underlying physical contrast, optimal signal acquisition, and image reconstruction approaches. We report on a method for hybrid acquisition and reconstruction of three-dimensional pulse-echo ultrasound and optoacoustic images in real time based on passive ultrasound generation with an optical absorber, thus avoiding the hardware complexity of active ultrasound generation. In this way, complete hybrid datasets are generated with a single laser interrogation pulse, resulting in simultaneous rendering of ultrasound and optoacoustic images at an unprecedented rate of 10 volumetric frames per second. Performance is subsequently showcased in phantom experiments and in-vivo measurements from a healthy human volunteer, confirming general clinical applicability of the method.

  14. Enumeration of respiring Pseudomonas spp. in milk within 6 hours by fluorescence in situ hybridization following formazan reduction.

    Science.gov (United States)

    Kitaguchi, Akiko; Yamaguchi, Nobuyasu; Nasu, Masao

    2005-05-01

    Respiring Pseudomonas spp. in milk were quantified within 6 h by fluorescence in situ hybridization (FISH) with vital staining. FISH with an oligonucleotide probe based on 16S rRNA sequences was used for the specific detection of Pseudomonas spp. at the single cell level. 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was used to estimate bacterial respiratory activity. The numbers of respiring Pseudomonas cells as determined by FISH with CTC staining (CTC-FISH) were almost the same or higher than the numbers of CFU as determined by the conventional culture method.

  15. Focused upon hybridization: rapid and high sensitivity detection of DNA using isotachophoresis and peptide nucleic acid probes.

    Science.gov (United States)

    Ostromohov, Nadya; Schwartz, Ortal; Bercovici, Moran

    2015-09-15

    We present a novel assay for rapid and high sensitivity detection of nucleic acids without amplification. Utilizing the neutral backbone of peptide nucleic acids (PNA), our method is based on the design of low electrophoretic mobility PNA probes, which do not focus under isotachophoresis (ITP) unless bound to their target sequence. Thus, background noise associated with free probes is entirely eliminated, significantly improving the signal-to-noise ratio while maintaining a simple single-step assay requiring no amplification steps. We provide a detailed analytical model and experimentally demonstrate the ability to detect targets as short as 17 nucleotides (nt) and a limit of detection of 100 fM with a dynamic range of 5 decades. We also demonstrate that the assay can be successfully implemented for detection of DNA in human serum without loss of signal. The assay requires 15 min to complete, and it could potentially be used in applications where rapid and highly sensitive amplification-free detection of nucleic acids is desired.

  16. The application of fluorescence in situ hybridization in different ploidy levels cross-breeding of lily.

    Directory of Open Access Journals (Sweden)

    Qing Wang

    Full Text Available 21 crossing were conducted between Asiatic Lily with different ploidy levels, the results showed that the interploidy hybridization between diploid and tetraploid lilies was not as successful as intraploidy hybridization. Regardless of male sterility, triploid lilies could be used as female parents in the hybridization which the progenies were aneuploidy. 3x×4x crosses could be cultured more successfully than 3x×2x crosses. 45S rDNA was mapped on the chromosomes of seven Lilium species and their progenies using fluorescence in situ hybridization (FISH. FISH revealed six to sixteen 45S rDNA gene loci, and normally the sites were not in pairs. The asymmetry indexes of LA (Longiflorum hybrids × Asiatic hybrids hybrids was higher than Asiatic hybrids, the evolution degree was LA hybrids > Asiatic hybrids. 45S rDNA distributed variably on chromosome 1-10 and 12 among Asiatic hybrids. Chromosome 1 had invariable sites of 45S rDNA in all Asiatic hybrids, which could be considered as the characteristic of Asiatic hybrids. LA hybrid 'Freya' had two sites of 45S rDNA on one homologous chromosome 5, and also it could be found in the progenies. The karyotype and fluorescence in situ hybridization with 45S rDNA as probe were applied to identify the different genotypes of 9 hybrids. Typical chromosomes with parental signal sites could be observed in all the genotypes of hybrids, it was confirmed that all the hybrids were true.

  17. Comprehensive meiotic segregation analysis of a 4-breakpoint t(1;3;6) complex chromosome rearrangement using single sperm array comparative genomic hybridization and FISH.

    Science.gov (United States)

    Hornak, Miroslav; Vozdova, Miluse; Musilova, Petra; Prinosilova, Petra; Oracova, Eva; Linkova, Vlasta; Vesela, Katerina; Rubes, Jiri

    2014-10-01

    Complex chromosomal rearrangements (CCR) represent rare structural chromosome abnormalities frequently associated with infertility. In this study, meiotic segregation in spermatozoa of an infertile normospermic carrier of a 4-breakpoint t(1;3;6) CCR was analysed. A newly developed array comparative genomic hybridization protocol was used, and all chromosomes in 50 single sperm cells were simultaneously examined. Three-colour FISH was used to analyse chromosome segregation in 1557 other single sperm cells. It was also used to measure an interchromosomal effect; sperm chromatin structure assay was used to measure chromatin integrity. A high-frequency of unbalanced spermatozoa (84%) was observed, mostly arising from the 3:3 symmetrical segregation mode. Array comparative genomic hybridization was used to detect additional aneuploidies in two out of 50 spermatozoa (4%) in chromosomes not involved in the complex chromosome rearrangement. Significantly increased rates of diploidy and XY disomy were found in the CCR carrier compared with the control group (P < 0.001). Defective condensation of sperm chromatin was also found in 22.7% of spermatozoa by sperm chromatin structure assay. The results indicate that the infertility in the man with CCR and normal spermatozoa was caused by a production of chromosomally unbalanced, XY disomic and diploid spermatozoa and spermatozoa with defective chromatin condensation.

  18. A feasibility assessment of automated FISH image and signal analysis to assist cervical cancer detection

    Science.gov (United States)

    Wang, Xingwei; Li, Yuhua; Liu, Hong; Li, Shibo; Zhang, Roy R.; Zheng, Bin

    2012-02-01

    Fluorescence in situ hybridization (FISH) technology provides a promising molecular imaging tool to detect cervical cancer. Since manual FISH analysis is difficult, time-consuming, and inconsistent, the automated FISH image scanning systems have been developed. Due to limited focal depth of scanned microscopic image, a FISH-probed specimen needs to be scanned in multiple layers that generate huge image data. To improve diagnostic efficiency of using automated FISH image analysis, we developed a computer-aided detection (CAD) scheme. In this experiment, four pap-smear specimen slides were scanned by a dual-detector fluorescence image scanning system that acquired two spectrum images simultaneously, which represent images of interphase cells and FISH-probed chromosome X. During image scanning, once detecting a cell signal, system captured nine image slides by automatically adjusting optical focus. Based on the sharpness index and maximum intensity measurement, cells and FISH signals distributed in 3-D space were projected into a 2-D con-focal image. CAD scheme was applied to each con-focal image to detect analyzable interphase cells using an adaptive multiple-threshold algorithm and detect FISH-probed signals using a top-hat transform. The ratio of abnormal cells was calculated to detect positive cases. In four scanned specimen slides, CAD generated 1676 con-focal images that depicted analyzable cells. FISH-probed signals were independently detected by our CAD algorithm and an observer. The Kappa coefficients for agreement between CAD and observer ranged from 0.69 to 1.0 in detecting/counting FISH signal spots. The study demonstrated the feasibility of applying automated FISH image and signal analysis to assist cyto-geneticists in detecting cervical cancers.

  19. 荧光原位杂交技术在检测胎儿染色体亚显微结构异常中的应用%Application of the fluorescent in situ hybridization (FISH) on the prenatal diagnosis of the subtle chromosomal aberrations

    Institute of Scientific and Technical Information of China (English)

    尹爱华; 刘舒; 潘小英; 傅文婷; 王继成; 卢建; 杨洁霞

    2012-01-01

    Objective To study the diagnostic value of the fluorescent in situ hybridization on detection of subtle chromosomal aberrations in cultured amniotic fluid cells. Methods Amniocenteses were performed in a pregnant women of 18 gestational weeks, and metaphase FISH was followed because the results of amniotic fluid karyotype and ultrasound were disagree with each other. The metaphase chromosomes were hybridized in situ with the human centromere probes of chromosome 18,X,Y and site specific probe of chromosome 13,21. The treated slides were examined and taken photos under the carl zeiss fluoromicroscope. Result Subtle translocation of chromosome Y was detected by FISH. Conclusion Our result indicated that it is very helpful for the diagnosis of subtle chromosomal aberrations by combining FISH with traditional karyotype analysis.%目的 探讨荧光原位杂交(FISH)技术在诊断培养后的羊水细胞染色体亚显微结构异常中的应用价值.方法 对1例18孕周,经典细胞遗传学羊水染色体核型分析结果与B超检查结果有不符合的胎儿,应用FISH的18号、X、Y染色体着丝粒探针和13、21号染色体位点特异性探针,对培养后的羊水中期细胞标本进行检测.结果 共分析了22个独立细胞克隆的分裂象,发现胎儿染色体存在两种核型嵌合,结果记为:mos 45,X[20]/46,XY[2];FISH检测发现此胎儿核型存在Y染色体亚显微小片段易位.结论 FISH技术结合传统细胞遗传学核型分析,对于诊断染色体亚显微结构异常非常重要.

  20. Design of species-specific oligonucleotide probes for the detection of Bacteroides and Parabacteroides by fluorescence in situ hybridization and their application to the analysis of mouse caecal Bacteroides-Parabacteroides microbiota.

    Science.gov (United States)

    Momose, Y; Park, S H; Miyamoto, Y; Itoh, K

    2011-07-01

    To develop species-specific monitoring techniques for rapid detection of Bacteroides and Parabacteroides inhabiting the mouse intestine by fluorescence in situ hybridization. The specificity of oligonucleotide probes was evaluated by fluorescence whole-cell hybridization. Oligonucleotide probes specific for each species hybridized only with the target bacteria. Using these probes, caecal Bacteroides-Parabacteroides microbiota of conventional mice and specific pathogen-free (SPF) mice from three different breeders were analysed. It was shown that Bacteroides acidifaciens Group-1, Group-2 and Group-3 were dominant in conventional mice and SPF mice from two out of three breeders. Bacteroides vulgatus and Parabacteroides distasonis were detected in one of these two SPF breeding colonies in addition to Bact. acidifaciens. SPF mice of the remaining breeder harboured characteristic Bacteroides-Parabacteroides microbiota, consisting of Bacteroides sp. ASF519 and Bacteroides caccae. Bacteroides acidifaciens is the dominant and most typical species in the mouse Bacteroides-Parabacteroides microbiota. The Group-3 was identified as a novel group and revealed to occupy a major niche together with Bact. acidifaciens Group-1 and Group-2. The species-specific probe set developed in this study was the efficient tool for rapid detection of target bacterial groups inhabiting the mouse intestine. The results of this study provide important new information on the mouse Bacteroides-Parabacteroides community. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

  1. [Physical localization of ribosomal genes and chromosome DAPI banding by in situ hybridization in Medicago sativa L].

    Science.gov (United States)

    Chen, Jian-Min; Hong, Yi-Huan; Wang, You-Ping; Bowley, Steve; Wan, Jian-Min

    2006-02-01

    Physical localization of ribosomal genes in diploid and tetraploid alfalfa (Medicago. sativa) was studied using fluorescent in situ hybridization (FISH). It was revealed that 45s gene was only located at nucleolus organizer region (NOR) with a single locus in both diploid and tetraploid alfalfa, while 5s gene had 2-3 loci on chromosomes. Using the genomic DNA from M. coerulea and M. falcata as probe to hybridize with tetraploid species in alfalfa, both diploid species were successfully hybridized with tetraploid chromosomes, only showing the difference in hybridization signals in different numbers of chromosomes. Chromosomes of alfalfa exhibited DAPI banding by FISH analysis. In general, the patterns of distribution of DAPI banding were consistent with C-banding for M. coerulea. The possible origination of tetraploid alfalfa was discussed based on DAPI banding patterns and FISH analysis for ribosomal genes .

  2. [Fluorescent in situ hybridization in clinical cytogenetics].

    Science.gov (United States)

    Michalová, K

    1995-02-01

    During the last few years molecular biology methods expanded into human cytogenetics. This is in close connection with advanced technologies of DNA probes preparation and possibilities of their non-radioactive labelling by means of enzymatic incorporation of modified nucleotides as well as their hybridization with complementary DNA of chromosomes and interphase nuclei. FISH became a useful method in the clinical research. We present the short review of FISH methodologies and their applications for studies of translocation, deletions, amplifications and other chromosomal rearrangements in genetic and oncologic patients. The sensitivity of these methods is approximately 1-10 kb and therefore precise localization of genes on chromosomes is possible. Except gene mapping FISH can be used for comparative genomic mapping (CGH) and for identification of chromosomal changes of tumor cells.

  3. Development of a new deep-sea hybrid Raman insertion probe and its application to the geochemistry of hydrothermal vent and cold seep fluids

    Science.gov (United States)

    Zhang, Xin; Du, Zengfeng; Zheng, Ronger; Luan, Zhendong; Qi, Fujun; Cheng, Kai; Wang, Bing; Ye, Wangquan; Liu, Xiaorui; Lian, Chao; Chen, Changan; Guo, Jinjia; Li, Ying; Yan, Jun

    2017-05-01

    Hydrothermal vent fluids, cold seep fluids, their associated chemosynthetic communities, and the biogeochemical anaerobic oxidation of methane (AOM) play very important roles in the biogeochemical sulfur and carbon cycles in the ocean. Based on our previous success developing and deploying a deep-sea sediment pore water Raman probe, we developed a new deep-sea hybrid Raman insertion probe (RiP) designed to operate at temperatures up to 450 °C that can be inserted directly into high-temperature fluids emerging from hydrothermal vents. By routinely exchanging the various tips and optics of the probe, we can analyze the geochemistry of hydrothermal vent fluids, cold seep fluids, and sediment pore water profiles (0-60 cm) in situ. The instrument ensemble also includes a new deep-sea laser Raman spectrometer in a custom-designed, 6000-m titanium pressure housing, which is powered, controlled and deployed by the remotely operated vehicle (ROV) Faxian down to a maximum water depth of 4500 m. The new RiP was deployed at the Izena Hole hydrothermal area in the middle Okinawa Trough back-arc basin; the Papua-Australia-Canada-Manus (PACManus) hydrothermal vent area in the Manus back-arc basin, Papua New Guinea; and a cold seep field at Formosa Ridge in the northern South China Sea. The Raman peaks of CO2, CH4, H2S, HS-, SO42- and S8 were obtained in situ from high-temperature hydrothermal vents (290 °C), low-temperature cold seep fluids (2 °C) and the surrounding sediment pore water. Dissolved CH4 and S8 were identified for the first time in the fluids under the lush chemosynthetic communities of the cold seep. Several sediment pore water profiles collected near the cold seep were characterized by the loss of SO42- and increased CH4, H2S and HS- peaks. Additionally, the in situ pH range of the pore water profile was between 6.95 and 7.22. Thus, the RiP system provides a very useful tool for investigating the geochemistry of hydrothermal vent and cold seep fluids.

  4. Ordering in bio-inorganic hybrid nanomaterials probed by in situ scanning transmission X-ray microscopy

    Science.gov (United States)

    Lee, Jonathan R. I.; Bagge-Hansen, Michael; Tunuguntla, Ramya; Kim, Kyunghoon; Bangar, Mangesh; Willey, Trevor M.; Tran, Ich C.; Kilcoyne, David A.; Noy, Aleksandr; van Buuren, Tony

    2015-05-01

    Phospholipid bilayer coated Si nanowires are one-dimensional (1D) composites that provide versatile bio-nanoelectronic functionality via incorporation of a wide variety of biomolecules into the phospholipid matrix. The physiochemical behaviour of the phospholipid bilayer is strongly dependent on its structure and, as a consequence, substantial modelling and experimental efforts have been directed at the structural characterization of supported bilayers and unsupported phospholipid vesicles; nonetheless, the experimental studies conducted to date have exclusively involved volume-averaged techniques, which do not allow for the assignment of spatially resolved structural variations that could critically impact the performance of the 1D phospholipid-Si NW composites. In this manuscript, we use scanning transmission X-ray microscopy (STXM) to probe bond orientation and bilayer thickness as a function of position with a spatial resolution of ~30 nm for Δ9-cis 1,2-dioleoyl-sn-glycero-3-phosphocholine layers prepared Si NWs. When coupled with small angle X-ray scattering measurements, the STXM data reveal structural motifs of the Si NWs that give rise to multi-bilayer formation and enable assignment of the orientation of specific bonds known to affect the order and rigidity of phospholipid bilayers.Phospholipid bilayer coated Si nanowires are one-dimensional (1D) composites that provide versatile bio-nanoelectronic functionality via incorporation of a wide variety of biomolecules into the phospholipid matrix. The physiochemical behaviour of the phospholipid bilayer is strongly dependent on its structure and, as a consequence, substantial modelling and experimental efforts have been directed at the structural characterization of supported bilayers and unsupported phospholipid vesicles; nonetheless, the experimental studies conducted to date have exclusively involved volume-averaged techniques, which do not allow for the assignment of spatially resolved structural

  5. Development of a sequential multicolor-FISH approach with 13 chromosome-specific painting probes for the rapid identification of river buffalo (Bubalus bubalis, 2n = 50) chromosomes.

    Science.gov (United States)

    Pauciullo, Alfredo; Perucatti, Angela; Iannuzzi, Alessandra; Incarnato, Domenico; Genualdo, Viviana; Di Berardino, Dino; Iannuzzi, Leopoldo

    2014-08-01

    The development of new molecular techniques (array CGH, M-FISH, SKY-FISH, etc.) has led to great advancements in the entire field of molecular cytogenetics. However, the application of these methods is still very limited in farm animals. In the present study, we report, for the first time, the production of 13 river buffalo (Bubalus bubalis, 2n = 50) chromosome-specific painting probes, generated via chromosome microdissection and the DOP-PCR procedure. A sequential multicolor-FISH approach is also proposed on the same slide for the rapid identification of river buffalo chromosome/arms, namely, 1p-1q, 2p-2q, 3p-3q, 4p-4q, 5p-5q, 18, X, and Y, using both conventional and late-replicating banded chromosome preparations counterstained by DAPI. The provided 'bank' of chromosome-specific painting probes is useful for any further cytogenetic investigation not only for the buffalo breeds, but also for other species of the family Bovidae, such as cattle, sheep, and goats, for chromosome abnormality diagnosis, and, more generally, for evolutionary studies.

  6. PNA-based fluorescence in situ hybridization for identification of bacteria in clinical samples

    DEFF Research Database (Denmark)

    Fazli, Mustafa; Bjarnsholt, Thomas; Høiby, Niels;

    2014-01-01

    Fluorescence in situ hybridization with PNA probes (PNA-FISH) that target specific bacterial ribosomal RNA sequences is a powerful and rapid tool for identification of bacteria in clinical samples. PNA can diffuse readily through the bacterial cell wall due to its uncharged backbone, and PNA-FISH....... In all these cases, bacteria can be identified in biofilm aggregates, which may explain their recalcitrance to antibiotic treatment.......Fluorescence in situ hybridization with PNA probes (PNA-FISH) that target specific bacterial ribosomal RNA sequences is a powerful and rapid tool for identification of bacteria in clinical samples. PNA can diffuse readily through the bacterial cell wall due to its uncharged backbone, and PNA......-FISH can be performed with high specificity due to the extraordinary thermal stability of RNA-PNA hybrid complexes. We describe a PNA-FISH procedure and provide examples of the application of PNA-FISH for the identification of bacteria in chronic wounds, cystic fibrosis lungs, and soft tissue fillers...

  7. [Evaluation of peptide nucleic acid fluorescent in situ hybridization (PNA FISH) method in the identifi cation of Candida species isolated from blood cultures].

    Science.gov (United States)

    Aydemir, Gonca; Koç, Ayşe Nedret; Atalay, Mustafa Altay

    2016-04-01

    In recent years, increased number of patients who are hospitalized in intensive care units, received immunosuppressive therapy and treated with broad-spectrum antibiotics that can lead an increase in the incidence of systemic candidiasis. In these patients, the most common clinical manifestation is candidemia. Since the identification of Candida species isolated from blood cultures is time consuming by conventional (morphological and biochemical) methods, rapid, reliable and accurate methods are needed. For this purpose novel systems have been developed to identify the agent directly. The aim of this study was to evaluate the peptide nucleic acid fluorescent in situ hybridization (PNA FISH) method for the identification of Candida species by comparing with the conventional methods. A total of 50 patients who were admitted to Erciyes University Medical Faculty Hospital clinics and followed with prediagnosis of systemic fungal infections whose blood cultures were positive for the yeasts between July 2011 and July 2012 were included in the study. The conventional identification of Candida isolates was performed by considering macroscopic and microscopic morphology, germ tube test, cycloheximide sensitivity, urease activity and carbohydrate assimilation patterns with API 20C AUX (bioMerieux, France) test. PNA FISH method was conducted by the use of a commercial kit namely Yeast Traffic Light(®) PNA FISH (AdvanDx, USA). According to morphological and biochemical characteristics (conventional methods), 19 (38%) out of 50 Candida isolates were identified as C.albicans, 12 (24%) as C.glabrata, five (10%) as C.parapsilosis, five (10%) as C.kefyr, four (8%) as C.krusei, two (4%) as C.guilliermondii, two (4%) as C.tropicalis and one (2%) as C.lusitaniae. On the other hand, 24 (48%) of the isolates were identified as C.albicans/C.parapsilosis (with green fluorescence), 16 (32%) as C.glabrata/C.krusei (with red fluorescence) and one (%2) as C.tropicalis (with yellow

  8. Lower hybrid frequency range waves generated by ion polarization drift due to electromagnetic ion cyclotron waves: Analysis of an event observed by the Van Allen Probe B

    Science.gov (United States)

    Khazanov, G. V.; Boardsen, S.; Krivorutsky, E. N.; Engebretson, M. J.; Sibeck, D.; Chen, S.; Breneman, A.

    2017-01-01

    We analyze a wave event that occurred near noon between 07:03 and 07:08 UT on 23 February 2014 detected by the Van Allen Probes B spacecraft, where waves in the lower hybrid frequency range (LHFR) and electromagnetic ion cyclotron (EMIC) waves are observed to be highly correlated, with Pearson correlation coefficient of 0.86. We assume that the correlation is the result of LHFR wave generation by the ions' polarization drift in the electric field of the EMIC waves. To check this assumption the drift velocities of electrons and H+, He+, and O+ ions in the measured EMIC wave electric field were modeled. Then the LHFR wave linear instantaneous growth rates for plasma with these changing drift velocities and different plasma compositions were calculated. The time distribution of these growth rates, their frequency distribution, and the frequency dependence of the ratio of the LHFR wave power spectral density (PSD) parallel and perpendicular to the ambient magnetic field to the total PSD were found. These characteristics of the growth rates were compared with the corresponding characteristics of the observed LHFR activity. Reasonable agreement between these features and the strong correlation between EMIC and LHFR energy densities support the assumption that the LHFR wave generation can be caused by the ions' polarization drift in the electric field of an EMIC wave.

