Sample records for fluorescence in situ hybridization

  1. Routine fluorescence in situ hybridization in soil

    Microsoft Academic Search

    J. Bertaux; U. Gloger; M. Schmid; A. Hartmann; S. Scheu

    2007-01-01

    The use of fluorescence in situ hybridization (FISH) to identify and enumerate soil bacteria has long been hampered by the autofluorescence of soil particles masking the bacterial signals and because the need of counting hundreds of bacteria in order to achieve statistically reliable data is time consuming. Recently, it was demonstrated that Nycodenz facilitates FISH in soil by concentrating bacteria

  2. Molecular cytogenetics using fluorescence in situ hybridization

    SciTech Connect

    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.

  3. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...Automated fluorescence in situ hybridization (FISH) enumeration systems. 866.4700 ...fluorescence in situ hybridization (FISH) enumeration systems. (a) Identification. An automated FISH enumeration system is a device...

  4. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...Automated fluorescence in situ hybridization (FISH) enumeration systems. 866.4700 ...fluorescence in situ hybridization (FISH) enumeration systems. (a) Identification . An automated FISH enumeration system is a device...

  5. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...Automated fluorescence in situ hybridization (FISH) enumeration systems. 866.4700 ...fluorescence in situ hybridization (FISH) enumeration systems. (a) Identification . An automated FISH enumeration system is a device...

  6. Supernumerary ring chromosome 17 identified by fluorescent in situ hybridization

    SciTech Connect

    Fagan, K. [Hunter Area Pathology Service, New South Wales (Australia)] [Hunter Area Pathology Service, New South Wales (Australia); Edwards, M. [Western Suburbs Hospital, New South Wales (Australia)] [Western Suburbs Hospital, New South Wales (Australia)

    1997-04-14

    We present a patient with multiple anomalies and severe developmental delay. A small supernumerary ring chromosome was found in 40% of her lymphocyte cells at birth. The origin of the marker chromosome could not be determined by GTG banding, but fluorescent in situ hybridization (FISH) later identified the marker as deriving from chromosome 17. 20 refs., 2 figs., 1 tab.

  7. FISH-ing for Genes: Modeling Fluorescence "in situ" Hybridization

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton Buck

    2006-01-01

    Teaching methods of genetic analysis such as fluorescence in situ hybridization (FISH) can be an important part of instructional units in biology, microbiology, and biotechnology. Experience, however, indicates that these topics are difficult for many students. The authors of this article describe how they created an activity that effectively…

  8. Sperm aneuploidy testing using fluorescence in situ hybridization.

    PubMed

    Emery, Benjamin R

    2013-01-01

    Sperm aneuploidy screening has been used as a tool in diagnosis and determining treatment options for male factor infertility since the development of human sperm karyotyping by injection into hamster and mouse oocytes in the 1970s. From these studies and subsequent work with interphase chromosome analysis, at risk populations of men with teratozoospermia, oligozoospermia, and men with translocations, have since been identified. The current technique is an application of fluorescent in situ hybridization (FISH) on interphase sperm nuclei with careful enumeration of the labeled chromosomes to determine sperm ploidy. Typically, five to seven chromosomes are evaluated in individual ejaculates to determine the percent of aneuploid sperm present. This protocol will detail the procedures for: preparation of specimens, exposure of the sperm nuclei to the FISH probes, hybridization, destaining, and scoring criteria. PMID:22992912

  9. Visualization of mitotic chromosomes in filamentous fungi by fluorescence staining and fluorescence in situ hybridization

    Microsoft Academic Search

    Masatoki Taga; Minoru Murata

    1994-01-01

    Mitotic chromosomes of the plant pathogenic filamentous fungi Botrytis cinerea and Alternaria alternata were observed. Chromosomes prepared by the germ tube burst method were stained with the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI) to yield figures with good resolution. Using this method, component chromosomes were clearly distinguished and the chromosome number could be determined. Fluorescence in situ hybridization (FISH) was also successfully

  10. Human cDNA mapping using fluorescence in situ hybridization

    SciTech Connect

    Korenberg, J.R.

    1993-03-04

    Genetic mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach generated 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

  11. Sexing of Dog Sperm by Fluorescence In Situ Hybridization

    PubMed Central

    OI, Maya; YAMADA, Keisuke; HAYAKAWA, Hiroyuki; SUZUKI, Hiroshi

    2012-01-01

    Abstract Effective preselection of sex has been accomplished in several species of livestock and also in humans using the flow cytometric sperm sorting method. A guaranteed high sorting accuracy is a key prerequisite for the widespread use of sperm sexing. The standard validation method is flow cytometric remeasurement of the DNA content of the sexed sperm. Since this method relies on the same instrument that produced the original sperm separation, it is not truly independent. Therefore, to be able to specifically produce either male or female offspring in the dog, we developed a method of direct visualization of sex chromosomes in a single sperm using fluorescence in situ hybridization (FISH) as a validation method. Denaturation of canine spermatozoa by immersion in 1 M NaOH for 4 min yielded consistent hybridization results with over 97% hybridization efficiency and a good preservation of sperm morphology. There was no significant difference between the theoretical ratio (50:50) and the observed ratio of X- and Y-chromosome-bearing spermatozoa in any of the three dogs. In addition, the mean purities of flow-sorted sex chromosomes in spermatozoa of the three dogs were 90.8% for the X chromosome fraction and 89.6% for the Y chromosome fraction. This sorting was evaluated by using the dual color FISH protocol. Therefore, our results demonstrated that the FISH protocol worked reliably for both unsorted and sexed sperm samples. PMID:23059640

  12. Pallister-Killian syndrome detected by fluorescence in situ hybridization

    SciTech Connect

    Butler, M.G.; Dev, V.G. [Genetics Associates, Nashville, TN (United States)

    1995-07-03

    The Pallister-Killian syndrome is a rare cytogenetic condition first described in 1977 by Pallister et al. in 3 adults; the first affected child was reported in 1981. This syndrome (also known as Pallister mosaic aneuploidy syndrome or isochromosome 12p mosaicism) is characterized by postnatal growth retardation, seizures, hypotonia, deafness, profound mental retardation, minimal speech development, and a distinctive facial appearance (high prominent forehead, ocular hypertelorism, sparse anterior scalp hair, prominent lower lip, large ears with thick protruding lobules, cupid-bow shaped upper lip, and a long philtrum). A chromosome 12 abnormality (tetrasomy 12p) has been reported in skin biopsies from these patients but this chromosome anomaly is usually not found (or in only a small proportion, e.g., <0.5%, of blood cells) in peripheral blood. We report on an additional patient with Pallister-Killian syndrome confirmed with fluorescence in situ hybridization (FISH) using an alpha satellite DNA probe for chromosome 12. This report further illustrates the application of FISH in identifying the source of chromosomal markers of unknown origin in infants with multiple congenital anomalies specifically before the natural history of a condition allows for definitive diagnosis based on clinical findings. 9 refs., 2 figs.

  13. Fluorescence in situ hybridization of three oncogenes on a leiomyoma.

    PubMed

    Faruqi, Shamim Ahmad; Saquib, Mohammad; Harsch, Christopher; Noumoff, Joel S

    2012-08-01

    Using fluorescence in situ hybridization technique, expression of three oncogenes, C-myc, RARa, and cyclin-D was tested on a uterine leiomyoma. C-myc and RARa were amplified in approximately 30% and 90% of the cells, respectively. Numerous small signals of C-myc were indicative of the presence of double minutes. Amplification of RARa is being reported for the first time in a leiomyoma. Cyclin-D was normal in diploid cells while it was highly amplified in polyploid cells. Low levels of amplified C-myc and cyclin-D cells seem to be the reason for this tumor to be benign, while RARa could not be effective without the association of some other gene such as PML. Information presented here are significant toward developing new curative strategies such as gene-specific drugs and molecular manipulation to stop the activity of cancer gene. Further study may elucidate that how fibroids grow and maintain their rare benign nature. PMID:22593003

  14. Detection of dengue group viruses by fluorescence in situ hybridization

    PubMed Central

    2012-01-01

    Background Dengue fever (DF) and dengue hemorrhagic fever (DHF) represent a global challenge in public health. It is estimated that 50 to 100 million infections occur each year causing approximately 20,000 deaths that are usually linked to severe cases like DHF and dengue shock syndrome. The causative agent of DF is dengue virus (genus Flavivirus) that comprises four distinct serotypes (DENV-1 to DENV-4). Fluorescence in situ hybridization (FISH) has been used successfully to detect pathogenic agents, but has not been implemented in detecting DENV. To improve our understanding of DENV infection and dissemination in host tissues, we designed specific probes to detect DENV in FISH assays. Methods Oligonucleotide probes were designed to hybridize with RNA from the broadest range of DENV isolates belonging to the four serotypes, but not to the closest Flavivirus genomes. Three probes that fit the criteria defined for FISH experiments were selected, targeting both coding and non-coding regions of the DENV genome. These probes were tested in FISH assays against the dengue vector Aedes albopictus (Diptera: Culicidae). The FISH experiments were led in vitro using the C6/36 cell line, and in vivo against dissected salivary glands, with epifluorescence and confocal microscopy. Results The three 60-nt oligonucleotides probes DENV-Probe A, B and C cover a broad range of DENV isolates from the four serotypes. When the three probes were used together, specific fluorescent signals were observed in C6/36 infected with each DENV serotypes. No signal was detected in either cells infected with close Flavivirus members West Nile virus or yellow fever virus. The same protocol was used on salivary glands of Ae. albopictus fed with a DENV-2 infectious blood-meal which showed positive signals in the lateral lobes of infected samples, with no significant signal in uninfected mosquitoes. Conclusion Based on the FISH technique, we propose a way to design and use oligonucleotide probes to detect arboviruses. Results showed that this method was successfully implemented to specifically detect DENV in a mosquito cell line, as well as in mosquito salivary glands for the DENV-2 serotype. In addition, we emphasize that FISH could be an alternative method to detect arboviruses in host tissues, also offering to circumvent the discontinuity of antibodies used in immunofluorescent assays. PMID:23110979

  15. Visualization of mitotic chromosomes in filamentous fungi by fluorescence staining and fluorescence in situ hybridization.

    PubMed

    Taga, M; Murata, M

    1994-10-01

    Mitotic chromosomes of the plant pathogenic filamentous fungi Botrytis cinerea and Alternaria alternata were observed. Chromosomes prepared by the germ tube burst method were stained with the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI) to yield figures with good resolution. Using this method, component chromosomes were clearly distinguished and the chromosome number could be determined. Fluorescence in situ hybridization (FISH) was also successfully applied to the specimens, revealing one ribosomal RNA gene cluster, or nucleolus organizer region (NOR) in the genome of each fungus. A long attenuated chromatid thread expanding from a condensed metaphase chromosome, which had been called a thread-like structure in B. cinerea, was proved to be an NOR. This is the first report of the successful application of FISH to the chromosomes of filamentous fungi. PMID:7859561

  16. Reliability of chromogenic in situ hybridization for detecting HER2 gene status in breast cancer: comparison with fluorescence in situ hybridization and assessment of interobserver reproducibility

    Microsoft Academic Search

    Yun Gong; Michael Gilcrease; Nour Sneige

    2005-01-01

    Accurate determination of HER-2 status is important in the management of patients with breast cancer, especially in determining their eligibility for trastuzumab therapy. Fluorescence in situ hybridization (FISH) has been regarded as the gold standard method for detecting HER-2 gene amplification. Recently, chromogenic in situ hybridization (CISH), in which HER-2 is detected by a peroxidase reaction and the gene copies

  17. Nature of B chromosomes in the harvest mouse Reithrodontomys megalotis by fluorescence in situ hybridization (FISH)

    Microsoft Academic Search

    John A. Peppers; Lara E. Wiggins; Robert J. Baker

    1997-01-01

    Using fluorescence in situ hybridization, we examined the characteristics of two types of B chromosomes in harvest mice of the genus Reithrodontomys. B chromosomes were interrogated with rDNA, telomeric repeat, LINE element and centromeric heterochromatin probes. The two types of B chromosomes share the following features: (a) telomeres present on the ends of both arms; (b) hybridization to LINE probes;

  18. Enumeration of viable E. coli in rivers and wastewaters by fluorescent in situ hybridization

    Microsoft Academic Search

    Tamara Garcia-Armisen; Pierre Servais

    2004-01-01

    A combination of direct viable count (DVC) and fluorescent in situ hybridization (FISH) procedures was used to enumerate viable Escherichia coli in river waters and wastewaters. A probe specific for the 16S rRNA of E. coli labeled with the CY3 dye was used; enumeration of hybridized cells was performed by epifluorescence microscopy. Data showed that the method was able to

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  20. Aneuploidy detection in human sperm nuclei using fluorescence in situ hybridization

    Microsoft Academic Search

    Janet M. Holmes; Renee H. Martin

    1993-01-01

    Fluorescence in situ hybridization (FISH) was performed on human interphase sperm nuclei to determine the utility of this technique for aneuploidy detection. Repetitive DNA sequences specific for chromosomes 1, 12 and X were biotinylated and hybridized with mature sperm, which had been treated with cetyltrimethylammonium bromide and dithiothreitol to render them accessible to the probes. Detection of bound probe was

  1. Multiple and sensitive fluorescence in situ hybridization with rhodamine-, fluorescein-, and coumarin-labeled DNAs

    Microsoft Academic Search

    J. Wiegant; C. C. Wiesmeijer; J. M. N. Hoovers; E. Schuuring; A. d’Azzo; J. Vrolijk; H. J. Tanke; A. K. Raap

    1993-01-01

    We have tested the use of several newly developed red, green, and blue fluorescent dUTPs in direct, multiple, and sensitive fluorescence in situ hybridization procedures. Among the ones tested, the tetramethylrhodamine-dUTP proved to give the best sensitivity; using conventional epifiuorescence microscopy, cosmids could be visualized with a hybridization efficiency of 90%. Fluorescein-dUTP permitted visual cosmid detection with 50% efficiency, and,

  2. Fluorescent in situ hybridization as an aid to introducing alien genetic variation into wheat

    Microsoft Academic Search

    T. E. Miller; S. M. Reader; K. A. Purdie; S. Abbo; R. P. Dunford; I. P. King

    1995-01-01

    Summary  Fluorescent in situ hybridization (FISH) has been used to assess the occurrence and frequency of wheat-alien chromosome pairing in a wheat\\/Thinopyrum bessarabicum hybrid and in wheat\\/rye hybrids with different levels of chromosome pairing by examining pollen mother cells at metaphase\\u000a I of meiosis. The use of FISH to identify the presence and size of alien chromatin in a wheat background

  3. Fluorescent in situ hybridization for sex chromosome determination before and after fertilization in mice

    Microsoft Academic Search

    J. J. Whyte; R. M. Roberts; C. S. Rosenfeld

    2007-01-01

    In mice, the relative numbers of male and female pups per litter not only can vary but can probably change over the course of pregnancy in response to numerous environmental and physiological factors. As such, a technique is required to determine gender at several developmental stages. Here we describe a robust and accurate fluorescent in situ hybridization (FISH) procedure for

  4. Subsurface methanogenic activity in the Iberian Pyrite Belt detected by fluorescence in situ hybridization

    NASA Astrophysics Data System (ADS)

    Oggerin, M.; Escudero, C.; Rodriguez, N.; Amils, R.

    2014-04-01

    The IPBSL is a drilling project designed to investigate the subsurface geomicrobiology of the Iberian Pyrite Belt (Southwestern, Spain), a geological and mineralogical terrestrial analogue of Mars. Fluorescence in situ hybridization (CARDFISH) has been used to detect the presence of methanogenic archaea in samples from uncontaminated cores of the IPBSL.

  5. SPATIAL DISTRIBUTION OF SPERM-DERIVED CHROMATIN IN ZYGOTES DETERMINED BY FLUORESCENCE IN SITU HYBRIDIZATION

    EPA Science Inventory

    Fluorescence in situ hybridization was used to determine the spatial distribution of chromatin in zygote pronuclei. ybrid system involving golden hamster eggs and individual human sperm permitted use of DNA probes specific for the entire human chromosome 4, for the heterochromati...

  6. Direct Visualization of Propionibacterium acnes in Prostate Tissue by Multicolor Fluorescent In Situ Hybridization Assay

    Microsoft Academic Search

    Oleg A. Alexeyev; Ingrid Marklund; Beverley Shannon; Irina Golovleva; Jan Olsson; Charlotte Andersson; Irene Eriksson; Ronald Cohen; Fredrik Elgh

    2007-01-01

    Prostate tissues from patients with prostate cancer and benign prostatic hyperplasia (BPH) frequently contain histological inflammation, and a proportion of these patients show evidence of Propionibacterium acnes infection in the prostate gland. We developed a multicolor fluorescent in situ hybridization (FISH) assay targeting P. acnes 23S rRNA along with a 14-kb region of the P. acnes genome. This assay was

  7. ERBB2 Amplification in Breast Cancer Analyzed by Fluorescence in situ Hybridization

    Microsoft Academic Search

    Olli-P. Kallioniemi; Anne Kallioniemi; Wayne Kurisu; Ann Thor; Ling-Chun Chen; Helene S. Smith; Frederic M. Waldman; Dan Pinkel; Joe W. Gray

    1992-01-01

    We illustrate the use of fluorescence in situ hybridization (FISH) for analysis of ERBB2 oncogene copy number, the level of amplification (here defined as the ratio of ERBB2 copy number to copy number of chromosome 17 centromeres), and the distribution of amplified genes in breast cancer cell lines and uncultured primary breast carcinomas. The relative ERBB2 copy number determined by

  8. Simultaneous Detection of Multiple Genetic Aberrations in Single Cells by Spectral Fluorescence in Situ Hybridization1

    Microsoft Academic Search

    Marilyn L. Slovak; Lucene Tcheurekdjian; Feiyu F. Zhang; Joyce L. Murata-Collins

    2001-01-01

    Spectral fluorescence in situ hybridization (S-FISH) is a novel molec- ular cytogenetic approach that detects multiple disease-specific chromo- somal aberrations in interphase nuclei using combinatorial fluorescence and digital imaging microscopy. A panel of six centromeric probes for chromosomes 7, 8, 9, 10, X, and Y, using a unique two-dye combination of four fluorophores, was developed to assess ploidy in breast

  9. FISH - (Fluoresence In Situ Hybridization)

    NSDL National Science Digital Library

    Darryl Leja (National Human Genome Research Institute REV)

    2005-04-04

    Fluorescence in situ hybridization (FISH) is a process which vividly paints chromosomes or portions of chromosomes with fluorescent molecules. This technique is useful for identifying chromosomal abnormalities and gene mapping.

  10. Evolution of Chromosome 6 of Solanum Species Revealed by Comparative Fluorescence in Situ Hybridization Mapping

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Comparative genome mapping is an important tool in evolutionary research. Here we demonstrate a comparative fluorescent in situ hybridization (FISH) mapping strategy. A set of 13 bacterial artificial chromosome (BAC) clones derived from potato chromosome 6 was used for FISH mapping in seven differen...

  11. Fluorescence In Situ Hybridization Analysis of Renal Oncocytoma Reveals Frequent Loss of Chromosomes Y and 1

    Microsoft Academic Search

    James A. Brown; Satoru Takahashi; Antonio Alcaraz; Thomas J. Borell; Kari L. Anderl; Junqi Qian; Diane L. Persons; David G. Bostwick; Michael M. Lieber; Robert B. Jenkins

    1996-01-01

    PurposeCytogenetic studies of a small number of renal oncocytomas have indicated that loss of chromosomes 1 and Y may be involved in the pathogenesis of this tumor. To evaluate these observations further we selected paraffin embedded renal oncocytoma specimens from 20 male and 10 female patients for fluorescence in situ hybridization analysis.

  12. De Novo nonreciprocal translocation 1;8 confirmed by fluorescent in situ hybridization

    SciTech Connect

    Wiley, J.E.; Stout, C.; Palmer, S.M. [East Carolina Univ. School of Medicine, Greenville, NC (United States)] [and others

    1995-07-17

    Constitutional nonreciprocal translocations are extremely rare, and even their existence is controversial. We report on a newborn infant with a de novo nonreciprocal translocation between chromosomes 1 and 8 resulting in 1q42.3 deletion syndrome. Fluorescent in situ hybridization with whole chromosome paints confirmed the conventional cytogenetic diagnosis. 3 refs., 2 figs., 1 tab.

  13. Estimates of aneuploidy using multicolor fluorescence in situ hybridization on human sperm

    Microsoft Academic Search

    F. Z. Bischoff; D. D. Nguyen; K. J. Burt; L. G. Shaffer

    1994-01-01

    Single color fluorescence in situ hybridization (FISH) has been utilized on sperm to estimate nondisjunction rates for chromosomes 1, 12, 15, 16, X and Y. Using single-color FISH, one cannot distinguish nonhybridization from nullisomy nor disomy from diploidy. In order to provide an internal control, a multicolor FISH strategy was employed. Satellite probes specific for 13 human chromosomes were used

  14. An Improved Protocol for Quantification of Freshwater Actinobacteria by Fluorescence In Situ Hybridization

    Microsoft Academic Search

    Raju Sekar; Annelie Pernthaler; Jakob Pernthaler; Falk Warnecke; Thomas Posch; Rudolf Amann

    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

  15. Application of Fluorescence In Situ Hybridization (FISH) to Fish Genetics and Genome Mapping

    Microsoft Academic Search

    Ruth B. Phillips

    2001-01-01

    The various applications of the technique of fluorescence in situ hybridization (FISH) to fish genetics will be reviewed for fishes being used as model organisms to study human disease, including those species for which major genome projects have been initiated. ``FISH on fish'' has been used to map highly repetitive sequences including centromere-specific sequences and sex-specific sequences, moderately repetitive sequences

  16. Heteromorphic Variant 18ph+ Analyzed by Sequential CBG and Fluorescence in situ Hybridization

    Microsoft Academic Search

    A. Sensi; C. Giunta; A. Bonfatti; R. Gruppioni; M. Rubmini; F. Fontana

    1994-01-01

    An 18ph+ variant formerly detected by QFQ\\/CBG was subsequently analyzed by CBG and fluorescence in situ hybridization. It was found to be the consequence of amplification of alphoid DNA specific for centromeric heterochromatin of chromosome 18.Copyright © 1994 S. Karger AG, Basel

  17. Mapping pachytene chromosomes of coffee using a modified protocol for fluorescence in situ hybridization

    PubMed Central

    Iacia, Ana Amélia Sanchez; Pinto-Maglio, Cecília A. F.

    2013-01-01

    Fluorescence in situ hybridization (FISH) is the most direct method for physically mapping DNA sequences on chromosomes. Fluorescence in situ hybridization mapping of meiotic chromosomes during the pachytene stage is an important tool in plant cytogenetics, because it provides high-resolution measurements of physical distances. Fluorescence in situ hybridization mapping of coffee pachytene chromosomes offers significant advantages compared with FISH mapping of somatic chromosomes, because pachytene chromosomes are 30 times longer and provide additional cytological markers. However, the application of this technique to pachytene chromosomes has been complicated by problems in making preparations of meiotic chromosomes and by difficulties in the application of standard FISH protocols. We have been able to overcome most of these obstacles in applying the FISH technique to the pachytene chromosomes of coffee plants. Digesting the external callose layer surrounding the pollen mother cells (PMCs) in conjunction with other procedures permitted suitable pachytene chromosomes to be obtained by increasing cell permeability, which allowed the probe sequences to enter the cells. For the first time, hybridization signals were registered on coffee pachytene chromosomes using the FISH technique with a repetitive sequence as a probe. We obtained slides on which 80 % of the PMCs had hybridization signals, resulting in FISH labelling with high efficiency. The procedure does not seem to be dependent on the genotype, because hybridization signals were detected in genetically different coffee plants. These findings enhance the possibilities for high-resolution physical mapping of coffee chromosomes. PMID:24244840

  18. Identification of mosaicism in Prader-Willi syndrome using fluorescent in situ hybridization

    SciTech Connect

    Mowery-Rushton, P.A.; Surti, U. [Univ. of Pittsburgh, PA (United States)] [Univ. of Pittsburgh, PA (United States); Hanchett, J.M. [Rehabilitation Inst., Pittsburgh, PA (United States)] [and others] [Rehabilitation Inst., Pittsburgh, PA (United States); and others

    1996-12-30

    We report on our findings of 4 patients with mosaicism for a deletion of chromosome 15, most commonly associated with Prader-Willi syndrome (PWS). We examined a series of typical and atypical PWS patients in order to identify cytogenetically undetected deletions, using fluorescence in situ hybridization. In 4 of the patients analyzed we detected a deletion in 14-60% of peripheral blood leukocytes, using four commercially available probes. Our results indicate that mosaicism may play a role in the etiology of some PWS cases. These findings may be especially useful in patients who display discrepancies between clinical phenotype and established diagnostic criteria. Methylation and microsatellite polymorphism analyses of 2 patients with low-level mosaicism failed to identify the deletion. We propose that fluorescence in situ hybridization is the most effective method for detecting somatic mosaicism, since a large number of cells can be individually examined for the presence or absence of a specific deletion. 47 refs., 5 figs., 3 tabs.

  19. Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

    PubMed Central

    Vyboh, Kishanda; Ajamian, Lara; Mouland, Andrew J.

    2012-01-01

    Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1. FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF. PMID:22588480

  20. Numerical chromosome abnormalities in spermatozoa of fertile and infertile men detected by fluorescence in situ hybridization

    Microsoft Academic Search

    Norio Miharu; Robert G. Best; S. Robert Young

    1994-01-01

    Fluorescence in situ hybridization (FISH) with single-color chromosome-specific probes was used to study the rates of disomy for chromosome 1, 16, X, and Y in sperm of fertile and infertile subjects. Diploidy rates were studied using a two-color cocktail of probes for chromosomes 17 and 18 in the same sperm samples. Two-color methodology was not available at the outset of

  1. Quantitative Fluorescence In Situ Hybridization of Aureobasidium pullulans on Microscope Slides and Leaf Surfaces

    Microsoft Academic Search

    SHUXIAN LI; RUSSELL N. SPEAR; JOHN H. ANDREWS

    1997-01-01

    A 21-mer oligonucleotide probe designated Ap665, directed at the 18S rRNA of Aureobasidium pullulans and labelled with five molecules of fluorescein isothiocyanate, was applied by fluorescence in situ hybridization (FISH) to populations of the fungus on slides and apple leaves from growth chamber seedlings and orchard trees. In specificity tests that included Ap665 and a similarly labelled universal probe and

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

    SciTech Connect

    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.

  3. Fluorescent In Situ Hybridization to Detect Transgene Integration into Plant Genomes

    NASA Astrophysics Data System (ADS)

    Schwarzacher, Trude

    Fluorescent chromosome analysis technologies have advanced our understanding of genome organization during the last 30 years and have enabled the investigation of DNA organization and structure as well as the evolution of chromosomes. Fluorescent chromosome staining allows even small chromosomes to be visualized, characterized by their composition and morphology, and counted. Aneuploidies and polyploidies can be established for species, breeding lines, and individuals, including changes occurring during hybridization or tissue culture and transformation protocols. Fluorescent in situ hybridization correlates molecular information of a DNA sequence with its physical location on chromosomes and genomes. It thus allows determination of the physical position of sequences and often is the only means to determine the abundance and distribution of DNA sequences that are difficult to map with any other molecular method or would require segregation analysis, in particular multicopy or repetitive DNA. Equally, it is often the best way to establish the incorporation of transgenes, their numbers, and physical organization along chromosomes. This chapter presents protocols for probe and chromosome preparation, fluorescent in situ hybridization, chromosome staining, and the analysis of results.