  9. Chromosome-specific DNA Repeat Probes

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. Hybridization Capture Using RAD Probes (hyRAD, a New Tool for Performing Genomic Analyses on Collection Specimens.

    Directory of Open Access Journals (Sweden)

    Tomasz Suchan

    Full Text Available In the recent years, many protocols aimed at reproducibly sequencing reduced-genome subsets in non-model organisms have been published. Among them, RAD-sequencing is one of the most widely used. It relies on digesting DNA with specific restriction enzymes and performing size selection on the resulting fragments. Despite its acknowledged utility, this method is of limited use with degraded DNA samples, such as those isolated from museum specimens, as these samples are less likely to harbor fragments long enough to comprise two restriction sites making possible ligation of the adapter sequences (in the case of double-digest RAD or performing size selection of the resulting fragments (in the case of single-digest RAD. Here, we address these limitations by presenting a novel method called hybridization RAD (hyRAD. In this approach, biotinylated RAD fragments, covering a random fraction of the genome, are used as baits for capturing homologous fragments from genomic shotgun sequencing libraries. This simple and cost-effective approach allows sequencing of orthologous loci even from highly degraded DNA samples, opening new avenues of research in the field of museum genomics. Not relying on the restriction site presence, it improves among-sample loci coverage. In a trial study, hyRAD allowed us to obtain a large set of orthologous loci from fresh and museum samples from a non-model butterfly species, with a high proportion of single nucleotide polymorphisms present in all eight analyzed specimens, including 58-year-old museum samples. The utility of the method was further validated using 49 museum and fresh samples of a Palearctic grasshopper species for which the spatial genetic structure was previously assessed using mtDNA amplicons. The application of the method is eventually discussed in a wider context. As it does not rely on the restriction site presence, it is therefore not sensitive to among-sample loci polymorphisms in the restriction sites

  11. Identification of the chromosome complement and the spontaneous 1R/1V translocations in allotetraploid Secale cereale × Dasypyrum villosum hybrids through cytogenetic approaches.

    Science.gov (United States)

    Książczyk, Tomasz; Apolinarska, Barbara; Kulak-Książczyk, Sylwia; Wiśniewska, Halina; Stojałowski, Stefan; Łapiński, Mirosław

    2011-08-01

    Genome modifications that occur at the initial interspecific hybridization event are dynamic and can be consolidated during the process of stabilization in successive generations of allopolyploids. This study identifies the number and chromosomal location of ribosomal DNA (rDNA) sites between Secale cereale, Dasypyrum villosum, and their allotetraploid S. cereale × D. villosum hybrids. For the first time, we show the advantages of FISH to reveal chromosome rearrangements in the tetraploid Secale × Dasypyrum hybrids. Based on the specific hybridization patterns of ribosomal 5S, 35S DNA and rye species-specific pSc200 DNA probes, a set of genotypes with numerous Secale/Dasypyrum translocations of 1R/1V chromosomes were identified in successive generations of allotetraploid S. cereale × D. villosum hybrids. In addition we analyse rye chromosome pairs using FISH with chromosome-specific DNA sequences on S. cereale × D. villosum hybrids.

  12. Prenatal detection of aneuploidies using fluorescence in situ hybridization: A preliminary experience in an Indian set up

    Indian Academy of Sciences (India)

    Vaidehi Jobanputra; Kalol Kumar Roy; Kiran Kucheria

    2002-03-01

    Fluorescence in situ hybridization (FISH) is a powerful molecular cytogenetic technique which allows rapid detection of aneuploidies on interphase cells and metaphase spreads. The aim of the present study was to evaluate FISH as a tool in prenatal diagnosis of aneuploidies in high risk pregnancies in an Indian set up. Prenatal diagnosis was carried out in 88 high-risk pregnancies using FISH and cytogenetic analysis. Multicolour commercially available FISH probes specific for chromosomes 13, 18, 21, X and Y were used. Interphase FISH was done on uncultured cells from chorionic villus and amniotic fluid samples. FISH on metaphase spreads was done from cord blood samples. The results of FISH were in conformity with the results of cytogenetic analysis in all the normal and aneuploid cases except in one case of structural chromosomal abnormality. The hybridization efficiency of the 5 probes used for the detection of aneuploidies was 100%. Using these probes FISH assay yielded discrete differences in the signal profiles between cytogenetically normal and abnormal samples. The overall mean interphase disomic signal patterns of chromosomes 13, 18, 21, X and Y were 94.45%; for interphase trisomic signal pattern of chromosome 21 was 97.3%. Interphase FISH is very useful in urgent high risk cases. The use of FISH overcomes the difficulties of conventional banding on metaphase spreads and reduces the time of reporting. However, with the limited number of probes used, the conventional cytogenetic analysis serves as a gold standard at present. It should be employed as an adjunctive tool to conventional cytogenetics.

  13. Integrated karyotyping of sorghum by in situ hybridization of landed BACs.

    Science.gov (United States)

    Kim, Jeong-Soon; Childs, Kevin L; Islam-Faridi, M Nurul; Menz, Monica A; Klein, Robert R; Klein, Patricia E; Price, H James; Mullet, John E; Stelly, David M

    2002-04-01

    The reliability of genome analysis and proficiency of genetic manipulation are increased by assignment of linkage groups to specific chromosomes, placement of centromeres, and orientation with respect to telomeres. We have endeavored to establish means to enable these steps in sorghum (Sorghum bicolor (L.) Moench), the genome of which contains ca. 780 Mbp spread across n = 10 chromosomes. Our approach relies on fluorescence in situ hybridization (FISH) and integrated structural genomic resources, including large-insert genomic clones in bacterial artificial chromosome (BAC) libraries. To develop robust FISH probes, we selected sorghum BACs by association with molecular markers that map near the ends of linkage groups, in regions inferred to be high in recombination. Overall, we selected 22 BACs that encompass the 10 linkage groups. As a prelude to development of a multiprobe FISH cocktail, we evaluated BAC-derived probes individually and in small groups. Biotin- and digoxygenin-labeled probes were made directly from the BAC clones and hybridized in situ to chromosomes without using suppressive unlabelled C0t-1 DNA. Based on FISH-signal strength and the relative degree of background signal, we judged 19 BAC-derived probes to be satisfactory. Based on their relative position, and collective association with all 10 linkage groups, we chose 17 of the 19 BACs to develop a 17-locus probe cocktail for dual-color detection. FISH of the cocktail allowed simultaneous identification of all 10 chromosomes. The results indicate that linkage and physical maps of sorghum allow facile selection of BAC clones according to position and FISH-signal quality. This capability will enable development of a high-quality molecular cytogenetic map and an integrated genomics system for sorghum, without need of chromosome flow sorting or microdissection. Moreover, transgeneric FISH experiments suggest that the sorghum system might be applicable to other Gramineae.

  14. Development and application of small-subunit rRNA probes for assessment of selected Thiobacillus species and members of the genus Acidiphilium.

    Science.gov (United States)

    Peccia, J; Marchand, E A; Silverstein, J; Hernandez, M

    2000-07-01

    Culture-dependent studies have implicated sulfur-oxidizing bacteria as the causative agents of acid mine drainage and concrete corrosion in sewers. Thiobacillus species are considered the major representatives of the acid-producing bacteria in these environments. Small-subunit rRNA genes from all of the Thiobacillus and Acidiphilium species catalogued by the Ribosomal Database Project were identified and used to design oligonucleotide DNA probes. Two oligonucleotide probes were synthesized to complement variable regions of 16S rRNA in the following acidophilic bacteria: Thiobacillus ferrooxidans and T. thiooxidans (probe Thio820) and members of the genus Acidiphilium (probe Acdp821). Using (32)P radiolabels, probe specificity was characterized by hybridization dissociation temperature (T(d)) with membrane-immobilized RNA extracted from a suite of 21 strains representing three groups of bacteria. Fluorochrome-conjugated probes were evaluated for use with fluorescent in situ hybridization (FISH) at the experimentally determined T(d)s. FISH was used to identify and enumerate bacteria in laboratory reactors and environmental samples. Probing of laboratory reactors inoculated with a mixed culture of acidophilic bacteria validated the ability of the oligonucleotide probes to track specific cell numbers with time. Additionally, probing of sediments from an active acid mine drainage site in Colorado demonstrated the ability to identify numbers of active bacteria in natural environments that contain high concentrations of metals, associated precipitates, and other mineral debris.

  15. Fluorescence in situ hybridization in uncultured amniocytes for detection of aneuploidy in 4210 prenatal cases

    Institute of Scientific and Technical Information of China (English)

    JIA Chan-wei; WANG Shu-yu; MA Yan-min; LAN Yong-lian; SI Yan-mei; YU Lan; ZHOU Li-ying

    2011-01-01

    Background Almost all reported fluorescence in situ hybridization (FISH) kits for prenatal diagnosis use probes from foreign (non-Chinese) countries. The aim of this study was to analyze the reliability of domestic (Chinese) FISH probe sets to detect aneuploidies of chromosomes 13, 18, 21, X, and Y related to prenatal diagnosis in 4210 cases.Methods Cytogenetic karyotyping was carded out as a standard prenatal diagnostic test, and amniotic fluid cell interphase FISH analysis was performed using two sets of probes (centromeric probes for chromosomes 18, X, and Y,and locus-specific probes for chromosomes 13 and 21) provided by GP Medical Technologies, Beijing, China. Then we compared the two results and found the performance characteristics for informative FISH results of aneuploidies by the domestic kit probes.Results In 4210 cases, 4126 cases generated karyotype results and 133 abnormal karyotypes (including 97 aneuploidies) were found. The FISH results of 98 cases (among them, 31 cases gave normal cytogenetic results) were uninformative. The rate of abnormal cases was 3.2% (133/4126). For the abnormal karyotypes, the rate of aneuploidy was 72.9% (97/133). Among the 97 aneuploidies, there were 58 cases of trisomy 21 (58/97, 59.8%), four cases of trisomy 13, 23 cases of trisomy 18, and 12 cases of sex chromosomal aneuploidies. The total concordance of the two methods was 97.9% (95/97; two cases were mosaics that had a low percentage of abnormal cells), and the concordance of trisomy 21, 13, and 18 by the two methods was 100%.Conclusions The two sets of the domestic FISH kit probes are reliable for prenatal diagnosis. The results demonstrate that FISH is a rapid and accurate clinical method for prenatal identification of chromosome aneuploidies.

  16. Effect of tannic acid-fish scale gelatin hydrolysate hybrid nanoparticles on intestinal barrier function and α-amylase activity.

    Science.gov (United States)

    Wu, Shao-Jung; Ho, Yi-Cheng; Jiang, Shun-Zhou; Mi, Fwu-Long

    2015-07-01

    Practical application of tannic acid is limited because it readily binds proteins to form insoluble aggregates. In this study, tannic acid was self-assembled with fish scale gelatin hydrolysates (FSGH) to form stable colloidal complex nanoparticles. The nanoparticles prepared from 4 mg ml(-1) tannic acid and 4 mg ml(-1) FSGH had a mean particle size of 260.8 ± 3.6 nm, and showed a positive zeta potential (20.4 ± 0.4 mV). The nanoparticles acted as effective nano-biochelators and free radical scavengers because they provided a large number of adsorption sites for interaction with heavy metal ions and scavenging free radicals. The maximum adsorption capacity for Cu(2+) ions was 123.5 mg g(-1) and EC50 of DPPH radical scavenging activity was 21.6 ± 1.2 μg ml(-1). Hydroxyl radical scavenging effects of the nanoparticles were investigated by electron spin resonance spectroscopy. The copper-chelating capacity and free radical scavenging activity of the nanoparticles were associated with their capacity to inhibit Cu(2+) ion-induced barrier impairment and hyperpermeability of Caco-2 intestinal epithelial tight junction (TJ). However, α-amylase inhibitory activity of the nanoparticles was significantly lower than that of free tannic acid. The results suggest that the nanoparticles can ameliorate Cu(2+) ion induced intestinal epithelial TJ dysfunction without severely inhibiting the activity of the digestive enzymes.

  17. A radiation hybrid map of the European sea bass (Dicentrarchus labrax) based on 1581 markers: Synteny analysis with model fish genomes.

    Science.gov (United States)

    Guyon, Richard; Senger, Fabrice; Rakotomanga, Michaelle; Sadequi, Naoual; Volckaert, Filip A M; Hitte, Christophe; Galibert, Francis

    2010-10-01

    The selective breeding of fish for aquaculture purposes requires the understanding of the genetic basis of traits such as growth, behaviour, resistance to pathogens and sex determinism. Access to well-developed genomic resources is a prerequisite to improve the knowledge of these traits. Having this aim in mind, a radiation hybrid (RH) panel of European sea bass (Dicentrarchus labrax) was constructed from splenocytes irradiated at 3000 rad, allowing the construction of a 1581 marker RH map. A total of 1440 gene markers providing ~4400 anchors with the genomes of three-spined stickleback, medaka, pufferfish and zebrafish, helped establish synteny relationships with these model species. The identification of Conserved Segments Ordered (CSO) between sea bass and model species allows the anticipation of the position of any sea bass gene from its location in model genomes. Synteny relationships between sea bass and gilthead seabream were addressed by mapping 37 orthologous markers. The sea bass genetic linkage map was integrated in the RH map through the mapping of 141 microsatellites. We are thus able to present the first complete gene map of sea bass. It will facilitate linkage studies and the identification of candidate genes and Quantitative Trait Loci (QTL). The RH map further positions sea bass as a genetic and evolutionary model of Perciformes and supports their ongoing aquaculture expansion.

  18. Use of DBD-FISH for the study of cervical cancer progression.

    Science.gov (United States)

    Cortés-Gutiérrez, Elva I; Fernández, Jose Luis; Dávila-Rodríguez, Martha I; López-Fernández, Carmen; Gosálvez, Jaime

    2015-01-01

    DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) is a procedure to detect and quantify DNA breaks in single cells, either in the whole genome or within specific DNA sequences. This methodology combines microgel embedding of cells and DNA unwinding procedures with the power of FISH coupled to digital image analysis. Cells trapped within an agarose matrix are lysed and immersed in an alkaline unwinding solution that produces single-stranded DNA motifs beginning at the ends of internal DNA strand breaks. After neutralization, the microgel is dehydrated and the cells are incubated with fluorescently labeled DNA probes. The amount of hybridized probe at a target sequence correlates with the amount of single-stranded DNA generated during the unwinding step, which is in turn proportional to the degree of local DNA breakage. A general view of the technique is provided, emphasizing its versatility for evaluating the association between DNA damage and progressive stages of cervical neoplasia.

  19. Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms

    Directory of Open Access Journals (Sweden)

    Thurnheer Thomas

    2011-01-01

    Full Text Available Abstract Background The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotrophia/Granulicatella. Results As lactobacilli are known for notorious resistance to probe penetration, probe-specific assay protocols were experimentally developed to provide maximum cell wall permeability, probe accessibility, hybridization stringency, and fluorescence intensity. The new assays were then applied in a pilot study to three biofilm samples harvested from variably demineralized bovine enamel discs that had been carried in situ for 10 days by different volunteers. Best probe penetration and fluorescent labeling of reference strains were obtained after combined lysozyme and achromopeptidase treatment followed by exposure to lipase. Hybridization stringency had to be established strictly for each probe. Thereafter all probes showed the expected specificity with reference strains and labeled the anticipated morphotypes in dental plaques. Applied to in situ grown biofilms the set of probes detected only Lactobacillus fermentum and bacteria of the Lactobacillus casei group. The most cariogenic biofilm contained two orders of magnitude higher L. fermentum cell numbers than the other biofilms. Abiotrophia/Granulicatella and streptococci from the mitis group were found in all samples at high levels, whereas Streptococcus mutans was detected in only one sample in very low numbers. Conclusions Application of these new group- and species-specific FISH probes to oral biofilm-forming lactic acid bacteria will allow a clearer understanding of the supragingival biome, its spatial architecture and of structure-function relationships implicated during plaque homeostasis and caries development. The probes should prove of value far beyond the field of

  20. Deletion of small nuclear ribonucleoprotein polypeptide N (SNRPN) in Prader-Willi syndrome detected by fluorescence in situ hybridization: Two sibs with the typical phenotype without a cytogenetic deletion in chromosome 15q

    Energy Technology Data Exchange (ETDEWEB)

    Ishikawa, Tatsuya; Kibe, Tetsuya; Wada, Yoshiro [Nagoya City Univ. Medical School (Japan)

    1996-04-24

    The small nuclear ribonucleoprotein polypeptide N (SNRPN) gene is regarded as one of the candidates for Prader-Willi syndrome (PWS). We describe two sibs with typical PWS presenting deletion of SNRPN detected by fluorescence in situ hybridization (FISH). Neither a cytogenetically detectable 15q12 deletion nor a deletion for the D15S11, D15S10, and GABRB3 cosmid probes were found in either patient. This implies a smaller deletion limited to the PWS critical region. FISH with a SNRPN probe will permit analysis of PWS patients with limited deletions not detectable with other probes. 22 refs., 1 fig.

  1. Fluorescence in situ hybridization rapidly detects three different pathogenic bacteria in urinary tract infection samples.

    Science.gov (United States)

    Wu, Qing; Li, Yan; Wang, Ming; Pan, Xiao P; Tang, Yong F

    2010-11-01

    The detection of pathogenic bacteria in urine is an important criterion for diagnosing urinary tract infections (UTIs). By using fluorescence in situ hybridization (FISH) with rRNA-targeted, fluorescently labeled oligonucleotide probes, bacterial pathogens present in urine samples were identified within 3-4 h. In this study, three probes that are specific for Escherichia coli, Enterococcus faecalis and Staphylococcus aureus were designed based on the conserved 16S RNA sequences, whereas probe Eub338 broadly recognizes all bacteria. We collected a total of 1000 urine samples, and 325 of these samples tested positive for a UTI via traditional culturing techniques; additionally, all 325 of these samples tested positive with the Eub338 probe in FISH analysis. FISH analyses with species-specific probes were performed in parallel to the test the ability to differentiate among several pathogenic bacteria. The samples for these experiments included 76 E. coli infected samples, 32 E. faecalis infected samples and 9 S. aureus infected samples. Compared to conventional methods of bacterial identification, the FISH method produced positive results for >90% of the samples tested. FISH has the potential to become an extremely useful diagnostic tool for UTIs because it has a quick turnaround time and high accuracy.

  2. Cryptic Translocation Identification in Human and Mouse using Several Telomeric Multiplex FISH (TM-FISH) Strategies

    Energy Technology Data Exchange (ETDEWEB)

    Henegariu, O; Artan, S; Greally, J M; Chen, X-N; Korenberg, J R; Vance, G H; Stubbs, L; Bray-Ward, P; Ward, D C

    2003-08-19

    Experimental data published in recent years showed that up to 10% of all cases with mild to severe idiopathic mental retardation may result from small rearrangements of the subtelomeric regions of human chromosomes. To detect such cryptic translocations, we developed a ''telomeric'' multiplex FISH assay, using a set of previously published and commercially available subtelomeric probes. This set of probes includes 41 cosmid/PAC/P1 clones located from less than 100kb to about 1 Mb from the end of the chromosomes. Similarly, a published mouse probe set, comprised of BACs hybridizing to the closest known marker toward the centromere and telomere of each mouse chromosome, was used to develop a mouse-specific ''telomeric'' M-FISH. Three different combinatorial labeling strategies were used to simultaneously detect all human sub-telomeric regions on one slide. The simplest approach uses only three fluors, and can be performed in laboratories lacking sophisticated imaging equipment or personnel highly trained in cytogenetics. A standard fluorescence microscope equipped with only three filters is sufficient. Fluor-dUTPs and labeled probes can be custom-made, thus dramatically reducing costs. Images can be prepared using generic imaging software (Adobe Photoshop), and analysis performed by simple visual inspection.

  3. Exploring the origin of the D genome of oat by fluorescence in situ hybridization.

    Science.gov (United States)

    Luo, Xiaomei; Zhang, Haiqin; Kang, Houyang; Fan, Xing; Wang, Yi; Sha, Lina; Zhou, Yonghong

    2014-09-01

    Further understanding of the origin of cultivated oat would accelerate its genetic improvement. In particular, it would be useful to clarify which diploid progenitor contributed the D genome of this allohexaploid species. In this study, we demonstrate that the landmarks produced by fluorescence in situ hybridization (FISH) of species of Avena using probes derived from Avena sativa can be used to explore the origin of the D genome. Selected sets of probes were hybridized in several sequential experiments performed on exactly the same chromosome spreads, with multiple probes of cytological preparations. Probes pITS and A3-19 showed there might be a similar distribution of pITS between the Ac and D genomes. These results indicated that the Ac genome is closely related to the D genome, and that Avena canariensis (AcAc) could be the D-genome donor of cultivated oat.

  4. Application of inter-fluorecence in situ hybridization of chromosome 13/21α satellite probe in amniotic cells for prenatal diagnosis trisomy 21 syndrome%染色体13/21α卫星探针用于产前诊断21三体综合征

    Institute of Scientific and Technical Information of China (English)

    李文典; 吴玉萍; 叶兹礼; 周玉球; 肖鸽飞; 朱兰芳; 刘莉

    2001-01-01

    目的探讨应用染色体13/21α卫星探针荧光原位杂交(FISH)技术行产前诊断21三体综合征的价值。方法选择10例经产前细胞遗传学检查证实为孕正常胎儿孕妇的羊水细胞(对照组)、3例证实为孕21三体胎儿孕妇的羊水细胞(观察组),用13/21α卫星探针对未经培养的羊水细胞间期核进行FISH杂交。结果两组总杂交率分别为36.7%和38.6%,差异无显著性(P>0.05)。对照组和观察组含4个杂交信号的核平均百分比分别为36.5%和3.9%,含5个杂交信号的核平均百分比分别为4.0%和36.1%,差异有极显著性(P<0.01),含5个信号的核百分比<36.1%可作为21三体综合征的诊断标准。结论 13/21α卫星探针间期FISH 用于未培养的羊水细胞可以快速、准确地在产前诊断21三体综合征。%Objective To investigate the prenatal diagnosis of trisomy 21 syndrome using chromosome 13/21α satellite probe fluorescence in situ hybridization (FISH) on uncultured interphase cells from amniotic fluid. Methods The interphase amniocytes of 10 fetuses who were detected normal and 3 fetus who were detected trisomy by prenatal cytogenetic diagnosis were selected. We did FISH which used chromosome 13/21α satellite probe directly on the uncultured amniocytes of these 13 samples. Results The total rate of the hybridization was 36.7% and 38.6% in control group and observation group respectively, showed no significantly difference. There were four signals in the nucleus, two groups were 36.5% and 3.9% respectively,there were five signals in the nucleus, two groups were 4.0%and 36.1% respectively. The control group and observation group showed significantly difference by the statistical χ2 values (P<0.01).Trisomy 21 syndrome was diagnosed when nucleus of five signals accounted for more than 36.1%. Conclusion FISH with Chromosome13/21α satellite probe is a valuable method for rapid prenatal diagnosis of trisomy 21

  5. Tailoring bifunctional hybrid organic–inorganic nanoadsorbents by the choice of functional layer composition probed by adsorption of Cu2+ ions

    Science.gov (United States)

    Tomina, Veronika V; Melnyk, Inna V; Zub, Yuriy L; Kareiva, Aivaras; Vaclavikova, Miroslava; Kessler, Vadim G

    2017-01-01

    Spherical silica particles with bifunctional (≡Si(CH2)3NH2/≡SiCH3, ≡Si(CH2)3NH2/≡Si(CH2)2(CF2)5CF3) surface layers were produced by a one-step approach using a modified Stöber method in three-component alkoxysilane systems, resulting in greatly increased contents of functional components. The content of functional groups and thermal stability of the surface layers were analyzed by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, and 13C and 29Si solid-state NMR spectroscopy revealing their composition and organization. The fine chemical structure of the surface in the produced hybrid adsorbent particles and the ligand distribution were further investigated by electron paramagnetic resonance (EPR) and electron spectroscopy of diffuse reflectance (ESDR) spectroscopy using Cu2+ ion coordination as a probe. The composition and structure of the emerging surface complexes were determined and used to provide an insight into the molecular structure of the surfaces. It was demonstrated that the introduction of short hydrophobic (methyl) groups improves the kinetic characteristics of the samples during the sorption of copper(II) ions and promotes fixation of aminopropyl groups on the surface of silica microspheres. The introduction of long hydrophobic (perfluoroctyl) groups changes the nature of the surface, where they are arranged in alternately hydrophobic/hydrophilic patches. This makes the aminopropyl groups huddled and less active in the sorption of metal cations. The size and aggregation/morphology of obtained particles was optimized controlling the synthesis conditions, such as concentrations of reactants, basicity of the medium, and the process temperature. PMID:28243572

  6. Tailoring bifunctional hybrid organic-inorganic nanoadsorbents by the choice of functional layer composition probed by adsorption of Cu(2+) ions.

    Science.gov (United States)

    Tomina, Veronika V; Melnyk, Inna V; Zub, Yuriy L; Kareiva, Aivaras; Vaclavikova, Miroslava; Seisenbaeva, Gulaim A; Kessler, Vadim G

    2017-01-01

    Spherical silica particles with bifunctional (≡Si(CH2)3NH2/≡SiCH3, ≡Si(CH2)3NH2/≡Si(CH2)2(CF2)5CF3) surface layers were produced by a one-step approach using a modified Stöber method in three-component alkoxysilane systems, resulting in greatly increased contents of functional components. The content of functional groups and thermal stability of the surface layers were analyzed by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, and (13)C and (29)Si solid-state NMR spectroscopy revealing their composition and organization. The fine chemical structure of the surface in the produced hybrid adsorbent particles and the ligand distribution were further investigated by electron paramagnetic resonance (EPR) and electron spectroscopy of diffuse reflectance (ESDR) spectroscopy using Cu(2+) ion coordination as a probe. The composition and structure of the emerging surface complexes were determined and used to provide an insight into the molecular structure of the surfaces. It was demonstrated that the introduction of short hydrophobic (methyl) groups improves the kinetic characteristics of the samples during the sorption of copper(II) ions and promotes fixation of aminopropyl groups on the surface of silica microspheres. The introduction of long hydrophobic (perfluoroctyl) groups changes the nature of the surface, where they are arranged in alternately hydrophobic/hydrophilic patches. This makes the aminopropyl groups huddled and less active in the sorption of metal cations. The size and aggregation/morphology of obtained particles was optimized controlling the synthesis conditions, such as concentrations of reactants, basicity of the medium, and the process temperature.

  7. Tailoring bifunctional hybrid organic–inorganic nanoadsorbents by the choice of functional layer composition probed by adsorption of Cu2+ ions

    Directory of Open Access Journals (Sweden)

    Veronika V. Tomina

    2017-02-01

    Full Text Available Spherical silica particles with bifunctional (≡Si(CH23NH2/≡SiCH3, ≡Si(CH23NH2/≡Si(CH22(CF25CF3 surface layers were produced by a one-step approach using a modified Stöber method in three-component alkoxysilane systems, resulting in greatly increased contents of functional components. The content of functional groups and thermal stability of the surface layers were analyzed by diffuse reflectance infrared Fourier transform (DRIFT spectroscopy, and 13C and 29Si solid-state NMR spectroscopy revealing their composition and organization. The fine chemical structure of the surface in the produced hybrid adsorbent particles and the ligand distribution were further investigated by electron paramagnetic resonance (EPR and electron spectroscopy of diffuse reflectance (ESDR spectroscopy using Cu2+ ion coordination as a probe. The composition and structure of the emerging surface complexes were determined and used to provide an insight into the molecular structure of the surfaces. It was demonstrated that the introduction of short hydrophobic (methyl groups improves the kinetic characteristics of the samples during the sorption of copper(II ions and promotes fixation of aminopropyl groups on the surface of silica microspheres. The introduction of long hydrophobic (perfluoroctyl groups changes the nature of the surface, where they are arranged in alternately hydrophobic/hydrophilic patches. This makes the aminopropyl groups huddled and less active in the sorption of metal cations. The size and aggregation/morphology of obtained particles was optimized controlling the synthesis conditions, such as concentrations of reactants, basicity of the medium, and the process temperature.

  8. An Improved Protocol for Quantification of Freshwater Actinobacteria by Fluorescence In Situ Hybridization

    Science.gov (United States)

    Sekar, Raju; Pernthaler, Annelie; Pernthaler, Jakob; Warnecke, Falk; Posch, Thomas; Amann, Rudolf

    2003-01-01

    We tested a previously described protocol for fluorescence in situ hybridization of marine bacterioplankton with horseradish peroxidase-labeled rRNA-targeted oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) in plankton samples from different lakes. The fraction of Bacteria detected by CARD-FISH was significantly lower than after FISH with fluorescently monolabeled probes. In particular, the abundances of aquatic Actinobacteria were significantly underestimated. We thus developed a combined fixation and permeabilization protocol for CARD-FISH of freshwater samples. Enzymatic pretreatment of fixed cells was optimized for the controlled digestion of gram-positive cell walls without causing overall cell loss. Incubations with high concentrations of lysozyme (10 mg ml−1) followed by achromopeptidase (60 U ml−1) successfully permeabilized cell walls of Actinobacteria for subsequent CARD-FISH both in enrichment cultures and environmental samples. Between 72 and >99% (mean, 86%) of all Bacteria could be visualized with the improved assay in surface waters of four lakes. For freshwater samples, our method is thus superior to the CARD-FISH protocol for marine Bacteria (mean, 55%) and to FISH with directly fluorochrome labeled probes (mean, 67%). Actinobacterial abundances in the studied systems, as detected by the optimized protocol, ranged from 32 to >55% (mean, 45%). Our findings confirm that members of this lineage are among the numerically most important Bacteria of freshwater picoplankton. PMID:12732568

  9. Peptide nucleic acid fluorescence in situ hybridization for identification of Listeria genus, Listeria monocytogenes and Listeria ivanovii.

    Science.gov (United States)

    Zhang, Xiaofeng; Wu, Shan; Li, Ke; Shuai, Jiangbing; Dong, Qiang; Fang, Weihuan

    2012-07-02

    A fluorescent in situ hybridization (FISH) method in conjunction with fluorescin-labeled peptide nucleic acid (PNA) probes (PNA-FISH) for detection of Listeria species was developed. In silico analysis showed that three PNA probes Lis-16S-1, Lm-16S-2 and Liv-16S-5 were suitable for specific identification of Listeria genus, Listeria monocytogenes and Listeria ivanovii, respectively. These probes were experimentally verified by their reactivity against 19 strains of six Listeria species (excluding newly described species Listeria marthii and Listeria rocourtiae) and eight other bacterial species. The PNA-FISH method was optimized as 30 min of hybridization with 0.2% Triton X-100 in the solution and used to identify 85 Listeria strains from individual putative Listeria colonies on PALCAM agar plates streaked from selectively enriched cultures of 780 food or food-related samples. Of the 85 Listeria strains, thirty-seven were identified as L. monocytogenes with the probe Lm-16S-2 and two as L. ivanovii with the probe Liv-16S-5 which was in agreement with the results obtained by the API LISTERIA method. Thus, the PNA-FISH protocol has the potential for identification of pathogenic Listeria spp. from food or food-related samples.

  10. Surface Enhanced Raman Scattering Based in Situ Hybridization Strategy for Telomere Length Assessment.

    Science.gov (United States)

    Zong, Shenfei; Chen, Chen; Wang, Zhuyuan; Zhang, Yizhi; Cui, Yiping

    2016-02-23

    Assessing telomere length is of vital importance since telomere length is closely related with several fatal diseases such as atherosclerosis and cancer. Here, we present a strategy to assess/measure telomere length, that is, surface enhanced Raman scattering (SERS) based in situ hybridization (SISH). The SISH method uses two kinds of SERS nanoprobes to hybridize in situ with telomeres and centromeres, respectively. The telomere specific SERS nanoprobe is called the Telo-probe, while the centromere specific SERS nanoprobe is called the Centro-probe. They are composed of metal nanoparticles (NPs), Raman reporter molecules and specially designed DNA strands. With longer telomeres, more Telo-probes will hybridize with them, resulting in a stronger SERS signal. To exclude possible influence of the SERS intensity by external factors (such as the nanoprobe concentration, the cell number or different batches of nanoprobes), centromeres are used as the inner control, which can be recognized by Centro-probes. Telomere length is evaluated using a redefined telomere-to-centromere ratio (T/C ratio). The calculation method for T/C ratio in SISH method is more reliable than that in fluorescent in situ hybridization (FISH). In addition, unlike FISH method, the SISH method is insensitive to autofluorescence. Moreover, SISH method can be used to analyze single telomeres. These features make SISH an excellent alternative strategy for telomere length measurement.

  11. Mesoscale hybrid calibration artifact

    Science.gov (United States)

    Tran, Hy D.; Claudet, Andre A.; Oliver, Andrew D.

    2010-09-07

    A mesoscale calibration artifact, also called a hybrid artifact, suitable for hybrid dimensional measurement and the method for make the artifact. The hybrid artifact has structural characteristics that make it suitable for dimensional measurement in both vision-based systems and touch-probe-based systems. The hybrid artifact employs the intersection of bulk-micromachined planes to fabricate edges that are sharp to the nanometer level and intersecting planes with crystal-lattice-defined angles.

  12. Chromosome painting by GISH and multi-color FISH

    Science.gov (United States)

    Fluorescent in situ hybridization (FISH) is a powerful cytogenetic technique for identifying chromosomes and mapping specific genes and DNA sequences on individual chromosomes. Genomic in situ hybridization (GISH) and multi-color FISH (mc-FISH) represent two special types of FISH techniques. Both ...

  13. FISH Loci of 18-26s rDNA in Four Gossypium Species%四个棉种18-26s rDNA荧光原位杂交

    Institute of Scientific and Technical Information of China (English)

    Kunbo WANG; Chunying WANG; Shu BIE; Guoli SONG; Maoxue LI

    2002-01-01

    @@ Detection of specific nucleic acid sequences such as RNA or DNA in chromosomes by in situ hybridization has important applications in many areas of biology. The genes encoding 18-26s rRNA are located nucleus organizer regions (NORs) in plant chromosomes. Fluorescent in situ hybridization ( FISH ) with 18-26s rDNA as probe to somatic chromosomes may directly provide insight into genetic mapping and then,by comparisons with karyotypes, physical loci of NORs of the genome.

  14. Application of dual color fluorescence in situ hybridization (D—FISH) to the diagnosis of a 49,XXXXY chromosomal abnormality

    Institute of Scientific and Technical Information of China (English)

    LiuYZ; ZengX

    2002-01-01

    Objective:To study the technique of D-FISH and its application in the diagnosis of a 49.XXXXY chromosomal abnormality.Methods:Biotin-labeled alpha satellite X chromosome DNA(pBamX7) probe and digoxi-genin-labeled Y chromosome long arm terminal repetitive sequence (pY3.4) probe in situ hybridized with pre-treated slides of peripheral blood chromosome and interphase nucleus.After washing,the slides were treated with avidin-FITC,rhodamine-FITC and anti-avidin,amplified with an additional layer and counter-stained with DAPI in an antifade solution.The hybridization signals and chromosomal or interphase nucleus settings were observed respectively with WIB,WIG and WU filters under fluorescent microscope (Olympus AX-70) and the number of metaphase chromosome and interphase nucleus in the peripheral blood was counted.Results:The biotin-labeled pBamX7 probe showed 4 green hybridization signal and the digoxigenin-labeled pY3.4 probe showed 1 red hybridization signal.The chromosome or cytoplasm counter-stained with DAPI showed blue.The positive rate of X chromosome hybridization signal for the 350 metaphase chromosomes and interphase nucleus was 91.43% and 92.57%,respectively,while that of the Y chromosome hybridization signal was 99.5% and 99.8%,respectively.Conclusion:D-FISH is a valuable technique in diagnosing 49,XXXXY chromosomal abnormality and other sex chromosomal abnormalities.

  15. Formation of diploid and triploid hybrid groupers (hybridization of Epinephelus coioides ♀ × Epinephelus lanceolatus ♂) and their 5S gene analysis.

    Science.gov (United States)

    Huang, Wen; Qin, Qinbo; Yang, Huirong; Li, Shuisheng; Hu, Chaoqun; Wang, Yude; Zhang, Yong; Liu, Shaojun; Lin, Haoran

    2016-10-07

    Interspecies hybridization is widely used to achieve heterosis or hybrid vigor, which has been observed and harnessed by breeders for centuries. Natural allopolyploid hybrids generally exhibit more superior heterosis than both the diploid progenies and their parental species. However, polyploid formation processes have been long ignored, the genetic basis of heterosis in polyploids remains elusive. In the present study, triploid hybrids had been demonstrated to contain two sets of chromosomes from mother species and one set from father species. Cellular polyploidization process in the embryos had been traced. The triploid hybrids might be formed by failure formation of the second polarized genome during the second meiosis stage. Four spindle centers were observed in anaphase stage of the first cell division. Three spindle centers were observed in side of cell plate after the first cell division. The 5S rDNA genes of four types of groupers were cloned and analyzed. The diploid and triploid hybrids had been proved to contain the tandem chimera structures which were recombined by maternal and paternal monomer units. The results indicated that genome re-fusion had occurred in the hybrid progenies. To further elucidate the genetic patterns of diploid and triploid hybrids, fluorescence chromosome location had been carried out, maternal 5S gene (M-386) were used as the probe. The triploid hybrids contained fewer fluorescence loci numbers than the maternal species. The results indicated that participation of paternal 5S gene in the triploid hybrid genome had degraded the match rates of M-386 probe. Our study is the first to investigate the cellular formation processes of natural allopolyploids in hybrid fish, the cellular polyploidization process may be caused by failure formation of the second polarized genome during the meiosis, and our results will provide the molecular basis of hybrid vigor in interspecies hybridization.

  16. In situ hybridization to somatic chromosomes in Drosophila.

    Science.gov (United States)

    Dernburg, Abby F

    2011-09-01

    In situ hybridization was originally developed as a technique for visualizing and physically mapping specific sequences on Drosophila melanogaster polytene chromosomes. Hybridization techniques can also be used to localize sequences on smaller, diploid chromosomes, such as condensed mitotic chromosomes. Variations of the method also allow the hybridization of probes to chromosomes within intact cells and tissues, rather than to chromosomes isolated from their cellular context and flattened on slides. This article presents methods for hybridizing fluorescent probes to chromosomes in whole-mount Drosophila tissues. These methods allow the investigation of nuclear organization even at stages where chromosomes are decondensed (as in interphase) or, for other reasons, cannot be discriminated in the light microscope. Consequently, they are useful for addressing a variety of cell biological questions. In addition to enhancing our understanding of somatic chromosome organization, this experimental approach has also revealed interactions among meiotic chromosomes in Drosophila females, which spend much of meiosis in a compact ball called the karyosome. Fluorescent in situ hybridization (FISH) methods can also be used to karyotype individual nuclei using chromosome-specific markers. With appropriate fixation conditions, hybridization to chromosomal DNA can be performed in conjunction with immunostaining, allowing the colocalization of cellular or chromosomal proteins.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Directory of Open Access Journals (Sweden)

    Rhein Andreas P

    2008-12-01

    Full Text Available Abstract 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.

  20. Simultaneous RNA-DNA FISH.

    Science.gov (United States)

    Lai, Lan-Tian; Meng, Zhenyu; Shao, Fangwei; Zhang, Li-Feng

    2016-01-01

    A highly useful tool for studying lncRNAs is simultaneous RNA-DNA FISH, which reveals the localization and quantitative information of RNA and DNA in cellular contexts. However, a simple combination of RNA FISH and DNA FISH often generates disappointing results because the fragile RNA signals are often damaged by the harsh conditions used in DNA FISH for denaturing the DNA. Here, we describe a robust and simple RNA-DNA FISH protocol, in which amino-labeled nucleic acid probes are used for RNA FISH. The method is suitable to detect single-RNA molecules simultaneously with DNA.

  1. Chromosome studies of Astyanax jacuhiensis Cope, 1894 (Characidae) from the Tramandai River Basin, Brazil, using in situ hybridization with the 18S rDNA probe, DAPI and CMA3 staining.

    Science.gov (United States)

    da Silva, Laura Lahr Lourenço; Giuliano-Caetano, Lucia; Dias, Ana Lúcia

    2012-01-01

    The genus Astyanax comprises 86 species of fish distributed in Brazilian river basins and is considered of the Incertae sedis group within the family Characidae. This study presents an analysis of 12 specimens of Astyanax jacuhiensis from the Tramandai River Basin, RS Brazil: 6 from the Maquiné River and 6 from the Quadros Lagoon. All specimens showed a diploid number equal to 50 chromosomes with different karyotypic formula between the two localities. The population from the Maquiné River showed 10m+26sm+6st+8a (FN=92). Fish from the Quadros Lagoon showed 12m+20sm+6st+12a (FN=88). AgNORs were evidenced in the short arm of one acrocentric chromosome pair in both populations, confirmed by FISH with the 18S rDNA probe. CMA3 fluorochrome corresponded with the AgNOR sites, while DAPI staining was negative in these regions. C banding revealed that heterochromatin was weakly distributed, mainly in the pericentromeric and terminal regions in most chromosomes. Analyses of male gonadal tissue were conducted with the objective of characterizing the meiotic chromosome behavior in A. jacuhiensis. The following stages were evidenced: spermatogonial with 50 chromosomes, pachytene and metaphase I with 25 bivalents, and metaphase II with 25 chromosomes, thus confirming the diploid number of the species. Chromosomal abnormalities were not observed. This study shows preliminary data on A. jacuhiensis from the Tramandai River Basin, contributing with more chromosomal information for this group of fish.

  2. A 16S rRNA-targeted Probe for Detection of Lactobacilli and Enterococci in Faecal Samples by Fluorescent In Situ Hybridization

    OpenAIRE

    Harmsen, Hermie J. M.; Elfferich, Peter; Schut, Frits; Welling, Gjalt W

    2011-01-01

    A group-specific 16S rRNA-targeted oligonucleotide probe S-G-Lab-0158-a-A20 (Lab158) was designed and validated to quantify species of the phylogenetic group lactobacilli-enterococci. The Lab158 probe detects nearly all species of the genera Lactobacillus, Enterococcus, Pediococcus, Weissella, Vagococcus, Leuconostoc and Oenococcus. The specificity of the probe was tested on various species of the target group and on a range of common intestinal bacteria. For these experiments, procedures to ...

  3. Facile semi-automated forensic body fluid identification by multiplex solution hybridization of NanoString® barcode probes to specific mRNA targets.

    Science.gov (United States)

    Danaher, Patrick; White, Robin Lynn; Hanson, Erin K; Ballantyne, Jack

    2015-01-01

    A DNA profile from the perpetrator does not reveal, per se, the circumstances by which it was transferred. Body fluid identification by mRNA profiling may allow extraction of contextual 'activity level' information from forensic samples. Here we describe the development of a prototype multiplex digital gene expression (DGE) method for forensic body fluid/tissue identification based upon solution hybridization of color-coded NanoString(®) probes to 23 mRNA targets. The method identifies peripheral blood, semen, saliva, vaginal secretions, menstrual blood and skin. We showed that a simple 5 min room temperature cellular lysis protocol gave equivalent results to standard RNA isolation from the same source material, greatly enhancing the ease-of-use of this method in forensic sample processing. We first describe a model for gene expression in a sample from a single body fluid and then extend that model to mixtures of body fluids. We then describe calculation of maximum likelihood estimates (MLEs) of body fluid quantities in a sample, and we describe the use of likelihood ratios to test for the presence of each body fluid in a sample. Known single source samples of blood, semen, vaginal secretions, menstrual blood and skin all demonstrated the expected tissue-specific gene expression for at least two of the chosen biomarkers. Saliva samples were more problematic, with their previously identified characteristic genes exhibiting poor specificity. Nonetheless the most specific saliva biomarker, HTN3, was expressed at a higher level in saliva than in any of the other tissues. Crucially, our algorithm produced zero false positives across this study's 89 unique samples. As a preliminary indication of the ability of the method to discern admixtures of body fluids, five mixtures were prepared. The identities of the component fluids were evident from the gene expression profiles of four of the five mixtures. Further optimization of the biomarker 'CodeSet' will be required

  4. Fast-FISH Detection and Semi-Automated Image Analysis of Numerical Chromosome Aberrations in Hematological Malignancies

    Directory of Open Access Journals (Sweden)

    Arif Esa

    1998-01-01

    Full Text Available A new fluorescence in situ hybridization (FISH technique called Fast-FISH in combination with semi-automated image analysis was applied to detect numerical aberrations of chromosomes 8 and 12 in interphase nuclei of peripheral blood lymphocytes and bone marrow cells from patients with acute myelogenous leukemia (AML and chronic lymphocytic leukemia (CLL. Commercially available α-satellite DNA probes specific for the centromere regions of chromosome 8 and chromosome 12, respectively, were used. After application of the Fast-FISH protocol, the microscopic images of the fluorescence-labelled cell nuclei were recorded by the true color CCD camera Kappa CF 15 MC and evaluated quantitatively by computer analysis on a PC. These results were compared to results obtained from the same type of specimens using the same analysis system but with a standard FISH protocol. In addition, automated spot counting after both FISH techniques was compared to visual spot counting after standard FISH. A total number of about 3,000 cell nuclei was evaluated. For quantitative brightness parameters, a good correlation between standard FISH labelling and Fast-FISH was found. Automated spot counting after Fast-FISH coincided within a few percent to automated and visual spot counting after standard FISH. The examples shown indicate the reliability and reproducibility of Fast-FISH and its potential for automatized interphase cell diagnostics of numerical chromosome aberrations. Since the Fast-FISH technique requires a hybridization time as low as 1/20 of established standard FISH techniques, omitting most of the time consuming working steps in the protocol, it may contribute considerably to clinical diagnostics. This may especially be interesting in cases where an accurate result is required within a few hours.