  4. Visualization of lncRNA by single-molecule fluorescence in situ hybridization.

    PubMed

    Dunagin, Margaret; Cabili, Moran N; Rinn, John; Raj, Arjun

    2015-01-01

    Single-molecule RNA fluorescence in situ hybridization is a technique that holds great potential for the study of long noncoding RNA. It enables quantification and spatial resolution of single RNA molecules within cells via hybridization of multiple, labeled nucleic acid probes to a target RNA. It has recently become apparent that single-molecule RNA FISH probes targeting noncoding RNA are more prone to off-target binding yielding spurious results than when targeting mRNA. Here we present a protocol for the application of single-molecule RNA FISH to the study of noncoding RNA as well as an experimental procedure for validating legitimate signals. PMID:25555572

  5. Detection of sex chromosome aneuploidy in dog spermatozoa by triple color fluorescence in situ hybridization.

    PubMed

    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. PMID:24962971

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

    PubMed

    Jensen, Henrik Elvang; Jensen, Louise Kruse; Barington, Kristiane; Pors, Susanne Elisabeth; Bjarnsholt, Thomas; Boye, Mette

    2015-01-01

    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 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 samples mounted on glass slides. Two different methods are presented: one is illustrated with the use of peptide nucleic acid (PNA) that is carried out directly on glass slides (Method I), whereas the other is exemplified by using a DNA probe in a Shandon rack (Method II). In the two methods, both PNA and DNA probes can be used. PMID:25399100

  7. Combination of Fluorescent In Situ Hybridization and Microautoradiography—a New Tool for Structure-Function Analyses in Microbial Ecology

    Microsoft Academic Search

    NATUSCKA LEE; PER HALKJÆR NIELSEN; KJÆR HOLM ANDREASEN; STEFAN JURETSCHKO; JEPPE LUND NIELSEN; KARL-HEINZ SCHLEIFER; MICHAEL WAGNER

    1999-01-01

    A new microscopic method for simultaneously determining in situ the identities, activities, and specific sub- strate uptake profiles of individual bacterial cells within complex microbial communities was developed by combining fluorescent in situ hybridization (FISH) performed with rRNA-targeted oligonucleotide probes and microautoradiography. This method was evaluated by using defined artificial mixtures of Escherichia coli and Herpetosiphon aurantiacus under aerobic incubation

  8. Fluorescence in situ hybridization (FISH) on maize metaphase chromosomes with quantum dot-labeled DNA conjugates.

    PubMed

    Ma, Lu; Wu, Sheng-Mei; Huang, Jing; Ding, Yi; Pang, Dai-Wen; Li, Lijia

    2008-04-01

    Semiconductor nanocrystals, also called quantum dots (QDs), are novel inorganic fluorophores which are brighter and more photostable than organic fluorophores. In the present study, highly dispersive QD-labeled oligonucleotide (TAG)(8) (QD-deoxyribonucleic acid [DNA]) conjugates were constructed via the metal-thiol bond, which can be used as fluorescence in situ hybridization (FISH) probes. FISH analysis of maize metaphase chromosomes using the QD-DNA probes showed that the probes could penetrate maize chromosomes and nuclei and solely hybridized to complementary target DNAs. Compared with the conventional organic dyes such as Cy3 and fluorescein isothiocyanate, this class of luminescent labels bound with oligonucleotides is brighter and more stable against photobleaching on the chromosomes after FISH. These results suggest that QD fluorophores may be a more stable and useful fluorescent label for FISH applications in plant chromosome mapping considering their size-tunable luminescence spectra. PMID:18046569

  9. Identification of two Skeletonema costatum-like diatoms by fluorescence in situ hybridization

    NASA Astrophysics Data System (ADS)

    Zhang, Baoyu; Chen, Guofu; Wang, Guangce; Lu, Douding

    2010-03-01

    A harmful algae bloom (HAB) is a dense aggregation of algae in a marine or aquatic environment that can result in significant environmental problems. To forecast the occurrence of HAB, development of a rapid and precise detection method is urgently required. In this study, two Skeletonema costatum-like diatoms (SK-1 and SK-2), were identified morphologically under a light microscope, and detected using fluorescent in situ hybridization (FISH). Strain SK-1 was isolated from a frequently HAB affected area of the East China Sea, and strain SK-2 from an aquatic farm in Qingdao, China. Fluorescent DNA probes were designed that were complementary to the ITS sequence (including 5.8S rDNA) of strain SK-1. After hybridization, strong green fluorescence was observed in cells of strain SK-1 under an epifluorescence microscope; however, no such fluorescence was observed with strain SK-2, which indicates that probes hybridized only the DNA of the target strain, SK-1, in species-specific manner, and that the two strains do not belong to a same species. This finding was confirmed by ITS sequence analysis. The FISH technique used in this study was sensitive, simple, and rapid, and is a promising tool for detecting target HAB species in natural environments.

  10. Catalyzed Reported Deposition-Fluorescence In Situ Hybridization Protocol To Evaluate Phagotrophy in Mixotrophic Protists

    PubMed Central

    Medina-Sánchez, Juan M.; Felip, Marisol; Casamayor, Emilio O.

    2005-01-01

    We describe a catalyzed reported deposition-fluorescence in situ hybridization (CARD-FISH) protocol particularly suited to assess the phagotrophy of mixotrophic protists on prokaryotes, since it maintains cell and plastid integrity, avoids cell loss and egestion of prey, and allows visualization of labeled prey against plastid autofluorescence. This protocol, which includes steps such as Lugol's-formaldehyde-thiosulfate fixation, agarose cell attachment, cell wall permeabilization with lysozyme plus achromopeptidase, and signal amplification with Alexa-Fluor 488, allowed us to detect almost 100% of planktonic prokaryotes (Bacteria and Archaea) and, for the first time, to show archaeal cells ingested by mixotrophic protists. PMID:16269774

  11. Classification of fluorescence in situ hybridization images using belief networks

    Microsoft Academic Search

    Roy Malka; Boaz Lerner

    2004-01-01

    The structure and parameters of a belief network are learned in order to classify images enabling the detection of genetic abnormalities. We compare a structure learned from the data to another structure obtained utilizing expert knowledge and to the naive Bayesian classifier and study quantization in comparison to density estimation in parameter learning.

  12. Preparation of Amniocytes for Interphase Fluorescence In Situ Hybridization (FISH).

    PubMed

    Schwartz, Stuart

    2015-01-01

    FISH has been used to detect and clarify deletions and/or other structural rearrangements, and also has applications in interphase analysis. This unit describes preparation of uncultured amniotic fluid cells for FISH analysis. Cells are swollen, and then slides are prepared by standard methods. The cells are then fixed and permeabilized for subsequent FISH. An alternate protocol describes attachment of amniocytes to a glass or plastic surface followed by hypotonic swelling, fixation, and permeabilization for subsequent FISH. Interphase FISH analysis of amniotic fluid cells is also described. © 2015 by John Wiley & Sons, Inc. PMID:25827350

  13. Multiplexed miRNA Fluorescence In Situ Hybridization for Formalin-Fixed Paraffin-Embedded Tissues

    PubMed Central

    Renwick, Neil; Cekan, Pavol; Bognanni, Claudia; Tuschl, Thomas

    2015-01-01

    Multiplexed miRNA fluorescence in situ hybridization (miRNA FISH) is an advanced method for visualizing differentially expressed miRNAs, together with other reference RNAs, in archival tissues. Some miRNAs are excellent disease biomarkers due to their abundance and cell-type specificity. However, these short RNA molecules are difficult to visualize due to loss by diffusion, probe mishybridization, and signal detection and signal amplification issues. Here, we describe a reliable and adjustable method for visualizing and normalizing miRNA signals in formalin-fixed paraffin-embedded (FFPE) tissue sections. PMID:25218385

  14. Characterization of supernumerary ring marker chromosomes by fluorescence in situ hybridization (FISH)

    SciTech Connect

    Blennow, E.; Nordenskjoeld, M. (Karolinska Hospital (Sweden)); Asadi, E. (Region Hospital, Oerebro (Sweden)); Anneren, G.; Berggren, E.; Nordenskjoeld, M.

    1993-08-01

    Five cases with small supernumerary ring chromosomes are characterized at the molecular level. Routine chromosome banding analysis was insufficient for identification of the ring chromosomes, and none of them was DA/DAPI positive. Fluorescence in situ hybridization utilizing repetitive centromeric probes for all chromosomes has determined that one of these five ring chromosomes originates in each of chromosomes 4, 7, 8, 9, and 20. Chromosome painting with chromosome-specific libraries has confirmed this and excluded the involvement of additional chromosomes in the rearrangements. 30 refs., 3 figs., 2 tabs.

  15. Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids.

    PubMed

    Silvia, Fontenete; Joana, Barros; Pedro, Madureira; Céu, Figueiredo; Jesper, Wengel; Filipe, Azevedo Nuno

    2015-05-01

    In the past few years, several researchers have focused their attention on nucleic acid mimics due to the increasing necessity of developing a more robust recognition of DNA or RNA sequences. Fluorescence in situ hybridization (FISH) is an example of a method where the use of these novel nucleic acid monomers might be crucial to the success of the analysis. To achieve the expected accuracy in detection, FISH probes should have high binding affinity towards their complementary strands and discriminate effectively the noncomplementary strands. In this study, we investigate the effect of different chemical modifications in fluorescent probes on their ability to successfully detect the complementary target and discriminate the mismatched base pairs by FISH. To our knowledge, this paper presents the first study where this analysis is performed with different types of FISH probes directly in biological targets, Helicobacter pylori and Helicobacter acinonychis. This is also the first study where unlocked nucleic acids (UNA) were used as chemistry modification in oligonucleotides for FISH methodologies. The effectiveness in detecting the specific target and in mismatch discrimination appears to be improved using locked nucleic acids (LNA)/2'-O-methyl RNA (2'OMe) or peptide nucleic acid (PNA) in comparison to LNA/DNA, LNA/UNA, or DNA probes. Further, the use of LNA modifications together with 2'OMe monomers allowed the use of shorter fluorescent probes and increased the range of hybridization temperatures at which FISH would work. PMID:25840566

  16. Identification of the pathogenic ciliate Pseudocohnilembus persalinus (Oligohymenophorea: Scuticociliatia) by fluorescence in situ hybridization.

    PubMed

    Zhan, Zifeng; Stoeck, Thorsten; Dunthorn, Micah; Xu, Kuidong

    2014-02-01

    Many scuticociliatid ciliates are regarded as devastating pathogens in aquaculture. Among these, Pseudocohnilembus persalinus is a facultative pathogen that often results in refractory diseases of mariculture fish. Although traditional silver staining methods have been successfully used to identify these ciliates, their identification is hampered by their small size and their morphological similarity to closely related species. We designed an alternative method of identification of P. persalinus using an SSU-rDNA targeted oligonucleotide probe labeled with a fluorochrome, and optimized in a fluorescence in situ hybridization (FISH) protocol. The assay results in a clear identification by strong fluorescence signals from the oligonucleotide probe. The method can be used for quick and early detection of P. persalinus infections on host fish, as well as other susceptible organisms in aquiculture water. It may also be used in studies of the geographical distribution of this scuticociliate. PMID:24287159

  17. Exploring the origin of the D genome of oat by fluorescence in situ hybridization.

    PubMed

    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. PMID:25478818

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

    SciTech Connect

    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.

  19. Nature of B chromosomes in the harvest mouse Reithrodontomys megalotis by fluorescence in situ hybridization (FISH).

    PubMed

    Peppers, J A; Wiggins, L E; Baker, R J

    1997-11-01

    Using fluorescence in situ hybridization, we examined the characteristics of two types of B chromosomes in harvest mice of the genus Reithrodontomys. B chromosomes were interrogated with rDNA, telomeric repeat, LINE element and centromeric heterochromatin probes. The two types of B chromosomes share the following features: (a) telomeres present on the ends of both arms; (b) hybridization to LINE probes; (c) absence of hybridization to the ribosomal gene probes; (d) C-band-positive centromeric regions; and (e) euchromatic arms. They differ as follows: (a) the larger B element hybridizes to the centromeric heterochromatin (pMeg-1) probe whereas the smaller B element does not; (b) the amount of C-band-positive material is reduced in the smaller B chromosome relative to that present on the larger B chromosome; and (c) the smaller element is reduced in size by about a third. It is concluded that the larger B chromosome arose as a leftover centromere from centric fusion, whereas the smaller element has a different origin perhaps as an intact fragment or as an amplified region from the A chromosomes. The presence of euchromatic regions on B chromosomes may account for their survival in the karyotype. PMID:9421265

  20. Fluorescence intensity profiles of in situ hybridization signals depict genome architecture within human interphase nuclei.

    PubMed

    Iourov, I Y; Vorsanova, S G; Yurov, Y B

    2008-01-01

    An approach towards construction of two-dimensional (2D) and three-dimensional (3D) profiles of interphase chromatin architecture by quantification of fluorescence in situ hybridization (FISH) signal intensity is proposed. The technique was applied for analysis of signal intensity and distribution within interphase nuclei of somatic cells in different human tissues. Whole genomic DNA, fraction of repeated DNA sequences (Cot 1) and cloned satellite DNA were used as probes for FISH. The 2D and 3D fluorescence intensity profiles were able to depict FISH signal associations and somatic chromosome pairing. Furthermore, it allowed the detection of replicating signal patterns, the assessment of hybridization efficiency, and comparative analysis of DNA content variation of specific heterochromatic chromosomal regions. The 3D fluorescence intensity profiles allowed the analysis of intensity gradient within the signal volume. An approach was found applicable for determination of assembly of different types of DNA sequences, including classical satellite and alphoid DNA, gene-rich (G-negative bands) and gene-poor (G-positive bands) chromosomal regions as well as for assessment of chromatin architecture and targeted DNA sequence distribution within interphase nuclei. We conclude the approach to be a powerful additional tool for analysis of interphase genome architecture and chromosome behavior in the nucleus of human somatic cells. PMID:19140435

  1. Fluorescent in situ hybridization analyses of human oocytes in trisomy 18 and 21

    SciTech Connect

    Cheng, E.Y.; Chen, Y.J.; Gartler, S.M. [Univ. of Washington School of Medicine, Seattle, WA (United States)

    1994-09-01

    The commonly accepted view of synapsis is that only 2 homologues can synapse at any one site and that this restriction applies to polyploids as well. However, triple synapsis has been observed is some triploid plants and in triploid chicken. In humans, triple synapsis of the long arm of chromosome 21 was detected in sperm of a trisomic 21 individual. More recently, studies of oocytes from trisomic 21 and 18 fetuses also indicated extensive triple synapsis along the entire length of the chromosomes. To further investigate this question, we undertook an evaluation of trivalent synapsis in fetal oocytes from 2 trisomic 21 and 2 trisomic 18 fetuses using fluorescent in situ hybridization (FISH) with whole chromosome probes. Oocytes were hybridized with whole chromosome probes obtained from ONCOR, Inc. after fixation with methanol and acetic acid. Slides were scored for the distribution of prophase stages, hybridization efficiency, and hybridization characteristics of chromosomes 18 and 21 in the trisomic 18 and 21 fetuses respectively. Fifty-eight per cent (379/650) of pachytenes analyzed for chromosome 18 contained a conspicous trivalent and 319 (48%) of these nuclei contained a single, thick, continuous fluorescent signal consistent with complete triple synapsis along the entire length of all 3 chromosomes. Sixteen per cent (104/650) of pachytene contained 2 signals consistent with a bivalent and a univalent, and 9 cells contained 3 thin signals consistent with asynapsis of all 3 chromosomes. The remaining 158 pachytenes had unusual pairing configurations that we could not classify, but they most likely represent trivalents with partial pairing between different homologues. In the 2 trisomic 21 fetuses, the majority (143/232) of pachytenes also contained one signal while only 52 cells contained a bivalent and univalent. Five cells contained 3 separate signals. These results confirm the existence of triple synapsis in human meiosis.

  2. Substrate uptake in extremely halophilic microbial communities revealed by microautoradiography and fluorescence in situ hybridization.

    PubMed

    Rosselló-Mora, Ramon; Lee, Natuschka; Antón, Josefa; Wagner, Michael

    2003-10-01

    The combination of fluorescence in situ hybridization and microautoradiography (FISH-MAR approach) was applied to brine samples of a solar saltern crystallizer pond from Mallorca (Spain) where the simultaneous occurrence of Salinibacter spp. and the conspicuous square Archaea had been detected. Radioactively labeled bicarbonate, acetate, glycerol, and an amino acid mixture were tested as substrates for the microbial populations inhabiting such brines. The results indicated that hitherto uncultured 'square Archaea' do actively incorporate amino acids and acetate. However, Salinibacter spp. only showed amino acid incorporation in pure culture, but no evidence of such activity in their natural environment could be demonstrated. No glycerol incorporation was observed for any component of the microbial community. PMID:12820037

  3. Chromogenic in situ hybridization is a reliable alternative to fluorescence in situ hybridization for diagnostic testing of 1p and 19q loss in paraffin-embedded gliomas.

    PubMed

    Lass, Ulrike; Hartmann, Christian; Capper, David; Herold-Mende, Christel; von Deimling, Andreas; Meiboom, Maren; Mueller, Wolf

    2013-05-01

    Recent studies imply the importance of rapid and reliable diagnostic assessment of 1p/19q status in oligodendroglial tumors. To date, fluorescence in situ hybridization (FISH) is the most commonly applied technique. FISH, however, has several technical shortcomings that are suboptimal for diagnostic applications: results must be viewed in a fluorescence microscope, results are usually evaluated by a single investigator only, and signal fading excludes physical archiving. Also, in gliomas, the distinction of diffusely infiltrating tumor cells from reactively altered normal tissue may be challenging in fluorescence microscopy. Dual-color chromogenic in situ hybridization (CISH) has started to replace FISH in some diagnostic tests performed in pathology. Here, we present the first single institute experience with a side-by-side analysis of 1p/19q FISH and CISH in a series of 42 consecutive gliomas. FISH and CISH produced identical results for 1p and 19q in 93% of cases (n?=?39/42). Discrepant results were reevaluated by repeated FISH and a polymerase chain reaction (PCR)-based microsatellite marker analysis for loss of heterozygosity. Reevaluation confirmed CISH data in all three cases. We conclude that CISH is a reliable alternative in 1p/19q testing in paraffin-embedded tissues likely to be more sensitive to detect 1p/19q status than FISH analysis. PMID:23107103

  4. Detection of micro-organisms in soil after in situ hybridization with rRNA-targeted, fluorescently labelled oligonucleotides

    Microsoft Academic Search

    DITTMAR HAHN; RUDOLF I. AMANN; WOLFGANG LUDWIG; ANTOON D. L. AKKERMANS; KARL-HEINZ SCHLEIFER

    1992-01-01

    rRNA sequences were used as targets for synthetic oligonucleotides labelled with the fluorescent dye tetramethylrhodamine isothiocyanate (Tritc) for in situ hybridizations to detect micro-organisms directly in soils that have different contents of soil minerals and organic material. Introduced Pseudomonas aeruginosa cells were directly fixed in soils and applied to slides after separation of large soil minerals only. Remaining soil minerals

  5. Ribosomal DNA Location in the Scarab Beetle Thorectes Intermedius (Costa) (Coleoptera: Geotrupidae) Using Banding and Fluorescent in-situ Hybridization

    Microsoft Academic Search

    R. Vitturi; M. S. Colomba; R. Barbieri; M. Zunino

    1999-01-01

    Mitotic metaphase chromosomes of the scarab beetle Thorectes intermedius (Costa) (Coleoptera Scarabaeoidea: Geotrupidae) were analyzed using various banding methods and fluorescent in-situ hybridization (FISH) with a ribosomal probe. The results obtained indicate that silver and CMA3 staining are unable to localize the chromosome sites of nucleolar organizer regions (NORs). Such an inadequacy is a consequence of the extensive silver and

  6. Fluorescence in-situ hybridization (FISH) as a tool for visualization and enumeration of Campylobacter in broiler ceca

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Food-borne human pathogens are typically detected and enumerated by either cultural methods or PCR-based approaches. Fluorescence in-situ hybridization (FISH) is a standard microscopy tool for microbial ecology but has not been widely used for food safety applications despite important advantages o...

  7. Detection of trisomy 12 on ovarian sex cord stromal tumors by fluorescence in situ hybridization.

    PubMed

    Taruscio, D; Carcangiu, M L; Ward, D C

    1993-06-01

    Trisomy of chromosome 12 has been frequently described in various neoplasms, particularly in tumors of the female genitourinary tract. Fluorescence in situ hybridization with a centromeric repetitive DNA probe, specific for chromosome 12, was done to detect such cytogenic changes on frozen-tissue sections from 10 cases of ovarian sex cord stromal tumors. The case series was composed by granulosa cell tumors (four cases), fibromas (four cases), thecoma (one case), and Sertoli-Leydig cell tumor (one case). In granulosa cell tumors, the range of trisomy was 12 to 32% and in fibromas 8 to 22%, whereas in the single case of thecoma trisomy was present in 8% and in the Sertoli-Leydig cell tumor in 4% of the nuclei examined. These results represent an additional series of cases of trisomy 12 in ovarian neoplasms, namely, in ovarian sex cord stromal tumors. PMID:8269283

  8. Simultaneous visualization of Propionibacterium acnes and Propionibacterium granulosum with immunofluorescence and fluorescence in situ hybridization.

    PubMed

    Jahns, Anika C; Oprica, Cristina; Vassilaki, Ismini; Golovleva, Irina; Palmer, Ruth H; Alexeyev, Oleg A

    2013-10-01

    Propionibacterium acnes (P. acnes) and Propionibacterium granulosum (P. granulosum) are common skin colonizers that are implicated as possible contributing factors in acne vulgaris development. We have established direct visualization tools for the simultaneous detection of these closely related species with immunofluorescence assay and fluorescence in situ hybridization (FISH). As proof of principle, we were able to distinguish P. acnes and P. granulosum bacteria in multi-species populations in vitro as well as in a mock skin infection model upon labelling with 16S rRNA probes in combinatorial FISH as well as with antibodies. Furthermore, we report the co-localization of P. acnes and P. granulosum in the stratum corneum and hair follicles from patients with acne vulgaris as well as in healthy individuals. Further studies on the spatial distribution of these bacteria in skin structures in various skin disorders are needed. PMID:23896347

  9. Identification of supernumerary ring chromosome 1 mosaicism using fluorescence in situ hybridization

    SciTech Connect

    Chen, H.; Tuck-Muller, C.M.; Wertelecki, W. [Univ. of South Alabama, Mobile, AL (United States)] [and others

    1995-03-27

    We report on a 15-year-old black boy with severe mental retardation, multiple congenital anomalies, and a supernumerary ring chromosome mosaicism. Fluorescence in situ hybridization with a chromosome 1 painting probe (pBS1) identified the ring as derived from chromosome 1. The karyotype was 46,XY/47,XY,+r(1)(p13q23). A review showed 8 reports of ring chromosome 1. In 5 cases, the patients had a non-supernumerary ring chromosome 1 resulting in partial monosomies of the short and/or long arm of chromosome 1. In 3 cases, the presence of a supernumerary ring resulted in partial trisomy of different segments of chromosome 1. In one of these cases of the supernumerary ring was composed primarily of the centromere and the heterochromatic region of chromosome 1, resulting in normal phenotype. Our patient represents the third report of a supernumerary ring chromosome 1 resulting in abnormal phenotype. 28 refs., 5 figs., 1 tab.

  10. Enumeration of methanogens with a focus on fluorescence in situ hybridization

    NASA Astrophysics Data System (ADS)

    Kumar, Sanjay; Dagar, Sumit Singh; Mohanty, Ashok Kumar; Sirohi, Sunil Kumar; Puniya, Monica; Kuhad, Ramesh C.; Sangu, K. P. S.; Griffith, Gareth Wyn; Puniya, Anil Kumar

    2011-06-01

    Methanogens, the members of domain Archaea are potent contributors in global warming. Being confined to the strict anaerobic environment, their direct cultivation as pure culture is quite difficult. Therefore, a range of culture-independent methods have been developed to investigate their numbers, substrate uptake patterns, and identification in complex microbial communities. Unlike other approaches, fluorescence in situ hybridization (FISH) is not only used for faster quantification and accurate identification but also to reveal the physiological properties and spatiotemporal dynamics of methanogens in their natural environment. Aside from the methodological aspects and application of FISH, this review also focuses on culture-dependent and -independent techniques employed in enumerating methanogens along with associated problems. In addition, the combination of FISH with micro-autoradiography that could also be an important tool in investigating the activities of methanogens is also discussed.

  11. Detecting genomic aberrations by fluorescence in situ hybridization with quantum dots-labeled probes.

    PubMed

    Jiang, Zhengran; Li, Ruiyun; Todd, Nevins W; Stass, Sanford A; Jiang, Feng

    2007-12-01

    Detection of genomic alterations of cancer genes by fluorescent in situ hybridization (FISH) will provide important information for cancer diagnosis and therapy. To effectively and reliably detect the genomic changes, we prepared novel FISH probes by directly conjugating genomic DNA of genes to semiconductor quantum dot fluorophores (QDs). The generated QD-genomic probes are substantially more photostable than the probes labeled with organic dye and show high intensity in both metaphase and interphase cell. The directly labeling probes allow detection of genomic targets in a fast and simple FISH procedure with high sensitivity and specificity. Furthermore, application of the QD-genomic probes in lung cancer specimens can reliably visualize gene amplification in cancer cells. These results suggest that the QD-FISH probes may offer an effective approach to analyze cancer-related genomic aberrations in basic research and clinical applications. PMID:18283800

  12. The design of a microscopic system for typical fluorescent in-situ hybridization applications

    NASA Astrophysics Data System (ADS)

    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.

  13. Partial trisomy 13q identified by sequential fluorescence in situ hybridization

    SciTech Connect

    Gopal Rao, V.V.N.; Carpenter, N.J.; Gucsavas, M. [Institute of Medical Genetics, Tulsa, OK (United States)] [and others

    1995-07-31

    We report on a 19-month-old boy with partial trisomy 13q resulting from a probable balanced translocation involving chromosomes 1 and 13. The infant presented with omphalocele, malrotation, microcephaly with overriding skull bones, micrognathia, apparently low-set ears, rocker-bottom feet, and congenital heart disease, findings suggestive of trisomy 13. Karyotypic studies from peripheral blood lymphocytes documented an unbalanced karyotype 46,XY,-1,+der. The mother`s chromosomes were normal, and the father was not available. Conventional cytogenetic techniques were unable to identify the extra material on the terminal 1q. Using fluorescence in situ hybridization (FISH) on the GTL-banded metaphases, the extra material on 1q was identified as the terminal long arm of 13, thus resulting in partial trisomy 13 (q32-qter). 8 refs., 2 figs., 1 tab.

  14. Development of single-cell array for large-scale DNA fluorescence in situ hybridization

    PubMed Central

    Liu, Yingru; Kirkland, Brett; Shirley, James; Wang, Zhibin; Zhang, Peipei; Stembridge, Jacquelyn; Wong, Wilson; Takebayashi, Shin-ichiro; Gilbert, David M.; Lenhert, Steven

    2013-01-01

    DNA fluorescence in situ hybridization (FISH) is a powerful cytogenetic assay, but conventional sample-preparation methods for FISH do not support large-scale high-throughput data acquisition and analysis, which are potentially useful for several biomedical applications. To address this limitation, we have developed a novel FISH sample-preparation method based on generating a centimetre-sized cell array, in which all cells are precisely positioned and separated from their neighbours. This method is simple and easy and capable of patterning nonadherent human cells. We have successfully performed DNA FISH on the single-cell arrays, which facilitate analysis of FISH results with the FISH-FINDER computer program. PMID:23370691

  15. Fluorescence in situ hybridization analysis of minute marker chromosomes in leukemia with monosomy 7.

    PubMed

    Viguié, F; Prigent, Y; Ramond, S; Baumelou, E; Cadiou, M; Dreyfus, F; Zittoun, R

    1995-07-01

    Monosomy 7 was detected in bone marrow cells from three patients, one with myeloid leukemia, and two others with myelodysplastic syndrome following previous chemotherapy. Fluorescence in situ hybridization (FISH), carried out with an alphoid DNA probe specific for chromosome 7 centromere, showed that a small marker chromosome present in the tumor cells' karyotype of the three patients, was derived from the missing chromosome 7. In two cases, the marker was a ring chromosome, whereas in the third case it was a tiny dot-like chromosome, unnoticed at first examination on R-banded metaphases. In the three cases, the marker was lost in a proportion of tumor cells. FISH experiments suggested that the marker centromere had undergone structural alterations, with a fluorescence pattern distinct from a normal one. On the whole, these data suggest that: firstly, leukemia-associated monosomy 7 results, in a proportion of cases, from a structural event rather than from simple loss of a whole chromosome 7; secondly, interpretation of interphase FISH must be cautious in monosomy 7 evaluation; and thirdly structural alteration of the chromosome 7 derivative alphoid DNA could explain its propensity to segregate unequally and to be lost at mitosis. PMID:7630189

  16. Determination of the ruminant origin of bone particles using fluorescence in situ hybridization (FISH)

    PubMed Central

    Lecrenier, M. C.; Ledoux, Q.; Berben, G.; Fumière, O.; Saegerman, C.; Baeten, V.; Veys, P.

    2014-01-01

    Molecular biology techniques such as PCR constitute powerful tools for the determination of the taxonomic origin of bones. DNA degradation and contamination by exogenous DNA, however, jeopardise bone identification. Despite the vast array of techniques used to decontaminate bone fragments, the isolation and determination of bone DNA content are still problematic. Within the framework of the eradication of transmissible spongiform encephalopathies (including BSE, commonly known as “mad cow disease”), a fluorescence in situ hybridization (FISH) protocol was developed. Results from the described study showed that this method can be applied directly to bones without a demineralisation step and that it allows the identification of bovine and ruminant bones even after severe processing. The results also showed that the method is independent of exogenous contamination and that it is therefore entirely appropriate for this application. PMID:25034259

  17. X chromosome aneuploidy in infertile women: Analysis by interphase fluorescent in situ hybridization

    SciTech Connect

    Morris, M.A.; Moix, I.; Mermillod, B. [Univ. and Cantonal Hospital, Geneva (Switzerland)] [and others

    1994-09-01

    Up to 1 in 3 couples have a problem of infertility at some time in their lives. Sex chromosome anomalies are found in 5-10% of couples, with mosaic aneuploidy being a common finding in primary infertility. Recurrent spontaneous abortion (RSA), in contrast, is frequently associated with autosomal structural anomalies. We hypothesized that low-level mosaic X chromosome aneuploidy was associated with primary infertility but not with RSA. Three groups were studied: women from couples with primary infertillity (n=26); women with three or more spontaneous abortions (n=22); and age-matched normally fertile women (at least two pregnancies; n=28). Interphase fluorescent in situ hybridization (FISH) was used to determine X chromosome ploidy in 100 nuclei per patient, using a contig of three cosmids from MAO locus (kindly donated by W. Berger, Nijmegen). A control probe (chr. 15 centromere) was simultaneously hybridized, and only nuclei containing two control signals were scored for the X chromosome. The mean numbers of nuclei with two X chromosome signals were the same in all groups (Welch equality of means test: p>0.97). However, there is a significant difference between the variances of the primary infertile and RSA groups (Levene`s test: p=0.025 after Bonferrone correction for multiple testing). This provides preliminary support for the hypothesis of an association between primary infertility and low-level mosaic X chromosome aneuploidy.

  18. Intranuclear Relocalization of Matrix Binding Sites during T Cell Activation Detected by Amplified Fluorescence in Situ Hybridization

    Microsoft Academic Search

    Shutao Cai; Terumi Kohwi-Shigematsu

    1999-01-01

    We describe a method for analyzing the nuclear localization of specific DNA sequences, with special emphasis on their binding status to the nuclear matrix, depending on the developmental stage of the cells. This method employs high-resolution fluorescence in situ hybridization procedures. For our studies, it was important to examine the nuclear localization of a particular gene locus. Previously, however, it

  19. Fluorescence In Situ Hybridization for Rapid Identification of Achromobacter xylosoxidans and Alcaligenes faecalis Recovered from Cystic Fibrosis Patients

    PubMed Central

    Wellinghausen, Nele; Wirths, Beate; Poppert, Sven

    2006-01-01

    Achromobacter xylosoxidans is frequently isolated from the respiratory secretions of cystic fibrosis (CF) patients, but identification with biochemical tests is unreliable. We describe fluorescence in situ hybridization assays for the rapid identification of Achromobacter xylosoxidans and Alcaligenes faecalis. Both assays showed high sensitivities and high specificities with a collection of 155 nonfermenters from CF patients. PMID:16954289

  20. StellarisTM fluorescence in situ hybridization (FISH) probes: a powerful tool for mRNA detection

    E-print Network

    Cai, Long

    StellarisTM fluorescence in situ hybridization (FISH) probes: a powerful tool for mRNA detection The StellarisTM FISH technology is a new mRNA detection method that enables simultaneous detection, localization transcription To supplement Stellaris assays designed for custom targets, positive control probe sets targeting

  1. Identification of Dekkera bruxellensis (Brettanomyces) from Wine by Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes

    Microsoft Academic Search

    HENRIK STENDER; CLETUS KURTZMAN; JENS J. HYLDIG-NIELSEN; D. Sorensen; ADAM BROOMER; KENNETH OLIVEIRA; HEATHER PERRY-O' KEEFE; ANDREW SAGE; BARBARA YOUNG; JAMES COULL

    2001-01-01

    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

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

    SciTech Connect

    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.

  3. In Situ Detection of Freshwater Fungi in an Alpine Stream by New Taxon-Specific Fluorescence In Situ Hybridization Probes

    Microsoft Academic Search

    Christiane Baschien; Werner Manz; Thomas R. Neu; Ludmila Marvanova; Ulrich Szewzyk

    2008-01-01

    New rRNA-targeting oligonucleotide probes permitted thefluorescence in situ hybridization (FISH) identification of freshwater fungi in an Austrian second-order alpine stream. Based on computer-assisted comparative sequence analysis, nine taxon-specific probes were designed and evaluated by whole-fungus hybridizations. Oligonucleotide probe MY1574, specific for a wide range of Eumycota, and the genus (Tetracladium)-specific probe TCLAD1395, as well as the species-specific probes ALacumi1698 (Alatospora

  4. In situ hybridization of phytoplankton using fluorescently labeled rRNA probes.

    PubMed

    Groben, René; Medlin, Linda

    2005-01-01

    Phytoplankton are one of the major components of ecosystem processes and play an important role in many biogeochemical cycles in the marine and freshwater environment. Despite their importance, many microalgae are poorly described and little is known of broad spatial and temporal scale trends in their abundance and distribution. Reasons for this are that microalgae are often small, lack distinct morphological features, and are unculturable, which make analyses difficult. It is now possible by using molecular biological techniques to advance our knowledge of aquatic biodiversity and to understand how biodiversity supports ecosystem structure, dynamics, and resilience. We present in this chapter a brief review of the progress that has been made in analyzing microalgae from populations to the species level. The described methods range from DNA fingerprinting techniques, such as random amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLPs), and simple sequence repeats (SSRs), to microsatellites, which are used in population studies, to sequence analysis, which help to reconstruct the evolutionary history of organisms and to examine relationships at various taxonomic levels. Special emphasis is given to the application of molecular probes for the identification and characterization of microalgal taxa. The fast and secure identification of phytoplankton, especially of toxic species, is important from an ecological and economical point of view and whole-cell hybridization with specific fluorochrome-labeled probes followed by fluorescence microscopy or flow cytometry offers a fast method for this purpose. In this context, we present a detailed protocol for fluorescence in situ hybridization (FISH) of ribosomal RNA (rRNA) probes that can be applied to many algal cell types and discuss practical considerations of its use. PMID:15865974

  5. Development of a PNA Probe for Fluorescence In Situ Hybridization Detection of Prorocentrum donghaiense

    PubMed Central

    Chen, Guofu; Zhang, Chunyu; Zhang, Baoyu; Wang, Guangce; Lu, Douding; Xu, Zhong; Yan, Peishen

    2011-01-01

    Prorocentrum donghaiense is a common but dominant harmful algal bloom (HAB) species, which is widely distributed along the China Sea coast. Development of methods for rapid and precise identification and quantification is prerequisite for early-stage warning and monitoring of blooms due to P. donghaiense. In this study, sequences representing the partial large subunit rDNA (D1–D2), small subunit rDNA and internal transcribed spacer region (ITS-1, 5.8S rDNA and ITS-2) of P. donghaiense were firstly obtained, and then seven candidate DNA probes were designed for performing fluorescence in situ hybridization (FISH) tests on P. donghaiense. Based on the fluorescent intensity of P. donghaiense cells labeled by the DNA probes, the probe DP0443A displayed the best hybridization performance. Therefore, a PNA probe (PP0443A) analogous to DP0443A was used in the further study. The cells labeled with the PNA probe displayed more intensive green fluorescence than that labeled with its DNA analog. The PNA probe was used to hybridize with thirteen microalgae belonging to five families, i.e., Dinophyceae, Prymnesiophyceae, Raphidophyceae, Chlorophyceae and Bacillariophyceae, and showed no visible cross-reaction. Finally, FISH with the probes PP0443A and DP0443A and light microscopy (LM) analysis aiming at enumerating P. donghaiense cells were performed on the field samples. Statistical comparisons of the cell densities (cells/L) of P. donghaiense in the natural samples determined by FISH and LM were performed using one-way ANOVA and Duncan's multiple comparisons of the means. The P. donghaiense cell densities determined by LM and the PNA probe are remarkably higher than (p<0.05) that determined by the DNA probe, while no significant difference is observed between LM and the PNA probe. All results suggest that the PNA probe is more sensitive that its DNA analog, and therefore is promising for the monitoring of harmful algal blooms of P. donghaiense in the future. PMID:22022408

  6. Locus/Chromosome aberrations in intraductal papillary mucinous neoplasms analyzed by fluorescence in situ hybridization.

    PubMed

    Miyabe, Katsuyuki; Hori, Yasuki; Nakazawa, Takahiro; Hayashi, Kazuki; Naitoh, Itaru; Shimizu, Shuya; Kondo, Hiromu; Nishi, Yuji; Yoshida, Michihiro; Umemura, Shuichiro; Kato, Akihisa; Ohara, Hirotaka; Joh, Takashi; Inagaki, Hiroshi

    2015-04-01

    Locus and chromosome abnormalities have not been well clarified in intraductal papillary mucinous neoplasms (IPMNs). The aim of this study was to retrospectively examine these abnormalities using fluorescence in situ hybridization. IPMNs (n=28) were histopathologically classified into noninvasive IPMN (n=17) and IPMN with an associated invasive carcinoma (invasive IPMN, n=11) groups. Noninvasive IPMNs possessed non-neoplastic and noninvasive spots in their tissues, and invasive IPMN cases possessed non-neoplastic, noninvasive, and invasive spots. Non-neoplastic (n=28), noninvasive (n=28), and invasive (n=11) spots were then analyzed for aneuploidy of chromosomes 3, 6, 7, 8, 17, and 18 and deletions of p16 and p53 loci. Polysomy 6 and p16 deletion were significantly more frequent in noninvasive than in non-neoplastic spots. Polysomy 7, polysomy 18, p16 deletion, and p53 deletion were significantly more frequent in invasive than in noninvasive spots. Detection of polysomy 7 and p53 deletion gave a high diagnostic accuracy for invasive IPMN (sensitivity, 90.9%; specificity, 94.1%; and accuracy, 92.5%). Our study suggests that: (1) polysomy 6 and p16 deletion may contribute to adenomatous change of IPMN; (2) polysomy 7, polysomy 18, p16 deletion, and p53 deletion play roles in malignant transformation of noninvasive IPMN; and (3) polysomy 7 and p53 deletion may be excellent diagnostic markers for invasive IPMN. PMID:25517961

  7. Identification of pathogens in mastitis milk samples with fluorescent in situ hybridization.

    PubMed

    Gey, Annerose; Werckenthin, Christiane; Poppert, Sven; Straubinger, Reinhard K

    2013-05-01

    Traditionally, the bacteriological examination of mastitis milk samples is performed by culture followed by biochemical tests on the cultured bacteria to allow identification of the causative pathogen. Depending on the species involved, this classic identification is time-consuming compared to other techniques such as fluorescent in situ hybridization (FISH), a culture-independent method that utilizes oligonucleotides (labeled with a fluorophore) that are specific to a string of target DNA/RNA. In the current study, the applicability of FISH was evaluated for the detection of mastitis pathogens directly in milk samples. To remove interfering lipids and proteins from mastitis milk samples prior to FISH, a previously published enzymatic treatment with savinase was evaluated. FISH was performed using oligonucleotides specific for Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, and Trueperella (Arcanobacterium) pyogenes. The enzymatic pretreatment and the sensitivity of FISH were evaluated using spiked whole milk samples and mastitis milk samples with bacterial loads of less than 10(3) up to 10(8) colony-forming units (CFU)/ml. Bacteria were reliably detected in milk samples with bacterial numbers of 10(6) CFU/ml or higher. However, bacteria present in numbers below 10(6) CFU/ml were not detectable in all cases. The ability of FISH to identify mastitis-causing pathogens directly in milk samples, and therefore earlier than classical culture methods, can supplement the classic diagnostic procedures for mastitis milk samples. PMID:23632662

  8. Fluorescence in situ hybridization (CARD-FISH) of microorganisms in hydrocarbon contaminated aquifer sediment samples.

    PubMed

    Tischer, Karolin; Zeder, Michael; Klug, Rebecca; Pernthaler, Jakob; Schattenhofer, Martha; Harms, Hauke; Wendeberg, Annelie

    2012-12-01

    Groundwater ecosystems are the most important sources of drinking water worldwide but they are threatened by contamination and overexploitation. Petroleum spills account for the most common source of contamination and the high carbon load results in anoxia and steep geochemical gradients. Microbes play a major role in the transformation of petroleum hydrocarbons into less toxic substances. To investigate microbial populations at the single cell level, fluorescence in situ hybridization (FISH) is now a well-established technique. Recently, however, catalyzed reporter deposition (CARD)-FISH has been introduced for the detection of microbes from oligotrophic environments. Nevertheless, petroleum contaminated aquifers present a worst case scenario for FISH techniques due to the combination of high background fluorescence of hydrocarbons and the presence of small microbial cells caused by the low turnover rates characteristic of groundwater ecosystems. It is therefore not surprising that studies of microorganisms from such sites are mostly based on cultivation techniques, fingerprinting, and amplicon sequencing. However, to reveal the population dynamics and interspecies relationships of the key participants of contaminant degradation, FISH is an indispensable tool. In this study, a protocol for FISH was developed in combination with cell quantification using an automated counting microscope. The protocol includes the separation and purification of microbial cells from sediment particles, cell permeabilization and, finally, CARD-FISH in a microwave oven. As a proof of principle, the distribution of Archaea and Bacteria was shown in 60 sediment samples taken across the contaminant plume of an aquifer (Leuna, Germany), which has been heavily contaminated with several ten-thousand tonnes of petroleum hydrocarbons since World War II. PMID:22425347

  9. Comparative gene mapping in cattle, Indian muntjac, and Chinese muntjac by fluorescence in situ hybridization.

    PubMed

    Murmann, Andrea E; Mincheva, Antoaneta; Scheuermann, Markus O; Gautier, Mathieu; Yang, Fentang; Buitkamp, Johannes; Strissel, Pamela L; Strick, Reiner; Rowley, Janet D; Lichter, Peter

    2008-11-01

    The Indian muntjac (Muntiacus muntjak vaginalis) has a karyotype of 2n = 6 in the female and 2n = 7 in the male. The karyotypic evolution of Indian muntjac via extensive tandem fusions and several centric fusions are well documented by molecular cytogenetic studies mainly utilizing chromosome paints. To achieve higher resolution mapping, a set of 42 different genomic clones coding for 37 genes and the nucleolar organizer region were used to examine homologies between the cattle (2n = 60), human (2n = 46), Indian muntjac (2n = 6/7) and Chinese muntjac (2n = 46) karyotypes. These genomic clones were mapped by fluorescence in situ hybridization (FISH). Localization of genes on all three pairs of M. m. vaginalis chromosomes and on the acrocentric chromosomes of M. reevesi allowed not only the analysis of the evolution of syntenic regions within the muntjac genus but also allowed a broader comparison of synteny with more distantly related species, such as cattle and human, to shed more light onto the evolving genome organization. PMID:18283540

  10. Permeabilization of mycolic-acid-containing actinomycetes for in situ hybridization with fluorescently labelled oligonucleotide probes

    Microsoft Academic Search

    S. J. Macnaughton; A. G. O'Donnelll; T. M. Embley

    1994-01-01

    The application of whole-cell hybridization using labelled oligonucleotide probes in microbial systematics and ecology is limited by difficulties in permeabilizing many Gram-positive organisms. In this investigation paraformaldehyde treatment, acid methanolysis and acid hydrolysis were evaluated as a means of permeabilizing mycolic-acid-containing actinomycetes prior to hybridization with a fluorescently labelled oligonucleotide probe designed to bind to a conserved sequence of bacterial

  11. Novel Repeated DNA Sequences in Safflower (Carthamus tinctorius L.) (Asteraceae): Cloning, Sequencing, and Physical Mapping by Fluorescence in situ Hybridization

    Microsoft Academic Search

    S. N. Raina; S. SHARMA; T. SASAKUMA; M. KISHII; S. VAISHNAVI

    2005-01-01

    Two novel repetitive DNA sequences, pCtKpnI-1 and pCtKpnI-2, were isolated from Carthamus tinctorius (2n ¼ 2x ¼ 24) and cloned. Both represent tandemly repeated sequences. The pCtKpnI-1 and pCtKpnI-2 clones constitute repeat units of 343-345 bp and 367 bp, respectively, with 63% sequence heterogeneity between the two. Fluorescence in situ hybridization (FISH) was employed on metaphase chromo- somes of C.

  12. Detection of a variable number of ribosomal DNA loci by fluorescent in situ hybridization in Populus species.

    PubMed

    Prado, E A; Faivre-Rampant, P; Schneider, C; Darmency, M A

    1996-10-01

    Fluorescent in situ hybridization (FISH) was applied to related Populus species (2n = 19) in order to detect rDNA loci. An interspecific variability in the number of hybridization sites was revealed using as probe an homologous 25S clone from Populus deltoides. The application of image analysis methods to measure fluorescence intensity of the hybridization signals has enabled us to characterize major and minor loci in the 18S-5.8S-25S rDNA. We identified one pair of such rDNA clusters in Populus alba; two pairs, one major and one minor, in both Populus nigra and P. deltoides; and three pairs in Populus balsamifera, (two major and one minor) and Populus euroamericana (one major and two minor). FISH results are in agreement with those based on RFLP analysis. The pBG13 probe containing 5S sequence from flax detected two separate clusters corresponding to the two size classes of units that coexist within 5S rDNA of most Populus species. Key words : Populus spp., fluorescent in situ hybridization, FISH, rDNA variability, image analysis. PMID:18469951

  13. Interphase fluorescence in situ hybridization for trisomy 12 on archival ovarian sex cord-stromal tumors.

    PubMed

    Shashi, V; Golden, W L; von Kap-Herr, C; Andersen, W A; Gaffey, M J

    1994-12-01

    Trisomy 12 is a nonrandom chromosomal abnormality found in a large proportion of ovarian sex cord-stromal tumors (OSCTs), including thecoma-fibromas (TFs) and granulosa cell tumors (GCTs). The prognostic significance of trisomy 12 in these tumors, however, is unknown. A series of 16 OSCTs, obtained from patients with long-term follow-up, was analyzed for the presence of trisomy 12 by interphase fluorescence in situ hybridization on paraffin-embedded sections. Sections of the contralateral nonneoplastic ovary were available in five cases and utilized as controls. Evidence of trisomy 12 was detected in 9 of 10 TFs, and contrary to previous reports, in only one of six GCTs. One TF with trisomy 12 was a malignant variant that resulted in the death of the patient in 5 months, but the remaining TFs with trisomy 12 were cytologically and clinically benign in those with follow-up available. The single GCT with trisomy 12 was a nonaggressive, stage 1 lesion without evidence of recurrence after 264 months, whereas those GCTs without trisomy 12 included one stage 2 tumor and a cytologically atypical GCT with tumor necrosis and an elevated number of mitotic figures. The evidence suggests that the great majority of OSCTs with trisomy 12 is clinically benign, but not all benign OSCTs have trisomy 12. We conclude that the presence of trisomy 12 is of limited prognostic usefulness in OSCTs. PMID:7835773

  14. The importance of using fluorescence in situ hybridization for the diagnosis of Smith-Magenis syndrome

    SciTech Connect

    Juyal, R.C.; Greenberg, F.; Lupski, J.R. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1994-09-01

    Smith-Magenis syndrome (SMS) is a clinically recognizable multiple congenital anomaly/mental retardation syndrome associated with deletion of chromosome 17p11.2. Quality metaphase preparations are required for unambiguous detection of the deletion. We and others have reported cases of SMS due to mosaicism for del(17)(p11.2). Examination of peripheral blood lymphocyte cultures of a patient with the SMS phenotype at 850 band level of resolution revealed a low level mosaicism (11%) for the deletion. Examination of fibroblasts at relatively low resolution revealed the deletion in all cells. In a second study, we reported molecular evidence for mosaicism in the unaffected mother of an SMS patient who demonstrated mosaicism (55%) for the deletion at a resolution level of < 500 bands. We now report a different SMS patient who was initially diagnosed as mosaic del(17)(p11.2) in two different cytogenetic laboratories. A third blinded cytogenetic study yielded a questionable diagnosis. Fluorescence in situ hybridization (FISH) conducted in two different laboratories with two different markers shown to be within the deletion region and a control marker from chromosome 17 demonstrated a deletion in 20/20 and 25/25 metaphases scored, respectively. It appears the latter patient may harbor a very small deletion and that FISH is a more reliable test for the Smith-Magenis deletion. Furthermore, FISH should be used to confirm or refute mosaicism seen in routine cytogenetics studies.

  15. Fluorescence in situ hybridization (FISH): an increasingly demanded tool for biomarker research and personalized medicine

    PubMed Central

    2014-01-01

    Extensive studies of the genetic aberrations related to human diseases conducted over the last two decades have identified recurrent genomic abnormalities as potential driving factors underlying a variety of cancers. Over the time, a series of cutting-edge high-throughput genetic tests, such as microarrays and next-generation sequencing, have been developed and incorporated into routine clinical practice. Although it is a classical low-throughput cytogenetic test, fluorescence in situ hybridization (FISH) does not show signs of fading; on the contrary, it plays an increasingly important role in detecting specific biomarkers in solid and hematologic neoplasms and has therefore become an indispensable part of the rapidly developing field of personalized medicine. In this article, we have summarized the recent advances in FISH application for both de novo discovery and routine detection of chromosomal rearrangements, amplifications, and deletions that are associated with the pathogenesis of various hematopoietic and non-hematopoietic malignancies. In addition, we have reviewed the recent developments in FISH methodology as well. PMID:24499728

  16. Fluorescent in situ hybridization of human sperm: diagnostics, indications, and therapeutic implications.

    PubMed

    Ramasamy, Ranjith; Besada, Stefan; Lamb, Dolores J

    2014-12-01

    Male factor infertility is a relatively common condition, affecting at least 6% of men of reproductive age. Typically, men with unknown genetic abnormalities resort to using assisted reproductive techniques (ART) to achieve their reproductive goals. Infertile men who father biological children using ART could have a higher incidence of aneuploidy, which is a deviation from the normal haploid or diploid chromosomal state. Aneuploidy can be evaluated using fluorescent in situ hybridization (FISH), a cytogenetic assay that gives an estimate of the frequencies of chromosomal abnormalities. The chromosomes that are generally analyzed in FISH (13, 18, 21, X, and Y) are associated with aneuploidies that are compatible with life. The technique is indicated for various reasons but primarily in [1] men who despite normal semen parameters suffer recurrent pregnancy loss, and [2] men with normal semen parameters, who are undergoing in vitro fertilization but still experiencing recurrent implantation failure. As a screening tool, the technique can help in reproductive and genetic counseling of affected couples, or those who have previously experienced failure of ART. A qualitative analysis of FISH study results allows couples to make informed reproductive choices. Given the increasing clinical use of FISH in various infertility diagnoses, and the development of novel adjunct technologies, one can expect much progress in the areas of preimplantation genetic screening, diagnostics, and therapeutics. PMID:25439797

  17. Potential clinical impact of three-dimensional visualization for fluorescent in situ hybridization image analysis

    NASA Astrophysics Data System (ADS)

    Li, Zheng; Li, Shibo; Bin, Zheng; Zhang, Roy; Li, Yuhua; Tian, Huimin; Chen, Wei; Liu, Hong

    2012-05-01

    Chromosomal translocation is strong indication of cancers. Fluorescent in situ hybridization (FISH) can effectively detect this translocation and achieve high accuracy in disease diagnosis and prognosis assessment. For this purpose, whole chromosome paint probes are utilized to image the configuration of DNA fragments. Although two-dimensional (2-D) microscopic images are typically used in FISH signal analysis, we present a case where the translocation occurs in the depth direction where two probed FISH signals are overlapped in the projected image plane. Thus, the translocation cannot be identified. However, when imaging the whole specimen with a confocal microscope at 27 focal planes with 0.5-?m step interval, the translocation can be clearly identified due to the free rotation capability by the three-dimensional (3-D) visualization. Such a translocation detection error of using 2-D images might be critical in detecting and diagnosing early or subtle disease cases where detecting a small number of abnormal cells can make diagnostic difference. Hence, the underlying implication of this report suggests that utilizing 3-D visualization may improve the overall accuracy of FISH analysis for some clinical cases. However, the clinical efficiency and cost of using 3-D versus 2-D imaging methods are also to be assessed carefully.

  18. Interphase fluorescence in situ hybridization signal detection by computing intensity variance along the optical axis

    NASA Astrophysics Data System (ADS)

    Li, Zheng; Zheng, Bin; Ren, Liqiang; Liu, Hong

    2014-02-01

    Fluorescence in situ Hybridization technology is a commonly used tool to detect chromosome aberrations, which are often pathologically significant. Since manual FISH analysis is a tedious and time-consuming procedure, reliable and robust automated image acquisition and analysis are in demand. Under high magnification objective lenses such as 60x and 100x, the depth of field will often be too small and the FISH probes may not always lie in the same focal plane. A statistical variance based automated FISH analysis method is developed in order to address this problem. On a stack of slices at consecutive image planes with a step size d, the statistical variance alone the z-axis is calculated to form a 2-D matrix. Since pixels shift dramatically to high intensity at FISH probe location, the probes will manifest high peak values in the matrix. A computer-aided detection scheme based on top-hat transform is applied to the matrix to detect FISH probe signals. This study demonstrates a simple and robust method for FISH probe detection as well as a way of 2- D representation of 3-D data.

  19. Molecular cytogenetic characterization of the DiGeorge syndrome region using fluorescence in situ hybridization

    SciTech Connect

    Lindsay, E.A. (St. Mary's Hosptial, London (United Kingdom) Imperial Cancer Research Fund, London (United Kingdom)); Halford, S.; Wadey, R.; Scambler, P.J. (St. Mary's Hospital, London (United Kingdom)); Baldini, A. (Instituto di Genetica Molecolare del CNR, Alghero (Italy))

    1993-08-01

    DiGeorge syndrome (DGS) is a developmental defect characterized by cardiac defects, facial dysmorphism, and mental retardation. Several studies have described a critical region for DGS at 22q11, within which the majority of DGS patients have deletions. The authors have isolated nine cosmid and three YAC clones using previously described and newly isolated probes that have been shown to be deleted in many DGS patients. Using fluorescence in situ hybridization and digital imaging, they have mapped and ordered these clones relative to the breakpoints of two balanced translocations at 22q11 (one in a DGS patient and one in the unaffected parent of a DGS child). The data indicate that the breakpoint in the unaffected individual distally limits the DGS critical region (defined as the smallest region of overlap), while proximally the region is limited by repeat-rich DNA. The critical region includes the balanced translocation breakpoint of the DGS patient that presumably disrupts the gene causing this syndrome.

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

    NASA Technical Reports Server (NTRS)

    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.

  1. Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method.