  5. Meiotic analysis and FISH with rDNA and rice BAC probes of the Thai KPS 01-01-25 sugarcane cultivar

    NARCIS (Netherlands)

    Thumjamras, Sarut; Iamtham, Siriluck; Prammanee, Siripatr; Jong, de Hans

    2016-01-01

    The interspecific sugarcane hybrid “KPS 01-01-25” is one of Thailand’s most successful cultivars, but its genetics and genomic constitution are greatly complicated due to the highly polyploid nature of this crop. Here we analyzed the crop’s karyotype, studied chromosome pairing at meiosis I and p

  6. Distinguishing between incomplete lineage sorting and genomic introgressions: complete fixation of allospecific mitochondrial DNA in a sexually reproducing fish (Cobitis; Teleostei, despite clonal reproduction of hybrids.

    Directory of Open Access Journals (Sweden)

    Lukas Choleva

    Full Text Available Distinguishing between hybrid introgression and incomplete lineage sorting causing incongruence among gene trees in that they exhibit topological differences requires application of statistical approaches that are based on biologically relevant models. Such study is especially challenging in hybrid systems, where usual vectors mediating interspecific gene transfers--hybrids with Mendelian heredity--are absent or unknown. Here we study a complex of hybridizing species, which are known to produce clonal hybrids, to discover how one of the species, Cobitis tanaitica, has achieved a pattern of mito-nuclear mosaic genome over the whole geographic range. We appplied three distinct methods, including the method using solely the information on gene tree topologies, and found that the contrasting mito-nuclear signal might not have resulted from the retention of ancestral polymorphism. Instead, we found two signs of hybridization events related to C. tanaitica; one concerning nuclear gene flow and the other suggested mitochondrial capture. Interestingly, clonal inheritance (gynogenesis of contemporary hybrids prevents genomic introgressions and non-clonal hybrids are either absent or too rare to be detected among European Cobitis. Our analyses therefore suggest that introgressive hybridizations are rather old episodes, mediated by previously existing hybrids whose inheritance was not entirely clonal. Cobitis complex thus supports the view that the type of resulting hybrids depends on a level of genomic divergence between sexual species.

  7. Karyotype variability in tropical maize sister inbred lines and hybrids compared with KYS standard line.

    Science.gov (United States)

    Mondin, Mateus; Santos-Serejo, Janay A; Bertäo, Mônica R; Laborda, Prianda; Pizzaia, Daniel; Aguiar-Perecin, Margarida L R

    2014-01-01

    Maize karyotype variability has been extensively investigated. The identification of maize somatic and pachytene chromosomes has improved with the development of fluorescence in situ hybridization (FISH) using tandemly repeated DNA sequences as probes. We identified the somatic chromosomes of sister inbred lines that were derived from a tropical flint maize population (Jac Duro [JD]), and hybrids between them, using FISH probes for the 180-bp knob repeat, centromeric satellite (CentC), centromeric satellite 4 (Cent4), subtelomeric clone 4-12-1, 5S ribosomal DNA and nucleolus organizing region DNA sequences. The observations were integrated with data based on C-banded mitotic metaphases and conventional analysis of pachytene chromosomes. Heterochromatic knobs visible at pachynema were coincident with C-bands and 180-bp FISH signals on somatic chromosomes, and most of them were large. Variation in the presence of some knobs was observed among lines. Small 180-bp knob signals were invariant on the short arms of chromosomes 1, 6, and 9. The subtelomeric 4-12-1 signal was also invariant and useful for identifying some chromosomes. The centromere location of chromosomes 2 and 4 differed from previous reports on standard maize lines. Somatic chromosomes of a JD line and the commonly used KYS line were compared by FISH in a hybrid of these lines. The pairing behavior of chromosomes 2 and 4 at pachytene stage in this hybrid was investigated using FISH with chromosome-specific probes. The homologues were fully synapsed, including the 5S rDNA and CentC sites on chromosome 2, and Cent4 and subtelomeric 4-12-1 sites on chromosome 4. This suggests that homologous chromosomes could pair through differential degrees of chromatin packaging in homologous arms differing in size. The results contribute to current knowledge of maize global diversity and also raise questions concerning the meiotic pairing of homologous chromosomes possibly differing in their amounts of repetitive DNA.

  8. Combining M-FISH and Quantum Dot technology for fast chromosomal assignment of transgenic insertions

    Directory of Open Access Journals (Sweden)

    Yusuf Mohammed

    2011-12-01

    Full Text Available Abstract Background Physical mapping of transgenic insertions by Fluorescence in situ Hybridization (FISH is a reliable and cost-effective technique. Chromosomal assignment is commonly achieved either by concurrent G-banding or by a multi-color FISH approach consisting of iteratively co-hybridizing the transgenic sequence of interest with one or more chromosome-specific probes at a time, until the location of the transgenic insertion is identified. Results Here we report a technical development for fast chromosomal assignment of transgenic insertions at the single cell level in mouse and rat models. This comprises a simplified 'single denaturation mixed hybridization' procedure that combines multi-color karyotyping by Multiplex FISH (M-FISH, for simultaneous and unambiguous identification of all chromosomes at once, and the use of a Quantum Dot (QD conjugate for the transgene detection. Conclusions Although the exploitation of the unique optical properties of QD nanocrystals, such as photo-stability and brightness, to improve FISH performance generally has been previously investigated, to our knowledge this is the first report of a purpose-designed molecular cytogenetic protocol in which the combined use of QDs and standard organic fluorophores is specifically tailored to assist gene transfer technology.

  9. Detection of sex chromosome aneuploidy in dog spermatozoa by triple color fluorescence in situ hybridization.

    Science.gov (United States)

    Komaki, Haruna; Oi, Maya; Suzuki, Hiroshi

    2014-09-01

    With the development of a direct visualization of sex chromosome in a single sperm by fluorescence in situ hybridization (FISH) technique, the frequency of aberration (aneuploidy) in spermatozoa in several mammals has been investigated. However, there is no report in the incidence of X-Y aneuploidy in the sperm population of dogs. Therefore, in this study, the aneuploidy in dog spermatozoa was examined by multicolor FISH using specific molecular probes for canine sex chromosomes and autosome. Semen from eight male Labrador retrievers was used as specimen. For decondensation of sperm nuclei, the specimen was treated with 1 M NaOH for 4 minutes at room temperature. Probes for chromosomes X, Y, and 1, labeled with SpectrumGreen, Cy3 and Cy5, respectively, were hybridized with decondensed spermatozoa. Fluorescence in situ hybridization signals in sperm heads were clearly detected in each specimen, regardless of the sperm donor. The FISH signal of at least one of the three probes was detected in all sperm heads examined. There was no significant difference between the theoretical ratio (50:50) and the observed ratio of X and Y chromosomes in spermatozoa of all the eight dogs. Mean percentage of sex chromosome aneuploidy was 0.127% (ranged between 0% and 0.316%). This percentage of canine sex chromosome aneuploidy was lower than the one reported in cattle, horses, river buffalo, and goats sperm, but higher than that observed in mice and sheep.

  10. Mobile Probing and Probes

    DEFF Research Database (Denmark)

    2013-01-01

    Mobile probing is a method, developed for learning about digital work situations, as an approach to discover new grounds. The method can be used when there is a need to know more about users and their work with certain tasks, but where users at the same time are distributed (in time and space......). Mobile probing was inspired by the cultural probe method, and was influenced by qualitative interview and inquiry approaches. The method has been used in two subsequent projects, involving school children (young adults at 15-17 years old) and employees (adults) in a consultancy company. Findings point...... to mobile probing being a flexible method for uncovering the unknowns, as a way of getting rich data to the analysis and design phases. On the other hand it is difficult to engage users to give in depth explanations, which seem easier in synchronous dialogs (whether online or face2face). The development...

  11. Mobile Probing and Probes

    DEFF Research Database (Denmark)

    2012-01-01

    Mobile probing is a method, which has been developed for learning about digital work situations, as an approach to discover new grounds. The method can be used when there is a need to know more about users and their work with certain tasks, but where users at the same time are distributed (in time...... and space). Mobile probing was inspired by the cultural probe method, and was influenced by qualitative interview and inquiry approaches. The method has been used in two subsequent projects, involving school children (young adults at 15-17 years old) and employees (adults) in a consultancy company. Findings...... point to mobile probing being a flexible method for uncovering the unknowns, as a way of getting rich data to the analysis and design phases. On the other hand it is difficult to engage users to give in depth explanations, which seem easier in synchronous dialogs (whether online or face2face...

  12. Mobile Probing and Probes

    DEFF Research Database (Denmark)

    2012-01-01

    Mobile probing is a method, which has been developed for learning about digital work situations, as an approach to discover new grounds. The method can be used when there is a need to know more about users and their work with certain tasks, but where users at the same time are distributed (in time...... and space). Mobile probing was inspired by the cultural probe method, and was influenced by qualitative interview and inquiry approaches. The method has been used in two subsequent projects, involving school children (young adults at 15-17 years old) and employees (adults) in a consultancy company. Findings...... point to mobile probing being a flexible method for uncovering the unknowns, as a way of getting rich data to the analysis and design phases. On the other hand it is difficult to engage users to give in depth explanations, which seem easier in synchronous dialogs (whether online or face2face...

  13. 膀胱移行细胞癌各分期分级中荧光原位杂交检测敏感性比较%Evaluation on significance of fluorescence in situ hybridization (FISH) for detection of bladder cancer

    Institute of Scientific and Technical Information of China (English)

    徐秀红; 杜雨; 张道新; 田野

    2011-01-01

    Objective In order to look for a better non - invasive method for diagnosing bladder cancer, the fluorescence in situ hybridization ( FISH ) technique had been applied in diagnosis of bladder cancer. Methods FISH and cytological analysis were performed simultaneously on urine - exfoliated cells from 138 patients with hematuria. Chromosomal abnormalities and gene mutation were determined by FISH with probes for chromosomes 3,7, 17 and pl6. The sensitivity of FISH and cytological examination in diagnosing transitional cell carcinoma of bladder was studied and compared. Results Big and irregular cell nuclei and position effect variegation were seen in some cells ( P < 0. 05 ). In stage Tl bladder cancer, the sensitivity of FISH and cytological analysis was 83 . 6 % and 31.3% respectively, while in stage T2 - T4 , their sensitivity was 100 % and 55. 6 % respectively. Their sensitivity was 36. 3 % and 9.1% respectively with grade Gl versus 95. 2 % and 42. 9 % respectively with grade G2, and the sensitivity was 100 % and 50 % respectively with grade G3 ( P < 0. 05 ) in a comparable manner. There was significant difference in total sensitivity for diagnosis and staging of transitional cell carcinoma of bladder between FISH and cytological examination ( P < 0. 05 ). Conclusion FISH is more sensitive than cytological examination, hence it may be applied as a valuable method for diagnosing transitional cell carcinoma of bladder.%目的 评价荧光原位杂交(FISH)在诊断膀胱移行细胞癌各分期分级中的敏感性.方法 应用荧光标记的3、7、17号染色体及p16基因探针检测138例血尿患者尿液中脱落细胞的染色体及基因畸变情况,根据诊断标准计算FISH诊断膀胱移行细胞癌的敏感性.同时留取上述患者尿液送病理科行尿找瘤细胞检查,并比较两种方法对膀胱移行细胞癌的诊断效能.结果 FISH图像中细胞核大且不规则者更易观察到染色体基因异常情况,并可见具有较多染

  14. FISH diagnosis of partial trisomy 13 and tetrasomy 13 in a patient with severe trigonocephaly (C) phenotype

    Energy Technology Data Exchange (ETDEWEB)

    Chu, T.W.; Teebi, A.S.; Gibson, L.; Breg, W.R.; Yang-Feng, T.L.

    1994-08-01

    An infant girl with manifestations resembling Opitz trigonocephaly (C) syndrome who died at age 6 days was found to have a complex chromosome abnormality with t(13;18)(q22;q23) and a recombinant chromosome 13 involving duplicated segments of 13q. Precise characterization was possible with the application of fluorescence in situ hybridization (FISH) using chromosome specific probes. The patient`s phenotype is compared to that of other syndromes involving trigonocephaly. 20 refs., 3 figs., 3 tabs.

  15. Leptin stimulates hepatic growth hormone receptor and insulin-like growth factor gene expression in a teleost fish, the hybrid striped bass.

    Science.gov (United States)

    Won, Eugene T; Douros, Jonathan D; Hurt, David A; Borski, Russell J

    2016-04-01

    Leptin is an anorexigenic peptide hormone that circulates as an indicator of adiposity in mammals, and functions to maintain energy homeostasis by balancing feeding and energy expenditure. In fish, leptin tends to be predominantly expressed in the liver, another important energy storing tissue, rather than in fat depots as it is in mammals. The liver also produces the majority of circulating insulin-like growth factors (IGFs), which comprise the mitogenic component of the growth hormone (GH)-IGF endocrine growth axis. Based on similar regulatory patterns of leptin and IGFs that we have documented in previous studies on hybrid striped bass (HSB: Morone saxatilis×Morone chrysops), and considering the co-localization of these peptides in the liver, we hypothesized that leptin might regulate the endocrine growth axis in a manner that helps coordinate somatic growth with energy availability. Using a HSB hepatocyte culture system to simulate autocrine or paracrine exposure that might occur within the liver, this study examines the potential for leptin to modulate metabolism and growth through regulation of IGF gene expression directly, or indirectly through the regulation of GH receptors (GHR), which mediate GH-induced IGF expression. First, we verified that GH (50nM) has a classical stimulatory effect on IGF-1 and additionally show it stimulates IGF-2 transcription in hepatocytes. Leptin (5 and/or 50nM) directly stimulated in vitro GHR2 gene expression within 8h of exposure, and both GHR1 and GHR2 as well as IGF-1 and IGF-2 gene expression after 24h. Cells were then co-incubated with submaximal concentrations of leptin and GH (25nM each) to test if they had a synergistic effect on IGF gene expression, possibly through increased GH sensitivity following GHR upregulation by leptin. In combination, however, the treatments only had an additive effect on stimulating IGF-1 mRNA despite their capacity to increase GHR mRNA abundance. This suggests that leptin's stimulatory

  16. Detection of Lawsonia intracellularis in formalinfixed Porcine Intestinal Tissue Samples: Comparison of Immunofluorescense and In-situ Hybridization, and Evaluation of the Effectf of Controlled Autolysis

    DEFF Research Database (Denmark)

    Jensen, Tim Kåre; Boesen, H. T.; Vigre, Håkan;

    2010-01-01

    Two methods, an immunofluorescence assay (IFA; with a Lawsonia intracellularis-specific monoclonal antibody) and fluorescent in-situ hybridization (FISH; with a specific oligonucleotide probe targeting 16S ribosomal RNA of the bacterium), were compared for their ability to detect L. intracellularis...... (the cause of porcine proliferative enteritis [PE]) in formalin-fixed samples of intestinal tissue. Of 69 intestinal samples with gross lesions of PE, 63 were positive by both FISH and IFA, but six were positive only by IFA. This indicated that the sensitivity of FISH was 91% that of IFA. However, both...

  17. Heavy ion-induced chromosomal aberrations analyzed by fluorescence in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Durante, M.; Gialanella, G.; Grossi, G.; Pugliese, M. [Univ. ``Federico II``, Naples (Italy). Dept. of Physics]|[INFN, Naples (Italy); Cella, L.; Greco, O. [Univ. ``Federico II``, Naples (Italy). Dept. of Physics; Furusawa, Y. [NIRS, Chiba (Japan); George, K.; Yang, T.C. [NASA Lyndon B. Johnson Space Center, Houston, TX (United States)

    1997-09-01

    We have investigated the effectiveness of heavy ions in the induction of chromosomal aberrations in mammalian cells by the recent technique of fluorescence in situ hybridization (FISH) with whole-chromosome probes. FISH-painting was used both in metaphase and interphase (prematurely condensed) chromosomes. The purpose of our experiments was to address the following problems: (a) the ratio of different types of aberrations as a function of radiation quality (search for biomarkers); (b) the ratio between aberrations scored in interphase and metaphase as a function of radiation quality (role of apoptosis); (c) differences between cytogenetic effects produced by different ions at the same LET (role of track structure). (orig./MG)

  18. BIOTECHNOLOGY OF THE FISH AQUACULTURE

    Directory of Open Access Journals (Sweden)

    L. P. Buchatsky

    2013-12-01

    Full Text Available The latest progress in biotechnology on fish aquaculture and different modern methods of investigations for increasing of fish productivity in aquaculture are analyzed. Except for the applied aspect, the use of modern biotechnological methods of investigations opens new possibilities for fundamental researches of sex-determining mechanisms, polyploidy, distant hybridization, and developmental biology of bony fishes. Review contains examples of utilizing modern biotechnology methods to obtain transgenic fishes with accelerated growth and for designing surrogate fishes. Methods for receiving unisexual shoals of salmon and sturgeon female fishes with the view of obtaining a large quantity of caviar, as well as receiving sterile (triploid fishes are analyzed. Great attention is given to androgenesis, particularly to disperm one, in connection with the problem of conserving rare and vanishing fish species using only sperm genetic material. Examples how distant hybrids may be obtained with the use of disperm androgenesis and alkylated DNA are given. Methods of obtaining fish primordium germ cells, recent developments in cultivation of fish stem cells and their use in biotechnology, as well as ones of transplantation of oogonium and spermatogonium to obtain surrogate fishes. The examples of successful experiments on spermatogonial xenotransplantation and characteristic of antifreezing fish proteins and also the prospect of their practical usage are given.

  19. Fish Allergy

    Science.gov (United States)

    ... Old Feeding Your 1- to 2-Year-Old Fish Allergy KidsHealth > For Parents > Fish Allergy Print A ... From Home en español Alergia al pescado About Fish Allergy A fish allergy is not exactly the ...

  20. Modified cIg-FISH protocol for multiple myeloma in routine cytogenetic laboratory practice.

    Science.gov (United States)

    Gole, Leena; Lin, Adeline; Chua, Constance; Chng, Wee Joo

    2014-01-01

    The International Myeloma Working Group recommends that fluorescence in situ hybridization (FISH) be performed on specifically identified plasma cells (PC). This is because chromosomal abnormalities are not frequently detected by traditional karyotyping due to the low proliferative rate of PC in multiple myeloma (MM). Conventional FISH enhances the sensitivity but lacks the specificity, as it does not distinguish PC from other hematopoetic cells. To fulfill this recommendation, PC need to be selected either by flow cytometry or immunomagnetic bead-based PC sorting or by concomitant labeling of the cytoplasmic immunoglobulin light chain, which allows for unambiguous identification. These techniques require expertise, time, and funding and are not easily incorporated into the routine workflow of the cytogenetic laboratory. We have modified and refined the technique using fixed cell pellets to achieve nicely separated and easily identifiable PC. With immunostaining and subsequent FISH (i.e., cytoplasmic immunoglobulin FISH, cIg-FISH), this technique can be easily incorporated into every cytogenetic laboratory. Twenty samples from patients with MM were subjected to routine FISH, cIg-FISH, and chromosomal karyotyping and the results were compared. Three FISH probes, which enabled detection of the t(4;14), t(14;16) and deletion of TP53, were used to validate this modified technique successfully. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Amplification of HER2 and TOP2A and deletion of TOP2A genes in breast cancer investigated by new FISH probes

    DEFF Research Database (Denmark)

    Olsen, Karen E; Knudsen, Helle; Rasmussen, Birgitte

    2004-01-01

    New strategies for improving treatment of patients with breast carcinoma have focused on the HER2 oncoprotein with regard to response to traditional therapy regimes and the effect of a new drug specifically directed against the protein. Furthermore, the status of the topoisomerase IIalpha (TOP2A......). In addition, the HercepTest was evaluated as a screening tool for choosing cases for FISH investigation of TOP2A gene aberrations. The HercepTest score 3+ identified HER2 gene amplification in 27 of 30 amplified tumours (sensitivity of 0.90) with a false-negative rate of 0.10 and a false-positive rate of 0.......06. TOP2A gene amplification or deletion was found in 20 cases. Sixteen (80%) of these carcinomas were in the HercepTest 3+ group, but four tumours had alterations in the TOP2A gene with normal HER2 status. Traditionally, in the FISH technique the result has been based on counting 60 cells. However, we...

  2. Insights into food preference in hybrid F1 of Siniperca chuatsi (♀) × Siniperca scherzeri (♂) mandarin fish through transcriptome analysis.

    Science.gov (United States)

    He, Shan; Liang, Xu-Fang; Sun, Jian; Li, Ling; Yu, Ying; Huang, Wei; Qu, Chun-Mei; Cao, Liang; Bai, Xiao-Li; Tao, Ya-Xiong

    2013-09-05

    As economically relevant traits, feeding behavior and food preference domestication determine production cost and profitability. Although there are intensive research efforts on feeding behavior and food intake, little is known about food preference. Mandarin fish accept only live prey fish and refuse dead prey fish or artificial diets. Very little is currently known about the genes regulating this unique food preference. Using transcriptome sequencing and digital gene expression profiling, we identified 1,986 and 4,526 differentially expressed genes in feeders and nonfeeders of dead prey fish, respectively. Up-regulation of Crbp, Rgr and Rdh8, and down-regulation of Gc expression, consistent with greater visual ability in feeders, could promote positive phototaxis. Altered expressions of period, casein kinase and Rev-erbα might reset circadian phase. Down-regulation of orexigenic and up-regulation of anorexigenic genes in feeders were associated with lower appetite. The mRNA levels of Creb, c-fos, C/EBP, zif268, Bdnf and Syt were dramatically decreased in feeders, which might result in significant deficiency in memory retention of its natural food preference (live prey fish). There were roughly 100 times more potential SNPs in feeders than in nonfeeders. In summary, differential expression in the genes identified shed new light on why mandarin fish only feed on live prey fish, with pathways regulating retinal photosensitivity, circadian rhythm, appetite control, learning and memory involved. We also found dramatic difference in SNP abundance in feeders vs nonfeeders. These differences together might account for the different food preferences. Elucidating the genes regulating the unique food preference (live prey fish) in mandarin fish could lead to a better understanding of mechanisms controlling food preference in animals, including mammals.

  3. BIOTECHNOLOGY OF THE FISH AQUACULTURE

    OpenAIRE

    L. P. Buchatsky

    2013-01-01

    The latest progress in biotechnology on fish aquaculture and different modern methods of investigations for increasing of fish productivity in aquaculture are analyzed. Except for the applied aspect, the use of modern biotechnological methods of investigations opens new possibilities for fundamental researches of sex-determining mechanisms, polyploidy, distant hybridization, and developmental biology of bony fishes. Review contains examples of utilizing modern biotechnology methods to obtain ...

  4. Mobile Probing and Probes

    DEFF Research Database (Denmark)

    2013-01-01

    to mobile probing being a flexible method for uncovering the unknowns, as a way of getting rich data to the analysis and design phases. On the other hand it is difficult to engage users to give in depth explanations, which seem easier in synchronous dialogs (whether online or face2face). The development...