    PubMed

    Machado, António; Castro, Joana; Cereija, Tatiana; Almeida, Carina; Cerca, Nuno

    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

  2. Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method

    PubMed Central

    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

  3. Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization

    PubMed Central

    Williamson, Iain; Berlivet, Soizik; Eskeland, Ragnhild; Boyle, Shelagh; Illingworth, Robert S.; Paquette, Denis

    2014-01-01

    Although important for gene regulation, most studies of genome organization use either fluorescence in situ hybridization (FISH) or chromosome conformation capture (3C) methods. FISH directly visualizes the spatial relationship of sequences but is usually applied to a few loci at a time. The frequency at which sequences are ligated together by formaldehyde cross-linking can be measured genome-wide by 3C methods, with higher frequencies thought to reflect shorter distances. FISH and 3C should therefore give the same views of genome organization, but this has not been tested extensively. We investigated the murine HoxD locus with 3C carbon copy (5C) and FISH in different developmental and activity states and in the presence or absence of epigenetic regulators. We identified situations in which the two data sets are concordant but found other conditions under which chromatin topographies extrapolated from 5C or FISH data are not compatible. We suggest that products captured by 3C do not always reflect spatial proximity, with ligation occurring between sequences located hundreds of nanometers apart, influenced by nuclear environment and chromatin composition. We conclude that results obtained at high resolution with either 3C methods or FISH alone must be interpreted with caution and that views about genome organization should be validated by independent methods. PMID:25512564

  4. Microfluidic fluorescence in situ hybridization and flow cytometry (?FlowFISH).

    PubMed

    Liu, Peng; Meagher, Robert J; Light, Yooli K; Yilmaz, Suzan; Chakraborty, Romy; Arkin, Adam P; Hazen, Terry C; Singh, Anup K

    2011-08-21

    We describe an integrated microfluidic device (?FlowFISH) capable of performing 16S rRNA fluorescence in situ hybridization (FISH) followed by flow cytometric detection for identifying bacteria in natural microbial communities. The device was used for detection of species involved in bioremediation of Cr(vi) and other metals in groundwater samples from a highly-contaminated environmental site (Hanford, WA, USA). The ?FlowFISH seamlessly integrates two components: a hybridization chamber formed between two photopolymerized membranes, where cells and probes are electrophoretically loaded, incubated and washed, and a downstream cross structure for electrokinetically focusing cells into a single-file flow for flow cytometry analysis. The device is capable of analyzing a wide variety of bacteria including aerobic, facultative and anaerobic bacteria and was initially tested and validated using cultured microbes, including Escherichia coli, as well as two strains isolated from Hanford site: Desulfovibrio vulgaris strain RCH1, and Pseudomonas sp.strain RCH2 that are involved in Cr(vi) reduction and immobilization. Combined labeling and detection efficiencies of 74-97% were observed in experiments with simple mixtures of cultured cells, confirming specific labeling. Results obtained were in excellent agreement with those obtained by conventional flow cytometry confirming the accuracy of ?FlowFISH. Finally, the device was used for analyzing water samples collected on different dates from the Hanford site. We were able to monitor the numbers of Pseudomonas sp. with only 100-200 cells loaded into the microchip. The ?FlowFISH approach provides an automated platform for quantitative detection of microbial cells from complex samples, and is ideally suited for analysis of precious samples with low cell numbers such as those found at extreme environmental niches, bioremediation sites, and the human microbiome. PMID:21755095

  5. Microfluidic fluorescence in situ hybridization and flow cytometry (µFlowFISH)

    PubMed Central

    Liu, Peng; Meagher, Robert J.; Light, Yooli Kim; Yilmaz, Suzan; Chakraborty, Romy; Arkin, Adam P.; Hazen, Terry C.; Singh, Anup K.

    2011-01-01

    We describe an integrated microfluidic device (µFlowFISH) capable of performing 16S rRNA fluorescence in situ hybridization (FISH) followed by flow cytometric detection for identifying bacteria in natural microbial communities. The device was used for detection of species involved in bioremediation of Cr(VI) and other metals in groundwater samples from a highly-contaminated environmental site (Hanford, WA, USA). The µFlowFISH seamlessly integrates two components: a hybridization chamber formed between two photopolymerized membranes, where cells and probes are electrophoretically loaded, incubated and washed; and a downstream cross structure for electrokinetically focusing cells into a single-file flow for flow cytometry analysis. The device is capable of analyzing a wide variety of bacteria including aerobic, facultative and anaerobic bacteria and was initially tested and validated using cultured microbes, including Escherichia coli, as well as two strains isolated from Hanford site: Desulfovibrio vulgaris strain RCH1, and Pseudomonas sp. strain RCH2 that are involved in Cr(VI) reduction and immobilization. Combined labeling and detection efficiencies of 74–97% were observed in experiments with simple mixtures of cultured cells confirmed specific labeling. Results obtained were in excellent agreement with those obtained by conventional flow cytometry confirming the accuracy of µFlowFISH. Finally, the device was used for analyzing water samples collected on different dates from the Hanford Site. We were able to monitor the numbers of Pseudomonas sp. with only 100–200 cells loaded into the microchip. The µFlowFISH approach provides an automated platform for quantitative detection of microbial cells from complex samples, and is ideally suited for analysis of precious samples with low cell numbers such as those found at extreme environmental niches, bioremediation sites, and the human microbiome. PMID:21755095

  6. Characterization by fluorescence and electron microscopy in situ hybridization of a double Y isochromosome

    SciTech Connect

    Fetni, R.; Lemieux, N.; Richer, C.L. [Universite de Montreal, Quebec (Canada)] [and others] [Universite de Montreal, Quebec (Canada); and others

    1996-06-14

    A patient with mixed gonadal dysgenesis and Y isochromosomes I(Y) is described. Lymphocyte cultures from peripheral blood contained a high proportion of 45,X cells and several other cell lines with two different marker chromosomes (mars). These markers had either a monocentric (mar1) or a dicentric appearance (mar2). Following high-resolution GTG, RBG, QFQ, and CBG bandings, five cell lines were identified; 45,X/46,X, + mar1/46,X, + mar2/47,X, + mar1x2/47,X + mar2x2. The percentages were 66/6/26/1/1%, respectively. Chromosome banding analyses were insufficient for characterization of the markers. In situ hybridization of specific probes for the Y centromere and its short arm showed, both in fluorescence and electron microscopy (ENT), two different Y rearrangements. Mar1 is an isochromosome for the short arm i(Yp) and mar2 is a dicentric which was shown by EM to be a double isochromosome Yp, inv dup i(Yp). The breakpoint producing mar1 is within the centromere and the one producing mar2 is within one of the short arms of the Y isochromosome. The findings of different cell populations in peripheral blood lymphocytes indicate the postzygotic instability of this i(Yp). 24 refs., 3 figs., 1 tab.

  7. Mapping of a human LIM protein (CLP) to human chromosome 11p15.1 by fluorescence in situ hybridization

    SciTech Connect

    Fung, Y.W.; Wang, R.X.; Heng, H.H.Q.; Liew, C.C. [Univ. of Toronto (Canada)] [Univ. of Toronto (Canada)

    1995-08-10

    Using fluorescence in situ hybridization (FISH), we have localized the gene for cardiac LIM protein to human chromosome 11, between regions p14.3 and p15.2, most likely on 11p15.1. It is likely that CLP plays a regulatory role in muscle-specific gene expression in cardiac and skeletal muscles. The FISH data of human CLP provide a gene-associated marker for genetic mapping. 10 refs., 1 fig.

  8. Rapid Identification of Staphylococcus aureus Directly from Blood Cultures by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes

    Microsoft Academic Search

    Kenneth Oliveira; Gary W. Procop; Deborah Wilson; James Coull; Henrik Stender

    2002-01-01

    A new fluorescence in situ hybridization (FISH) method with peptide nucleic acid (PNA) probes for identification of Staphylococcus aureus directly from positive blood culture bottles that contain gram-positive cocci in clusters (GPCC) is described. The test (the S. aureus PNA FISH assay) is based on a fluorescein- labeled PNA probe that targets a species-specific sequence of the 16S rRNA of

  9. Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes for Rapid Identification of Candida albicans Directly from Blood Culture Bottles

    Microsoft Academic Search

    Susan Rigby; Gary W. Procop; Gerhard Haase; Deborah Wilson; Cletus Kurtzman; Kenneth Oliveira; Sabina Von Oy; Jens J. Hyldig-Nielsen; James Coull; Henrik Stender

    2002-01-01

    A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C.

  10. Localization of the human OB gene (OBS) to chromosome 7q32 by fluorescence in situ hybridization

    SciTech Connect

    Geffroy, S.; Duban, B.; Martinville, B. de [Universitaire de Lille (France)] [and others] [Universitaire de Lille (France); and others

    1995-08-10

    An important gene involved in the pathogenesis of obesity is the product of the human homologue of the murine obese gene (gene symbol OBS). Using fluorescence in situ hybridization (FISH), we have localized the human OB gene to human chromosome 7, specifically to region 7q32.1. The FISH data of human OBS provide a gene-associated marker for genetic mapping. 8 refs., 1 fig.

  11. Accurate characterization of porcine bivariate flow karyotype by PCR and fluorescence in situ hybridization.

    PubMed

    Yerle, M; Schmitz, A; Milan, D; Chaput, B; Monteagudo, L; Vaiman, M; Frelat, G; Gellin, J

    1993-04-01

    The 19 chromosomal pairs of the swine karyotype are resolved into 18 peaks denoted A to Q and Y by dual-beam flow cytometry. The chromosomal content of six peaks has previously been determined by analyzing male/female differences, karyotypes of animals carrying translocations, and PCR studies of genes with known assignments. For the remaining chromosomes, putative assignments to flow peaks were deduced from comparison of DNA contents, determined by flow cytometry, and chromosomal size. We present here the complete characterization of the pig bivariate flow karyotype using the PARM-PCR technique combined with fluorescence in situ hybridization. Chromosome-specific probes were generated by PCR amplification of 300 sorted chromosomes with primers under nonspecific conditions and used to paint chromosomes by FISH. The chromosomal content of each peak was identified: peaks A (chromosome 1), B (13), C (6), D (2), E (14), F (3), G (7), H (9,4,X), H1 (9), I (15), J (8), K (5), L (10), M (12), N (16), O (11), P (17), Q (18), Y (Y). We were able to characterize perfectly the pig bivariate flow karyotype. Such techniques could be applied to any other species. PMID:8486390

  12. Investigation of chromosomal aberrations in Egyptian hepatocellular carcinoma patients by fluorescence in situ hybridization

    PubMed Central

    Aly, Magdy S.; Bahnassy, Abeer A.; Abdel-Rahman, Zekri N.

    2010-01-01

    BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is a very common and highly malignant tumor, associated mainly with chronic viral hepatitis, cirrhosis of any cause, aflatoxin exposure and ethanol consumption. Cytogenetic analysis on HCC has been limited because of poor hepatocyte growth in vitro. Conventional cytogenetic studies have demonstrated frequent abnormalities of specific chromosomes in HCC. Molecular cytogenetic approaches have been applied only rarely in the characterization of HCC. The main aim of this study was to evaluate genetic aberrations of different chromosomes in HCC. The study included 35 patients with HCC, who have been diagnosed and treated at National Cancer Institute, Cairo University, Egypt. The clinico-pathologic features of the studied patient were collected from patient’s files. MATERIALS AND METHODS: Interphase cytogenetics by fluorescence in situ hybridization with the use of a panel of centromere-associated DNA probes for chromosomes 1, 4, 8, 9, 13, 17, 20 and Y were performed on paraffin-embedded HCC specimens. RESULTS: The most common chromosomal aberrations detected were gain of chromosomes 8 in 12 cases (34.28%), 17 in 6 cases (17.14%). Loss of chromosome Y was detected in 6 of male cases (30%). Monosomy 4 was also detected in 5 cases (14.28%). Negative correlation could be detected only between chromosome 4 and 8. (r = -0.381, P < 0.05). Correlations between gain or loss of chromosomes and the different clinicopathologic parameters in the patients investigated, indicated negative correlation between: chromosome Y and age and chromosome 1 and cirrhosis. CONCLUSION: Gains and losses of DNA found in this study probably involve oncogenes and tumor suppressor genes that play a role in the puzzle of hepatocarcinogenesis. PMID:21031057

  13. Evaluation of fluorescence in situ hybridization for the detection of bacteria in feline inflammatory liver disease.

    PubMed

    Twedt, David C; Cullen, John; McCord, Kelly; Janeczko, Stephanie; Dudak, Julie; Simpson, Kenny

    2014-02-01

    The etiopathogenesis of feline inflammatory liver disease (ILD) is unclear. Therefore, we sought to determine the presence and distribution of bacteria within the livers of cats with ILD using eubacterial fluorescence in situ hybridization (FISH). Histopathology from 39 cats with ILD and 19 with histologically normal livers (C) were classified using World Small Animal Veterinary Association guidelines. Hepatic sections were examined by 16 and 23S ribosomal RNA FISH. Antibodies against cytokeratins and factor VIIIa were used to distinguish bile ducts and vascular structures. Histopathologic findings included non-specific reactive hepatitis (12), neutrophilic cholangitis (NC; 12), lymphocytic cholangitis (seven), cholestasis/obstruction (three), probable lymphoma (three) and acute hepatitis (two). Bacteria were observed in 21/39 ILD and 3/19 C (P = 0.0054). In 8/39 ILD and 2/19 C bacteria were restricted to the outer liver capsule (P = 0.29) and may represent contaminants. The prevalence of intrahepatic bacteria was higher (P = 0.008) in ILD (13/31) than C (1/17). Bacteria in ILD were more frequently (P <0.0001) localized to portal vessels, venous sinusoids and parenchyma (12/13) than bile duct (1/13). Bacterial colonization was highest in Escherichia coli-positive NC cats. Concurrent non-hepatic disease, predominantly pancreatic and intestinal (8/10 cats biopsied), was present in all 13 cats with intrahepatic bacteria. Bacterial culture was positive (predominantly E coli and Enterococcus species) in 11/23 (48%) samples, and concurred with FISH in 15/23 cases. The presence of intrahepatic bacteria in 13/31 (41%) cats with ILD suggests a role in etiopathogenesis. The distribution of bacteria within the liver supports the possibility of colonization via either enteric translocation or hematogenous seeding. PMID:23884636

  14. Smith-Magenis syndrome deletion: A case with equivocal cytogenetic findings resolved by fluorescence in situ hybridization

    Microsoft Academic Search

    Ramesh C. Juyal; P. I. Patel; F. Greenberg; James R. Lupski; Barbara J. Trask; Ger van den Engh; Elizabeth A. Lindsay; Heather Christy; Ken-Shiung Chen; Antonio Baldini; Lisa G. Shaffer

    1995-01-01

    The availability of markers for the 17p11.2 region has enabled the diagnosis of Smith-Magenis syndrome (SMS) by fluorescence in situ hybridization (FISH). SMS is typically associated with a discernible deletion of band 17p11.2 upon cytogenetic analysis at a resolution of 400-550 bands. We present a case that illustrates the importance of using FISH to confirm a cytogenetic diagnosis of del(17)(p11.2).

  15. Fluorescence in situ hybridization investigation of potentially pathogenic bacteria involved in neonatal porcine diarrhea

    PubMed Central

    2014-01-01

    Background Neonatal diarrhea is a multifactorial condition commonly present on pig farms and leads to economic losses due to increased morbidity and mortality of piglets. Immature immune system and lack of fully established microbiota at birth predispose neonatal piglets to infection with enteric pathogens. The microorganisms that for decades have been associated with enteritis and diarrhea in suckling piglets are: rotavirus A, coronavirus, enterotoxigenic Escherichia coli (ETEC), Clostridium perfringens type C, Cryptosporidium spp., Giardia spp., Cystoisospora suis and Strongyloides ransomi. However, in recent years, the pig industry has experienced an increased number of neonatal diarrhea cases in which the above mentioned pathogens are no longer detected. Potentially pathogenic bacteria have recently received focus in the research on the possible etiology of neonatal diarrhea not caused by common pathogens. The primary aim of this study was to investigate the role of E. coli, Enterococcus spp., C. perfringens and C. difficile in the pathogenesis of neonatal porcine diarrhea with no established casual agents. Fluorescence in situ hybridization with oligonucleotide probes was applied on the fixed intestinal tissue samples from 51 diarrheic and 50 non-diarrheic piglets collected from four Danish farms during outbreaks of neonatal diarrhea not caused by well-known enteric pathogens. Furthermore, an association between the presence of these bacteria and histological lesions was evaluated. Results The prevalence of fluorescence signals specific for E. coli, C. perfringens and C. difficile was similar in both groups of piglets. However, Enterococcus spp. was primarily detected in the diarrheic piglets. Furthermore, adherent bacteria were detected in 37 % diarrheic and 14 % non-diarrheic piglets. These bacteria were identified as E. coli and Enterococcus spp. and their presence in the intestinal mucosa was associated with histopathological changes. Conclusions The results of this study showed that simultaneous colonization of the intestinal mucosa by adherent non-ETEC E. coli and Enterococcus spp. can be involved in the pathogenesis of neonatal porcine diarrhea. These bacteria should be considered in diagnosis of diarrhea in piglets, when detection of common, well-known enteric agents is unsuccessful. PMID:24628856

  16. Recognition of individual genes in a single bacterial cell by fluorescence in situ hybridization--RING-FISH.

    PubMed

    Zwirglmaier, K; Ludwig, W; Schleifer, K-H

    2004-01-01

    Fluorescence in situ hybridization (FISH) using rRNA targeted oligonucleotide probes is a standard method for identification of microorganisms in environmental samples. Apart from its value as a phylogenetic marker ribosomal RNA has always been the favoured target molecule for FISH because of its abundance in all cells, whereas plasmids and DNA were regarded as unsuitable targets because of their low copy number. Here we present an improved FISH technique, which is based on polynucleotide probes. It goes beyond the detection of high copy intracellular nucleic acids such as rRNA (up to 10(4)-10(5) copies per cell) and allows for the first time the in situ detection of individual genes or gene fragments on plasmids (10(1)-10(3) copies per cell) and chromosomal DNA (<10 copies per cell) in a single cell. Using E. coli as model organism we were able to detect in situ cells harbouring the antibiotic resistance gene beta lactamase on high, medium and low copy plasmids as well as the chromosomal encoded housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, we detected the prepilin peptidase gene xpsO in the plant pathogen Xanthomonas campestris in situ. Because of the characteristic hybridization signal obtained with this method--a halo-like, ring-shaped concentration of fluorescence in the cell periphery--we coined the term RING-FISH (recognition of individual genes) to differentiate it from conventional FISH. PMID:14651613

  17. Identification of autosomal supernumerary chromosome markers (SMCs) by fluorescent in situ hybridization (FISH).

    PubMed

    Kolialexi, A; Kitsiou, S; Fryssira, H; Sofocleous, C; Kouvidi, E; Tsangaris, G Th; Salavoura, K; Mavrou, A

    2006-01-01

    Supernumerary marker chromosomes (SMCs) are rare chromosomal abnormalities resulting in partial trisomy of specific genomic regions with characteristic phenotypic effects. Twenty six cases with autosomal SMCs are reported. Four were identified prenatally and 22 postnatally in children, aged from 8 days to 15 years, who were referred for genetic evaluation because of various congenital anomalies and developmental delay. In 22 of the 26 cases, the SMCs were de novo, in two they were familial and in another two a 11;22 reciprocal translocation was revealed in the mothers. In only one patient was the SMC present in a mosaic form. Sequential fluorescent in situ hybridization studies (FISH) using Whole Chromosome Paint (WCP) probes were performed in order to determine the chromosomal origin of the SMCs. Sixteen of them originated from chromosome 15, five were shown to be an isochromosome 18p and one was derived from chromosome 22, but did not contain the DiGeorge/ VCFS critical region. In two instances, the SMCs were derivatives of chromosome 13 and in two the SMCs resulted from a 11;22 maternal translocation and contained material from both chromosomes 11 and 22. Molecular investigation of two of the patients with an SMC[15] revealed three copies of the SNRPN gene, but the diagnosis of PW/AS due to possible imprinting was excluded in both patients by a methylation-specific PCR. FISH and molecular studies have greatly facilitated the characterization of marker chromosomes. As more SMCs are classified, better genetic counseling and risk evaluation can be achieved. PMID:16900777

  18. Chromosomal organization and fluorescence in situ hybridization of the human Sirtuin 6 gene.

    PubMed

    Mahlknecht, Ulrich; Ho, Anthony D; Voelter-Mahlknecht, Susanne

    2006-02-01

    Sirtuin 6 (SIRT6) is a member of the sirtuin deacetylases (sirtuins), which are derivatives of the yeast Silent information regulator 2 (Sir2) protein. SIR2 and its mammalian derivatives play a central role in epigenetic gene silencing, recombination, metabolism, cell differentiation and in the regulation of aging. In contrast to most sirtuins, SIRT6 lacks NAD+-dependent protein deacetylase activity. We have isolated and characterized the human Sirt6 genomic sequence, which spans a region of 8,427 bp and which has one single genomic locus. Determination of the exon-intron splice junctions found the full-length SIRT6 protein to consist of 8 exons ranging in size from 60 bp (exon 4) to 838 bp (exon 8). The human Sirt6 open reading frame encodes a 355-aa protein with a predictive molecular weight of 39.1 kDa and an isoelectric point of 9.12. Characterization of the 5' flanking genomic region, which precedes the Sirt6 open reading frame, revealed a TATA- and CCAAT-box less promoter with an approximately 300-bp long CpG island. A number of AML-1 and GATA-x transcription factor binding sites were found which remain to be further evaluated experimentally. Fluorescence in situ hybridization analysis localized the human Sirt6 gene to chromosome 19p13.3; a region which is frequently affected by chromosomal alterations in acute leukemia. Human SIRT6 appears to be most predominantly expressed in bone cells and in the ovaries while, in the bone marrow, it is practically absent. The functional characteristics of SIRT6 are essentially unknown at present and remain to be elucidated. PMID:16391800

  19. Fluorescence in situ hybridization and chromosomal organization of the human Sirtuin 7 gene.

    PubMed

    Voelter-Mahlknecht, Susanne; Letzel, Stephan; Mahlknecht, Ulrich

    2006-04-01

    Sirtuin 7 (SIRT7) is a member of the sirtuin family of protein deacetylases and is, therefore, a derivative of yeast Silent information regulator 2 (SIR2). SIR2 and its mammalian orthologs play an important role in epigenetic gene silencing, DNA recombination, cellular differentiation and metabolism, and the regulation of aging. In contrast to most sirtuins, SIRT7 does not exert characteristic NAD+-dependent deacetylase activity. We have isolated and characterized the human Sirt7 genomic sequence, which spans a region of 6.2 kb and which has one single genomic locus. Determination of the exon/intron splice junctions found the full-length SIRT7 protein to consist of 10 exons ranging in size from 71 bp (exon 4) to 237 bp (exon 7). The human Sirt7 open reading frame encodes a 400-aa protein with a predictive molecular weight of 44.9 kDa and an isoelectric point of 9.80. Characterization of the 5' flanking genomic region, which precedes the Sirt7 open reading frame, revealed a TATA- and CCAAT-box less promoter that lacks CpG islands. A number of AML-1 and GATA-x transcription factor binding sites were found, which remain to be further evaluated experimentally. Fluorescence in situ hybridization analysis localized the human Sirt7 gene to chromosome 17q25.3; a region which is frequently affected by chromosomal alterations in acute leukemias and lymphomas. Human SIRT7 appears to be most predominantly expressed in the blood and in CD33+ myeloid bone marrow precursor cells, while the lowest levels are found in the ovaries and skeletal muscle. Functional characteristics of SIRT7 are essentially unknown at present and remain to be further elucidated. PMID:16525639

  20. Rapid-prenatal diagnosis through fluorescence in situ hybridization for preventing aneuploidy related birth defects

    PubMed Central

    Fauzdar, Ashish; Chowdhry, Mohit; Makroo, R. N.; Mishra, Manoj; Srivastava, Priyanka; Tyagi, Richa; Bhadauria, Preeti; Kaul, Anita

    2013-01-01

    BACKGROUND AND OBJECTIVE: Women with high-risk pregnancies are offered prenatal diagnosis through amniocentesis for cytogenetic analysis of fetal cells. The aim of this study was to evaluate the effectiveness of the rapid fluorescence in situ hybridization (FISH) technique for detecting numerical aberrations of chromosomes 13, 21, 18, X and Y in high-risk pregnancies in an Indian scenario. MATERIALS AND METHODS: A total of 163 samples were received for a FISH and/or a full karyotype for prenatal diagnosis from high-risk pregnancies. In 116 samples both conventional culture techniques for getting karyotype through G-banding techniques were applied in conjunction to FISH test using the AneuVysion kit (Abbott Molecular, Inc.), following standard recommended protocol to compare the both the techniques in our setup. RESULTS: Out of 116 patients, we got 96 normal for the five major chromosome abnormality and seven patients were found to be abnormal (04 trisomy 21, 02 monosomy X, and 01 trisomy 13) and all the FISH results correlated with conventional cytogenetics. To summarize the results of total 163 patients for the major chromosomal abnormalities analyzed by both/or cytogenetics and FISH there were 140 (86%) normal, 9 (6%) cases were abnormal and another 4 (2.5%) cases were suspicious mosaic and 10 (6%) cases of culture failure. The diagnostic detection rate with FISH in 116 patients was 97.5%. There were no false-positive and false-negative autosomal or sex chromosomal results, within our established criteria for reporting FISH signals. CONCLUSION: Rapid FISH is a reliable and prompt method for detecting numerical chromosomal aberrations and has now been implemented as a routine diagnostic procedure for detection of fetal aneuploidy in India. PMID:23901191

  1. A pre-breeding screening program for transgenic boars based on fluorescence in situ hybridization assay.

    PubMed

    Bou, Gerelchimeg; Sun, Mingju; Lv, Ming; Zhu, Jiang; Li, Hui; Wang, Juan; Li, Lu; Liu, Zhongfeng; Zheng, Zhong; He, Wenteng; Kong, Qingran; Liu, Zhonghua

    2014-08-01

    For efficient transgenic herd expansion, only the transgenic animals that possess the ability to transmit transgene into next generation are considered for breeding. However, for transgenic pig, practically lacking a pre-breeding screening program, time, labor and money is always wasted to maintain non-transgenic pigs, low or null transgenic transmission pigs and the related fruitless gestations. Developing a pre-breeding screening program would make the transgenic herd expansion more economical and efficient. In this technical report, we proposed a three-step pre-breeding screening program for transgenic boars simply through combining the fluorescence in situ hybridization (FISH) assay with the common pre-breeding screening workflow. In the first step of screening, combined with general transgenic phenotype analysis, FISH is used to identify transgenic boars. In the second step of screening, combined with conventional semen test, FISH is used to detect transgenic sperm, thus to identify the individuals producing high quality semen and transgenic sperm. In the third step of screening, FISH is used to assess the in vitro fertilization embryos, thus finally to identify the individuals with the ability to produce transgenic embryos. By this three-step screening, the non-transgenic boars and boars with no ability to produce transgenic sperm or transgenic embryos would be eliminated; therefore only those boars could produce transgenic offspring are maintained and used for breeding and herd expansion. It is the first time a systematic pre-breeding screening program is proposed for transgenic pigs. This program might also be applied in other transgenic large animals, and provide an economical and efficient strategy for herd expansion. PMID:24788205

  2. LINE-1 elements: analysis by fluorescence in-situ hybridization and nucleotide sequences.

    PubMed

    Waters, Paul D; Dobigny, Gauthier; Waddell, Peter J; Robinson, Terence J

    2008-01-01

    Long-interspersed nuclear element-1 (LINE-1) is a non-terminal repeat transposon that constitutes a major component of the mammalian genome. LINE-1 has a dynamic evolutionary history characterized by the rise, fall, and replacement of subfamilies. The distribution of LINE-1 elements can be viewed from a chromosomal perspective using fluorescence in-situ hybridization (FISH), as well as at the sequence level. We have designed LINE-1 primers from regions conserved among mouse, rat, rabbit, and human L1, which were able to amplify part of ORF2 from all eutherian (placental) mammals tested thus far. The product generated can be used as a FISH painting probe to examine the genomic distribution of L1 in different species. It can also be cloned and sequenced for phylogenetic analysis. Although FISH patterns resulting from LINE-1 chromosome painting and bioinformatic analyses have shown that this element accumulates in AT-rich regions of the genomes of mouse and human, our PCR amplified LINE-1 probe suggests that this is not a universal phenomenon, and that the patterns displayed in laurasiatherian, afrotherian and xenarthran species are less prominent. The "banding" like distribution of LINE-1 observed in human and mouse, therefore, appears to reflect aspects of genome architecture unique to Euarchontoglires (Supraprimates), the superordinal clade to which they belong. By sequencing the cloned amplicons used for FISH experiments and supplementing these with L1 sequences obtained from public databases, analysis by parsimony, distance-based, maximum likelihood, and "hierarchical Bayesian" or "marginal likelihood" methods provides a powerful adjunct to the FISH data. Using this approach, relatively intact LINE-1 from most placental orders tend to reflect accepted eutherian evolutionary relationships. This suggests that there were often only closely related copies active near branch points in the tree, that inactive copies tended to become extinct quite readily, and that for many orders recently active copies belong to a single lineage of this LINE. PMID:18629670

  3. Identification of a centromeric exchange of acrocentric chromosomes by fluorescence in situ hybridization

    SciTech Connect

    Yu, C.W.; Immken, L.; Curry, C.J.R. [UCSF, Fresno, CA (United States)] [and others

    1994-09-01

    Exchanges of the peri-centromeric area of acrocentric chromosomes are difficult to identify using the conventional cytogenetic techniques. Fluorescence in situ hybridization (FISH) provides a new way for precisely identifying such rearrangements. Here we report a case of centromeric rearrangement in an amniotic fluid specimen with an extra marker chromosome. M.G., a 41-year-old G1, was referred for advanced maternal age. Chromosome studies revealed a 47,XX +mar karyotype. The marker appeared to be bi-satallited with a single C band. Chromosome studies from the parents were normal. The parents elected to terminate the pregnancy. Anatomical examination of the abortus revealed a very short neck, posteriorly rotated ears, high set cecum, absent hepatic lobation and low abdominal kidneys with short ureters. FISH studies with alpha-satellite probes of 13/21, 14/22, and 15, and the DiGeorge probe, indicated that there is a translocation of 21 alpha-satellite to the 22, and that the marker chromosome probably consists of 14/22 alpha-satellite material. FISH analysis of the parents chromosome revealed that father had the translocation of 21 alpha-satellite to the 22 as well. Exchanges of centromeric material among the acrocentric chromosomes may not be an uncommon event in humans. Although it probably has no clinical significance, it may result in non-disjunction or marker chromosome formation from an uncommon satellite association. With the use of FISH techniques, exchanges involving the centromeric regions of acrocentric chromosomes can be identified.

  4. Assignment of the human ubiquitous receptor gene (UNR) to 19q13.3 using fluorescence in situ hybridization

    SciTech Connect

    Le Beau, M.M.; Song, C.; Davis, E.M. [Univ. of Chicago, IL (United States)] [and others] [Univ. of Chicago, IL (United States); and others

    1995-03-01

    Human UNR cDNAs were used to screen a Lambda FIX II human male placenta genomic library. Phage DNA from clones hybridizing to UNR cDNA was characterized by Southern hybridization and restriction mapping, and two different clones (hG10 and hG12) with inserts of 15-20 kb were chosen for fluorescence in situ hybridization (FISH) analysis. Biotin-labeled probes were prepared from phage DNA by nick-translation using Bio-11-dUTP. FISH was performed as described previously. Hybridization was detected with fluorescien-conjugated avidin, and chromosomes were identified by staining with 4,6-diamidino-2-phenylindole-dihydrochloride (DAPI).

  5. Direct visualization of the novel pathogen, Spiroplasma eriocheiris, in the freshwater crayfish Procambarus clarkii (Girard) using fluorescence in situ hybridization.

    PubMed

    Ding, Z F; Xia, S Y; Xue, H; Tang, J Q; Ren, Q; Gu, W; Meng, Q G; Wang, W

    2014-08-29

    Spiroplasma eriocheiris is the first spiroplasma strain known to be pathogenic to freshwater crustaceans. It has caused considerable economic losses both in the freshwater crayfish Procambarus clarkii (Girard) and in some other crustaceans. The monitoring of the pathogen in crustacean populations and study of its behaviour in the laboratory require the development of reliable diagnostic tools. In this article, we improved microscopic identification of S. eriocheiris by combining in situ hybridization with specific fluorescently labelled oligonucleotide probes. The established fluorescence in situ hybridization (FISH) allowed simultaneous visualization, identification and localization of S. eriocheiris in the tissues of diseased crayfish P. clarkii and exhibited low background autofluorescence and ideal signal-to-noise ratio. With the advantages of better tissue penetration, potentially more specific and stable, we designed three species-specific oligonucleotide probes utilizing the sequences of 16S-23S rRNA intergenic spacer regions (ISRs) of S. eriocheiris. Positive hybridization signals were visualized in haemocytes and connective tissues of hepatopancreas, cardiac muscle and gill from diseased crayfish. This unique distribution pattern matched the pathological changes when diagnosed by H&E staining and indicated that S. eriocheiris probably spread throughout the tissues in P. clarkii by hemokinesis. This assay will facilitate our understanding of the pathogenesis of S. eriocheiris and enhance the early diagnosis of the novel pathogen. PMID:25167936

  6. QUANTITATIVE IMAGING AND STATISTICAL ANALYSIS OF FLUORESCENCE IN SITU HYBRIDIZATION (FISH) OF AUREOBASIDIUM PULLULANS. (R823845)

    EPA Science Inventory

    Abstract Image and multifactorial statistical analyses were used to evaluate the intensity of fluorescence signal from cells of three strains of A. pullulans and one strain of Rhodosporidium toruloides , as an outgroup, hybridized with either a universal o...

  7. High resolution multicolor fluorescence in situ hybridization using cyanine and fluorescein dyes: rapid chromosome identification by directly fluorescently labeled alphoid DNA probes.

    PubMed

    Yurov, Y B; Soloviev, I V; Vorsanova, S G; Marcais, B; Roizes, G; Lewis, R

    1996-03-01

    We tested DNA probes directly labeled by fluorescently labeled nucleotides (Cy3-dCTP, Cy5-dCTP, FluorX-dCTP) for high resolution uni- and multicolor detection of human chromosomes and analysis of centromeric DNA organization by in situ hybridization. Alpha-satellite DNA probes specific to chromosomes 1, 2, 3, 4 + 9, 5 + 19, 6, 7, 8, 10, 11, 13 + 21, 14 + 22, 15, 16, 17, 18, 20, 22, X and Y were suitable for the accurate identification of human chromosomes in metaphase and interphase cells. Cy3-labeled probes had several advantages: (1) a high level of fluorescence (5-10 times more compared with fluorescein-labeled probes); (2) a low level of fluorescence in solution, allowing the detection of target chromosomes in situ during hybridization without the washing of slides; and (3) high resistance to photobleaching during prolonged (1-2 h) exposure to strong light, thus allowing the use of a high energy mercury lamp or a long integration time during image acquisition in digital imaging microscopy for the determination of weak signals. For di- and multicolor fluorescence in situ hybridization (FISH), we successfully used different combinations of directly fluorophorated probes with preservation of images by conventional microscopy or by digital imaging microscopy. FluorX and Cy3 dyes allowed the use of cosmid probes for mapping in a one-step hybridization experiment. Cyanine-labeled fluorophorated DNA probes offer additional possibilities for rapid chromosome detection during a simple 15-min FISH procedure, and can be recommended for basic research and clinical studies, utilizing FISH. PMID:8786090

  8. Whole-mount fluorescent in situ hybridization staining of the colonial tunicate Botryllus schlosseri.

    PubMed

    Langenbacher, Adam D; Rodriguez, Delany; Di Maio, Alessandro; De Tomaso, Anthony W

    2015-01-01

    Botryllus schlosseri is a colonial ascidian with characteristics that make it an attractive model for studying immunology, stem cell biology, evolutionary biology, and regeneration. Transcriptome sequencing and the recent completion of a draft genome sequence for B. schlosseri have revealed a large number of genes, both with and without vertebrate homologs, but analyzing the spatial and temporal expression of these genes in situ has remained a challenge. Here, we report a robust protocol for in situ hybridization that enables the simultaneous detection of multiple transcripts in whole adult B. schlosseri using Tyramide Signal Amplification in conjunction with digoxigenin- and dinitrophenol-labeled RNA probes. Using this protocol, we have identified a number of genes that can serve as markers for developing and mature structures in B. schlosseri, permitting analysis of phenotypes induced in loss-of-function experiments. PMID:25179474

  9. Use of fluorescence in situ hybridization to detect and monitor transfected and amplified sequences in recombinant CHO cells

    SciTech Connect

    Pallavicini, M.G.; DeTeresa, P.S.; Wurm, F.M.

    1988-08-04

    Fluorescence In Situ Hybridization (FISH) can be used routinely to detect and monitor foreign DNA sequences in recombinant mammalian cells. Non-radioactive hybridization and detection of foreign DNA can be performed within 1-2 days, in contrast to weeks often required for autoradiographic analysis. Since cells can be evaluated on an individual basis, cell-to-cell variation in integration patterns of foreign DNA in populations of cells can be assessed. The effects of culture conditions on DNA integration patterns are also easily monitored by FISH. FISH technology is a useful tool, therefore, for monitoring of foreign genes in recombinant mammalian cells. Further improvements of the method and accumulation of more data from cells grown in and selected for various concentration of MTX will allow us to achieve a better understanding of the mechanisms responsible for integration and amplification of transfected DNA in CHO cells. 16 refs., 5 figs.

  10. A high-resolution karyotype of Brassica rapa ssp. pekinensis revealed by pachytene analysis and multicolor fluorescence in situ hybridization

    Microsoft Academic Search

    Dal-Hoe Koo; Prikshit Plaha; Yong Pyo Lim; Yoonkang Hur; Jae-Wook Bang

    2004-01-01

    A molecular cytogenetic map of Chinese cabbage ( Brassica rapa ssp. pekinensis, 2 n=20) was constructed based on the 4?-6-diamino-2-phenylindole dihydrochloride-stained mitotic metaphase and pachytene chromosomes and multicolor fluorescence in situ hybridization (McFISH), using three repetitive DNA sequences, 5S rDNA, 45S rDNA, and C11-350H. The lengths of mitotic metaphase chromosomes ranged from 1.46 ?m to 3.30 ?m. Five 45S and three 5S

  11. [Identification of fungal pathogens in tissue samples using fluorescence in situ hybridization].

    PubMed

    Rickerts, V

    2013-11-01

    Deep fungal infections are associated with significant mortality despite the availability of new antifungal agents. The identification of causative fungi is important to define successful antifungal therapies as agents differ in the in vitro susceptibility. Characterization of tissue morphology and cultivation from tissue provide important clues to patient management. Molecular techniques such as PCR-based assays are increasingly being used to identify agents of invasive fungal infections. However, potential contamination limits the use when ubiquitous fungi are targeted. Hybridization with fluorescently labeled probes targeting the ribosomal RNA of fungi is emerging as an alternative identification strategy. Using conserved or variable regions of the rRNA as targets, group or species-specific probes can be synthesized to identify fungal pathogens and localize them in the infectious process. These techniques have been successfully applied to deep fungal infections due to different agents in various organ samples. PMID:24071866

  12. Improved signal recognition for interphase fluorescent in-situ hybridization using a non-ionic detergent (NP-40) pretreatment

    SciTech Connect

    Zhu, H.M.; Day-Salvatore, D.L.; Sciorra, L.J. [Univ. of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, NJ (United States)] [and others

    1994-09-01

    We have reported that the non-ionic detergent ethylphenolpoly (ethyleneglycolether)x known as Noniet-P40 (Shell International Petroleum) can gently disrupt cell membranes, resulting in cells with varying degrees of free chromatin release. The extent of this phenomena is dependent upon the concentration of NP-40 and the detergent`s exposure time to the cells. Treated cells can range from halos of DNA around the cells to fully extended free chromatin configurations. We have demonstrated that these treated cells are excellent targets for many different fluorescently labelled probes used for in situ hybridization studies. Recently, we have compared NP-40 harvested lymphocytes with normally harvested cells to see if we could improve upon the number of cells showing discreet signals in interphase fluorescent in situ hybridization. Preliminary work has shown that using a trisomy 21 cell line, one can get a statistically significant improvement with NP-40 pretreatment cells over control levels, in the number of cells having three discreet signals in interphase {open_quotes}FISH{close_quotes}. Such a pretreatment is simple to perform and may be of value when the number of cells available for analysis is low, as in the search for fetal cells from maternal circulation.

  13. Fluorescence in situ hybridization techniques (FISH) to detect changes in CYP19a gene expression of Japanese medaka (Oryzias latipes)

    SciTech Connect

    Park, June-Woo [Department of Zoology, National Food Safety and Toxicology Center and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Tompsett, Amber [Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan (Canada); Zhang, Xiaowei [Department of Zoology, National Food Safety and Toxicology Center and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan (Canada); Newsted, John L. [Department of Zoology, National Food Safety and Toxicology Center and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); ENTRIX, Inc., Okemos, MI 48823 (United States); Jones, Paul D.; Au, Doris [Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong (China); Kong, Richard; Wu, Rudolf S.S. [School of Environmental Science, Nanjing University, Nanjing (China); Giesy, John P. [Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan (Canada); Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong (China); School of Environmental Science, Nanjing University, Nanjing (China); ENTRIX, Inc., Saskatoon, Saskatchewan (Canada)], E-mail: JGiesy@aol.com; Hecker, Markus [Department of Zoology, National Food Safety and Toxicology Center and Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong (China); ENTRIX, Inc., Saskatoon, Saskatchewan (Canada)

    2008-10-15

    The aim of this study was to develop a sensitive in situ hybridization methodology using fluorescence-labeled riboprobes (FISH) that allows for the evaluation of gene expression profiles simultaneously in multiple target tissues of whole fish sections of Japanese medaka (Oryzias latipes). To date FISH methods have been limited in their application due to autofluorescence of tissues, fixatives or other components of the hybridization procedure. An optimized FISH method, based on confocal fluorescence microscopy was developed to reduce the autofluorescence signal. Because of its tissue- and gender-specific expression and relevance in studies of endocrine disruption, gonadal aromatase (CYP19a) was used as a model gene. The in situ hybridization (ISH) system was validated in a test exposure with the aromatase inhibitor fadrozole. The optimized FISH method revealed tissue-specific expression of the CYP19a gene. Furthermore, the assay could differentiate the abundance of CYP19a mRNA among cell types. Expression of CYP19a was primarily associated with early stage oocytes, and expression gradually decreased with increasing maturation. No expression of CYP19a mRNA was observed in other tissues such as brain, liver, or testes. Fadrozole (100 {mu}g/L) caused up-regulation of CYP19a expression, a trend that was confirmed by RT-PCR analysis on excised tissues. In a combination approach with gonad histology, it could be shown that the increase in CYP19a expression as measured by RT-PCR on a whole tissue basis was due to a combination of both increases in numbers of CYP19a-containing cells and an increase in the amount of CYP19a mRNA present in the cells.

  14. Rapid identification of the animal pathogens Streptococcus uberis and Arcanobacterium pyogenes by fluorescence in situ hybridization (FISH).

    PubMed

    Werckenthin, Christiane; Gey, Annerose; Straubinger, Reinhard K; Poppert, Sven

    2012-05-01

    Fluorescence in situ hybridization (FISH) has been reported to be an easy and rapid identification method for many human pathogens, but applications for common veterinary pathogens are lacking. Gene probes for FISH of the animal pathogens Streptococcus uberis and Arcanobacterium pyogenes were designed to provide probes for a specific identification of these bacteria from cultures. Specific FISH probes for these species have so far not been published. Both probes recognized all isolates of the target species correctly. With the S. uberis probe SUB 196 no false-positive results were obtained for reference strains as well as for clinical isolates. Probe APYO 183 for A. pyogenes produced false-positive reactions with so far rarely described Arcanobacterium species from animals at standard hybridization conditions. In order to avoid any incorrect classifications of microorganisms as A. pyogenes, two non-labelled competitor probes were designed and successfully evaluated. PMID:22033042

  15. Subcellular localization of low-abundance human immunodeficiency virus nucleic acid sequences visualized by fluorescence in situ hybridization

    SciTech Connect

    Lawrence, J.B.; Marselle, L.M.; Byron, K.S.; Johnson, C.V.; Sullivan, J.L.; Singer, R.H. (Univ. of Massachusetts Medical Center, Worcester (USA))

    1990-07-01

    Detection and subcellular localization of human immunodeficiency virus (HIV) were investigated using sensitive high-resolution in situ hybridization methodology. Lymphocytes infected with HIV in vitro or in vivo were detected by fluorescence after hybridization with either biotin or digoxigenin-labeled probes. At 12 hr after infection in vitro, a single intense signal appeared in the nuclei of individual cells. Later in infection, when cytoplasmic fluorescence became intense, multiple nuclear foci frequently appeared. The nuclear focus consisted of newly synthesized HIV RNA as shown by hybridization in the absence of denaturation and by susceptibility to RNase and actinomycin D. Virus was detected in patient lymphocytes and it was shown that a singular nuclear focus also characterizes cells infected in vivo. The cell line 8E5/LAV containing one defective integrated provirus revealed a similar focus of nuclear RNA, and the single integrated HIV genome was unequivocally visualized on a D-group chromosome. This demonstrates an extremely sensitive single-cell assay for the presence of a single site of HIV transcription in vitro and in vivo and suggests that it derives from one (or very few) viral genomes per cell. In contrast, productive Epstein-Barr virus infection exhibited many foci of nuclear RNA per cell.

  16. The human tissue transglutaminase gene maps on chromosome 20q12 by in situ fluorescence hybridization

    SciTech Connect

    Gentile, V.; Davies, P.J.A. (Univ. of Texas, Houston, TX (United States)); Baldini, A. (Baylor College of Medicine, Houston, TX (United States))

    1994-03-15

    A cDNA encoding for the human tissue transglutaminase gene has been used to identify the chromosomal localization of the corresponding structural gene. The precise chromosomal and subregional localizations have been established by using in situ fluorescence mapping with a recombinant [lambda]-Zap phage containing the full cDNA coding sequence. The study showed that the human tissue transglutaminase gene is localized on chromosome 20 and, more precisely, within the band 20q12. To date, this is the third member of the transglutaminase gene family to be mapped. Human factor XIIIa (plasma transglutaminase), human keratinocyte transglutaminase (type I), and human tissue transglutaminase (type II) genes, although codifying for homologous enzymes, are localized on three different chromosomes. 16 refs., 1 fig.

  17. Physical mapping of rRNA genes in Medicago sativa and M. glomerata by fluorescent in situ hybridization.

    PubMed

    Falistocco, E

    2000-01-01

    Fluorescent in situ hybridization (FISH) was applied to diploid and tetraploid subspecies of alfalfa (Medicago sativa L.) to investigate the distribution of rRNA genes and to utilize the sites of 18S-5.8S-25S rDNA and 5S rDNA sequences as markers for studying the genome evolution within the species. Medicago glomerata Balb., the species considered to be the ancestor of alfalfa, was included in this study in order to obtain more information on the phylogenetics of alfalfa. Simultaneous in situ hybridization was performed with the probes pTa71 and pXVI labeled with digoxigenin and biotin, respectively. In the diploid taxa, M. glomerata, M. sativa ssp. coerulea Schmalh and ssp. falcata Arcangeli, the 18S-5.8S-25S rDNA sequences were mapped to two sites corresponding to the secondary constrictions of the nucleolar chromosome pair, while 5S rDNA appeared to be distributed in two pairs of sites. Chromosomes carrying 5S loci could be distinguished on the basis of their morphological characteristics. The number of rDNA sites detected in the tetraploid M. sativa ssp. falcata and ssp. sativa (L.) L. & L. were twice the number found in the respective diploid ssp. falcata and ssp. coerulea. The results of this study show that the distribution of ribosomal genes was maintained during the evolutionary steps from the primitive diploid to the cultivated alfalfa. Modifications of the number of rRNA loci were not observed. The importance of in situ hybridization for improving karyotype analysis in M. sativa L. is discussed. PMID:10833055

  18. Pallister-Killian syndrome: A mild case diagnosed by fluorescence in situ hybridization. Review of the literature and expansion of the phenotype

    SciTech Connect

    Bielanska, M.M.; Khalifa, M.M.; Duncan, A.M.V. [Queen`s Univ., Kingston, Ontario (Canada)] [Queen`s Univ., Kingston, Ontario (Canada)

    1996-10-16

    Pallister-Killian syndrome (PKS) is a rare disorder characterized by a specific combination of anomalies, mental retardation and mosaic presence of a supernumerary isochromosome 12p which is tissue-limited. We report an atypical case of PKS with a mild phenotype. Fluorescence in situ hybridization (FISH) was used to demonstrate that the supernumerary marker chromosome identified in the patient`s fibroblasts was an isochromosome 12p. This study broadens the spectrum of PKS phenotype. It also illustrates the usefulness of fluorescence in situ hybridization in diagnosis of patients with chromosomal abnormalities and mild or atypical clinical findings. 40 refs., 2 figs., 1 tab.

  19. Nonrandom gain of chromosome 7 in central neurocytoma: a chromosomal analysis and fluorescence in situ hybridization study.

    PubMed

    Taruscio, D; Danesi, R; Montaldi, A; Cerasoli, S; Cenacchi, G; Giangaspero, F

    1997-01-01

    Central neurocytoma is a benign, slow-growing neoplasm with favourable prognosis. Biomolecular analysis has failed to demonstrate significant alterations, and no cytogenetic alterations have been reported. In this study we demonstrate chromosome 7 gain in three of nine neurocytomas (33%). Traditional cytogenetic analysis performed in four of the nine cases identified trisomy 7 as the sole chromosomal abnormality in one case. Interphase cytogenetics utilizing fluorescent in situ hybridization (FISH) on cell suspensions from formalin-fixed paraffin-embedded tumour tissue performed in all nine cases detected trisomy 7 in two more cases and tetrasomy in another. Our results suggest that chromosome 7 gain is a feature of neuroectodermal tumorigenesis, possibly conferring growth advantage on the neoplastic cells. FISH on interphase nuclei is a valuable adjunct in the genetic evaluation of rare central nervous system neoplasms with low baseline proliferative activity. PMID:9037315

  20. Improvement of ciliate identification and quantification: a new protocol for fluorescence in situ hybridization (FISH) in combination with silver stain techniques.

    PubMed

    Fried, Johannes; Ludwig, Wolfgang; Psenner, Roland; Schleifer, Karl Heinz

    2002-12-01

    A new protocol for taxon specific probe based fluorescent in situ hybridization was developed for the identification and quantification of ciliates in microbial communities. Various fixatives and experimental parameters were evaluated and optimized with respect to cell permeability and morphological preservation. Optimum results were adaption by obatined of a modified fixation method using Bouin's solution. Furthermore, conventional staining procedures such as different Protargol stain techniques and a silver nitrate impregnation method were modified and can now be applied in combination with fluorescence in situ hybridization. The new protocol allows a rapid and reliable identification as well as quantification of ciliates based upon classical morphological aspects and rRNA based phylogenetic relationships performed in one experiment. Furthermore, a set of specific probes targeting different regions of the 18S rRNA was designed for Glaucoma scintillans Ehrenberg, 1830 and tested by applying this new approach of combining in situ cell hybridization with conventional staining techniques. PMID:12583717

  1. Detection of Panulirus argus Virus 1 (PaVI) in the Caribbean spiny lobster using fluorescence in situ hybridization (FISH).

    PubMed

    Li, Caiwen; Shields, Jeffrey D; Small, Hamish J; Reece, Kimberly S; Hartwig, Carmony L; Cooper, Roland A; Ratzlaff, Robert E

    2006-10-27

    Panulirus argus Virus 1 (PaV1) is the first virus known to be pathogenic to a wild lobster. It infects the Caribbean spiny lobster P. argus from the Florida Keys, and has a predilection for juveniles. The monitoring of the virus in wild populations and study of its behavior in the laboratory require the development of reliable diagnostic tools. A sensitive and specific fluorescence in situ hybridization (FISH) assay was developed for detection of PaV1. The lower detection limit using a 110 bp DNA probe in a dot-blot hygridization for PaV1 DNA was 10 pg of cloned template PaV1 DNA and 10 ng of genomic DNA extracted from the hemolymph of diseased spiny lobster. The fluorescein (FITC)-labeled probe specifically hybridized to PaVl-infected cells in the hepatopancreas, hindgut, gills, heart, foregut, and nerve tissues. FITC staining was observed around the inner periphery of the nuclear membrane, with lighter staining in a more dispersed pattern within the nucleus. The probe did not hybridize with host tissues of uninfected spiny lobsters, nor did it cross-react with 4 other virus samples tested. This assay will facilitate our understanding of the pathogenesis of the viral disease and help in monitoring efforts directed at determining the prevalence of PaV1 in juvenile nurseries for this lobster. PMID:17190197

  2. Rapid Identification of Staphylococcus aureus in Blood Cultures by a Combination of Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes and Flow Cytometry

    Microsoft Academic Search

    Hanna Hartmann; Henrik Stender; Andrea Schafer; Ingo B. Autenrieth; Volkhard A. J. Kempf

    2005-01-01

    Fluorescence in situ hybridization (FISH) using peptide nucleic acid probes (PNAs) allows the identification of Staphylococcus aureus from human blood culture samples. We present data revealing that the combination of PNA FISH and flow cytometry is a possible approach for the noncultural identification of staphylococci in blood cultures.

  3. Visualization and Enumeration of Marine Planktonic Archaea and Bacteria by Using Polyribonucleotide Probes and Fluorescent In Situ Hybridization

    PubMed Central

    DeLong, Edward F.; Taylor, Lance Trent; Marsh, Terence L.; Preston, Christina M.

    1999-01-01

    Fluorescent in situ hybridization (FISH) using rRNA-specific oligonucleotide probes has emerged as a popular technique for identifying individual microbial cells. In natural samples, however, the signal derived from fluor-labeled oligonucleotide probes often is undetectable above background fluorescence in many cells. To circumvent this difficulty, we applied fluorochrome-labeled polyribonucleotide probes to identify and enumerate marine planktonic archaea and bacteria. The approach greatly enhanced the sensitivity and applicability of FISH with seawater samples, allowing confident identification and enumeration of planktonic cells to ocean depths of 3,400 m. Quantitative whole-cell hybridization experiments using these probes accounted for 90 to 100% of the total 4?,6-diamidino-2-phenylindole (DAPI)-stained cells in most samples. As predicted in a previous study (R. Massana, A. E. Murray, C. M. Preston, and E. F. DeLong, Appl. Environ. Microbiol. 63:50–56, 1997), group I and II marine archaea predominate in different zones in the water column, with maximal cell densities of 105/ml. The high cell densities of archaea, extending from surface waters to abyssal depths, suggest that they represent a large and significant fraction of the total picoplankton biomass in coastal ocean waters. The data also show that the vast majority of planktonic prokaryotes contain significant numbers of ribosomes, rendering them easily detectable with polyribonucleotide probes. These results imply that the majority of planktonic cells visualized by DAPI do not represent lysed cells or “ghosts,” as was suggested in a previous report. PMID:10584017

  4. Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes for Rapid Detection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis in Potable-Water Biofilms

    Microsoft Academic Search

    Markku J. Lehtola; Eila Torvinen; Ilkka T. Miettinen; C. William Keevil

    2006-01-01

    Here, we present for the first time a high-affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting Mycobacterium avium bacteria, including the opportunistically pathogenic subspecies M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum, by the fluorescence in situ hybrid- ization (FISH) method. There is evidence that M. avium subsp. avium especially is able to survive

  5. Evaluation of Peptide Nucleic Acid-Fluorescence In Situ Hybridization for Identification of Clinically Relevant Mycobacteria in Clinical Specimens and Tissue Sections

    Microsoft Academic Search

    Michael Lefmann; Birgitta Schweickert; Petra Buchholz; Ulf B. Gobel; Timo Ulrichs; Peter Seiler; Dirk Theegarten; Annette Moter

    2006-01-01

    With fluorescently labeled PNA (peptide nucleic acid) probes targeting 16S rRNA, we established a 3-h fluorescence in situ hybridization (FISH) procedure for specific visualization of members of the Mycobacterium tuberculosis complex, M. leprae, M. avium, and M. kansasii. Probe specificity was tested against a panel of 25 Mycobacterium spp. and 10 gram-positive organisms. After validation, probes were used to identify

  6. Human cDNA mapping using fluorescence in situ hybridization. Progress report, April 1, 1992--December 31, 1992

    SciTech Connect

    Korenberg, J.R.