  5. Retrospective study of trisomy 18 in chorionic villi with fluorescent in situ hybridization on archival direct preparations

    OpenAIRE

    Van Opstal, Diane; Berg, Cardi; Jahoda, M.; Brandenburg, Helen; Los, F.J.; in 't Veld, Peter

    1995-01-01

    textabstractTrisomy 18 in direct chorionic villus preparations needs further investigation since the chromosome abnormality may be confined to the placenta and may not represent the actual fetal karyotype. We performed, retrospectively, fluorescent in situ hybridization (FISH) with the chromosome 18 centromere probe (L1.84) on interphase nuclei of destained slides of all cases of full trisomy 18 (n=22) and mosaic trisomy 18 (n=8) detected among 7600 first-trimester chorionic villus samples du...

  6. Probe and method for DNA detection

    Science.gov (United States)

    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.

  7. Chromosomal localization of rDNA genes and genomic organization of 5S rDNA in Oreochromis mossambicus, O. urolepis hornorum and their hybrid

    Indian Academy of Sciences (India)

    Hua Ping Zhu; Mai Xin Lu; Feng Ying Gao; Zhang Han Huang; Li Ping Yang; Jain Fang Gui

    2010-08-01

    In this study, classical and molecular cytogenetic analyses were performed in tilapia fishes, Oreochromis mossambicus (XX/XY sex determination system), O. urolepis hornorum (WZ/ZZ sex determination system) and their hybrid by crossing O. mossambicus female × O. u. hornorum male. An identical karyotype (($2n = 44$, NF (total number of chromosomal arms) = 50) was obtained from three examined tilapia samples. Genomic organization analysis of 5S rDNA revealed two different types of 5S rDNA sequences, 5S type I and 5S type II. Moreover, fluorescence in situ hybridization (FISH) with 5S rDNA probes showed six positive fluorescence signals on six chromosomes of all the analysed metaphases from the three tilapia samples. Subsequently, 45S rDNA probes were also prepared, and six positive fluorescence signals were observed on three chromosome pairs in all analysed metaphases of the three tilapia samples. The correlation between 45 rDNA localization and nucleolar organizer regions (NORs) was confirmed by silver nitrate staining in tilapia fishes. Further, different chromosomal localizations of 5S rDNA and 45S rDNA were verified by two different colour FISH probes. Briefly, the current data provide an insights for hybridization projects and breeding improvement of tilapias.

  8. 10p Duplication characterized by fluorescence in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Wiktor, A.; Feldman, G.L.; Van Dyke, D.L.; Kratkoczki, P.; Ditmars, D.M. Jr. [Henry Ford Hospital, Detroit, MI (United States)

    1994-09-01

    We describe a patient with severe failure to thrive, mild-moderate developmental delay, cleft lip and palate, and other anomalies. Routine cytogenetic analysis documented a de novo chromosome rearrangement involving chromosome 4, but the origin of the derived material was unknown. Using chromosome specific painting probes, the karyotype was defined as 46,XY,der(4)t(4;10)(q35;p11.23). Characterization of the dup(10p) by fluorescence in situ hybridization (FISH) analysis provides another example of the usefulness of this technology in identifying small deletions, duplications, or supernumerary marker chromosomes. 19 refs., 4 figs.

  9. Probing the receptor recognition site of the FimH adhesin by fimbriae-displayed FimH-FocH hybrids

    DEFF Research Database (Denmark)

    Knudsen, Thomas Borch; Klemm, Per

    1998-01-01

    Type 1 fimbriae are surface organelles of Escherichia coli which mediate D-mannose-sensitive binding to different host surfaces.This binding is conferred by the minor fimbrial component FimH. The binding domain of the FimH adhesin has been studied byconstructing hybrids of FimH and a homologous...

  10. 16S rRNA-targeted probes for specific detection of Thermoanaerobacterium spp., Thermoanaerobacterium thermosaccharolyticum, and Caldicellulosiruptor spp. by fluorescent in situ hybridization in biohydrogen producing systems

    DEFF Research Database (Denmark)

    O-Thong, Sompong; Prasertsan, P.; Karakashev, Dimitar Borisov

    2008-01-01

    spp. were detected with the probes designed with coverage of 75%, 100% and 93%, respectively. Thermophilic (60 °C) hydrogen producing reactors, one fed with sucrose and another, fed with palm oil mill effluent comprised of following major groups of hydrogen producers: Thermoanaerobacterium spp. (49...

  11. Simultaneous labeling of single- and double-strand DNA breaks by DNA breakage detection-FISH (DBD-FISH).

    Science.gov (United States)

    Fernández, José Luis; Cajigal, Dioleyda; Gosálvez, Jaime

    2011-01-01

    DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) permits simultaneous and selective labeling of single- and double-strand DNA breaks in individual cells, either in the whole genome or within specific DNA sequences. In this technique, cells are embedded into agarose microgels, lysed and subjected to electrophoresis under nondenaturing conditions. Subsequently, the produced "comets" are exposed to a controlled denaturation step which transforms DNA breaks into single-stranded DNA regions, detected by hybridization with whole genome fluorescent probes or the probes to specific DNA sequences. This makes possible a targeted analysis of various chromatin areas for the presence of DNA breaks. The migration length of the DBD-FISH signal is proportional to the number of double strand breaks, whereas its fluorescence intensity depends on numbers of single-strand breaks.The detailed protocol for detection of two types of DNA breaks produced by ionizing radiation is presented. The technique can be used to determine intragenomic and intercellular heterogeneity in the induction and repair of DNA damage.

  12. A CARD-FISH protocol for the identification and enumeration of cyanobacterial akinetes in lake sediments.

    Science.gov (United States)

    Ramm, Jessica; Lupu, Achsa; Hadas, Ora; Ballot, Andreas; Rücker, Jacqueline; Wiedner, Claudia; Sukenik, Assaf

    2012-10-01

    Akinetes are the dormant cells of Nostocales (cyanobacteria) that enable the organisms to survive harsh environmental conditions while resting in bottom sediments. The germination of akinetes assists the dispersal and persistence of the species. The assessment of the akinete pool in lake sediments is essential to predict the bloom formation of the Nostocales population. We present here the implementation of an improved catalysed reporter deposition (CARD)-fluorescence in situ hybridization (FISH) protocol to assist the identification and quantification of akinetes in sediment samples. Several 16S rRNA gene oligonucleotide probes were evaluated for labelling akinetes of various species of Anabaena, Aphanizomenon and Cylindrospermopsis. Akinetes of all the taxa studied were successfully labelled and could be easily detected by their bright fluorescence signal. The probes' specificity was tested with 32 strains of different taxa. All six Cylindrospermopsis raciborskii strains were labelled with a specific probe for its 16S rRNA gene. A more general probe labelled 73% of the Anabaena and Aphanizomenon strains. The counting data of field samples obtained with CARD-FISH and the regular light microscopy approach did not differ significantly, confirming the suitability of both methods. The CARD-FISH approach was found to be less time-consuming because of better visibility of akinetes.

  13. Development of a fluorescence in situ hybridization protocol for the identification of micro-organisms associated with wastewater particles and flocs.

    Science.gov (United States)

    Ormeci, Banu; Linden, Karl G

    2008-11-01

    Fluorescence in situ hybridization (FISH) provides a unique tool to study micro-organisms associated with particles and flocs. FISH enables visual examination of micro-organisms while they are structurally intact and associated with particles. However, application of FISH to wastewater and sludge samples presents a specific set of problems. Wastewater samples generate high background fluorescence due to their organic and inorganic content making it difficult to differentiate a probe-conferred signal from naturally fluorescing particles with reasonable certainty. Furthermore, some of the FISH steps involve harsh treatment of samples, and are likely to disrupt the floc structure. This study developed a FISH protocol for studying micro-organisms that are associated with particles and flocs. The results indicate that choice of a proper fluorochrome and labeling technique is a key step in reducing the background fluorescence and non-specific binding, and increasing the intensity of the probe signal. Compared to other fluorochromes tested, CY3 worked very well and enabled the observation of particles and debris in red and probe signal from microbes in yellow. Fixation, hybridization, and washing steps disturbed the floc structure and particle-microbe association. Modifications to these steps were necessary, and were achieved by replacing centrifugation with filtration and employment of nylon filters. Microscope slides generated excellent quality images, but polycarbonate membrane filters performed better in preserving the floc structure.

  14. Detection of illegitimate rearrangement within the immunoglobulin locus on 14q32.3 in B-cell malignancies using end-sequenced probes

    DEFF Research Database (Denmark)

    Silahtaroglu, Asli; Poulsen, Tim; Giselø, Claus

    2001-01-01

    Translocation involving the immunoglobulin heavy chain (IGH) locus is a recurring event in B-cell oncogenesis. The aim of this study was to characterize clones from bacterial artificial chromosome (BAC) libraries and/or bacteriophage P1 artificial chromosome libraries spanning the IGH locus...... for detection of illegitimate rearrangement within the region by fluorescence in situ hybridization (FISH). In silico analysis of the IGH variable (IGHV) DNA sequence (NT_001716.v1) was performed to identify BAC probes located within the IGHV cluster. Clones of the constant (IGHC) cluster were found...... in the literature or at http://www.biologia.uniba.it/rmc/. Validation, orientation, and overlap of these probes were confirmed using interphase-, metaphase-, and fiber-FISH. We have identified seven BAC end-sequenced probes (3087C18, 47P23, 76N15, 12F16, 101G24, 112H5, and 151B17) covering 612 kb of the distal IGHV...

  15. The design of a microscopic system for typical fluorescent in-situ hybridization applications

    Science.gov (United States)

    Yi, Dingrong; Xie, Shaochuan

    2013-12-01

    Fluorescence in situ hybridization (FISH) is a modern molecular biology technique used for the detection of genetic abnormalities in terms of the number and structure of chromosomes and genes. The FISH technique is typically employed for prenatal diagnosis of congenital dementia in the Obstetrics and Genecology department. It is also routinely used to pick up qualifying breast cancer patients that are known to be highly curable by the prescription of Her2 targeted therapy. During the microscopic observation phase, the technician needs to count typically green probe dots and red probe dots contained in a single nucleus and calculate their ratio. This procedure need to be done to over hundreds of nuclei. Successful implementation of FISH tests critically depends on a suitable fluorescent microscope which is primarily imported from overseas due to the complexity of such a system beyond the maturity of the domestic optoelectrical industry. In this paper, the typical requirements of a fluorescent microscope that is suitable for FISH applications are first reviewed. The focus of this paper is on the system design and computational methods of an automatic florescent microscopy with high magnification APO objectives, a fast spinning automatic filter wheel, an automatic shutter, a cooled CCD camera used as a photo-detector, and a software platform for image acquisition, registration, pseudo-color generation, multi-channel fusing and multi-focus fusion. Preliminary results from FISH experiments indicate that this system satisfies routine FISH microscopic observation tasks.

  16. Probing into hybrid organic-molecule and InAs quantum-dots nanosystem with multistacked dots-in-a-well units

    DEFF Research Database (Denmark)

    Chen, Miaoxiang Max; Kobashi, Kazufumi

    2012-01-01

    Hybridizing air-stable organic-molecules with advanced III-V semiconductor quantum-dots (QDs) structures can be utilized to create a new generation of biochemical sensing devices. In order to enhance their optical performances, the active regions in these QDs structures commonly consist...... of multistacked dots-in-a-well (DWELL) units. The effects of grafted molecules on the performances of the QDs structures with multistacked DWELLs, however, still remain unclear. Here, we show the significant improvements in the optical properties of InAs QDs in a hybrid nanosystem obtained by grafting...... biocompatible diazonium salt compound (amine donor) atop InAs QDs structure. Since its interface between the QDs structure and molecular monolayer retains an uncontaminated and non-oxidized condition, the nanosystem is an ideal platform to study the intrinsic properties of charge-carrier transport inside...

  17. Probing the Salt Concentration Dependent Nucelobase Distribution in a Single-Stranded DNA-Single-Walled Carbon Nanotube Hybrid with Molecular Dynamics.

    Science.gov (United States)

    Ghosh, Soumadwip; Patel, Nisheet; Chakrabarti, Rajarshi

    2016-01-28

    The hybrids of single-walled carbon nanotube (SWCNT) and single stranded DNA (ssDNA) are novel nanoscale materials having remarkable applications in nanotechnology. The absorption of nucleobases on the surface of a SWCNT depends strongly on the ionic strength of the medium. In this paper, using atomistic molecular dynamics we have shown that at low salt concentration ssDNA wraps on the surface of SWCNT through hydrophobic π-π stacking between the DNA bases and the sp(2)-hybridized carbon atoms of the carbon nanotube. At high salt concentration, however, the DNA molecule adopts a partially folded structure and the ssDNA-SWCNT wrapping gets weakened significantly due to the self-stacking of the DNA bases. Our study can find relevance in CNT mediated gene delivery processes where subsequent unwrapping of the gene from its carrier is anticipated across the cell membrane regulated by an existing salt concentration gradient.

  18. Chaotic mixer improves microarray hybridization.

    Science.gov (United States)

    McQuain, Mark K; Seale, Kevin; Peek, Joel; Fisher, Timothy S; Levy, Shawn; Stremler, Mark A; Haselton, Frederick R

    2004-02-15

    Hybridization is an important aspect of microarray experimental design which influences array signal levels and the repeatability of data within an array and across different arrays. Current methods typically require 24h and use target inefficiently. In these studies, we compare hybridization signals obtained in conventional static hybridization, which depends on diffusional target delivery, with signals obtained in a dynamic hybridization chamber, which employs a fluid mixer based on chaotic advection theory to deliver targets across a conventional glass slide array. Microarrays were printed with a pattern of 102 identical probe spots containing a 65-mer oligonucleotide capture probe. Hybridization of a 725-bp fluorescently labeled target was used to measure average target hybridization levels, local signal-to-noise ratios, and array hybridization uniformity. Dynamic hybridization for 1h with 1 or 10ng of target DNA increased hybridization signal intensities approximately threefold over a 24-h static hybridization. Similarly, a 10- or 60-min dynamic hybridization of 10ng of target DNA increased hybridization signal intensities fourfold over a 24h static hybridization. In time course studies, static hybridization reached a maximum within 8 to 12h using either 1 or 10ng of target. In time course studies using the dynamic hybridization chamber, hybridization using 1ng of target increased to a maximum at 4h and that using 10ng of target did not vary over the time points tested. In comparison to static hybridization, dynamic hybridization reduced the signal-to-noise ratios threefold and reduced spot-to-spot variation twofold. Therefore, we conclude that dynamic hybridization based on a chaotic mixer design improves both the speed of hybridization and the maximum level of hybridization while increasing signal-to-noise ratios and reducing spot-to-spot variation.

  19. Generation of chicken Z-chromosome painting probes by microdissection for screening large-insert genomic libraries.

    Science.gov (United States)

    Zimmer, R; King, W A; Verrinder Gibbins, A M

    1997-01-01

    A strategy for rapid generation of chicken sex chromosome-Z painting probes has been developed using microdissection. Whole chromosome painting probes (WCPs) were prepared from 10-15 copies of mitotic metaphase chicken Z chromosomes. The microisolated chromosomes were subjected to PEG/proteinase K treatment in a collection drop to release DNA, which was then amplified using a degenerate oligonucleotide-primed shuttle PCR (DOP-Shuttle-PCR) strategy. Size distributions of the PCR products were analyzed by agarose gel electrophoresis and smears of DNA were revealed that ranged in size from 200-800 bp, without any evidence of preferential amplification. Both specificity and complexity of the probes have been analyzed by Southern blot and fluorescence in situ hybridization (FISH). Non-specific hybridization was efficiently blocked by using chicken competitor DNA. Analysis of the WCPs produced shows that collectively they provide uniform hybridization signals along the entire length of the chicken Z chromosome. To demonstrate one possible application of these complex probes, we screened a large-insert bacterial artificial chromosome (BAC) chicken genomic library to select Z chromosome-specific clones. To address specificity of the selected clones and to physically map them to the Z chromosome, FISH analysis was used. Of the 3 clones initially tested, one clone (C3) carrying a 250-kb insert mapped to the distal portion of the short arm of the chicken Z chromosome. Therefore, this technique has provided appropriate probes for screening large-insert genomic libraries. Further application of these probes includes the analysis of chromosome rearrangements, studies of cases of heteroploidy involving the Z chromosome, positional cloning of Z-linked genes and studies on mechanisms of sex-chromosome evolution in birds.

  20. Differentiation of Moraxella nonliquefaciens, M. lacunata, and M. bovis by using multilocus enzyme electrophoresis and hybridization with pilin-specific DNA probes.

    OpenAIRE

    1992-01-01

    Genetic relationships among strains of Moraxella nonliquefaciens, M. lacunata, and M. bovis were studied by using multilocus enzyme electrophoresis and DNA-DNA hybridization. The 74 isolates analyzed for electrophoretic variation at 12 enzyme loci were assigned to 59 multilocus genotypes. The multilocus genotypes were grouped in four major clusters, one representing strains of M. nonliquefaciens, two representing strains of M. lacunata, and one comprising strains of M. bovis and the single st...

  1. A simple and rapid fluorescence in situ hybridization microwave protocol for reliable dicentric chromosome analysis.

    Science.gov (United States)

    Cartwright, Ian M; Genet, Matthew D; Kato, Takamitsu A

    2013-03-01

    Fluorescence in situhybridization (FISH) is an extremely effective and sensitive approach to analyzing chromosome aberrations. Until recently, this procedure has taken multiple days to complete. The introduction of telomeric and centromeric peptide nucleic acid (PNA) probes has reduced the procedure's duration to several hours, but the protocols still call for a high temperature (80-90°C) step followed by 1-3 h of hybridization. The newest method to speed up the FISH protocol is the use of a microwave to shorten the heating element to less than a minute; however this protocol still calls for a 1-h hybridization period. We have utilized PNA centromere/telomere probes in conjunction with a microwave oven to show telomere and centromere staining in as little as 30 s. We have optimized the hybridization conditions to increase the sensitivity and effectiveness of the new protocol and can effectively stain chromosomes in 2 min and 30 s of incubation. We have found that our new approach to FISH produces extremely clear and distinct signals. Radiation-induced dicentric formation in mouse and human fibroblast cells was analyzed by two individual scorers and the observed dicentrics matched very well.

  2. Fish Hearing.

    Science.gov (United States)

    Blaxter, J. H. S.

    1980-01-01

    Provides related information about hearing in fish, including the sensory stimulus of sound in the underwater environment, mechanoreceptors in fish, pressure perception and the swimbladder, specializations in sound conduction peculiar to certain fish families. Includes numerous figures. (CS)

  3. Fish Allergy

    Science.gov (United States)

    ... in a clear and consistent manner, so that consumers with food allergies and their caregivers can be informed as ... the menu, cross-contact with fish is possible. Ethnic ... fish. Avoid foods like fish sticks and anchovies. Some individuals with ...

  4. FISH detection of chromosome 15 deletions in Prader-Willi and Angelman syndromes

    Energy Technology Data Exchange (ETDEWEB)

    Teshima, I.; Chadwick, D.; Chitayat, D. [Hospital for Sick Children and Univ. of Toronto, Ontario (Canada)

    1996-03-29

    We have evaluated fluorescence in situ hybridization (FISH) analysis for the clinical laboratory detection of the 15q11-q13 deletion seen in Prader-Willi syndrome (PWS) and Angelman syndrome (AS) using probes for loci D15S11, SNRPN, D15S10, and GABRB3. In a series of 118 samples from patients referred for PWS or AS, 29 had deletions by FISH analysis. These included two brothers with a paternally transmitted deletion detectable with the probe for SNRPN only. G-banding analysis was less sensitive for deletion detection but useful in demonstrating other cytogenetic alterations in four cases. Methylation and CA-repeat analyses of 15q11-q13 were used to validate the FISH results. Clinical findings of patients with deletions were variable, ranging from newborns with hypotonia as the only presenting feature to children who were classically affected. We conclude that FISH analysis is a rapid and reliable method for detection of deletions within 15q11-q13 and whenever a deletion is found, FISH analysis of parental chromosomes should also be considered. 41 refs., 4 figs., 2 tabs.

  5. Prenatal detection of chromosome aneuploidies in uncultured chorionic villus samples by FISH

    Energy Technology Data Exchange (ETDEWEB)

    Bryndorf, T.; Christensen, B.; Vad, M.; Philip, J. [Univ. of Copenhagen (Denmark)] [and others

    1996-10-01

    We developed a 1-d FISH assay for detection of numerical chromosome abnormalities in uncultured chorionic villus samples (CVS). Probes specific for chromosomes 13, 18, 21, X, and Y were used to determine ploidy by analysis of signal number in hybridized nuclei. Aneuploidy detection using this assay was directly compared with the results obtained by conventional cytogenetic analysis in a consecutive, clinical study of 2,709 CVS and placental samples. The FISH assay yielded discrete differences in the signal profiles between cytogenetically normal and abnormal samples. On the basis of these results, we generated FISH-assay cutoff values that discriminated between karyotypically normal and aneuploid samples. Samples with mosaicism and a single sample with possible heritable small chromosome X probe target were exceptions and showed poor agreement between FISH results and conventional cytogenetics. We conclude that the FISH assay may act as a more accurate and less labor-demanding alternative to {open_quotes}direct{close_quotes} CVS analysis. 22 refs., 1 fig., 4 tabs.

  6. The Interaction of the Solar Wind with Solar Probe Plus - 3D Hybrid Simulation. Report 2: The Study for the Distance 9.5Rs

    Science.gov (United States)

    Lipatov, Alexander S.; Sittler, Edward C.; Hartle, Richard E.; Cooper, John F.

    2010-01-01

    Our paper is a 2.5D and 3D numerical plasma models of the interaction of the solar wind (SW) with the Solar Probe Plus spacecraft (SPPSC). These results should be interpreted as a basic plasma model for which the derived SW interaction with spacecraft (SC) could have consequences for both plasma wave and electron plasma measurements on board SC in the inner heliosphere. We observe an excitation of the low frequency Alfven and whistler type wave directed by the magnetic field with an amplitude of the electromagnetic field oscillation about of (0.015-0.06) V/m. The compression waves and the jumps in an electric field with an amplitude of about 1.5 V/m and (12-18) V/m were also observed. The observed strong electromagnetic perturbations may be a crucial point in the electromagnetic measurements, which were planned in future Solar Probe Plus mission.

  7. Insight into octoploid strawberry (Fragaria) subgenome composition revealed by GISH analysis of pentaploid hybrids.

    Science.gov (United States)

    Liu, Bo; Poulsen, Elizabeth G; Davis, Thomas M

    2016-02-01

    As the product of interspecific hybridization between its two ancestral octoploid (2n = 8x = 56) species (Fragaria chiloensis and F. virginiana), the cultivated strawberry (F. ×ananassa) is among the most genomically complex of crop plants, harboring subgenomic components derived from as many as four different diploid ancestors. To physically visualize the octoploids' subgenome composition(s), we launched molecular cytogenetic studies using genomic in situ hybridization (GISH), comparative GISH (cGISH), and rDNA-FISH techniques. First, GISH resolution in Fragaria was tested by using diploid and triploid hybrids with predetermined genome compositions. Then, observation of an octoploid genome was implemented by hybridizing chromosomes of pentaploid (2n = 5x = 35) hybrids from F. vesca × F. virginiana with genomic DNA probes derived from diploids (2n = 2x = 14) F. vesca and F. iinumae, which have been proposed by phylogenetic studies to be closely related to the octoploids yet highly divergent from each other. GISH and cGISH results indicated that octoploid-derived gametes (n = 4x = 28) carried seven chromosomes with hybridization affinities to F. vesca, while the remaining 21 chromosomes displayed varying affinities to F. iinumae, indicating differing degrees of subgenomic contribution to the octoploids by these two putatively ancestral diploids. Combined rDNA-FISH revealed severe 25S rDNA loss in both the F. vesca- and F. iinumae-like chromosome groups, while only the prior group retained its 5S loci.

  8. Same-day prenatal diagnosis of common chromosomal aneuploidies using microfluidics-fluorescence in situ hybridization.

    Science.gov (United States)

    Ho, Sherry S Y; Chua, Cuiwen; Gole, Leena; Biswas, Arijit; Koay, Evelyn; Choolani, Mahesh

    2012-04-01

    Rapid molecular prenatal diagnostic methods, such as fluorescence in situ hybridization (FISH), quantitative fluorescence-PCR, and multiplex ligation-dependent probe amplification, can detect common fetal aneuploidies within 24 to 48 h. However, specific diagnosis or aneuploidy exclusion should be ideally available within the same day as fetal sampling to alleviate parental anxiety. Microfluidic technologies integrate different steps into a microchip, saving time and costs. We have developed a cost-effective, same-day prenatal diagnostic FISH assay using microfluidics. Amniotic fluids (1-4 mL from 40 pregnant women at 15-22 weeks of gestation) were fixed with Carnoy's before loading into the microchannels of a microfluidic FISH-integrated nanostructured device. The glass slides were coated with nanostructured titanium dioxide to facilitate cell adhesion. Pretreatment and hybridization were performed within the microchannels. Fifty nuclei were counted by two independent analysts, and all results were validated with their respective karyotypes. Of the 40 samples, we found three cases of fetal aneuploidies (trisomies 13, 18, and 21), whereas the remaining 37 cases were normal. Results were concordant with their karyotypes and ready to be released within 3 h of sample receipt. Microfluidic FISH, using 20-fold less than the recommended amount of probe, is a cost-effective method to diagnose common fetal aneuploidies within the same day of fetal sampling.

  9. Experimental improvements in combining CARD-FISH and flow cytometry for bacterial cell quantification.

    Science.gov (United States)

    Manti, Anita; Boi, Paola; Amalfitano, Stefano; Puddu, Alberto; Papa, Stefano

    2011-12-01

    Flow cytometry and Fluorescence In Situ Hybridization are common methods of identifying and quantifying bacterial cells. The combination of cytometric rapidity and multi-parametric accuracy with the phylogenetic specificity of oligonucleotide FISH probes has been regarded as a powerful and emerging tool in aquatic microbiology. In the present work, tests were carried out on E. coli pure culture and marine bacteria using an in-solution hybridization protocol revealing high efficiency hybridization signal for the first one and a lower for the second one. Other experiments were conducted on natural samples following the established CARD-FISH protocol on filter performed in a closed system, with the aim of improving cell detachment and detection. The hybridized cells were then subsequently re-suspended from the membrane filters by means of an optimized detachment procedure. The cytometric enumeration of hybridized marine bacteria reached 85.7%±18.1% of total events. The quality of the cytograms suggests that the procedures described may be applicable to the cytometric quantification of phylogenetic groups within natural microbial communities.

  10. Role of immunohistochemistry and fluorescence in-situ hybridization (FISH in the diagnosis of spindle and round cell tumors of the kidney

    Directory of Open Access Journals (Sweden)

    M. Abbas

    2015-09-01

    Conclusion: In summary we advise an immunohistochemical panel for round/spindle cell tumors of the kidney and for unclear cases we advise to add (FISH to get the correct diagnosis, as they are completely different regarding surgical approach and post-operative adjuvant therapy.

  11. In situ detection of denitrifying bacteria by mRNA-targeted nucleic acid probes and catalyzed reporter deposition

    DEFF Research Database (Denmark)

    Kofoed, Michael Vedel; Stief, Peter; Poulsen, Morten

    In situ detection of denitrifying bacteria by mRNA-targeted nucleic acid probes and catalyzed reporter deposition   Michael V.W. Kofoed, Peter Stief, Morten Poulsen, and Andreas Schramm Department of Biological Sciences, Microbiology, University of Aarhus, Denmark Denitrification, the sequential...... reduction of nitrate to dinitrogen gas, is essential for the removal of fixed nitrogen from natural and engineered ecosystems. However, community structure and activity dynamics of denitrifying bacteria in most systems are poorly understood, partially due to difficulties in identifying and quantifying...... and catalyzed fluorescent reporter deposition (CARD-FISH). The general feasibility of the approach was first tested with pure cultures of Pseudomonas stutzeri and various denitrifying and nitrate-reducing isolates. Detailed studies of probe specificity and hybridization conditions using Clone-FISH of nar...

  12. A three-hybrid system to probe in vivo protein-protein interactions: application to the essential proteins of the RD1 complex of M. tuberculosis.

    Directory of Open Access Journals (Sweden)

    Megha Tharad

    Full Text Available BACKGROUND: Protein-protein interactions play a crucial role in enabling a pathogen to survive within a host. In many cases the interactions involve a complex of proteins rather than just two given proteins. This is especially true for pathogens like M. tuberculosis that are able to successfully survive the inhospitable environment of the macrophage. Studying such interactions in detail may help in developing small molecules that either disrupt or augment the interactions. Here, we describe the development of an E. coli based bacterial three-hybrid system that can be used effectively to study ternary protein complexes. METHODOLOGY/PRINCIPAL FINDINGS: The protein-protein interactions involved in M. tuberculosis pathogenesis have been used as a model for the validation of the three-hybrid system. Using the M. tuberculosis RD1 encoded proteins CFP10, ESAT6 and Rv3871 for our proof-of-concept studies, we show that the interaction between the proteins CFP10 and Rv3871 is strengthened and stabilized in the presence of ESAT6, the known heterodimeric partner of CFP10. Isolating peptide candidates that can disrupt crucial protein-protein interactions is another application that the system offers. We demonstrate this by using CFP10 protein as a disruptor of a previously established interaction between ESAT6 and a small peptide HCL1; at the same time we also show that CFP10 is not able to disrupt the strong interaction between ESAT6 and another peptide SL3. CONCLUSIONS/SIGNIFICANCE: The validation of the three-hybrid system paves the way for finding new peptides that are stronger binders of ESAT6 compared even to its natural partner CFP10. Additionally, we believe that the system offers an opportunity to study tri-protein complexes and also perform a screening of protein/peptide binders to known interacting proteins so as to elucidate novel tri-protein complexes.

  13. Multicolor FISHs for simultaneous detection of genes and DNA segments on human chromosomes.

    Science.gov (United States)

    Shimizu, Nobuyoshi; Maekawa, Masahiko; Asai, Satoko; Shimizu, Yoshiko

    2015-12-01

    We have developed a convenient multicolor fluorescent in situ hybridization (FISH) (five-, four-, three-, and two-color FISHs) for detecting specific genes/DNA segments on the human chromosomes. As a foundation of multicolor FISH, we first isolated 80 bacterial artificial chromosome (BAC) probes that specifically detect the peri-centromeres (peri-CEN) and subtelomeres (subTEL) of 24 different human chromosomes (nos. 1~22, X, and Y) by screening our homemade BAC library (Keio BAC library) consisting of 200,000 clones. Five-color FISH was performed using human DNA segments specific for peri-CEN or subTEL, which were labeled with five different fluorescent dyes [7-diethylaminocoumarin (DEAC): blue, fluorescein isothiocyanate (FITC): green, rhodamine 6G (R6G): yellow, TexRed: red, and cyanine5 (Cy5): purple]. To observe FISH signals under a fluorescence microscope, five optic filters were carefully chosen to avoid overlapping fluorescence emission. Five-color FISH and four-color FISH enabled us to accurately examine the numerical anomaly of human chromosomes. Three-color FISH using two specific BAC clones, that distinguish 5' half of oncogene epidermal growth factor receptor (EGFR) from its 3' half, revealed the amplification and truncation of EGFR in EGFR-overproducing cancer cells. Moreover, two-color FISH readily detected a fusion gene in leukemia cells such as breakpoint cluster region (BCR)/Abelson murine leukemia viral oncogene homologue (ABL) on the Philadelphia (Ph') chromosome with interchromosomal translocation. Some other successful cases such as trisomy 21 of Down syndrome are presented. Potential applications of multicolor FISH will be discussed.

  14. Annual trends in catchability and fish stock assessments

    OpenAIRE

    2003-01-01

    A key assumption of many fish stock assessment models is that catchability is constant over time. We assume here that trends in catchability may occur through fishing power creeping. The tuning fleets, which are prone to fishing power development, may be identified using the "Hybrid" method. A range of catchability trends, including values derived from the "Hybrid" method, is then implemented to standardise the fishing effort of some tuning fleets used in the stock assessments performed by XS...

  15. Hybrid opto-chemical doping in Ag nanoparticle-decorated monolayer graphene grown by chemical vapor deposition probed by Raman spectroscopy

    Science.gov (United States)

    Maiti, R.; Haldar, S.; Majumdar, D.; Singha, A.; Ray, S. K.

    2017-02-01

    The novel opto-chemical doping effect in Ag nanoparticle-decorated monolayer graphene grown by chemical vapor deposition has been investigated using Raman spectroscopy for the first time. We used both noble metal nanoparticles and optical excitation, in a hybrid opto-chemical route, to tune the doping level in graphene. Metal nanoparticle-induced chemical effects and laser power-induced substrate effects alter the doping nature of graphene from p- to n-type. Compared with earlier studies, the proposed method significantly lowers the laser intensity required for optical power-dependent doping, resulting in prevention of damage to the sample due to local heating. Some other interesting observations are the enhanced peak intensity in the Raman spectrum of graphene, enhancement of the D-band intensity and the introduction of G-band splitting. This novel, cheap and easily implemented hybrid optical-chemical doping strategy could be very useful for tuning graphene plasmons on the widely used Si/SiO2 substrates for various photonic device applications.

  16. Subtelomeric rearrangements in Indian children with idiopathic intellectual disability/developmental delay: Frequency estimation & clinical correlation using fluorescence in situ hybridization (FISH)

    Science.gov (United States)

    Mohan, Shruthi; Koshy, Teena; Vekatachalam, Perumal; Nampoothiri, Sheela; Yesodharan, Dhanya; Gowrishankar, Kalpana; Kumar, Jeevan; Ravichandran, Latha; Joseph, Santhosh; Chandrasekaran, Anupama; Paul, Solomon F. D.

    2016-01-01

    Background & objectives: Subtelomeres are prone to deleterious rearrangements owing to their proximity to unique sequences on the one end and telomeric repetitive sequences, which increase their tendency to recombine, on the other end. These subtelomeric rearrangements resulting in segmental aneusomy are reported to contribute to the aetiology of idiopathic intellectual disability/developmental delay (ID/DD). We undertook this study to estimate the frequency of subtelomeric rearrangements in children with ID/DD. Methods: One hundred and twenty seven children with idiopathic ID/DD were tested for subtelomeric rearrangements using karyotyping and FISH. Blood samples were cultured, harvested, fixed and GTG-banded using the standard protocols. Results: Rearrangements involving the subtelomeres were observed in 7.8 per cent of the tested samples. Detection of rearrangements visible at the resolution of the karyotype constituted 2.3 per cent, while those rearrangements detected only with FISH constituted 5.5 per cent. Five deletions and five unbalanced translocations were detected. Analysis of parental samples wherever possible was informative regarding the inheritance of the rearrangement. Interpretation & conclusions: The frequency of subtelomeric rearrangements observed in this study was within the reported range of 0-35 per cent. All abnormal genotypes were clinically correlated. Further analysis with array technologies presents a future prospect. Our results suggest the need to test individuals with ID/DD for subtelomeric rearrangements using sensitive methods such as FISH. PMID:27934799

  17. Subtelomeric rearrangements in Indian children with idiopathic intellectual disability/developmental delay: Frequency estimation & clinical correlation using fluorescence in situ hybridization (FISH

    Directory of Open Access Journals (Sweden)

    Shruthi Mohan

    2016-01-01

    Full Text Available Background & objectives: Subtelomeres are prone to deleterious rearrangements owing to their proximity to unique sequences on the one end and telomeric repetitive sequences, which increase their tendency to recombine, on the other end. These subtelomeric rearrangements resulting in segmental aneusomy are reported to contribute to the aetiology of idiopathic intellectual disability/developmental delay (ID/DD. We undertook this study to estimate the frequency of subtelomeric rearrangements in children with ID/DD. Methods: One hundred and twenty seven children with idiopathic ID/DD were tested for subtelomeric rearrangements using karyotyping and FISH. Blood samples were cultured, harvested, fixed and GTG-banded using the standard protocols. Results: Rearrangements involving the subtelomeres were observed in 7.8 per cent of the tested samples. Detection of rearrangements visible at the resolution of the karyotype constituted 2.3 per cent, while those rearrangements detected only with FISH constituted 5.5 per cent. Five deletions and five unbalanced translocations were detected. Analysis of parental samples wherever possible was informative regarding the inheritance of the rearrangement. Interpretation & conclusions: The frequency of subtelomeric rearrangements observed in this study was within the reported range of 0-35 per cent. All abnormal genotypes were clinically correlated. Further analysis with array technologies presents a future prospect. Our results suggest the need to test individuals with ID/DD for subtelomeric rearrangements using sensitive methods such as FISH.

  18. DNA probe for lactobacillus delbrueckii

    Energy Technology Data Exchange (ETDEWEB)

    Delley, M.; Mollet, B.; Hottinger, H. (Nestle Research Centre, Lausanne (Switzerland))

    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.

  19. RNA Imaging with Multiplexed Error Robust Fluorescence in situ Hybridization

    Science.gov (United States)

    Moffitt, Jeffrey R.; Zhuang, Xiaowei

    2016-01-01

    Quantitative measurements of both the copy number and spatial distribution of large fractions of the transcriptome in single-cells could revolutionize our understanding of a variety of cellular and tissue behaviors in both healthy and diseased states. Single-molecule Fluorescence In Situ Hybridization (smFISH)—an approach where individual RNAs are labeled with fluorescent probes and imaged in their native cellular and tissue context—provides both the copy number and spatial context of RNAs but has been limited in the number of RNA species that can be measured simultaneously. Here we describe Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH), a massively parallelized form of smFISH that can image and identify hundreds to thousands of different RNA species simultaneously with high accuracy in individual cells in their native spatial context. We provide detailed protocols on all aspects of MERFISH, including probe design, data collection, and data analysis to allow interested laboratories to perform MERFISH measurements themselves. PMID:27241748

  20. Fish allergy and fish allergens

    DEFF Research Database (Denmark)

    Kuehn, A; Hilger, Christiane; Ollert, Markus

    2016-01-01

    but patients with this phenotype constitute an important sub-group among fish-allergic individuals. 2. Newly identified fish allergens, enolases, aldolases, and fish gelatin, are of high relevance as the majority of the fish-allergic individuals seem to develop specific IgE against these proteins. The present...

  1. Toward quality assurance for metaphase FISH: A multi-center experience

    Energy Technology Data Exchange (ETDEWEB)

    Dewald, G. [Mayo Clinic, Rochester, NY (United States); Higgins, R. [Abbott Northwestern Hospital, Minneapolis, MN (United States); Hirsch, B. [Univ. Minnesota Hospitals and Clinics, Minneapolis, MN (United States)] [and others

    1996-09-06

    Although fluorescent in situ hybridization (FISH) is rapidly becoming a part of clinical cytogenetics, no organization sponsors multi-center determinations of the efficacy of probes. We report on 23 laboratories that volunteered to provide slides and to use a probe for SNRPN and a control locus. Experiences with FISH for these laboratories during 1994 ranged from 0 to 645 utilizations (median = 84) involving blood, amniotic fluid and bone marrow. In an initial study of hybridization efficiency, the median percentage of metaphases from normal individuals showing two SNRPN and 2 control signals for slides prepared at each site was 97.0 (range = 74-100); for slides prepared by a central laboratory, it was 97.8 (range = 81.6-100). In a subsequent blind study, each laboratory attempted to score 5 metaphases from each of 23 specimens [8 with del(15)(q11.2{r_arrow}q12) and 15 with normal 15 chromosomes]. Of 529 challenges, the correct SNRPN pattern was found in 5 of 5 metaphases in 457 (86%) and in 4 of 5 in 33 (6%). Ambiguous, incomplete or no results were reported for 32 (6%) challenges. Seven (1%) diagnostic errors were made including 6 false positives and 1 false negative: 1 laboratory made 3 errors, 1 made 2, and 2 made 1 each. Most errors and inconsistencies seemed due to inexperience with FISH. The working time to process and analyze slides singly averaged 49.5 minutes; slides processed in batches of 4 and analyzed singly required 36.9 minutes. We conclude that proficiency testing for FISH using an extensive array of challenges is possible and that multiple centers can collaborate to test probes and to evaluate costs. 10 refs., 1 fig., 4 tabs.

  2. Prenatal detection of a rec (21),dup q,inv(21)(p11q22) utilizing FISH

    Energy Technology Data Exchange (ETDEWEB)

    Travers, H.; Weinstein, M.E. [Integrated Genetics, Miami, FL (United States); Lamb, A. [Integrated Genetics, Framingham, MA (United States)] [and others

    1994-09-01

    Region-specific probes to chromosomes 13, 18, 21, X, and Y have been used to determine ploidy level in uncultured anmiocytes. The case described here is the first reported instance of an observed trisomic hybridization pattern in a fetus with a recombinant chromosome 21. The 30-year-old G3P010 patient was referred at 13 weeks gestation due to abnormalities seen on ultrasound (cystic hygroma and edema surrounding the head and body). Amniotic fluid was submitted for aneuploidy detection for chromosomes 13, 18, 21, X & Y by FISH in addition to cytogenetic analysis. Molecular analysis with a chromosome-specific 21q22.3 probe showed a hybridization pattern in which 89% of hybridized nuclei had three signals, consistent with trisomy 21. Cytogenetic analysis showed a male karyotype with a modal number of 46; one no. 21 showed an abnormal G-banding pattern in the short arm. Subsequent metaphase analysis using the 21q22.3 probe showed hybridization to two regions on an F-group sized chromosome. Maternal chromosome analysis revealed a 46,XX,inv(21)(p11q22) karyotype. The abnormal chromosome 21 seen in the amniocyte cell represents a recombinant chromosome which has resulted from a meiotic crossover. This interpretion is consistent with the FISH results which indicate the presence of 3 copies of region 21q22.3. FISH analysis in this case was particularly helpful because of the short arms of chromosome no. 21 are highly polymorphic and this rearrangement was fairly subtle.

  3. Using Fluorescence in situ Hybridization to Identify DMD/BMD Deletion Carriers

    Institute of Scientific and Technical Information of China (English)

    Ren-li WANG; Yan-ping XIAO; Xiu-rong JIANG

    2003-01-01

    Objective To identify the deletions in Duchenne/Becker muscular dystrophy (DMD/BMD) by using fluorescence in situ hybridization (FISH) Methods The exon-specific cosmid DNA probes (representing 18 exons) were used to perform one-color FISH on metaphase and interphase preparations. The peripheral blood samples from 9 normal people (4 males and 5 females) and 5 females from independent deletion DMD/BMD families, as well as 2 amniotic fluid specimens and 2 chorionic villus samples (CVS) from normal pregnant females were analyzed.Results 72%~100% of peripheral blood lymphocyte metaphases or interphases, 60%~70% of amniocyte interphases, and 95~99% of chorionic villus cell interphases showed expected signals. One suspected female was identified as deletion carriers and two were excluded.Conclusion FISH in combination with other available techniques allows efficient screening of DMD/BMD deletion carriers, which also lay the ground work for prenatal diagnosis for potential fetal carriers.

  4. Fluorescence in situ hybridization for the identification of Treponema pallidum in tissue sections.

    Science.gov (United States)

    Petrich, Annett; Rojas, Pablo; Schulze, Julia; Loddenkemper, Christoph; Giacani, Lorenzo; Schneider, Thomas; Hertel, Moritz; Kikhney, Judith; Moter, Annette

    2015-10-01

    Syphilis is often called the great imitator because of its frequent atypical clinical manifestations that make the disease difficult to recognize. Because Treponema pallidum subsp. pallidum, the infectious agent of syphilis, is yet uncultivated in vitro, diagnosis is usually made using serology; however, in cases where serology is inconclusive or in patients with immunosuppression where these tests may be difficult to interpret, the availability of a molecular tool for direct diagnosis may be of pivotal importance. Here we present a fluorescence in situ hybridization (FISH) assay that simultaneously identifies and analyzes spatial distribution of T. pallidum in histological tissue sections. For this assay the species-specific FISH probe TPALL targeting the 16S rRNA of T. pallidum was designed in silico and evaluated using T. pallidum infected rabbit testicular tissue and a panel of non-syphilis spirochetes as positive and negative controls, respectively, before application to samples from four syphilis-patients. In a HIV positive patient, FISH showed the presence of T. pallidum in inguinal lymph node tissue. In a patient not suspected to suffer from syphilis but underwent surgery for phimosis, numerous T. pallidum cells were found in preputial tissue. In two cases with oral involvement, FISH was able to differentiate T. pallidum from oral treponemes and showed infection of the oral mucosa and tonsils, respectively. The TPALL FISH probe is now readily available for in situ identification of T. pallidum in selected clinical samples as well as T. pallidum research applications and animal models.

  5. Peptide Nucleic Acid-Fluorescence In Situ Hybridization for Detection of Staphylococci From Endophthalmitis Isolates: A Proof-of-Concept Study.

    Science.gov (United States)

    Patel, Nimesh; Miller, Darlene; Relhan, Nidhi; Flynn, Harry W

    2017-08-01

    Rapid identification of pathogens causing endophthalmitis may improve treatment outcomes through early administration of species-specific medication. The current study reports a new molecular application of peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) with Staphylococcus-specific molecular PNA probes for the potential rapid detection of common pathogens causing endophthalmitis. An experimental study was designed to evaluate the proof of concept at the microbiology laboratory of the Bascom Palmer Eye Institute. Stored culture-positive staphylococci endophthalmitis isolates obtained from prior vitreous samples (n = 15), along with broth as negative controls (n = 5) were used. Inoculum was prepared to a final concentration of 1 × 105 colony-forming units/mL to ensure that the isolates were viable. Smears of samples were fixed and hybridized using QuickFISH protocol with probes for Staphylococcus. With PNA-FISH technique, Staphylococcus aureus was identified in 9 of 10 samples and coagulase-negative staphylococci were identified in 10 of 10 samples. Detection time was 20 minutes. This study serves a proof of concept using a new microbial detection system with FISH probes, and may have the potential for clinical use in the rapid and accurate identification of isolates from patients with endophthalmitis.