    1993-03-04

    Genetic mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach generated 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

  7. Spatial distribution analyses of natural phyllosphere-colonizing bacteria on Arabidopsis thaliana revealed by fluorescence in situ hybridization.

    PubMed

    Remus-Emsermann, Mitja N P; Lücker, Sebastian; Müller, Daniel B; Potthoff, Eva; Daims, Holger; Vorholt, Julia A

    2014-07-01

    Bacterial colonizers of the aerial parts of plants, or phyllosphere, have been identified on a number of different plants using cultivation-dependent and independent methods. However, the spatial distribution at the micrometer scale of different main phylogenetic lineages is not well documented and mostly based on fluorescence-tagged model strains. In this study, we developed and applied a spatial explicit approach that allowed the use of fluorescence in situ hybridization (FISH) to study bacterial phylloplane communities of environmentally grown Arabidopsis thaliana. We found on average 5.4 × 10(6) bacteria?cm(-2) leaf surface and 1.5 × 10(8) bacteria?g(-1) fresh weight. Furthermore, we found that the total biomass in the phylloplane was normally distributed. About 31% of the bacteria found in the phylloplane did not hybridize to FISH probes but exhibited infrared autofluorescence indicative for aerobic anoxygenic phototrophs. Four sets of FISH probes targeting Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Bacteroidetes were sufficient to identify all other major contributors of the phylloplane community based on general bacterial probing. Spatial aggregation patterns were observed for all probe-targeted populations at distances up to 7 ?m, with stronger tendencies to co-aggregate for members of the same phylogenetic group. Our findings contribute to a bottom-up description of leaf surface community composition. PMID:24725362

  8. A view on Bartonella quintana endocarditis--confirming the molecular diagnosis by specific fluorescence in situ hybridization.

    PubMed

    Gescher, Dorothee Maria; Mallmann, Christian; Kovacevic, Dragoljub; Schmiedel, Dinah; Borges, Adrian C; Schweickert, Birgitta; Göbel, Ulf B; Moter, Annette

    2008-01-01

    Culture-negative endocarditis is a frequent problem in cardiology, especially if caused by fastidious organisms. Among these, the diagnostic tools for the detection of Bartonella quintana are still unsatisfactory. In a culture-negative case of suspected endocarditis undergoing aortic valve replacement, polymerase chain reaction amplification and sequencing of the 16S rRNA gene indicated B. quintana infection. To develop a new diagnostic tool, independent from culture and amplification techniques, we designed and optimized an oligonucleotide fluorescence in situ hybridization (FISH) probe specific for B. quintana and suitable for FISH. FISH succeeded in simultaneous visualization and identification of vital microorganisms directly within the aortic valve tissue and in fast and univocal diagnosis of B. quintana endocarditis. PMID:17889492

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

    SciTech Connect

    Dittmer, J. [Indiana Univ., Fort Wayne, IN (United States); [Lawrence Livermore National Lab., CA (United States)

    1996-05-01

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

  10. Detection of Ralstonia solanacearum, which causes brownrot of potato, by fluorescent in situ hybridization with 23S rRNA-targeted probes

    Microsoft Academic Search

    B. A. WULLINGS; Beuningen van A. R; J. D. Janse; A. D. L. Akkermans

    1998-01-01

    During the past few years, Ralstonia (Pseudomonas) solanacearum race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of five R. solanacea- rum strains and one Ralstonia pickettii strain were PCR amplified,

  11. Prolonged hybridization with a cRNA probe improves the signal to noise ratio for in-tube in situ hybridization for quantification of mRNA after fluorescence-activated cell sorting.

    PubMed

    Yamada, H; Yamakawa, N; Watanabe, M; Hidaka, Y; Iwatani, Y; Takano, T

    2012-07-01

    We developed an in-tube in situ hybridization method for mRNA quantification after fluorescence-activated cell sorting (FACS-mQ). A specific RNA in a particular cell type is stained with a cRNA probe and a fluorescent dye, which allows the stained cells to be selected by FACS without excessive RNA degradation. Our previous protocol required 4 h for hybridization with a cRNA probe, which might not produce enough fluorescence signal for sorting genes with low expressions. We determined the effect of prolonged hybridization for in-tube in situ hybridization on quantitative measurement of intracellular RNAs. During the hybridization step, the quantity of ACTB mRNA decreased gradually until 4 h, but remained constant from 4 to 16 h below 63.6° C. For flow cytometry, cells hybridization with cRNA probes for TG mRNA at 60° C for 16 h showed both increased signal and decreased background fluorescence compared to those hybridized for 4 h. These results indicate that when performing in-tube in situ hybridization, hybridization temperature can be raised to 63.6° C and the hybridization step can be extended up to 16 h without excessive intracellular RNA degradation. PMID:22443863

  12. Microfluidic extraction and stretching of chromosomal DNA from single cell nuclei for DNA fluorescence in situ hybridization

    PubMed Central

    Wang, Xiaozhu; Takebayashi, Shin-ichiro; Bernardin, Evans; Gilbert, David M.; Chella, Ravindran

    2012-01-01

    We have developed a novel method for genetic characterization of single cells by integrating microfluidic stretching of chromosomal DNA and fiber fluorescence in situ hybridization (FISH). In this method, individually isolated cell nuclei were immobilized in a microchannel. Chromosomal DNA was released from the nuclei and stretched by a pressure-driven flow. We analyzed and optimized flow conditions to generate a millimeter-long band of stretched DNA from each nucleus. Telomere fiber FISH was successfully performed on the stretched chromosomal DNA. Individual telomere fiber FISH signals from single cells could be resolved and their lengths measured, demonstrating the ability of the method to quantify genetic features at the level of single cells. PMID:22231286

  13. Histopathological characterization and fluorescence in situ hybridization of Cyprinid herpesvirus 2 in cultured Prussian carp, Carassius auratus gibelio in China.

    PubMed

    Ding, Zhengfeng; Xia, Siyao; Zhao, Ziming; Xia, Aijun; Shen, Meifang; Tang, Jianqing; Xue, Hui; Geng, Xuebing; Yuan, Sheng

    2014-09-01

    Cyprinid herpesvirus 2 (CyHV-2) is an emerging pathogen in the commercially exploited fish, Prussian carp (Carassius auratus gibelio), which has caused huge economic loss in China and appears to be spreading worldwide. In this article, CyHV-2 infection of Prussian carp was confirmed for the first time by polymerase chain reaction (PCR), which gave positive results from the tissue samples dissected from moribund fish including kidney, spleen, liver, and gill. Histological examination showed systemic inflammatory reactions in the infected tissues, with infiltration of hemocytes, hypertrophied nuclei, marginal chromatin and karyorrhexis, epithelial cell shedding, vacuolar degeneration and focal necrosis. Tissue alterations were also evaluated semi-quantitatively by the degree of tissue change. The values of degree of tissue change determined for kidney, spleen, liver, and gill were significantly greater than respective controls and kidney was the most severely damaged organ, with highest degree of tissue change value. In addition, a fluorescence in situ hybridization (FISH) based on oligonucleotide probes to detect the pathogen directly in the tissue, allowing pathogen-lesion correlation, was established. With the advantages of better tissue penetration, potentially more specific and stable, three oligonucleotide probes were designed. Positive reactions to the probes with intense green fluorescence were observed within the infected tissues where PCR and H&E analysis had suggested previously the presence of the virus within these lesions. The probes did not hybridize with host tissues of uninfected fish, nor did they cross-react with 3 other virus samples tested. The current research could facilitate the study of CyHV-2 infection mechanism in Prussian carp, and enhance the early diagnosis of the novel virus. PMID:24877901

  14. Food selection by bacterivorous protists: insight from the analysis of the food vacuole content by means of fluorescence in situ hybridization

    Microsoft Academic Search

    Jan Jezbera; Karel Hornak; Karel Simek

    A modified fluorescence in situ hybridization (FISH) method was used to analyze bacterial prey composition in protistan food vacuoles in both laboratory and natural populations. Under laboratory conditions, we exposed two bacterial strains (affiliated with b- and c-Proteobacteria - Aeromonas hydrophila and Pseudomonas fluorescens, respectively) to grazing by three protists: the flagel- lates Bodo saltans and Goniomonas sp., and the

  15. Assignment of the gastric inhibitory polypeptide receptor gene (GIPR) to chromosome bands 19q13.2-q13.3 by fluorescence in situ hybridization

    SciTech Connect

    Stoffel, M.; Fernald, A.A.; Bell, G.I.; Le Beau, M.M. [Univ. of Chicago, IL (United States)] [Univ. of Chicago, IL (United States)

    1995-08-10

    The gastric inhibitory polypeptide receptor gene (GIPR) was localized, using fluorescence in situ hybridization (FISH), to human chromosome bands 19q13.2-q13.3. Gastric inhibitory polypeptide (GIP) is a potent stimulator of insulin secretion and mutations in the GIPR gene may be related to non-insulin-dependent diabetes mellitus (NIDDM). 13 refs., 1 fig.

  16. Technical Note: Determination of the metabolically active fraction of benthic foraminifera by means of Fluorescent In Situ Hybridization (FISH)

    NASA Astrophysics Data System (ADS)

    Borrelli, C.; Sabbatini, A.; Luna, G. M.; Nardelli, M. P.; Sbaffi, T.; Morigi, C.; Danovaro, R.; Negri, A.

    2011-08-01

    Benthic foraminifera are an important component of the marine biota, but protocols for investigating their viability and metabolism are still extremely limited. Classical studies on benthic foraminifera have been based on direct counting under light microscopy. Typically, these organisms are stained with Rose Bengal, which binds proteins and other macromolecules, but does not allow discrimination between viable and recently dead organisms. The fluorescent in situ hybridization technique (FISH) represents a new and useful approach to identify living cells possessing an active metabolism. Our work is the first test of the suitability of the FISH technique, based on fluorescent probes targeting the 18S rRNA, to detect live benthic foraminifera. The protocol was applied on Ammonia group and Miliolids, as well as on agglutinated polythalamous (i.e., Leptohalysis scottii and Eggerella scabra) and soft-shelled monothalamous (i.e., Psammophaga sp. and saccamminid morphotypes) taxa. The results from FISH analyses were compared with those obtained, on the same specimens assayed with FISH, from microscopic analysis of the cytoplasm colour, presence of pigments and pseudopodial activity. Our results indicate that FISH targets only metabolically active foraminifera, and allows discerning from low to high cellular activity, validating the hypothesis that the intensity of the fluorescent signal emitted by the probe is dependent upon the physiological status of cells. These findings support the usefulness of this molecular approach as a key tool for obtaining information on the physiology of living foraminifera, both in field and experimental settings.

  17. Cultivation-Independent, Semiautomatic Determination of Absolute Bacterial Cell Numbers in Environmental Samples by Fluorescence In Situ Hybridization

    PubMed Central

    Daims, Holger; Ramsing, Niels B.; Schleifer, Karl-Heinz; Wagner, Michael

    2001-01-01

    Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has found widespread application for analyzing the composition of microbial communities in complex environmental samples. Although bacteria can quickly be detected by FISH, a reliable method to determine absolute numbers of FISH-stained cells in aggregates or biofilms has, to our knowledge, never been published. In this study we developed a semiautomated protocol to measure the concentration of bacteria (in cells per volume) in environmental samples by a combination of FISH, confocal laser scanning microscopy, and digital image analysis. The quantification is based on an internal standard, which is introduced by spiking the samples with known amounts of Escherichia coli cells. This method was initially tested with artificial mixtures of bacterial cultures and subsequently used to determine the concentration of ammonia-oxidizing bacteria in a municipal nitrifying activated sludge. The total number of ammonia oxidizers was found to be 9.8 × 107 ± 1.9 × 107 cells ml?1. Based on this value, the average in situ activity was calculated to be 2.3 fmol of ammonia converted to nitrite per ammonia oxidizer cell per h. This activity is within the previously determined range of activities measured with ammonia oxidizer pure cultures, demonstrating the utility of this quantification method for enumerating bacteria in samples in which cells are not homogeneously distributed. PMID:11722938

  18. Design and Performance of a 16S rRNA-Targeted Oligonucleotide Probe for Detection of Members of the Genus Bdellovibrio by Fluorescence In Situ Hybridization

    Microsoft Academic Search

    Khaled K. Mahmoud; Damian McNeely; Chelsea Elwood; Susan F. Koval

    2007-01-01

    A 16S rRNA-targeted, Cy3-labeled oligonucleotide probe was designed to detect members of the genus Bdellovibrio by fluorescence in situ hybridization. Specific hybridization conditions were established; however, the detection of bdellovibrios in environmental samples required enrichment, confirming that Bdellovibrio spp. are not present in large numbers in the environment. Bdellovibrio and like organisms (BALOs) are predatory gram-negative bacteria that are ubiquitous

  19. Fluorescence in situ hybridization mapping of human chromosome 19: Cytogenetic band location of 540 cosmids and 70 genes or DNA markers

    Microsoft Academic Search

    B. Trask; A. Fertitta; M. Christensen; A. Bergmann; A. Copeland; P. de Jong; H. Mohrenweiser; A. Olsen; A. Carrano; K. Tynan; J. Youngblom

    1993-01-01

    We report here the band location of 540 cosmids mapped to chromosome 19. The cosmids were mapped by fluorescence in situ hybridization (FISH) relative to chromosomal bands produced by DAPI\\/actinomycin staining. The cosmids are distributed throughout the chromosome, with a sampling bias for the q-arm. A detailed analysis of the distribution of three different subtelomeric and 22 pericentromeric chromosome 19

  20. Specific and Rapid Detection by Fluorescent In Situ Hybridization of Bacteria in Clinical Samples Obtained from Cystic Fibrosis Patients

    PubMed Central

    Hogardt, Michael; Trebesius, Karlheinz; Geiger, Anna M.; Hornef, Mathias; Rosenecker, Josef; Heesemann, Jürgen

    2000-01-01

    We report on the rapid and specific detection of bacteria commonly isolated from clinical specimens from cystic fibrosis (CF) patients by fluorescent in situ hybridization (FISH). On the basis of comparative sequence analysis, we designed oligonucleotide probes complementary to species-specific 16S rRNA regions of these microorganisms and demonstrated the specificities of the probes by hybridization of different remotely related as well as closely related reference strains. Furthermore, in a pilot project we investigated 75 sputum samples and 10 throat swab specimens from CF patients by FISH and detected Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia, Haemophilus influenzae, and Staphylococcus aureus within these specimens. The specificity of FISH was 100% in comparison to the results of conventional microbial culture. In contrast, the sensitivity of standard laboratory cultivation was moderately higher, since the limit for microscopic detection of bacteria within sputum samples by FISH was approximately 4 × 105 CFU/ml of sputum (resulting in a 90% sensitivity for FISH). Moreover, we demonstrated that FISH will be useful for the rapid detection of bacteria that cause acute pulmonary exacerbations in CF patients, as demonstrated in patients with H. influenzae, S. aureus, and P. aeruginosa exacerbations. Therefore, FISH is a valuable additional method for the rapid and specific detection of bacteria in clinical samples from CF patients, in particular, patients with pulmonary exacerbations. PMID:10655391

  1. Direct fluorescence in situ hybridization (FISH) in Escherichia coli with a target-specific quantum dot-based molecular beacon.

    PubMed

    Wu, Sheng-Mei; Tian, Zhi-Quan; Zhang, Zhi-Ling; Huang, Bi-Hai; Jiang, Peng; Xie, Zhi-Xiong; Pang, Dai-Wen

    2010-10-15

    Quantum dots (QDs) are inorganic fluorescent nanocrystals with excellent properties such as tunable emission spectra and photo-bleaching resistance compared with organic dyes, which make them appropriate for applications in molecular beacons. In this work, quantum dot-based molecular beacons (QD-based MBs) were fabricated to specifically detect ?-lactamase genes located in pUC18 which were responsible for antibiotic resistance in bacteria Escherichia coli (E. coli) DH5?. QD-based MBs were constructed by conjugating mercaptoacetic acid-quantum dots (MAA-QDs) with black hole quencher 2 (BHQ2) labeled thiol DNA vial metal-thiol bonds. Two types of molecular beacons, double-strands beacons and hairpin beacons, were observed in product characterization by gel electrophoresis. Using QD-based MBs, one-step FISH in tiny bacteria DH5? was realized for the first time. QD-based MBs retained their bioactivity when hybridizing with complementary target DNA, which showed excellent advantages of eliminating background noise caused by adsorption of non-specific bioprobes and achieving clearer focus of genes in plasmids pUC18, and capability of bacterial cell penetration and signal specificity in one-step in situ hybridization. PMID:20729070

  2. Chromosome analysis of nuclear power plant workers using fluorescence in situ hybridization and Giemsa assay.

    PubMed

    Hristova, Rositsa; Hadjidekova, Valeria; Grigorova, Mira; Nikolova, Teodora; Bulanova, Minka; Popova, Ljubomira; Staynova, Albena; Benova, Donka

    2013-09-01

    The aim of this study was to evaluate the genotoxic effects of ionizing radiation in vivo in exposed Bulgarian nuclear power plant workers by using classical cytogenetic and molecular cytogenetic analyses of peripheral lymphocytes. Chromosome analysis using fluorescence in situ hybrydization (FISH) and Giemsa techniques was undertaken on 63 workers and 45 administrative staff controls from the Bulgarian Nuclear Power Plant. Using the Giemsa method, the frequencies of cells studied with chromosome aberrations, dicentrics plus rings and chromosome fragments in the radiation workers were significantly higher compared with the control group (P = 0.044, P = 0.014, and P = 0.033, respectively). A significant association between frequencies of dicentrics plus rings and accumulated doses was registered (P < 0.01). In the present study, a FISH cocktail of whole chromosome paints for chromosomes 1, 4 and 11 was used. A significant association between frequency of translocations and accumulated doses was also observed (P < 0.001). Within the control group, a correlation was found between age and the spontaneous frequency of translocations. No correlation was found between smoking status and frequency of translocations. When compared with the control group, workers with accumulated doses up to 100 mSv showed no increase in genome translocation frequency, whereas workers with accumulated doses from 101 to 200 mSv showed a statistically significant doubling of genome translocation frequency (P = 0.009). Thus, in cases of chronic exposure and for purposes of retrospective dosimetry, the genome frequency of translocations is a more useful marker for evaluation of genotoxic effects than dicentric frequency. PMID:23536543

  3. [Estimation of the phylogenetic diversity of prokaryotic microorganisms in Sphagnum bogs with the use of fluorescence in situ hybridization (FISH)].

    PubMed

    Pankratov, T A; Belova, S E; Dedysh, S N

    2005-01-01

    The microbial population of Sphagnum bogs of northern Russia was analyzed with respect to the presence and cell numbers of representatives of particular phylogenetic groups of prokaryotes by means of in situ hybridization with fluorescently labeled rRNA-targeted oligonucleotide probes with broad detection spectra. The total number of cells that hybridized with universal Archaea- or Bacteria-specific probes varied, in peat samples of different bogs, from 45 to 83% of the number of cells revealed by DAPI staining. Down the bog profiles, the total number of prokaryotes and the fraction of archaea among them increased. Application of a set of oligonucleotide probes showed that the number of microorganisms belonging to such phylogenetic lineages of the domain Bacteria as the phyla Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, Acidobacteria, and Planctomycetes constituted, in total, 14.0-26.5% of the number of eubacteria detected in the samples. Among the bacteria identified in the peat samples, the most abundant were representatives of the classes Alphaproteobacteria and Betaproteobacteria and the phyla Acidobacteria, Bacteroidetes, and Actinobacteria. PMID:16400995

  4. Chromosomal structural rearrangement of Paeonia brownii and P. californica revealed by fluorescence in situ hybridization.

    PubMed

    Zhang, D; Sang, T

    1998-12-01

    Chromosomal structural rearrangement in Paeonia brownii and P. californica (2n = 10) was studied by in situ hybridization using 18S rDNA probes. Six major rDNA sites were detected in mitotic cells of P. californica; six major and two minor rDNA sites were found in P. brownii. Two cytotypes (A and B), with different chromosomal morphology and (or) rDNA locations, were observed in the population of P. californica. Cytotype A, with rDNA sites only on the short arms of chromosomes, was considered to be the normal cytotype. Both translocation and pericentric inversion may have occurred to give rise to cytotype B, in which one homolog of chromosome 4 has rDNA sites on both arms while its homolog has no rDNA sites: one homolog of chromosome 3 has a rDNA site on the long arm. Two rearranged cytotypes, C and D, were observed in the population of P. brownii. Given that the normal cytotype of P. brownii is most likely to have six major rDNA sites on the short arms of chromosomes 3, 4, and 5, and two minor sites on the short arms of chromosome 2, cytotype C may have resulted from a translocation between the short arm of one homolog of chromosome 2 and the long arm of one homolog of chromosome 4, and cytotype D may have resulted from a translocation between the short arm of one homolog of chromosome 3 and the long arm of one homolog of chromosome 4. These results supported previous observations, based on meiotic configurations, that chromosomal structural rearrangement occurred frequently in P. brownii and P. californica. PMID:9924793

  5. Fluorescence in situ Hybridization with Human Chromosome-Specific Libraries: Detection of Trisomy 21 and Translocations of Chromosome 4

    Microsoft Academic Search

    D. Pinkel; J. Landegent; C. Collins; J. Fuscoe; R. Segraves; J. Lucas; J. Gray

    1988-01-01

    Chromosomes can be specifically stained in metaphase spreads and interphase nuclei by in situ hybridization with entire chromosome-specific DNA libraries. Unlabeled human genomic DNA is used to inhibit the hybridization of sequences in the library that bind to multiple chromosomes. The target chromosome can be made at least 20 times brighter per unit length than the others. Trisomy 21 and

  6. DIG In Situ Hybridization Protocol (with detergent)

    E-print Network

    sensitivity, while BIOTIN is considerably less sensitive. Therefore I recommend that you use BIOTIN probes by fluorescence in situ hybridization. Histochem. Cell Biol. (1999) 111:435- 443. DAY ONE Add DIG, FITC, or BIOTIN:500 BIOTIN Rabbit anti-BIOTIN (Chemicon Cat. #AB1708) 1:500 FITC

  7. Detection of recombinant Pseudomonas putida in the wheat rhizosphere by fluorescence in situ hybridization targeting

    E-print Network

    Chen, Wilfred

    METHODS Detection of recombinant Pseudomonas putida in the wheat rhizosphere by fluorescence to achieve detection of recombinant Pseudomonas putida (TOM20) expressing toluene o- monooxygenase (tom to the respective targets, with minimal non- specific binding. The recombinant strain was inoculated into wheat

  8. Determination of the metabolically active fraction of benthic foraminifera by means of Fluorescent in situ Hybridization (FISH)

    NASA Astrophysics Data System (ADS)

    Borrelli, C.; Sabbatini, A.; Luna, G. M.; Morigi, C.; Danovaro, R.; Negri, A.

    2010-10-01

    Benthic foraminifera are an important component of the marine living biota, but protocols for investigating their viability and metabolism are still extremely limited. Classical studies on benthic foraminifera have been based on direct counting under light microscopy. Typically these organisms are stained with Rose Bengal, which binds proteins and other macromolecules, but this approach does not allow discriminating between viable and recently dead organisms. The fluorescent in situ hybridization technique (FISH) represents a potentially useful approach identifying living cells with active metabolism cells. In this work, we tested for the first time the suitability of the FISH technique based on fluorescent probes targeting the 18S rRNA, to detect these live benthic protists. The protocol was applied on the genus Ammonia, on the Miliolidae group and an attempt was made also with agglutinated species (i.e., Leptohalysis scottii and Eggerella scabra). In addition microscopic analysis of the cytoplasm colour, presence of pigments and, sometimes, those of pseudopodial activity where conducted. The results of the present study indicate that FISH targeted only live and metabolically active foraminifera. These results allowed to identify as "live", cells improperly classified as "dead" by means of the classical technique (Type I error) and vice versa to identify as dead the foraminifera without rRNA, but stained using Rose Bengal (Type II error). In addition, the comparative FISH analysis of starved and actively growing cells demonstrated that individuals with active metabolism were stained more intensively than starved cells. This finding supports the hypothesis that the physiological status of cells can be directly related with the intensity of the fluorescent signal emitted by the fluorescent probe. We conclude that the use of molecular approaches could represent a key tool for acquiring crucial information on living foraminifera specimens and for investigating their ecological role in marine sediments.

  9. Detection of sex chromosomal aneuploidies X-X, Y-Y, and X-Y in human sperm using two-chromosome fluorescence in situ hybridization

    SciTech Connect

    Wyrobek, A.J. [Lawrence Livermore National Lab., CA (United States); Robbins, W.A. [Lawrence Livermore National Lab., CA (United States)]|[Univ. of California, Berkeley, CA (United States); Pinkel, D.; Weier, H.U. [Univ. of California, San Francisco, CA (United States); Mehraein, Y. [Univ. of California, San Francisco, CA (United States)]|[Philipps Universitat, Marburg (Germany)

    1994-10-15

    Sex chromosome aneuploidy is the most common numerical chromosomal abnormality in humans at birth and a substantial portion of these abnormalities involve paternal chromosomes. An efficient method is presented for using air-dried smears of human semen to detect the number of X and Y chromosomes in sperm chromatin using two-chromosome fluorescence in situ hybridization. Air-dried semen smears were pre-treated with dithiothreitol and 3,4-diiodosalicylate salt to decondense the sperm chromatin and then were hybridized with repetitive sequence DNA probes that had been generated by PCR and differentially labeled. Hybridizations with X and Y specific probes showed the expected ratio of 50%X:50%Y bearing sperm. Sperm carrying extra fluorescence domains representing disomy for the X or Y chromosomes occurred at frequencies of {approximately} 4 per 10,000 sperm each. Cells carrying both X and Y fluorescence domains occurred at a frequency of {approximately} 6/10,000. Thus, the overall frequency of sperm that carried an extra sex chromosome was 1.4/1,000. The frequencies of sperm carrying sex chromosome aneuploidies determined by hybridization did not differ statistically from those reported from the same laboratory using the human-sperm/hamster-egg cytogenetic technique. Multi-chromosome fluorescence in situ hybridization to sperm is a promising method for assessing sex-ratio alterations in human semen and for determining the fraction of sperm carrying sex or other chromosome aneuploidies which may be transmissible to offspring. 44 refs., 1 fig., 3 tabs.

  10. Incidence of chromosome numerical changes in multiple myeloma: fluorescence in situ hybridization analysis using 15 chromosome-specific probes.

    PubMed Central

    Tabernero, D.; San Miguel, J. F.; Garcia-Sanz, M.; Nájera, L.; García-Isidoro, M.; Peréz-Simon, J. A.; Gonzalez, M.; Wiegant, J.; Raap, A. K.; Orfão, A.

    1996-01-01

    The presence of complex karotypes with frequent numerical and structural abnormalities has been reported in 20 to 50% of multiple myeloma (MM) patients. This variability is mainly due to the difficulty of conventional cytogenetics to obtain tumor metaphases representative of all possible neoplastic clones in MM. To gain insight into the real incidence of numerical chromosome changes in MM we have studied by fluorescence in situ hybridization technique 15 different human chromosomes, 1, 3, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, X, and Y, in a series of 52 MM patients. In all cases, the DNA index assessed by a propidium iodide/CD38 double-staining technique with flow cytometry was simultaneously investigated for correlation, with fluorescence in situ hybridization results. Additional aims of this study were 1) to analyze whether the abnormalities detected were common to all plasma cells or were present in only a subpopulation of tumor cells, 2) to explore changes caused by disease progression, and 3) to establish possible associations among the altered chromosomes. Although the overall incidence of numerical abnormalities was 67%, this frequency increased to 80% in the 41 cases in which 7 or more chromosomes were analyzed. Trisomies were significantly more common than monosomies (84% versus 16%). Chromosomes 9 and 15 were the most frequently altered (52% and 48% of cases, respectively), with all of their abnormalities corresponding to trisomies. The most frequent losses involved chromosomes 13 (26%) and X in females (32%). Other common numerical changes corresponded to chromosomes 1 (39%), 11 (37%), 6 (32%), 3 (31%), 18 (29%), 7 (28%), and 17 (22%). By contrast, chromosomes 8(13%), 10(8%), and 12(3%) were rarely altered. DNA aneuploidy by flow cytometry was detected in 67% of patients, and a high degree of correlation was observed between the DNA index obtained by flow cytometry and the chromosome index derived from fluorescence in situ hybridization studies, calculated according to two mathematical formulas (coefficient of correlation of 0.82 and 0.91 when at least 7 or 12 chromosomes were considered, respectively). The frequency of numeric chromosome aberrations was higher in those patients with progressive disease and, interestingly, trisomy of chromosome 8 was exclusively detected in this latter group of patients. Our study shows that, with the exception of chromosome 8, a possible marker of clonal evolution, the numeric chromosome changes are present in nearly all malignant plasma cells (r > 0.84). Finally, frequent associations between chromosomal aberrations were observed (ie, chromosomes 6, 7, 9, and 17; 7 and 15; and 11 and 17). By excluding them, it was found that two triple combinations of chromosome-specific probes, chromosomes 1 and 9 together with either chromosome 13 or 15, could be a useful marker for detection of residual disease, as it permits the identification of most MM patients displaying numerical changes. Images Figure 1 PMID:8686739

  11. Development of a flow-fluorescence in situ hybridization protocol for the analysis of microbial communities in anaerobic fermentation liquor

    PubMed Central

    2013-01-01

    Background The production of bio-methane from renewable raw material is of high interest because of the increasing scarcity of fossil fuels. The process of biomethanation is based on the inter- and intraspecific metabolic activity of a highly diverse and dynamic microbial community. The community structure of the microbial biocenosis varies between different biogas reactors and the knowledge about these microbial communities is still fragmentary. However, up to now no approaches are available allowing a fast and reliable access to the microbial community structure. Hence, the aim of this study was to originate a Flow-FISH protocol, namely a combination of flow cytometry and fluorescence in situ hybridization, for the analysis of the metabolically active microorganisms in biogas reactor samples. With respect to the heterogenic texture of biogas reactor samples and to collect all cells including those of cell aggregates and biofilms the development of a preceding purification procedure was indispensable. Results Six different purification procedures with in total 29 modifications were tested. The optimized purification procedure combines the use of the detergent sodium hexametaphosphate with ultrasonic treatment and a final filtration step. By this treatment, the detachment of microbial cells from particles as well as the disbandment of cell aggregates was obtained at minimized cell loss. A Flow-FISH protocol was developed avoiding dehydration and minimizing centrifugation steps. In the exemplary application of this protocol on pure cultures as well as biogas reactor samples high hybridization rates were achieved for commonly established domain specific oligonucleotide probes enabling the specific detection of metabolically active bacteria and archaea. Cross hybridization and autofluorescence effects could be excluded by the use of a nonsense probe and negative controls, respectively. Conclusions The approach described in this study enables for the first time the analysis of the metabolically active fraction of the microbial communities within biogas reactors by Flow-FISH. PMID:24304697

  12. Medical devices; hematology and pathology devices; classification of early growth response 1 gene fluorescence in-situ hybridization test system for specimen characterization. Final order.

    PubMed

    2014-09-01

    The Food and Drug Administration (FDA) is classifying early growth response 1 (EGR1) gene fluorescence in-situ hybridization (FISH) test system for specimen characterization into class II (special controls). The special controls that will apply to this device are identified in this order and will be part of the codified language for the early growth response 1 (EGR1) gene fluorescence in-site hybridization (FISH) test system for specimen characterization classification. The Agency is classifying the device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device. PMID:25195217

  13. Locked Nucleic Acid and Flow Cytometry-Fluorescence In Situ Hybridization for the Detection of Bacterial Small Noncoding RNAs

    PubMed Central

    Robertson, Kelly L.

    2012-01-01

    We describe the development and testing of a high-throughput method that enables the detection of small noncoding RNAs (ncRNAs) from single bacterial cells using locked nucleic acid probes (LNA) and flow cytometry-fluorescence in situ hybridization (flow-FISH). The LNA flow-FISH method and quantitative reverse transcription-PCR (qRT-PCR) were used to monitor the expression of three ncRNAs (6S, CsrB, and TPP-2) in Vibrio campbellii ATCC BAA-1116 cultures during lag phase, mid-log phase, and stationary phase. Both LNA flow-FISH and qRT-PCR revealed that CsrB and TPP-2 were highly expressed during lag phase but markedly reduced in mid-log phase and stationary phase, whereas 6S demonstrated no to little expression during lag phase but increased thereafter. Importantly, while LNA flow-FISH and qRT-PCR demonstrated similar overall expression trends, only LNA flow-FISH, which enabled the detection of ncRNAs in individual cells as opposed to the lysate-based ensemble measurements generated by qRT-PCR, was able to capture the cell-to-cell heterogeneity in ncRNA expression. As such, this study demonstrates a new method that simultaneously enables the in situ detection of ncRNAs and the determination of gene expression heterogeneity within an isogenic bacterial population. PMID:22057868

  14. Fluorescent in situ hybridization of pre-incubated blood culture material for the rapid diagnosis of histoplasmosis.

    PubMed

    da Silva, Roberto Moreira; da Silva Neto, João Ricardo; Santos, Carla Silvana; Cruz, Kátia Santana; Frickmann, Hagen; Poppert, Sven; Koshikene, Daniela; de Souza, João Vicente Braga

    2015-02-01

    Fluorescence in situ hybridization (FISH) has been shown to be useful for the detection of Candida and Cryptococcus species in blood culture materials. FISH procedures for the detection of Histoplasma capsulatum var. capsulatum have not been reported so far. This study describes the development and evaluation of fluorescently labeled rRNA-targeting FISH probes to detect and identify H. capsulatum in blood cultures. All three analyzed H. capsulatum reference strains and clinical isolates showed positive signals with the newly designed specific oligonucleotide probes for H. capsulatum, whereas negative reactions were observed for all three nontarget yeast species and the two nontarget bacteria. The assay was also successfully applied for detections of H. capsulatum cells in pre-incubated blood culture samples of patients with clinical suspicion of histoplasmosis (n = 33). The described FISH-based assay was shown to be easy to apply, sensitive, and specific (compared to polymerase chain reaction) for the detection and identification of H. capsulatum in this proof-of-principle analysis. Larger multicentric assessments are recommended for a thorough diagnostic evaluation of the procedure. PMID:25537280

  15. Triplex in-situ hybridization

    DOEpatents

    Fresco, Jacques R. (Princeton, NJ); Johnson, Marion D. (East Windsor, NJ)

    2002-01-01

    Disclosed are methods for detecting in situ the presence of a target sequence in a substantially double-stranded nucleic acid segment, which comprises: a) contacting in situ under conditions suitable for hybridization a substantially double-stranded nucleic acid segment with a detectable third strand, said third strand being capable of hybridizing to at least a portion of the target sequence to form a triple-stranded structure, if said target sequence is present; and b) detecting whether hybridization between the third strand and the target sequence has occured.

  16. Several chromosomes involved in translocations with chromosome 5 shown with fluorescence in situ hybridization in patients with malignant myeloid disorders.

    PubMed

    Bram, Susanne; Rödjer, Stig; Swolin, Birgitta

    2004-11-01

    In many patients with myelodysplastic syndromes or acute myeloid leukemia, complex chromosome aberrations can be seen, among which aberrations of chromosome 5 constitute a substantial part. With conventional cytogenetic technique, these aberrations are often identified as deletions or monosomy 5. We analyzed nine patients who, under conventional cytogenetic analysis, showed deletion or monosomy 5. We used fluorescence in situ hybridization with whole-chromosome painting probes to identify the counterpart chromosome and locus-specific identifiers for 5q31 and 5q33 approximately q34. A deletion of 5q was found concomitant with unbalanced translocations. Our results and cases from the literature showed that material from chromosome 5 could be translocated to almost all chromosomes. All patients but one had short survival; this one patient had a preserved 5q31 and 5q33 approximately q34 but a deletion of the q-arm more centromeric than these bands. In eight of the nine patients, further 14 translocations were revealed, not involving chromosome 5. PMID:15527906

  17. Quantification of cell-specific substrate uptake by probe-defined bacteria under in situ conditions by microautoradiography and fluorescence in situ hybridization.

    PubMed

    Nielsen, Jeppe Lund; Christensen, Dinna; Kloppenborg, Marie; Nielsen, Per Halkjaer

    2003-03-01

    A technique based on quantitative microautoradiography (QMAR) and fluorescence in situ hybridization (FISH) was developed and evaluated in order to determine the quantitative uptake of specific substrates in probe-defined filamentous bacteria directly in a complex system. The technique, QMAR-FISH, has a resolution of a single cell and is based on an improved fixation protocol and the use of an internal standard of bacteria with known specific radioactivity. The method was used to study the in situ ecophysiology of the filamentous bacteria 'Candidatus Meganema perideroedes' and Thiothrix sp. directly in an activated sludge system. The cellular uptake rate of tritium-labelled substrates revealed an average cell-specific uptake rate of 4.1 yen 10-15 mol of acetate cell-1 h-1 and 3.1 yen 10-15 mol of acetate cell-1 h-1 for the two filamentous species respectively. The two filamentous species had very similar activity in all cells along each filament. Surprisingly, the filaments within both probe-defined populations had threefold variation in activity between the different filaments, demonstrating a large variation in activity level within a single population in a complex system. The substrate affinity (Ks) for uptake of acetate of the cells within the two filamentous bacteria was determined by incubation with variable concentrations of labelled acetate. The Ks values of the 'Candidatus Meganema perideroedes' and the Thiothrix filamentous bacteria were determined to be 1.8 micro M and 2.4 micro M acetate respectively. PMID:12588299

  18. Fluorescence in situ hybridization panel for monitoring of minimal residual disease in patients with double minute chromosomes.

    PubMed

    Jeon, Yongbum; Kim, Seon Young; Kim, Miyoung; Park, Hyun-Kyung; Lee, Sang Ho; See, Cha Ja; Kwon, Jiseok; Lee, Dong Soon

    2014-04-01

    A double minute chromosome (dmin) is a small fragment of extrachromosomal DNA bearing amplified genes observed in malignancies. We investigated the incidence and characteristics of dmins in hematologic malignancies, and the quantitative changes during the treatment follow-up. Once a dmin was observed in conventional G-banding, it was characterized using fluorescence in situ hybridization (FISH) with the panel of MYC, NMYC, and MLL probes. Quantitative changes of malignant cells were measured using G-banding and FISH during the follow up. Dmins were observed in 1.23% of patients (6/489) at the initial diagnosis including 4 with MYC amplification, 1 with MLL and 1 with NMYC. All 6 had complex karyotypes and showed short overall survival (7.7 months). In follow-up specimens, FISH detected dmins in 11 cases out of which G-banding detected dmins in 9 cases. The number of dmins detected by FISH and G-banding did not correlate well. Amplification of NMYC in dmins is reported for the first time. A FISH panel composed of frequently amplified oncogenes (MYC, NMYC, and MLL) in dmins is useful for characterization of dmins. FISH is a sensitive method in detecting dmins and will be useful in monitoring of the minimal residual disease. PMID:24211232

  19. Aneuploidy detection for chromosomes 1, X and Y by fluorescence in situ hybridization in human sperm from oligoasthenoteratozoospermic patients

    SciTech Connect

    Pang, M.G.; Zackowski, J.L.; Acosta, A.A. [Eastern Virginia Medical School, Norfolk, VA (United States)] [and others

    1994-09-01

    Oligoasthenoteratozoospermic males (n=15) were investigated for infertility as compared with proven fertile donors. The oligoasthenoteratozoospermic population showed a mean sperm concentration of 9.7 x 10{sup 6}/ml (Range 4.2-19.7), mean motility of 38.5% (Range 10.6-76.8) and morphology (measured by the percentage of normal forms evaluated by strict criteria) with a mean of 3.49% (Range 1.5-5.0). Fluorescence in situ hybridization (FISH) using satellite DNA probes specific for chromosomes 1 (puc 1.77), X (alpha satellite), and Y (satellite-III at Yqh) was performed on human interphase sperm nuclei. DNA probes were either directly labelled with rhodamine-dUTP, FITC-dUTP, or biotinylated by nick translation. Hybridization and signal detection were done by routine laboratory protocols. Microscopic analysis was performed using a cooled CCD camera attached to an epi-fluorescent microscope. After hybridization, fertile donors yielded a frequency of 0.96% (n=12) nullisomic, 98.5% (n=1231) monosomic and 0.96% (n=12) disomic for chromosome 1, whereas oligoasthenoteratozoospermic males yielded a frequency of 16% (n=600) nullisomic, 74.5% (n=2792) monosomic and 9.9% (n=370) disomic. In addition, fertile donors yielded a frequency of 45.7% (n=633) monosomic and 0.7% (n=11) disomic for chromosome X, whereas oligoasthenoteratozoospermic males yielded a frequency of 38.7% (n=760) monosomic and 0.8% (n=13) disomic. Chromosome Y frequencies for fertile donors showed 44.6% (n=614) monosomic and 0.6% (n=2) disomic, whereas oligoasthenoteratozoospermic males yielded a frequency of 33.2% (n=701) monosomic and 0.8% (n=15) disomic. This suggests that the frequency of nullisomy for chromosome 1 is significantly higher (p<0.001) in sperm from oligoasthenoteratozoospermic makes versus sperm from our fertile donors. We conclude that FISH is a powerful tool to determine the frequency of aneuploidy in sperm from oligoasthenoteratozoospermic patients.

  20. Multicolor fluorescent in situ hybridization on post-mortem brain in schizophrenia as an approach for identification of low-level chromosomal aneuploidy in neuropsychiatric diseases

    Microsoft Academic Search

    Yuri B Yurov; Viktor M Vostrikov; Svetlana G Vorsanova; Victor V Monakhov; Ivan Y Iourov

    2001-01-01

    Fluorescence in situ hybridization (FISH) of DNA-DNA or DNA-RNA using post-mortem brain samples is one approach to study low-level chromosomal aneuploidy and selective expression of specific genes in the brain of patients with neuropsychiatric diseases. We have performed a pilot molecular-cytogenetic analysis of post-mortem brain of schizophrenic patients. Multicolor FISH on two post-mortem brain samples of normal individuals and six

  1. Detection of aneuploid human sperm by fluorescence in situ hybridization: Evidence for a donor difference in frequency of sperm disomic for chromosomes 1 and Y

    SciTech Connect

    Robbins, W.A. (Univ. of California, Berkeley (United States) Lawrence Livermore National Lab., CA (United States)); Segraves, R.; Pinkel, D. (Univ. of California, San Francisco (United States)); Wyrobek, A.J. (Lawrence Livermore National Lab., CA (United States))

    1993-04-01

    Fluorescence in situ hybridization with repetitive-sequence DNA probes was used to detect human sperm disomic for chromosomes 1 and Y in three healthy men. Data on these same men had been obtained previously, using the human-sperm/hamster-egg cytogenetic technique, providing a cytogenetic reference for validating sperm hybridization measurements. Air-dried smears were prepared from semen samples and treated with DTT and lithium diiodosalicylate to expand sperm chromatin. Hybridization with fluorescently tagged DNA probes for chromosomes 1 (pUC177) or Y (pY3.4) yielded average frequencies of sperm with two fluorescent domains of 14.2[+-]2.4/10,000 and 5.6[+-]1.6/10,000 sperm, respectively. These frequencies did not differ statistically from frequencies of hyperploidy observed for these chromosomes with the hamster technique. In addition, frequencies of disomic sperm from one donor were elevated [approximately]2.5-fold above those of other donors, for both chromosomes 1 (P = .045) and Y (P = .01), consistent with a trend found with the hamster technique. The authors conclude that fluorescence in situ hybridization to sperm chromosomes provides a valid and promising measure of the frequency of disomic human sperm. 43 refs., 1 fig., 4 tabs.

  2. An approach for quantitative assessment of fluorescence in situ hybridization (FISH) signals for applied human molecular cytogenetics.

    PubMed

    Iourov, Ivan Y; Soloviev, Ilia V; Vorsanova, Svetlana G; Monakhov, Viktor V; Yurov, Yuri B

    2005-03-01

    A number of applied molecular cytogenetic studies require the quantitative assessment of fluorescence in situ hybridization (FISH) signals (for example, interphase FISH analysis of aneuploidy by chromosome enumeration DNA probes; analysis of somatic pairing of homologous chromosomes in interphase nuclei; identification of chromosomal heteromorphism after FISH with satellite DNA probes for differentiation of parental origin of homologous chromosome, etc.). We have performed a pilot study to develop a simple technique for quantitative assessment of FISH signals by means of the digital capturing of microscopic images and the intensity measuring of hybridization signals using Scion Image software, commonly used for quantification of electrophoresis gels. We have tested this approach by quantitative analysis of FISH signals after application of chromosome-specific DNA probes for aneuploidy scoring in interphase nuclei in cells of different human tissues. This approach allowed us to exclude or confirm a low-level mosaic form of aneuploidy by quantification of FISH signals (for example, discrimination of pseudo-monosomy and artifact signals due to over-position of hybridization signals). Quantification of FISH signals was also used for analysis of somatic pairing of homologous chromosomes in nuclei of postmortem brain tissues after FISH with "classical" satellite DNA probes for chromosomes 1, 9, and 16. This approach has shown a relatively high efficiency for the quantitative registration of chromosomal heteromorphism due to variations of centromeric alphoid DNA in homologous parental chromosomes. We propose this approach to be efficient and to be considered as a useful tool in addition to visual FISH signal analysis for applied molecular cytogenetic studies. PMID:15750029

  3. Vidas UP-enzyme-linked fluorescent immunoassay based on recombinant phage protein and fluorescence in situ hybridization as alternative methods for detection of Salmonella enterica serovars in meat.

    PubMed

    Zadernowska, Anna; Chaj?cka-Wierzchowska, Wioleta; K??bukowska, Lucyna

    2014-09-01

    Several methods for the rapid and specific detection of Salmonella spp. in meat have been described. This study was conducted to evaluate the capability of the VIDAS(®) UP (SPT [Salmonella Phage Technology]), an enzyme-linked fluorescent immunoassay method, and fluorescence in situ hybridization (FISH) to complement the International Organization for Standardization Method 6579 (ISO) in detecting Salmonella spp. from beef, pork, and poultry meat samples. The meat was inoculated with a mixture of Salmonella spp. on three levels of contamination. It was also checked that the tests did not produce cross-reactions with other Enterobacteriaceae rods. On the basis of the results, the relative specificity, relative accordance, and relative sensitivity of the method were determined. In meat samples, Vidas UP and FISH detection results were in substantial agreement with ISO, with relative specificity, accordance, and sensitivity rates of 90%, 96.3%, and 100%, respectively, for Vidas UP and 100%, 100%, and 99.4%, respectively, for FISH. This is the first report on the evaluation of both Vidas UP and FISH compared to ISO for the rapid detection of Salmonella enterica serovars in meat. PMID:24971928

  4. Microbial populations identified by fluorescence in situ hybridization in a constructed wetland treating acid coal mine drainage

    SciTech Connect

    Nicomrat, D.; Dick, W.A.; Tuovinen, O.H. [Ohio State University, Wooster, OH (United States). Environmental Science Graduate Programme

    2006-07-15

    Microorganisms are an integral part of the biogeochemical processes in wetlands, yet microbial communities in sediments within constructed wetlands receiving acid mine drainage (AMD) are only poorly understood. The purpose of this study was to characterize the microbial diversity and abundance in a wetland receiving AMD using fluorescence in situ hybridization (FISH) analysis. Seasonal samples of oxic surface sediments, comprised of Fe(III) precipitates, were collected from two treatment cells of the constructed wetland system. The pH of the bulk samples ranged between pH 2.1 and 3.9. Viable counts of acidophilic Fe and S oxidizers and heterotrophs were determined with a most probable number (MPN) method. The MPN counts were only a fraction of the corresponding FISH counts. The sediment samples contained microorganisms in the Bacteria (including the subgroups of acidophilic Fe- and S-oxidizing bacteria and Acidiphilium spp.) and Eukarya domains. Archaea were present in the sediment surface samples at < 0.01% of the total microbial community. The most numerous bacterial species in this wetland system was Acidithiobacillus ferrooxidans, comprising up to 37% of the bacterial population. Acidithiobacillus thiooxidans was also abundant.

  5. Monitoring of chimerism using fluorescence in situ hybridization in a child with severe combined immune deficiency following bone marrow transplant

    SciTech Connect

    Wenger, S.L.; Chen, X.O.; Katz, A.J. [Children`s Hospital of Pittsburgh, PA (United States)]|[Univ. of Pittsburgh, PA (United States)

    1994-09-01

    A boy with severe combined immunodeficiency received a bone marrow transplant from his sister when he was approximately 3 years of age. His peripheral blood karyotype at age 3 and 4 years was 46,XX (20 cells analyzed). Because of a decline in antibody production at 19 years of age, the patient`s peripheral blood was analyzed again for suspected chimerism. His karyotype in phytohemagglutinin (PHA)-stimulated culture was 46,XX in 49 cells and 46,XY in one cell. Both metaphase and interphase cells were examined for sex chromosome constitution using X and Y dual-color alpha-satellite probes for fluorescence in situ hybridization (FISH). FISH results for metaphase cells showed 1/50 XY cells, but 38% of interphase cells showed the presence of both X and Y centromere. Pokeweed mitogen (PWM)-stimulated cultures grew poorly and were therefore analyzed using FISH only: 81% of interphase cells were 46,XX. The discrepancy between metaphase and interphase in the PHA-stimulated cultures most likely represents a failure of this boy`s own XY T-cells to be stimulated.

  6. Chromosome instability in ICF syndrome: Formation of micronuclei from multibranched chromosomes 1 demonstrated by fluorescence in situ hybridization

    SciTech Connect

    Sawyer, J.R.; Swanson, C.M.; Wheeler, G. [Univ. of Arkansas, Little Rock, AR (United States)] [and others

    1995-03-27

    We report on a new patient with immunodeficiency, centromeric heterochromatin instability, and facial anomalies (the ICF syndrome). Studies with traditional cytogenetic methods demonstrate that aberrations in this syndrome primarily involve the centromeric regions of chromosomes 1 and 16. We applied fluorescence in situ hybridization (FISH) using {open_quotes}painting{close_quotes} probes for chromosomes 1 and 16 to document the progression of centromeric instability from simple decondensation aberrations to the subsequent formation of complex multibranched chromosomes 1, and finally to the interphase aberrations of nuclear projections and micronuclei involving both chromosomes 1 and 16. The loss of the large multibranched chromosome 1 configurations from the cells as micronuclei suggests that the centromeric aberrations subsequently interfere with normal chromosome movement at anaphase in ICF syndrome. Circular areas of counterstained chromatin were observed by FISH in the micronuclei corresponding to the intertwined segments of centromeric heterochromatin seen involving multibranched chromosomes 1 in the patient`s G-banded chromosome study. The current hypothesis of recessive inheritance for this disorder suggests that the chromosomal aberrations are not a causative event in this syndrome; however, the chromosome aberrations are clearly an important basic diagnostic criterion. 22 refs., 3 figs.

  7. Fluorescence in situ hybridization and chromosomal organization of the sirtuin 4 gene (Sirt4) in the mouse.

    PubMed

    Mahlknecht, Ulrich; Voelter-Mahlknecht, Susanne

    2009-05-15

    The sirtuins (SIRT1-7), also being referred to as class III HDACs, exert NAD-dependent deacetylase and/or ADP-ribosyltransferase activities in various cellular compartments including the cell nucleus, the cytoplasm and the mitochondria. The sirtuins play a central role in epigenetic gene silencing, DNA repair and recombination, cell-cycle, microtubule organization, and in the regulation of aging. SIRT4 is a mitochondrial protein that lacks deacetylase activities but efficiently works as an ADP-ribosyltransferase. We have isolated and characterized the murine Sirt4 genomic sequence, which spans a region of 12kb and which has one single genomic locus. Determination of the exon-intron splice junctions established that SIRT4 is encoded by 6 exons. The 1648bp murine Sirt4 transcript encodes a 418 aa protein with a predictive molecular weight of 47.3kDa. Fluorescence in situ hybridization analysis identified a single genomic locus for murine Sirt4 gene on chromosome 5F and is neighbored by the PLA2G1B and PXN genes. PMID:19306844

  8. Quick fluorescent in situ hybridization protocol for Xist RNA combined with immunofluorescence of histone modification in X-chromosome inactivation.

    PubMed

    Yue, Minghui; Charles Richard, John Lalith; Yamada, Norishige; Ogawa, Akiyo; Ogawa, Yuya

    2014-01-01

    Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell level to detect the spatial dynamics of RNA localization with simultaneous insight into the localization of proteins, epigenetic modifications and other details which can be highlighted by immunofluorescence. X-chromosome inactivation is a paradigm for long non-coding RNA (lncRNA)-mediated gene silencing. X-inactive specific transcript (Xist) lncRNA accumulation (called an Xist cloud) on one of the two X-chromosomes in mammalian females is a critical step to initiate X-chromosome inactivation. Xist RNA directly or indirectly interacts with various chromatin-modifying enzymes and introduces distinct epigenetic landscapes to the inactive X-chromosome (Xi). One known epigenetic hallmark of the Xi is the Histone H3 trimethyl-lysine 27 (H3K27me3) modification. Here, we describe a simple and quick immuno-FISH protocol for detecting Xist RNA using RNA FISH with multiple oligonucleotide probes coupled with immunofluorescence of H3K27me3 to examine the localization of Xist RNA and associated epigenetic modifications. Using oligonucleotide probes results in a shorter incubation time and more sensitive detection of Xist RNA compared to in vitro transcribed RNA probes (riboprobes). This protocol provides a powerful tool for understanding the dynamics of lncRNAs and its associated epigenetic modification, chromatin structure, nuclear organization and transcriptional regulation. PMID:25489864

  9. Grazing of a Tetrahymena sp. on Adhered Bacteria in Percolated Columns Monitored by In Situ Hybridization with Fluorescent Oligonucleotide Probes

    PubMed Central

    Eisenmann, Heinrich; Harms, Hauke; Meckenstock, Rainer; Meyer, Elisabeth I.; Zehnder, Alexander J. B.

    1998-01-01

    Predation of attached Pseudomonas putida mt2 by the small ciliate Tetrahymena sp. was investigated with a percolated column system. Grazing rates were examined under static and dynamic conditions and were compared to grazing rates in batch systems containing suspended prey. The prey densities were 2 × 108 bacteria per ml of pore space and 2 × 108 bacteria per ml of suspension, respectively. Postingestion in situ hybridization of bacteria with fluorescent oligonucleotide probes was used to quantify ingestion. During 30 min, a grazing rate of 1,382 ± 1,029 bacteria individual?1 h?1 was obtained with suspended prey; this was twice the grazing rate observed with attached bacteria under static conditions. Continuous percolation at a flow rate of 73 cm h?1 further decreased the grazing rate to about 25% of the grazing rate observed with suspended prey. A considerable proportion of the protozoans fed on neither suspended bacteria nor attached bacteria. The transport of ciliates through the columns was monitored at the same time that predation was monitored. Less than 20% of the protozoans passed through the columns without being retained. Most of these organisms ingested no bacteria, whereas the retained protozoans grazed more efficiently. Retardation of ciliate transport was greater in columns containing attached bacteria than in bacterium-free columns. We propose that the correlation between grazing activity and retardation of transport is a consequence of the interaction between active predators and attached bacteria. PMID:9546161

  10. Quantitative Fluorescence In Situ Hybridization Analysis of Microbial Consortia from a Biogenic Gas Field in Alaska's Cook Inlet Basin

    PubMed Central

    Str?po?, Dariusz; Huizinga, Brad; Lidstrom, Ulrika; Ashby, Matt; Macalady, Jennifer L.