  6. Novel karyotype in the Ullrich-Turner syndrome - 45,X/46,X,r(X)/46,X,dic(X) - investigated with fluorescence in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Robson, L.; Jackson, J.; Cowell, C.; Sillence, D.; Smith, A. [Children`s Hospital, Camperdown (Australia)

    1994-04-15

    A 10-year-old girl with Ullrich-Turner syndrome was found to have the novel karyotype 45,X/46,X,r(X)(p11q11)/46,X,dic(X)(p11). Fluorescence in situ hybridization (FISH) with the {alpha} satellite X centromere probe established the origin of the small ring chromosome. Scanning a large number of cells by interphase FISH showed that the dicentric (X) was the least prevalent cell line. The common breakpoint of Xp11 suggests a sequence of errors as the mechanism whereby these 3 distinct cell lines have arisen. 11 refs., 4 figs., 1 tab.

  7. New insights on the origin of B chromosomes in Astyanax scabripinnis obtained by chromosome painting and FISH.

    Science.gov (United States)

    Vicari, Marcelo Ricardo; de Mello Pistune, Helena Flávia; Castro, Jonathan Pena; de Almeida, Mara Cristina; Bertollo, Luiz Antonio Carlos; Moreira-Filho, Orlando; Camacho, Juan Pedro M; Artoni, Roberto Ferreira

    2011-08-01

    Chromosome painting (CP) with a probe of B chromosome obtained by microdissection and fluorescence in situ hybridization (FISH) with probes of As51 satellite DNA, C( o )t-1 DNA, and 18S and 5S rDNA confirmed sharing of some repetitive DNA but not rDNA between A and B chromosomes in the fish Astyanax scabripinnis. Meiotic analysis revealed a pachytene B chromosome bivalent nearly half the size of its mitotic configuration, suggesting a self-pairing of B chromosome arms. Such an isochromosome nature of somatic B chromosome was further evidenced by CP and FISH. All the findings obtained suggest (i) intraspecific origin of B chromosome, and (ii) evolutionary enrichment of repetitive DNA classes, especially those contained in the C( o )t-1 and the As51 probes, in B chromosome. However, the precise origin of B chromosome in the present species remains to be elucidated by further molecular cytogenetic analysis because of painting of some A chromosome regions with the B chromosome-derived probe.

  8. Probing of Ehrlich ascites carcinoma cell using in situ aggregates of Au-NPs as SERS label created by plasmon exciting hybrid- TEM*11 laser mode

    Science.gov (United States)

    Kumar, R.; Mehta, D. S.; Saraswati, S.; Shakher, C.

    2012-02-01

    Apart from commonly employed target-specific labeling/adsorption of antibodies over Au-NPs surface for the creation of localized aggregates, an alternative approach using optical tweezers (OT) driven by hybrid-TEM*11 mode has been devised and exploited for in vitro detection of Ehrlich ascites carcinoma cells (EAC) relying on enhanced scattering. Intra-cavity generated spatially featured asymmetric (SFA) laser beam (λ = 532 nm) has effected simultaneous trapping of mice-EAC cells and in-situ crowd/assembly of incubated Au-NPs/small gold nano-aggregates (created from two or more individual Au-NPs). Relatively larger focus spot created by tightly focused SFA beam than frequently employed Gaussian-mode in OT has offered an extended working area and hence dilute heating has taken care of EAC cells. GNA improves significantly the sensitivity of diagnostics relying on scattered light and the safety and efficacy of therapeutic nanotechnologies for the diseases of cancer and vascular system in medicine.

  9. PEGylated hybrid ytterbia nanoparticles as high-performance diagnostic probes for in vivo magnetic resonance and X-ray computed tomography imaging with low systemic toxicity.

    Science.gov (United States)

    Liu, Zhen; Pu, Fang; Liu, Jianhua; Jiang, Liyan; Yuan, Qinghai; Li, Zhengqiang; Ren, Jinsong; Qu, Xiaogang

    2013-05-21

    Novel nanoparticulate contrast agents with low systemic toxicity and inexpensive character have exhibited more advantages over routinely used small molecular contrast agents for the diagnosis and prognosis of disease. Herein, we designed and synthesized PEGylated hybrid ytterbia nanoparticles as high-performance nanoprobes for X-ray computed tomography (CT) imaging and magnetic resonance (MR) imaging both in vitro and in vivo. These well-defined nanoparticles were facile to prepare and cost-effective, meeting the criteria as a biomedical material. Compared with routinely used Iobitridol in clinic, our PEG-Yb2O3:Gd nanoparticles could provide much significantly enhanced contrast upon various clinical voltages ranging from 80 kVp to 140 kVp owing to the high atomic number and well-positioned K-edge energy of ytterbium. By the doping of gadolinium, our nanoparticulate contrast agent could perform perfect MR imaging simultaneously, revealing similar organ enrichment and bio-distribution with the CT imaging results. The super improvement in imaging efficiency was mainly attributed to the high content of Yb and Gd in a single nanoparticle, thus making these nanoparticles suitable for dual-modal diagnostic imaging with a low single-injection dose. In addition, detailed toxicological study in vitro and in vivo indicated that uniformly sized PEG-Yb2O3:Gd nanoparticles possessed excellent biocompatibility and revealed overall safety.

  10. Design and analysis of mismatch probes for long oligonucleotide microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Ye; He, Zhili; Van Nostrand, Joy D.; Zhou, Jizhong

    2008-08-15

    Nonspecific hybridization is currently a major concern with microarray technology. One of most effective approaches to estimating nonspecific hybridizations in oligonucleotide microarrays is the utilization of mismatch probes; however, this approach has not been used for longer oligonucleotide probes. Here, an oligonucleotide microarray was constructed to evaluate and optimize parameters for 50-mer mismatch probe design. A perfect match (PM) and 28 mismatch (MM) probes were designed for each of ten target genes selected from three microorganisms. The microarrays were hybridized with synthesized complementary oligonucleotide targets at different temperatures (e.g., 42, 45 and 50 C). In general, the probes with evenly distributed mismatches were more distinguishable than those with randomly distributed mismatches. MM probes with 3, 4 and 5 mismatched nucleotides were differentiated for 50-mer oligonucleotide probes hybridized at 50, 45 and 42 C, respectively. Based on the experimental data generated from this study, a modified positional dependent nearest neighbor (MPDNN) model was constructed to adjust the thermodynamic parameters of matched and mismatched dimer nucleotides in the microarray environment. The MM probes with four flexible positional mismatches were designed using the newly established MPDNN model and the experimental results demonstrated that the redesigned MM probes could yield more consistent hybridizations. Conclusions: This study provides guidance on the design of MM probes for long oligonucleotides (e.g., 50 mers). The novel MPDNN model has improved the consistency for long MM probes, and this modeling method can potentially be used for the prediction of oligonucleotide microarray hybridizations.

  11. Design and analysis of mismatch probes for long oligonucleotide microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Deng, Ye; He, Zhili; Van Nostrand, Joy D.; Zhou, Jizhong

    2008-08-15

    Nonspecific hybridization is currently a major concern with microarray technology. One of most effective approaches to estimating nonspecific hybridizations in oligonucleotide microarrays is the utilization of mismatch probes; however, this approach has not been used for longer oligonucleotide probes. Here, an oligonucleotide microarray was constructed to evaluate and optimize parameters for 50-mer mismatch probe design. A perfect match (PM) and 28 mismatch (MM) probes were designed for each of ten target genes selected from three microorganisms. The microarrays were hybridized with synthesized complementary oligonucleotide targets at different temperatures (e.g., 42, 45 and 50 C). In general, the probes with evenly distributed mismatches were more distinguishable than those with randomly distributed mismatches. MM probes with 3, 4 and 5 mismatched nucleotides were differentiated for 50-mer oligonucleotide probes hybridized at 50, 45 and 42 C, respectively. Based on the experimental data generated from this study, a modified positional dependent nearest neighbor (MPDNN) model was constructed to adjust the thermodynamic parameters of matched and mismatched dimer nucleotides in the microarray environment. The MM probes with four flexible positional mismatches were designed using the newly established MPDNN model and the experimental results demonstrated that the redesigned MM probes could yield more consistent hybridizations. Conclusions: This study provides guidance on the design of MM probes for long oligonucleotides (e.g., 50 mers). The novel MPDNN model has improved the consistency for long MM probes, and this modeling method can potentially be used for the prediction of oligonucleotide microarray hybridizations.

  12. Antarctic Fishes.

    Science.gov (United States)

    Eastman, Joseph T.; DeVries, Arthur L.

    1986-01-01

    Explains the adaptations to Antarctic waters that Notothenioidei, a group of advanced bony fishes, have exhibited. Discusses the fishes' mechanisms of production of antifreeze properties and their capacities for neutral buoyancy in water. (ML)

  13. Probing the interfaces between the social sciences and social-ecological resilience: insights from integrative and hybrid perspectives in the social sciences

    Directory of Open Access Journals (Sweden)

    Samantha Stone-Jovicich

    2015-06-01

    Full Text Available Social scientists, and scholars in related interdisciplinary fields, have critiqued resilience thinking's oversimplification of social dimensions of coupled social-ecological systems. Resilience scholars have countered with "where is the ecology" in social analyses? My aim is to contribute to current efforts to strengthen inter- and transdisciplinary debate and inquiry between the social-ecological resilience community and the social sciences. I synthesize three social science perspectives, which stress the complex, dynamic, and multiscalar interconnections between the biophysical and social realms in explaining social-environmental change, and which place both the social and ecology centre stage in their analyses: materio-spatial world systems analysis, critical realist political ecology, and actor-network theory. By integrating, in a nondeterministic and nonessentialist manner, the biophysical environment into social inquiries (integrative approaches or by altogether abolishing the ecology/nature and human/culture divide (hybrid perspectives, these three social-science perspectives are well placed to foster stronger inter- and transdisciplinary ties with social-ecological resilience. Materio-spatial world systems analysis is highly compatible with resilience thinking. The emphasis on world systems structures and processes offers the potential to enrich resilience analyses of global environmental change, global governance and stewardship, planetary boundaries, and multiscale resilience. Critical realist political ecology offers avenues for more in-depth interdisciplinary inquiries around local/traditional/indigenous knowledge systems and power. It also challenges resilience scholars to incorporate critical analyses of resilience's core concepts and practices. Actor-network theory proposes a very different starting point for understanding and assessing social-ecological resilience. Its focus on "resilience-in-the-making" offers unique insights

  14. Fish Dishes.

    Science.gov (United States)

    Derby, Marie

    2003-01-01

    Describes an art project that was inspired by Greek pottery, specifically dishes shaped as fish. Explains that fourth-grade students drew a fish shape that was later used to create their clay version of the fish. Discusses how the students examined the pottery to make decisions about color and design. (CMK)

  15. Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes.

    Science.gov (United States)

    Burggraf, S; Mayer, T; Amann, R; Schadhauser, S; Woese, C R; Stetter, K O

    1994-09-01

    Two 16S rRNA-targeted oligonucleotide probes were designed for the archaeal kingdoms Euryachaeota and Crenarchaeota. Probe specificities were evaluated by nonradioactive dot blot hybridization against selected reference organisms. The successful application of fluorescent-probe derivatives for whole-cell hybridization required organism-specific optimizations of fixation and hybridization conditions to assure probe penetration and morphological integrity of the cells. The probes allowed preliminary grouping of three new hyperthermophilic isolates. Together with other group-specific rRNA-targeted oligonucleotide probes, these probes will facilitate rapid in situ monitoring of the populations present in hydrothermal systems and support cultivation attempts.

  16. Common descent of B chromosomes in two species of the fish genus Prochilodus (Characiformes, Prochilodontidae).

    Science.gov (United States)

    Voltolin, T A; Pansonato Alves, J C; Senhorini, J A; Foresti, F; Camacho, J P M; Porto-Foresti, F

    2013-01-01

    To ascertain the origin of B chromosomes in 2 fish species of the genus Prochilodus, i.e. P. lineatus and P. nigricans, we microdissected them and generated B-specific DNA probes. These probes were used to perform chromosome painting in both species and in 3 further ones belonging to the same genus (P. argenteus, P. brevis and P. costatus). Both probes hybridized with the B chromosomes in P. lineatus and P. nigricans, but with none of the chromosomes in the 5 species. This indicates that the B chromosomes have low similarity with DNAs located in the A chromosomes and suggests the possibility that the B chromosomes in the 2 species have a common origin. The most parsimonious explanation would imply intergeneric hybridization in an ancestor of P. lineatus and P. nigricans yielding the B chromosome as a byproduct, which remained in these 2 species after their phylogenetic origin, but was perhaps lost in other Prochilodus species. This hypothesis predicts that B chromosomes are old genomic elements in this genus, and this could be tested once a species from a relative genus would be found showing homology of its A chromosomes with the B-probes employed here, through a comparison of B chromosome DNA sequences with those in the A chromosomes of this other species.

  17. Fluorescence In Situ Hybridization for MicroRNA Detection in Archived Oral Cancer Tissues

    Directory of Open Access Journals (Sweden)

    Zonggao Shi

    2012-01-01

    Full Text Available The noncoding RNA designated as microRNA (miRNA is a large group of small single-stranded regulatory RNA and has generated wide-spread interest in human disease studies. To facilitate delineating the role of microRNAs in cancer pathology, we sought to explore the feasibility of detecting microRNA expression in formalin-fixed paraffin-embedded (FFPE tissues. Using FFPE materials, we have compared fluorescent in situ hybridization (FISH procedures to detect miR-146a with (a different synthetic probes: regular custom DNA oligonucleotides versus locked nucleic acid (LNA incorporated DNA oligonucleotides; (b different reporters for the probes: biotin versus digoxigenin (DIG; (c different visualization: traditional versus tyramide signal amplification (TSA system; (d different blocking reagents for endogenous peroxidase. Finally, we performed miR-146a FISH on a commercially available oral cancer tissue microarray, which contains 40 cases of oral squamous cell carcinoma (OSCC and 10 cases of normal epithelia from the human oral cavity. A sample FISH protocol for detecting miR-146a is provided. In summary, we have established reliable in situ hybridization procedures for detecting the expression of microRNA in FFPE oral cancer tissues. This method is an important tool for studies on the involvement of microRNA in oral cancer pathology and may have potential prognostic or diagnostic value.

  18. Probe Storage

    NARCIS (Netherlands)

    Gemelli, Marcellino; Abelmann, Leon; Engelen, Johan B.C.; Khatib, Mohammed G.; Koelmans, Wabe W.; Zaboronski, Olog; Campardo, Giovanni; Tiziani, Federico; Laculo, Massimo

    2011-01-01

    This chapter gives an overview of probe-based data storage research over the last three decades, encompassing all aspects of a probe recording system. Following the division found in all mechanically addressed storage systems, the different subsystems (media, read/write heads, positioning, data chan

  19. Cultural probes

    DEFF Research Database (Denmark)

    Madsen, Jacob Østergaard

    2016-01-01

    The aim of this study was thus to explore cultural probes (Gaver, Boucher et al. 2004), as a possible methodical approach, supporting knowledge production on situated and contextual aspects of occupation.......The aim of this study was thus to explore cultural probes (Gaver, Boucher et al. 2004), as a possible methodical approach, supporting knowledge production on situated and contextual aspects of occupation....

  20. Rapid Detection of Starved Escherichia coli with Respiratory Activity in Potable Water by Signal-Amplified in situ Hybridization Following Formazan Reduction.

    Science.gov (United States)

    Yamaguchi, Nobuyasu; Sasada, Makoto; Nasu, Masao

    2009-01-01

    The aim of this study was to develop a rapid method for the specific detection of respiring Escherichia coli (an indicator of fecal contamination) in potable water. Fluorescence in situ hybridization (FISH) with a rRNA-targeted oligonucleotide probe was used to detect E. coli cells and bacterial respiratory activity was estimated using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC). Fluorescent signals from hybridized cells were increased by optimized tyramide signal amplification (TSA). Respiring E. coli in potable ground water with low rRNA content were enumerated within 8 hours using signal-amplified in situ hybridization following formazan reduction (TSA-CTC-FISH), whereas these starved E. coli cells could not be detected by conventional FISH (FISH without signal amplification) which generated weak fluorescence. TSA-CTC-FISH can be used for simultaneous identification in situ based on phylogenetic information and the activity of individual bacterial cells in potable water. This method would be useful in the rapid monitoring of harmful or fecal indicator bacteria in potable water.

  1. Photonics at the frontiers. Generation of few-cycle light pulses via NOPCPA and real-time probing of charge transfer in hybrid photovoltaics

    Energy Technology Data Exchange (ETDEWEB)

    Herrmann, Daniel

    2011-11-11

    this thesis for the first time succeeded to resolve the photoinduced charge-transfer in the conjugate polymer polythiophene and in hybrid polythiophene/silicon solar cells in real time. Thereby a controverse debate about the nature of the primary photoexcitation in organic semiconductors is resolved: Excitons dissociate with 140 fs time constant to polarons (charge carriers). Deciding parameters (for instance structural order, charge-carrier mobility) for the efficiency of the generation and extraction of free charge carriers can be determined. Further ultrashort-time experiments at novel organic solar cells have here been begun and indicated.

  2. 应用FISH-AFLP技术分析平欧杂种榛主栽品种的遗传关系%Analysis of the Genetic Relationship of the Main Cultivars of Ping’ou Hybrid Hazelnut (C. heterophylla×C. avellana) by FISH-AFLP Markers

    Institute of Scientific and Technical Information of China (English)

    马庆华; 陈新; 赵天田; 刘庆忠; 王贵禧

    2013-01-01

    [目的]建立平欧杂种榛FISH-AFLP技术体系,并应用该技术分析平欧杂种榛主栽品种的遗传关系。[方法]以达维等10个主栽品种为试材,经 DNA 提取、PstⅠ/MseⅠ双酶切,进行 FISH-AFLP 反应,数据转换为“0-1”矩阵后,使用NTSYS pc 2.11F和Popgene 1.32软件进行数据分析并作图。[结果]从64对引物中,筛选出15对多态性强的PstⅠ/MseⅠ引物,共获得1739条谱带,引物平均多态带比率97.94%;平欧杂种榛主栽品种的相似性系数为0.7556-0.8543,当阈值为0.8398时,可分成4个AFLP群,其中,玉坠(84-310)、辽榛4号(85-41)和平欧69号(84-69)单独为1群,其他品种为1群;平欧杂种榛主栽品种的有效等位基因数、基因多样度、Shannon信息指数分别为1.3921、0.2482和0.3957,具有较高的遗传多样性;研究获得的特征条带可用于平欧杂种榛主栽品种的快速鉴别。[结论]由于平欧杂种榛的实生选优群体是多亲本组合的混合后代,因此,该群体是一个具有高度遗传多样性的群体,主栽品种间存在复杂的亲缘关系;研究构建的FISH-AFLP技术流程、筛选得到的引物以及分析得到的各项遗传参数,可为其他榛属植物的相关研究提供参考。%[Objective]The objective of this study is to establish FISH-AFLP analysis system for Ping’ou hybrid hazelnut and analyze the genetic relationship of the main cultivars.[Method]Ten main cultivars, such as Dawei, were selected as materials. High-quality genomic DNA was extracted with improved CTAB method, and the purified DNA samples were digested with PstⅠ/MseⅠ. After ligation reaction, the samples were used to perform FISH-AFLP detection. The information of all bands was converted into“0-1”matrix. NTSYS pc 2.11F and Popgene 1.32 software were used to conduct the data analysis and plotting.[Result]The results showed that:15 PstⅠ/MseⅠ primer pairs were selected from 64

  3. Male-to-female sex ratios of abnormalities detected by fluorescence in situ hybridization in a population of chronic lymphocytic leukemia patients

    Directory of Open Access Journals (Sweden)

    Eduardo S. Cantú

    2013-02-01

    Full Text Available Distorted sex ratios occur in hematologic disorders. For example, chronic lymphocytic leukemia (CLL displays disproportionate sex ratios with a large male excess. However, the underlying genetics for these disparities are poorly understood, and gender differences for specific cytogenetic abnormalities have not been carefully investigated. We sought to provide an initial characterization of gender representation in genetic abnormalities in CLL by using fluorescence in situ hybridization (FISH. We confirm the well known skewed male-to-female (M/F sex ratio of ~1.5 in our CLL study population, but also determine the genotypic M/F sex ratio values corresponding to specific FISH DNA probes. Genetic changes in CLL detectable by four FISH probes were statistically compared with respect to gender. Initial FISH evaluations of 4698 CLL patients were retrospectively examined and new findings of the genotypic M/F sex ratios for these probes are reported. This study represents the largest CLL survey conducted in the United States using FISH probes. The CLL database demonstrated that FISH abnormalities (trisomy 12, 13q14.3 deletion and 17p13.1 deletion probes had skewed M/F ratios of ~1.5. Also, by statistical analysis it was shown that ATM gene loss (11q22.3q23.1 deletion solely or with other abnormalities was considerably higher in males with an M/F ratio of 2.5 and significantly different from M/F ratios of 1.0 or 1.5. We hypothesize that interactions involving these autosomal abnormalities (trisomy 12, and deletions of 11q22.3, 13q14.3, and 17p13.1, and the sex chromosomes may provide the genetic basis for the altered phenotypic M/F ratio in CLL.

  4. Detection of illegitimate rearrangement within the immunoglobulin locus on 14q32.3 in B-cell malignancies using end-sequenced probes.

    Science.gov (United States)

    Poulsen, T S; Silahtaroglu, A N; Gisselø, C G; Gaarsdal, E; Rasmussen, T; Tommerup, N; Johnsen, H E

    2001-11-01

    Translocation involving the immunoglobulin heavy chain (IGH) locus is a recurring event in B-cell oncogenesis. The aim of this study was to characterize clones from bacterial artificial chromosome (BAC) libraries and/or bacteriophage P1 artificial chromosome libraries spanning the IGH locus for detection of illegitimate rearrangement within the region by fluorescence in situ hybridization (FISH). In silico analysis of the IGH variable (IGHV) DNA sequence (NT_001716.v1) was performed to identify BAC probes located within the IGHV cluster. Clones of the constant (IGHC) cluster were found in the literature or at http://www.biologia.uniba.it/rmc/. Validation, orientation, and overlap of these probes were confirmed using interphase-, metaphase-, and fiber-FISH. We have identified seven BAC end-sequenced probes (3087C18, 47P23, 76N15, 12F16, 101G24, 112H5, and 151B17) covering 612 kb of the distal IGHV cluster, which, together with probes covering the IGHC cluster (11771 and 998D24), could be used in interphase nuclei and metaphase chromosome analysis. A visual split of the IGHV and IGHC clusters indicating a translocation was analyzed by dual-color FISH in a series of 21 cell lines of different origins. Translocations were found, as expected, in eight of eight myelomas, four of four lymphomas, none of five leukemias, and none of four Epstein-Barr virus-transformed B-lymphoblastoid cell lines. To summarize, we have established a set of IGHV and IGHC probes that can be used for universal screening of illegitimate rearrangement within the IGH locus in B-cell malignancies. These probes allow for routine FISH analysis to detect this early central oncogenic event. Copyright 2001 Wiley-Liss, Inc.

  5. Breaking the host range: mandarin fish is susceptible to a vesiculovirus derived from snakehead fish.

    Science.gov (United States)

    Liu, Xiaodan; Wen, Yi; Hu, Xianqin; Wang, Wenwen; Liang, Xufang; Li, Jun; Vakharia, Vikram; Lin, Li

    2015-04-01

    Members of the genus Vesiculovirus, which belongs to the family Rhabdoviridae, can cause great economic loss in fish culture. In the present report, a vesiculovirus [named snakehead fish vesiculovirus (SHVV)] was isolated from diseased hybrid snakehead fish. SHVV shared 94 % nucleotide sequence identity at the genomic level with Siniperca chuatsi rhabdovirus (SCRV), which infects mandarin fish (S. chuatsi). We showed that SHVV was able to replicate and proliferate well in SSN-1 cells, which originate from striped snakehead fish (Channa striatus). Furthermore, mandarin fish was susceptible to SHVV by bath exposure, as well as by intraperitoneal injection. The infected fish showed typical clinical signs of rhabdovirus infection, including haemorrhage and oedema. Histopathological analysis revealed that extensive inflammation and necrosis were observed in the spleen, kidney, liver, heart and brain of the moribund mandarin fish. These results will shed new light on the epidemic of vesiculovirus infections among fish.

  6. Biphasic Chromatin Structure and FISH Signals Reflect Intranuclear Order

    Directory of Open Access Journals (Sweden)

    Jyoti P. Chaudhuri

    2005-01-01

    Full Text Available Background and Aim: One of the two parental allelic genes may selectively be expressed, regulated by imprinting, X-inactivation or by other less known mechanisms. This study aims to reflect on such genetic mechanisms. Materials and Methods: Slides from short term cultures or direct smears of blood, bone marrow and amniotic fluids were hybridized with FISH probes singly, combined or sequentially. Two to three hundred cells were examined from each preparation. Results and Aignificance: A small number of cells (up to about 5%, more frequent in leukemia cases, showed the twin features: (1 nuclei with biphasic chromatin, one part decondensed and the other condensed; and (2 homologous FISH signals distributed equitably in those two regions. The biphasic chromatin structure with equitable distribution of the homologous FISH signals may correspond to the two sets of chromosomes, supporting observations on ploidywise intranuclear order. The decondensed chromatin may relate to enhanced transcriptions or advanced replications. Conclusions: Transcriptions of only one of the two parental genomes cause allelic exclusion. Genomes may switch with alternating monoallelic expression of biallelic genes as an efficient genetic mechanism. If genomes fail to switch, allelic exclusion may lead to malignancy. Similarly, a genome-wide monoallelic replication may tilt the balance of heterozygosity resulting in aneusomy, initiating early events in malignant transformation and in predicting cancer mortality.

  7. Species-specific identification of Dekkera/Brettanomyces yeasts by fluorescently labeled DNA probes targeting the 26S rRNA.

    Science.gov (United States)

    Röder, Christoph; König, Helmut; Fröhlich, Jürgen

    2007-09-01

    Sequencing of the complete 26S rRNA genes of all Dekkera/Brettanomyces species colonizing different beverages revealed the potential for a specific primer and probe design to support diagnostic PCR approaches and FISH. By analysis of the complete 26S rRNA genes of all five currently known Dekkera/Brettanomyces species (Dekkera bruxellensis, D. anomala, Brettanomyces custersianus, B. nanus and B. naardenensis), several regions with high nucleotide sequence variability yet distinct from the D1/D2 domains were identified. FISH species-specific probes targeting the 26S rRNA gene's most variable regions were designed. Accessibility of probe targets for hybridization was facilitated by the construction of partially complementary 'side'-labeled probes, based on secondary structure models of the rRNA sequences. The specificity and routine applicability of the FISH-based method for yeast identification were tested by analyzing different wine isolates. Investigation of the prevalence of Dekkera/Brettanomyces yeasts in the German viticultural regions Wonnegau, Nierstein and Bingen (Rhinehesse, Rhineland-Palatinate) resulted in the isolation of 37 D. bruxellensis strains from 291 wine samples.

  8. The extent and position of homoeologous recombination in a distant hybrid of Alstroemeria: a molecular cytogenetic assessment of first generation backcross progenies.

    Science.gov (United States)

    Kamstra, S A; Kuipers, A G; De Jeu, M J; Ramanna, M S; Jacobsen, E

    1999-04-01

    To estimate the extent and position of homoeologous recombination during meiosis in an interspecific hybrid between two distantly related Alstroemeria species, the chromosome constitution of six first generation backcross (BC1) plants was analysed using sequential fluorescent in situ hybridization (FISH) and genomic in situ hybridization (GISH) analysis. Four different probes were used for the FISH analysis: two species-specific and two rDNA probes. The six BC1 plants were obtained from crosses between the hybrid A. aurea x A. inodora with its parent A. inodora. GISH clearly identified all chromosomes of both parental genomes as well as recombinant chromosomes. The sequential GISH and FISH analysis enabled the accurate identification of all individual chromosomes in the BC1 plants, resulting in the construction of detailed karyotypes of the plants. The identification of the recombinant chromosomes provided evidence which chromosomes of the two species are homoeologous. Two of the BC1 plants were aneuploid (2n=2x+1=17) and four triploid (2n=3x=24), indicating that both n and 2n gametes were functional in the F1 hybrid. Using GISH, it was possible to estimate homeologous recombination in two different types of gametes in the F1 hyrid. The positions of the crossover points ranged from highly proximal to distal and the maximum number of crossover points per chromosome arm was three. Compared with the aneuploid plants, the triploid plants (which received 2n gametes) clearly possessed fewer crossovers per chromosome, indicating reduced chromosome pairing/recombination prior to the formation of the 2n gametes. Besides homeologous recombination, evidence was found for the presence of structural rearrangements (inversion and translocation) between the chromosomes of the parental species. The presence of the ancient translocation was confirmed through FISH analysis of mitotic and meiotic chromosomes.

  9. Meiotic behaviour of individual chromosomes in allotriploid Alstroemeria hybrids.

    Science.gov (United States)

    Kamstra, S A; de Jong, J H; Jacobsen, E; Ramanna, M S; Kuipers, A G J

    2004-07-01

    Chromosome association and chiasma formation were studied in pollen mother cells at metaphase I of four allotriplod BC1 plants (2n=3x=24) obtained from the backcross of the hybrid Alstroemeria aurea x A. inodora with its parent A. inodora. We distinguished the chromosomes of both parental species by genomic in situ hybridization (GISH), whereas the individual chromosomes were identified on the basis of their multicolour FISH banding patterns obtained after a second hybridization with two species-specific satellite repeats as probes. All the four BC1 plants possessed two genomes of A. inodora and one of A. aurea. Variable numbers of recombinant chromosomes, resulting from meiotic recombination in the interspecific hybrid, were present in these plants. The homologous A. inodora chromosomes generally formed bivalents, leaving the homoeologous A. aurea chromosomes unassociated. High frequencies of trivalents were observed for the chromosome sets that contained recombinant chromosomes, even when the recombinant segments were small. Chromosome associations in the trivalents were restricted to homologous segments. The implications of the absence of homoeologous chromosome pairing on gamete constitution and prospects for introgression in Alstroemeria are discussed.

  10. Microbial detection in microfluidic devices through dual staining of quantum dots-labeled immunoassay and RNA hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Qing [Division of Environmental Science and Engineering, National University of Singapore, Blk E1A, 07-03, Engineering Drive 2, Singapore 117576 (Singapore); Zhu Liang [Division of Environmental Science and Engineering, National University of Singapore, Blk E1A, 07-03, Engineering Drive 2, Singapore 117576 (Singapore); Feng Hanhua [Institute of Microelectronics (Singapore); Ang, Simon [Department of Electrical Engineering, University of Arkansas, Fayetteville (United States); Chau, F.S. [Department of Mechanical Engineering, National University of Singapore (Singapore); Liu, W.-T. [Division of Environmental Science and Engineering, National University of Singapore, Blk E1A, 07-03, Engineering Drive 2, Singapore 117576 (Singapore)]. E-mail: cveliuwt@nus.edu.sg

    2006-01-18

    This paper reported the development of a microfludic device for the rapid detection of viable and nonviable microbial cells through dual labeling by fluorescent in situ hybridization (FISH) and quantum dots (QDs)-labeled immunofluorescent assay (IFA). The coin sized device consists of a microchannel and filtering pillars (gap = 1-2 {mu}m) and was demonstrated to effectively trap and concentrate microbial cells (i.e. Giardia lamblia). After sample injection, FISH probe solution and QDs-labeled antibody solution were sequentially pumped into the device to accelerate the fluorescent labeling reactions at optimized flow rates (i.e. 1 and 20 {mu}L/min, respectively). After 2 min washing for each assay, the whole process could be finished within 30 min, with minimum consumption of labeling reagents and superior fluorescent signal intensity. The choice of QDs 525 for IFA resulted in bright and stable fluorescent signal, with minimum interference with the Cy3 signal from FISH detection.

  11. Annual trends in catchability and fish stock assessments

    DEFF Research Database (Denmark)

    Marchal, P.; Ulrich, Clara; Korsbrekke, K.;

    2003-01-01

    A key assumption of many fish stock assessment models is that catchability is constant over time. We assume here that trends in catchability may occur through fishing power creeping. The tuning fleets, which are prone to fishing power development, may be identified using the "Hybrid" method...

  12. False positivity of ETV6/RUNX1 detected by FISH in healthy newborns and adults

    DEFF Research Database (Denmark)

    Kusk, Maria Schioldan; Lausten-Thomsen, Ulrik; Andersen, Mette Klarskov

    2014-01-01

    The leukemia-associated ETV6-RUNX1-translocation frequently emerges prenatally. Reverse-transcriptase PCR screening may indicate presence of ETV6-RUNX1 transcripts in random cord blood samples. Subsequent cell enrichment validation finds significantly lower levels than validation applying...... fluorescence in situ hybridization (FISH) (RUNX1-positive dilution series, healthy adults and random cord blood samples. The t(12;21) single fusion extra signal translocation probe and the ETV6 break apart probe...... gave false positive results mimicking ETV6-RUNX1-positive cell levels of 10(-3). This questions the paradigm that 1% of newborns have ETV6-RUNX1-positive cells at levels of 10(-3) to 10(-4)....

  13. Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method

    Science.gov (United States)

    Machado, António; Castro, Joana; Cereija, Tatiana; Almeida, Carina

    2015-01-01

    Bacterial vaginosis (BV) is one of most common vaginal infections. However, its diagnosis by classical methods reveals low specificity. Our goal was to evaluate the accuracy diagnosis of 150 vaginal samples with research gold standard methods and our Peptide Nucleic Acid (PNA) probes by Fluorescence in situ Hybridization (FISH) methodology. Also, we described the first PNA-FISH methodology for BV diagnosis, which provides results in approximately 3 h. The results showed a sensitivity of 84.6% (95% confidence interval (CI), from 64.3 to 95.0%) and a specificity of 97.6% (95% CI [92.6–99.4%]), demonstrating the higher specificity of the PNA-FISH method and showing false positive results in BV diagnosis commonly obtained by the classical methods. This methodology combines the specificity of PNA probes for Lactobacillus species and G. vaginalis visualization and the calculation of the microscopic field by Nugent score, allowing a trustful evaluation of the bacteria present in vaginal microflora and avoiding the occurrence of misleading diagnostics. Therefore, the PNA-FISH methodology represents a valuable alternative for BV diagnosis. PMID:25737820

  14. Spatial Exploration and Characterization of Endozoicomonas spp. Bacteria in Stylophora pistillata Using Fluorescence In Situ Hybridization

    KAUST Repository

    Alsheikh-­Hussain, Areej

    2011-12-12

    Studies of coral-­associated bacterial communities have repeatedly demonstrated that the microbial assemblages of the coral host are highly specific and complex. In particular, bacterial community surveys of scleractinian and soft corals from geographically diverse reefs continually uncover a high abundance of sequences affiliated with the Gammaproteobacteria genus Endozoicomonas. The role of these bacteria within the complex coral holobiont is currently unknown. In order to localize these cells and gain an understanding of their potential interactions within the coral, we developed a fluorescence in situ hybridization(FISH) approach for reef-­building coral tissues. Using a custom small-­subunit ribosomal RNA gene database, we developed two Endozoicomonas-­specific probes that cover almost all known coral-­associated Endozoicomonas sequences. Probe hybridization conditions were quantitatively evaluated against target and non-­target bacterial cultures using fluorescence microscopy. Using these experimentally tested conditions, probes were then hybridized to the branching coral Stylophora pistillata, obtained from the Red Sea, using whole mount and paraffin embedding techniques. This study allowed preliminary spatial exploration and characterization of Endozoicomonas in coral, which has provided insight into their functional role and interactions within the coral holobiont.

  15. Identification of Polyphosphate-Accumulating Organisms and Design of 16S rRNA-Directed Probes for Their Detection and Quantitation

    Science.gov (United States)

    Crocetti, Gregory R.; Hugenholtz, Philip; Bond, Philip L.; Schuler, Andrew; Keller, Jürg; Jenkins, David; Blackall, Linda L.

    2000-01-01

    Laboratory-scale sequencing batch reactors (SBRs) as models for activated sludge processes were used to study enhanced biological phosphorus removal (EBPR) from wastewater. Enrichment for polyphosphate-accumulating organisms (PAOs) was achieved essentially by increasing the phosphorus concentration in the influent to the SBRs. Fluorescence in situ hybridization (FISH) using domain-, division-, and subdivision-level probes was used to assess the proportions of microorganisms in the sludges. The A sludge, a high-performance P-removing sludge containing 15.1% P in the biomass, was comprised of large clusters of polyphosphate-containing coccobacilli. By FISH, >80% of the A sludge bacteria were β-2 Proteobacteria arranged in clusters of coccobacilli, strongly suggesting that this group contains a PAO responsible for EBPR. The second dominant group in the A sludge was the Actinobacteria. Clone libraries of PCR-amplified bacterial 16S rRNA genes from three high-performance P-removing sludges were prepared, and clones belonging to the β-2 Proteobacteria were fully sequenced. A distinctive group of clones (sharing ≥98% sequence identity) related to Rhodocyclus spp. (94 to 97% identity) and Propionibacter pelophilus (95 to 96% identity) was identified as the most likely candidate PAOs. Three probes specific for the highly related candidate PAO group were designed from the sequence data. All three probes specifically bound to the morphologically distinctive clusters of PAOs in the A sludge, exactly coinciding with the β-2 Proteobacteria probe. Sequential FISH and polyphosphate staining of EBPR sludges clearly demonstrated that PAO probe-binding cells contained polyphosphate. Subsequent PAO probe analyses of a number of sludges with various P removal capacities indicated a strong positive correlation between P removal from the wastewater as determined by sludge P content and number of PAO probe-binding cells. We conclude therefore that an important group of PAOs in EBPR

  16. Rapid generation of region-specific probes by chromosome microdissection: Application to the identification of chromosomal rearrangements

    Energy Technology Data Exchange (ETDEWEB)

    Trent, J.M.; Guan, X.Y.; Zang, J.; Meltzer, P.S. (Univ. of Michigan, Ann Arbor (United States))

    1993-01-01

    The authors present results using a novel strategy for chromosome microdissection and direct in vitro amplification of specific chromosomal regions, to identify cryptic chromosome alterations, and to rapidly generate region-specific genomic probes. First, banded chromosomes are microdissected and directly PCR amplified by a procedure which eliminates microchemistry (Meltzer, et al., Nature Genetics, 1:24, 1992). The resulting PCR product can be used for several applications including direct labeling for fluorescent in situ hybridization (FISH) to normal metaphase chromosomes. A second application of this procedure is the extremely rapid generation of chromosome region-specific probes. This approach has been successfully used to determine the derivation of chromosome segments unidentifiable by standard chromosome banding analysis. In selected instances these probes have also been used on interphase nuclei and provides the potential for assessing chromosome abnormalities in a variety of cell lineages. The microdissection probes (which can be generated in <24 hours) have also been utilized in direct library screening and provide the possibility of acquiring a significant number of region-specific probes for any chromosome band. This procedure extends the limits of conventional cytogenetic analysis by providing an extremely rapid source of numerous band-specific probes, and by enabling the direct analysis of essentially any unknown chromosome region.

  17. Improving comparability between microarray probe signals by thermodynamic intensity correction

    DEFF Research Database (Denmark)

    Bruun, G. M.; Wernersson, Rasmus; Juncker, Agnieszka

    2007-01-01

    different probes. It is therefore of great interest to correct for the variation between probes. Much of this variation is sequence dependent. We demonstrate that a thermodynamic model for hybridization of either DNA or RNA to a DNA microarray, which takes the sequence-dependent probe affinities...

  18. Le diagnostic anténatal de la trisomie 21 par l'hybridation in situ en fluorescence (FISH): à propos des premiers tests réalisés au Maroc

    Science.gov (United States)

    Lamzouri, Afaf; Natiq, Abdelhafid; Tajir, Mariam; Sendid, Mohamed; Sefiani, Abdelaziz

    2012-01-01

    Introduction Le but de cette étude était de présenter les premiers résultats de diagnostic anténatal de la trisomie 21 par la technique d'hybridation in situ en fluorescence (FISH) au Maroc et discuter son intérêt dans le diagnostic rapide de cette aneuploïdie. Méthodes Ce travail a été réalisé chez 23 femmes avec des grossesses à haut risque de trisomie 21. La moyenne d’âge des gestantes étaient de 37,43 ans avec des extrêmes de 21 et 43 ans. Toutes étaient musulmanes mariées, mariage légitimé par la Charia, dont trois mariages consanguins, sauf une originaire de la République Démocratique du Congo qui était chrétienne et concubine. La majorité des femmes étaient fonctionnaires et avaient un niveau de scolarisation moyen à élevé. Toutes les patientes ont bénéficié d'une consultation de génétique médicale au cours de laquelle il leur a été donné des informations sur la technique, son intérêt et ses limites. Il s'agit de femmes enceintes qui avaient soit un âge maternel élevé ou des signes d'appel échographiques et/ ou biochimiques. Une des patientes était porteuse d'une translocation robertsonienne t(14;21) équilibrée. Une amniocentèse a été réalisée chez toutes les gestantes et aucun avortement n'a était induit par ce geste invasif. L’âge gestationnel moyen à la première consultation était de 14 semaines d'aménorrhée (SA) et à l'amniocentèse était de 16 SA et 5 jours. L'analyse FISH a été réalisée, après consentement des couples, sur des cellules non cultivées à partir des échantillons de liquides amniotiques, en utilisant des sondes spécifiques du chromosome 21. Résultats Parmi les 23 patientes qui ont bénéficiées d'un diagnostic anténatal de la trisomie 21 par la technique FISH, nous avons pu rassurer 21 d'entre elles, et nous avons détecté deux cas de trisomie 21 fœtal. Conclusion La technique FISH permet un diagnostic anténatal rapide, en moins de 48h, de la trisomie 21 sur

  19. [Clinical value of interphase fluorescent in situ hybridization in diagnosis of core-binding factor acute myelocytic leukemia].

    Science.gov (United States)

    Yang, Hui; Fan, Lei; Qiu, Hai-Rong; Wang, Rong; Zhang, Jian-Fu; Wu, Yu-Jie; Li, Jian-Yong; Liu, Peng

    2011-10-01

    The purpose of this study was to evaluate the clinical value of interphase fluorescence in situ hybridization (I-FISH) in diagnosis of core-binding factor acute myelocytic leukemia (CBF AML). The cytogenetic characteristics in leukemia cells from 82 cases of AML-M(2) and 43 cases of AML-M(4)/M(5) were detected by using I-FISH with AML1-ETO double color double fusion probe and double color break point isolated gene probe CBFβ-MYH11, and the detected results were compared with results detected by conventional cytogenetic R banding technique (CC). The results indicated that AML1-ETO fusion gene was detected in 30.5% cases (25/82) by FISH, and t(8;21)(q22;q22) karyotypic aberrations was found in 28.0% cases (23/82) by CC method. Among 25 FISH positive cases, typical FISH positive signal pattern (1R1G2F) was displayed in 22 cases and atypical signal pattern (1R2G1F and 2R1G2F) was found in the other 3 cases. Among all 43 AML-M(4)/M(5) cases, the CBFβ-MYH11 fusion gene was detected in 23.3% cases (10/43) by FISH, which sensitivity was significant higher than that by CC method (2/43) (p < 0.05). It is concluded that some insufficiency of CC technique can be compensated by FISH, and combination of I-FISH with CC technique play a crucial role in diagnosis of CBF AML and in monitoring of minimal residual disease.

  20. FISHtrees 3.0: Tumor Phylogenetics Using a Ploidy Probe.

    Directory of Open Access Journals (Sweden)

    E Michael Gertz

    Full Text Available Advances in fluorescence in situ hybridization (FISH make it feasible to detect multiple copy-number changes in hundreds of cells of solid tumors. Studies using FISH, sequencing, and other technologies have revealed substantial intra-tumor heterogeneity. The evolution of subclones in tumors may be modeled by phylogenies. Tumors often harbor aneuploid or polyploid cell populations. Using a FISH probe to estimate changes in ploidy can guide the creation of trees that model changes in ploidy and individual gene copy-number variations. We present FISHtrees 3.0, which implements a ploidy-based tree building method based on mixed integer linear programming (MILP. The ploidy-based modeling in FISHtrees includes a new formulation of the problem of merging trees for changes of a single gene into trees modeling changes in multiple genes and the ploidy. When multiple samples are collected from each patient, varying over time or tumor regions, it is useful to evaluate similarities in tumor progression among the samples. Therefore, we further implemented in FISHtrees 3.0 a new method to build consensus graphs for multiple samples. We validate FISHtrees 3.0 on a simulated data and on FISH data from paired cases of cervical primary and metastatic tumors and on paired breast ductal carcinoma in situ (DCIS and invasive ductal carcinoma (IDC. Tests on simulated data show improved accuracy of the ploidy-based approach relative to prior ploidyless methods. Tests on real data further demonstrate novel insights these methods offer into tumor progression processes. Trees for DCIS samples are significantly less complex than trees for paired IDC samples. Consensus graphs show substantial divergence among most paired samples from both sets. Low consensus between DCIS and IDC trees may help explain the difficulty in finding biomarkers that predict which DCIS cases are at most risk to progress to IDC. The FISHtrees software is available at ftp://ftp.ncbi.nih.gov/pub/FISHtrees.

  1. Fish health and fish quality

    DEFF Research Database (Denmark)

    Ingerslev, Hans-Christian

    Aquaculture is an expanding worldwide industry producing an increasing amount of fish every year. The quality of the fish meat is dependent upon many biological and non-biological factors. Infectious diseases are known to cause bleedings and damage of the muscle tissue that may lead to scarring...... are poorly described in fish. The present work in this thesis focused on: 1) examination of potential changes in the quality regarding texture of the muscle tissue in rainbow trout (Oncorhynchus mykiss) after previous infection with the bacterial pathogens Yersinia ruckeri and Vibrio anguillarum; 2.....