    2012-01-01

    Filter-collected production water samples from a methane-rich gas field in the Cook Inlet basin of Alaska were investigated using whole-cell rRNA-targeted fluorescence in situ hybridization (FISH) and 16S rRNA tag pyrosequencing. Both techniques were consistent in determining the microbial community composition, including the archaeal or bacterial dominance of samples. The archaeal community is dominated by the obligate methylotrophic methanogen genus Methanolobus as well as the nutritional generalist methanogen genus Methanosarcina, which is capable of utilizing acetate, CO2, and methyl-bearing compounds. The most-abundant bacterial groups are Firmicutes, notably of the Acetobacterium genus, and Cytophaga-Flexibacter-Bacteroides species (CFBs) affiliated with the order Bacteroidales. We observed spatial variation among samples in both the percentage of members of Archaea compared to that of members of Bacteria and the dominant members of the bacterial community, differences which could not be explained with the available geochemical data. Based upon the microbial community composition and the isotopic signature of methane associated with the Cook Inlet basin site, we propose a simplified reaction network beginning with the breakdown of coal macromolecules, followed by fermentation and methylotrophic and acetoclastic methane production. PMID:22427501

  11. Fluorescent In Situ Hybridization on Isolated Tumor Cell Nuclei: A Sensitive Method for 1p and 19q Deletion Analysis in Paraffin-Embedded Oligodendroglial Tumor Specimens

    Microsoft Academic Search

    Ellen Gelpi; Inge M Ambros; Peter Birner; Andrea Luegmayr; Marcus Drlicek; Ingeborg Fischer; Reinhold Kleinert; Hans Maier; Michael Huemer; Brigitte Gatterbauer; Johann Anton; Karl Rössler; Herbert Budka; Peter F Ambros; Johannes A Hainfellner

    2003-01-01

    In oligodendroglial neoplasms, losses of chromosomal material at 1p and 19q associate with chemosensitivity and prolonged survival. Thus, 1p\\/19q testing is increasingly proposed for use in brain tumor diagnosis and prognostic assessment. Fluorescent in situ hybridization (FISH) is a classic technique for investigation of 1p\\/19q status in paraffin-embedded tissues. A major limitation of this method is truncation of tumor cell

  12. Fully Automated Fluorescent in situ Hybridization (FISH) Staining and Digital Analysis of HER2 in Breast Cancer: A Validation Study

    PubMed Central

    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 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% (? = 0.94) and 93.8% (? = 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. PMID:25844540

  13. Detection of sex chromosomal aneuploidies XX, Y-Y, and X-Y in human sperm using two-chromosome fluorescence in situ hybridization

    Microsoft Academic Search

    Andrew J. Wyrobek; Wendie A. Robbins; D. Pinkel; H. U. Weier; Y. Mehraein

    1994-01-01

    Sex chromosome aneuploidy is the most common numerical chromosomal abnormality in humans at birth and a substantial portion of these abnormalities involve paternal chromosomes. An efficient method is presented for using air-dried smears of human semen to detect the number of X and Y chromosomes in sperm chromatin using two-chromosome fluorescence in situ hybridization. Air-dried semen smears were pre-treated with

  14. Ecophysiological Interaction between Nitrifying Bacteria and Heterotrophic Bacteria in Autotrophic Nitrifying Biofilms as Determined by Microautoradiography-Fluorescence In Situ Hybridization

    Microsoft Academic Search

    Tomonori Kindaichi; Tsukasa Ito; Satoshi Okabe

    2004-01-01

    Ecophysiological interactions between the community members (i.e., nitrifiers and heterotrophic bacteria) in a carbon-limited autotrophic nitrifying biofilm fed only NH4 as an energy source were investigated by using a full-cycle 16S rRNA approach followed by microautoradiography (MAR)-fluorescence in situ hybridization (FISH). Phylogenetic differentiation (identification) of heterotrophic bacteria was performed by 16S rRNA gene sequence analysis, and FISH probes were designed

  15. X chromosome evolution in the suni and eland antelope: detection of homologous regions by fluorescence in situ hybridization and G-banding

    Microsoft Academic Search

    T. J. Robinson; W. R. Harrison; A. Ponce de León; F. F. B. Elder

    1997-01-01

    Comparative cytogenetics and fluorescence in situ hybridization (FISH) were used to track structural rearrangements in the X chromosomes of two African antelope species, the eland (Taurotragus oryx; tribe Tragelaphini) and the suni (Neotragus moschatus; tribe Neotragini). Using two microdissected cattle chromosome painting probes (one specific for Xp-containing sequences corresponding to Xp24?p12 of the cattle X, and one specific for Xq-containing

  16. Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes for Differentiation between Tuberculous and Nontuberculous Mycobacterium Species in Smears of Mycobacterium Cultures

    Microsoft Academic Search

    HENRIK STENDER; KAARE LUND; KENNETH H. PETERSEN; OLE F. RASMUSSEN; POONPILAS HONGMANEE; HÅKAN MIORNER; SVEN E. GODTFREDSEN

    1999-01-01

    TB PNA FISH is a new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for differentiation between species of the Mycobacterium tuberculosis complex (MTC) and nontubercu- lous mycobacteria (NTM) in acid-fast bacillus-positive (AFB1) cultures is described. The test is based on fluorescein-labelled PNA probes that target the rRNA of MTC or NTM species applied to smears

  17. Detection of Group B Streptococcus Bacteria in LIM Enrichment Broth by Peptide Nucleic Acid Fluorescent In Situ Hybridization (PNA FISH) and Rapid Cycle PCR ?

    PubMed Central

    Wilson, D. A.; Hall, G. S.; Procop, G. W.

    2010-01-01

    The sensitivity, specificity, and negative and positive predictive values for the detection of group B Streptococcus (GBS) in 206 LIM enrichment broths by the use of subculture, GBS peptide nucleic acid fluorescent in situ hybridization (PNA FISH), and GBS PCR were 96.9%, 100%, 98.6%, and 100%; 98.4%, 100%, 99.3%, and 100%; and 100%, 100%, 100%, and 100%, respectively. PMID:20200295

  18. Fluorescence in situ hybridization in diluted and flow cytometrically sorted boar spermatozoa using specific DNA direct probes labelled by nick translation

    Microsoft Academic Search

    I. Parrilla; J. M. V ´ azquez; M. Oliver-Bonet; J. Navarro; J. Y elamos; J. Roca; E. A. Mart ´ õnez

    2003-01-01

    Successful evaluation of X- and Y-chromosome-bearing sperm separation technology using flow cytometry-cell sorter is of great importance. Fluorescence in situ hybrid- ization (FISH), which allows for the detection of specific nucleic acid sequences on morphologically preserved spermatozoa, is an ideal method for quantitatively and qualitatively assessing the purity of sorted sperm samples. In this study specific pig DNA direct probes

  19. Physical Mapping of Ribosomal RNA Genes in Peonies (Paeonia, Paeoniaceae) by Fluorescent In situ Hybridization: Implications for Phylogeny and Concerted Evolution

    Microsoft Academic Search

    Daming Zhang; Tao Sang

    1999-01-01

    Physical maps of the 18S-5.8S-26S ribosomal RNA genes (rDNA) were generated by fluorescent in situ hybridization for five diploid Paeonia species, P. delavayi and P. rockii of section Moutan, and P. emodi, P. tenuifolia, and P. veitchii of section Paeonia. Of five pairs of mitotic chromosomes, rDNA loci were mapped near the telomeres of chromosomes 3, 4, and 5 of

  20. Mammary and vaginal myofibroblastomas are genetically related lesions: fluorescence in situ hybridization analysis shows deletion of 13q14 region.

    PubMed

    Magro, Gaetano; Righi, Alberto; Casorzo, Laura; Antonietta, Torrisi; Salvatorelli, Lucia; Kacerovská, Denisa; Kazakov, Dmitry; Michal, Michal

    2012-11-01

    Partial monosomy 13q, a chromosomal alteration originally reported in spindle cell lipoma, has also been documented in a few cases of mammary myofibroblastoma. Subsequently, a monoallelic loss of RB1 and FOXO1, located on 13q14, was identified in some cases of cellular angiofibroma, a benign stromal tumor of the lower female genital tract. This cytogenetic finding and the overlapping morphologic and immunohistochemical features shared by spindle cell lipoma, mammary myofibroblastoma, and cellular angiofibroma strongly suggest a histogenetic link among these tumors. Recently, we have emphasized morphologic and immunohistochemical similarities between mammary and vulvovaginal myofibroblastoma. The aim of the present study was to asses if these 2 tumors share the same chromosomal alteration. We studied the chromosome 13q14 region by fluorescence in situ hybridization analysis in a series of mammary and vaginal myofibroblastomas, with a readable signal in 7 of 13 mammary myofibroblastomas and 5 of 7 cases of vaginal myofibroblastomas. Despite histologic variation, most of the mammary (5/7) and vaginal (3/5) myofibroblastomas showed monoallelic deletion of FOXO1 in more than 22% of the cell populations. Our findings confirm that mammary myofibroblastoma is a tumor that exhibits chromosome abnormalities associated with the loss of the 13q14 region. In addition, we show for the first time that myofibroblastoma of the lower female genital tract also exhibits the same chromosomal abnormality, supporting the hypothesis that both tumors are in the spectrum of a single entity, likely arising from a common precursor cell. PMID:22575260

  1. Single-Gene Detection and Karyotyping Using Small-Target Fluorescence in Situ Hybridization on Maize Somatic Chromosomes

    PubMed Central

    Lamb, Jonathan C.; Danilova, Tatiana; Bauer, Matthew J.; Meyer, Julie M.; Holland, Jennifer J.; Jensen, Michael D.; Birchler, James A.

    2007-01-01

    Combined with a system for identifying each of the chromosomes in a genome, visualizing the location of individual genetic loci by fluorescence in situ hybridization (FISH) would aid in assembling physical and genetic maps. Previously, large genomic clones have been successfully used as FISH probes onto somatic chromosomes but this approach is complicated in species with abundant repetitive elements. In this study, repeat-free portions of sequences that were anchored to particular chromosomes including genes, gene clusters, large cDNAs, and portions of BACs obtained from public databases were used to label the corresponding physical location using FISH. A collection of probes that includes at least one marker on each chromosome in the maize complement was assembled, allowing a small-target karyotyping system to be developed. This set provides the foundation onto which additional loci could be added to strengthen further the ability to perform chromosomal identification in maize and its relatives. The probes were demonstrated to produce signals in several wild relatives of maize, including Zea luxurians, Z. diploperennis, and Tripsacum dactyloides. PMID:17237520

  2. Human cDNA mapping using fluorescence in situ hybridization. Progress report, April 1--December 31, 1992

    SciTech Connect

    Korenberg, J.R.

    1993-12-31

    The ultimate goal of this proposal is to create a cDNA map of the human genome. Mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach will generate 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

  3. Rapid identification of Acinetobacter spp. by fluorescence in situ hybridization (FISH) from colony and blood culture material

    PubMed Central

    Essig, A.; Hagen, R. M.; Riecker, M.; Jerke, K.; Ellison, D.; Poppert, S.

    2011-01-01

    Multi-drug-resistant strains of the Acinetobacter baumannii complex cause nosocomial infections. Rapid identification of Acinetobacter spp. is desirable in order to facilitate therapeutic or hygiene decisions. We evaluated a newly designed DNA probe that can be used under standard conditions in both a microwave oven and a slide chamber for the rapid identification of Acinetobacter spp. by fluorescence in situ hybridization (FISH). Using FISH, the new probe correctly identified 81/81 Acinetobacter spp. isolates and excluded 109/109 tested non-target organisms from agar culture. Furthermore, the new probe correctly identified 7/7 Acinetobacter spp. in 214 blood cultures determined to contain Gram-negative bacteria by Gram staining. Using either the microwave oven or slide chamber technique, the new probe was able to identify Acinetobacter spp. in 100% of the samples tested. FISH used in conjunction with our newly designed probe provides an easy, cheap, precise, and rapid method for the preliminary identification of Acinetobacter spp., especially in laboratories where more sophisticated methods like matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) are not available. PMID:24516735

  4. Rapid molecular cytogenetic analysis of X-chromosomal microdeletions: Fluorescence in situ hybridization (FISH) for complex glycerol kinase deficiency

    SciTech Connect

    Worley, K.C.; Lindsay, E.A.; McCabe, E.R.B. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1995-07-17

    Diagnosis of X-chromosomal microdeletions has relied upon the traditional methods of Southern blotting and DNA amplification, with carrier identification requiring time-consuming and unreliable dosage calculations. In this report, we describe rapid molecular cytogenetic identification of deleted DNA in affected males with the Xp21 contiguous gene syndrome (complex glycerol kinase deficiency, CGKD) and female carriers for this disorder. CGKD deletions involve the genes for glycerol kinase, Duchenne muscular dystrophy, and/or adrenal hypoplasia congenita. We report an improved method for diagnosis of deletions in individuals with CGKD and for identification of female carriers within their families using fluorescence in situ hybridization (FISH) with a cosmid marker (cosmid 35) within the glycerol kinase gene. When used in combination with an Xq control probe, affected males demonstrate a single signal from the control probe, while female carriers demonstrate a normal chromosome with two signals, as well as a deleted chromosome with a single signal from the control probe. FISH analysis for CGKD provides the advantages of speed and accuracy for evaluation of submicroscopic X-chromosome deletions, particularly in identification of female carriers. In addition to improving carrier evaluation, FISH will make prenatal diagnosis of CGKD more readily available. 17 refs., 2 figs.

  5. Interphase fluorescence in situ hybridization and reverse transcription polymerase chain reaction as a diagnostic aid for synovial sarcoma.

    PubMed Central

    Shipley, J.; Crew, J.; Birdsall, S.; Gill, S.; Clark, J.; Fisher, C.; Kelsey, A.; Nojima, T.; Sonobe, H.; Cooper, C.; Gusterson, B.

    1996-01-01

    Identification of the t(X;18)(p11.2;q11.2) that is associated with a high proportion of synovial sarcoma can be a useful diagnostic aid. The translocation results in fusion of the SYT gene on chromosome 18 to either the SSX1 or the SSX2 gene, two homologous genes within Xp11.2. Two-color interphase fluorescence in situ hybridization and reverse transcription polymerase chain reaction were assessed as approaches to identify the rearrangement in well characterized cases. The presence of the translocation, and the specific chromosome X gene disrupted, were inferred from the configuration of signals from chromosome-specific centromere probes, paints, and markers flanking each gene in preparations of interphase nuclei. Rearrangement was found in two cell lines and eight of nine tumor samples, including analysis of five touch imprints. This was consistent with cytogenetic data in four cases and reverse transcription polymerase chain reaction analysis using primers known to amplify both SYT-SSX1 and SYT-SSX2 transcripts. The transcripts were distinguished by restriction with LspI and SmaI. Contrary to previous suggestions, there was no obvious correlation between histological subtype and involvement of the SSX1 or SSX2 gene. These approaches could also be applied to the identification of tumor-free margins and metastatic disease. Images Figure 1 Figure 3 PMID:8579118

  6. Discriminating Multi-Species Populations in Biofilms with Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA FISH)

    PubMed Central

    Almeida, Carina; Azevedo, Nuno F.; Santos, Sílvio; Keevil, Charles W.; Vieira, Maria J.

    2011-01-01

    Background Our current understanding of biofilms indicates that these structures are typically composed of many different microbial species. However, the lack of reliable techniques for the discrimination of each population has meant that studies focusing on multi-species biofilms are scarce and typically generate qualitative rather than quantitative data. Methodology/Principal Findings We employ peptide nucleic acid fluorescence in situ hybridization (PNA FISH) methods to quantify and visualize mixed biofilm populations. As a case study, we present the characterization of Salmonella enterica/Listeria monocytogenes/Escherichia coli single, dual and tri-species biofilms in seven different support materials. Ex-situ, we were able to monitor quantitatively the populations of ?56 mixed species biofilms up to 48 h, regardless of the support material. In situ, a correct quantification remained more elusive, but a qualitative understanding of biofilm structure and composition is clearly possible by confocal laser scanning microscopy (CLSM) at least up to 192 h. Combining the data obtained from PNA FISH/CLSM with data from other established techniques and from calculated microbial parameters, we were able to develop a model for this tri-species biofilm. The higher growth rate and exopolymer production ability of E. coli probably led this microorganism to outcompete the other two [average cell numbers (cells/cm2) for 48 h biofilm: E. coli 2,1×108 (±2,4×107); L. monocytogenes 6,8×107 (±9,4×106); and S. enterica 1,4×106 (±4,1×105)]. This overgrowth was confirmed by CSLM, with two well-defined layers being easily identified: the top one with E. coli, and the bottom one with mixed regions of L. monocytogenes and S. enterica. Significance While PNA FISH has been described previously for the qualitative study of biofilm populations, the present investigation demonstrates that it can also be used for the accurate quantification and spatial distribution of species in polymicrobial communities. Thus, it facilitates the understanding of interspecies interactions and how these are affected by changes in the surrounding environment. PMID:21479268

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

    NASA Astrophysics Data System (ADS)

    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.

  8. Phylogenetic Composition, Spatial Structure, and Dynamics of Lotic Bacterial Biofilms Investigated by Fluorescent in Situ Hybridization and Confocal Laser Scanning Microscopy

    Microsoft Academic Search

    W. Manz; K. Wendt-Potthoff; T. R. Neu; U. Szewzyk; J. R. Lawrence

    1999-01-01

    The phylogenetic composition, three-dimensional structure and dynamics of bacterial communities in river biofilms generated\\u000a in a rotating annular reactor system were studied by fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Biofilms grew on independently removable polycarbonate\\u000a slides exposed in the reactor system with natural river water as inoculum and sole nutrient and carbon source. The microbial

  9. Use of microautoradiography combined with fluorescence in situ hybridization to determine dimethylsulfoniopropionate incorporation by marine bacterioplankton taxa.

    PubMed

    Vila, Maria; Simó, Rafel; Kiene, Ronald P; Pinhassi, Jarone; González, José M; Moran, Mary Ann; Pedrós-Alió, Carlos

    2004-08-01

    The fraction of planktonic heterotrophic bacteria capable of incorporating dissolved dimethylsulfoniopropionate (DMSP) and leucine was determined at two coastal sites by microautoradioagraphy (AU). In Gulf of Mexico seawater microcosm experiments, the proportion of prokaryotes that incorporated sulfur from [(35)S]DMSP ranged between 27 and 51% of 4',6-diamidino-2-phenylindole (DAPI)-positive cells, similar to or slightly lower than the proportion incorporating [(3)H]leucine. In the northwest Mediterranean coast, the proportion of cells incorporating sulfur from [(35)S]DMSP increased from 5 to 42% from January to March, coinciding with the development of a phytoplankton bloom. At the same time, the proportion of cells incorporating [(3)H]leucine increased from 21 to 40%. The combination of AU and fluorescence in situ hybridization (FISH) revealed that the Roseobacter clade (alpha-proteobacteria) accounted for 13 to 43% of the microorganisms incorporating [(35)S]DMSP at both sampling sites. Significant uptake of sulfur from DMSP was also found among members of the gamma-proteobacteria and Cytophaga-Flavobacterium groups. Roseobacter and gamma-proteobacteria exhibited the highest percentage of DAPI-positive cells incorporating (35)S from DMSP (around 50%). Altogether, the application of AU with [(35)S]DMSP combined with FISH indicated that utilization of S from DMSP is a widespread feature among active marine bacteria, comparable to leucine utilization. These results point toward DMSP as an important substrate for a broad and diverse fraction of marine bacterioplankton. PMID:15294798

  10. Molecular analysis of hematopoietic colonies derived from chronic myeloid leukemia patients: interphase fluorescence in situ hybridization compared with RT-PCR

    Microsoft Academic Search

    SFT Thijsen; GJ Schuurhuis; JW van Oostveen; AP Theijsmeijer; MMAC Langenhuijsen; GJ Ossenkoppele

    1997-01-01

    In this study we compared interphase fluorescence in situ hybridization (I-FISH) with reverse transcription polymerase chain reaction (RT-PCR) for the molecular analysis of hematopoietic colonies derived from patients with chronic myeloid leukemia (CML). Molecular analysis of individual colonies is often performed to monitor purging efficacy in CML. We harvested individual colony-forming unit granulocyte–macrophage (CFU-GM) colonies. One half was analyzed with

  11. Smith-Magenis syndrome deletion: A case with equivocal cytogenetic findings resolved by fluorescence in situ hybridization

    SciTech Connect

    Juyal, R.C.; Patel, P.I.; Greenberg, F. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1995-09-11

    The availability of markers for the 17p11.2 region has enabled the diagnosis of Smith-Magenis syndrome (SMS) by fluorescence in situ hybridization (FISH). SMS is typically associated with a discernible deletion of band 17p11.2 upon cytogenetic analysis at a resolution of 400-550 bands. We present a case that illustrates the importance of using FISH to confirm a cytogenetic diagnosis of del(17)(p11.2). Four independent cytogenetic analyses were performed with different conclusions. Results of low resolution analyses of amniocytes and peripheral blood lymphocytes were apparently normal, while high resolution analyses of peripheral blood samples in two laboratories indicated mosaicism for del(17)(p11.2). FISH clearly demonstrated a 17p deletion on one chromosome of all peripheral blood cells analyzed and ruled out mosaicism unambiguously. The deletion was undetectable by flow cytometric quantitation of chromosomal DNA content, suggesting that it is less than 2 Mb. We conclude that FISH should be used to detect the SMS deletion when routine chromosome analysis fails to detect it and to verify mosaicism. 23 refs., 3 figs., 1 tab.

  12. Improved Fluorescent In Situ Hybridization Method for Detection of Bacteria from Activated Sludge and River Water by Using DNA Molecular Beacons and Flow Cytometry

    Microsoft Academic Search

    Jeremy Lenaerts; Hilary M. Lappin-Scott; Jonathan Porter

    2007-01-01

    Fluorescent in situ hybridization (FISH) remains a key technique in microbial ecology. Molecular beacons (MBs) are self-reporting probes that have potential advantages over linear probes for FISH. MB-FISH strategies have been described using both DNA-based and peptide nucleic acid (PNA)-based approaches. Although recent reports have suggested that PNA MBs are superior, DNA MBs have some advantages, most notably cost. The

  13. Differentiation of Mycobacterium tuberculosis Complex and Nontuberculous Mycobacterial Liquid Cultures by Using Peptide Nucleic Acid-Fluorescence In Situ Hybridization Probes

    Microsoft Academic Search

    F. A. DROBNIEWSKI; P. G. MORE; G. S. HARRIS

    2000-01-01

    A blinded comparison of peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) with routine identification methods was performed on 74 consecutively positive mycobacterial liquid cultures. All Mycobac- terium tuberculosis cultures (48 of 48) and 22 of 27 (81.5%) nontuberculous cultures were correctly identified (including one mixed culture). Five isolates yielded no reaction with either probe and were identified as Mycobacterium xenopi,

  14. Fluorescence in situ hybridization as an ancillary tool in the diagnosis of ambiguous melanocytic neoplasms: a review of 804 cases.

    PubMed

    North, Jeffrey P; Garrido, Maria C; Kolaitis, Nicholas A; LeBoit, Philip E; McCalmont, Timothy H; Bastian, Boris C

    2014-06-01

    Previous studies have demonstrated the utility of fluorescence in situ hybridization (FISH) as an ancillary method in the diagnostic workup of histopathologically ambiguous melanocytic neoplasms. A combination of probes targeting 3 loci on chromosome 6 and 1 on 11q has been reported to distinguish unequivocal melanomas and nevi with a sensitivity and specificity of 87% and 96%, respectively. However, information on how FISH should be integrated into routine clinical testing is limited. We report our experience of FISH testing of 804 ambiguous melanocytic lesions performed as part of routine workup at University of California, San Francisco. The main category (47% of all cases) for which FISH testing was requested was Spitz tumors. Other categories included the distinction of possible melanoma from combined nevi (9%), acral or mucosal nevi (9%), Clark/dysplastic nevi (7%), and blue or deep penetrating nevi (6%) and to assess the possibility of nevoid melanoma (4%). Of the ambiguous tumors successfully tested, 88% received a more definitive benign or malignant final diagnosis. Of the 630 cases that tested negative by FISH, the final diagnosis was benign in 489 (78%) cases, ambiguous in 91 cases (14%), and malignant in 50 cases (8%). A positive FISH result was observed in 124 cases, with a final diagnosis of melanoma in 117 (94%). One (1%) FISH-positive case had an equivocal final diagnosis, and 6 (5%) were interpreted, despite the positive FISH result, as melanocytic nevi. We conclude that FISH testing can help reduce the number of equivocal diagnoses in ambiguous melanocytic neoplasms, in particular if FISH testing is positive, and discuss the challenges and limitations of FISH in clinical practice. PMID:24618603

  15. Utility of fluorescence in situ hybridization to detect MDM2 amplification in liposarcomas and their morphological mimics

    PubMed Central

    Kimura, Hiroaki; Dobashi, Yoh; Nojima, Takayuki; Nakamura, Hiroyuki; Yamamoto, Norio; Tsuchiya, Hiroyuki; Ikeda, Hiroko; Sawada-Kitamura, Seiko; Oyama, Takeru; Ooi, Akishi

    2013-01-01

    The atypical lipomatous tumor (ALT)/well-differentiated liposarcoma (WDLS) and the de-differentiated liposarcoma (DDLS) represent the most common category of liposarcomas. ALT/WDLSs and DDLSs are often difficult to distinguish from other tumors with similar morphological characteristics. In this study, we investigated whether the detection of amplified or overexpressed murine double-minute 2 (MDM2) can be a useful diagnostic ancillary aid. We used fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) to detect MDM2 amplification and protein overexpression, respectively, in 49 WDLSs, 5 DDLSs, 23 myxoid liposarcomas, 25 benign lipomatous tumors, and 75 spindle and pleomorphic sarcomas. MDM2 amplification was detected in 48 of 49 WDLSs, 5 of 5 DDLSs, 2 of 9 malignant peripheral nerve sheath tumors, and 2 of 10 myxofibrosarcomas. We did not detect MDM2 amplification in any of the benign lipomatous tumors. FISH-mediated detection of MDM2 amplification was the most valuable diagnostic aid for ALT/WDLS, as determined by using the Fisher exact test to compare two different diagnoses of 19 biopsies. On the contrary, unequivocal nuclear overexpression of MDM2 was found in only 10 of 50 ALT/WDLSs. The sensitivity and specificity of MDM2 amplification in distinguishing a DDLS from spindle and pleomorphic sarcomas were 100% and 95%, respectively, while those of MDM2 overexpression were 100% and 87%, respectively. In conclusion, our results indicate that FISH-mediated detection of MDM2 amplification is the most useful adjunct in the diagnosis of both ALT/WDLS and DDLS. However, IHC-mediated detection of MDM2 protein is useful only for the diagnosis of DDLS. PMID:23826411

  16. FISH in chips: turning microfluidic fluorescence in situ hybridization into a quantitative and clinically reliable molecular diagnosis tool.

    PubMed

    Perez-Toralla, Karla; Mottet, Guillaume; Guneri, Ezgi Tulukcuoglu; Champ, Jérôme; Bidard, François-Clément; Pierga, Jean-Yves; Klijanienko, Jerzy; Draskovic, Irena; Malaquin, Laurent; Viovy, Jean-Louis; Descroix, Stéphanie

    2015-02-01

    Microfluidic systems bear promise to provide new powerful tools for the molecular characterization of cancer cells, in particular for the routine detection of multiple cancer biomarkers using a minute amount of the sample. However, taking miniaturized cell-based assays into the clinics requires the implementation and validation of complex biological protocols on chip, as well as the development of disposable microdevices produced at a low cost. Based on a recently developed microfluidic chip made of Cyclic Olefin Copolymer for cell immobilization with minimal dead volume and controlled shear stress, we developed a protocol performed entirely in the liquid phase, allowing the immobilization and fixation of cells and their quantitative characterization by fluorescence in situ hybridization. We demonstrated first in cell lines and then in two clinical case studies the potential of this method to perform quantitative copy number measurement and clinical scoring of the amplification of the ERBB2 gene, a decisive biomarker for the prescription of HER2+ related targeted therapies. This validation was performed in a blind protocol in two clinical case studies, in reference to the gold standard and clinically used method based on glass slides. We obtained a comparable reproducibility and a minor difference in apparent amplification, which can be corrected by internal calibration. The method thus reaches the standard of robustness needed for clinical use. The protocol can be fully automated, and its consumption of samples and DNA probes is reduced as compared to glass slide protocols by a factor of at least 10. The total duration of the assay is divided by two. PMID:25474258

  17. Novel repeated DNA sequences in safflower (Carthamus tinctorius L.) (Asteraceae): cloning, sequencing, and physical mapping by fluorescence in situ hybridization.

    PubMed

    Raina, S N; Sharma, S; Sasakuma, T; Kishii, M; Vaishnavi, S

    2005-01-01

    Two novel repetitive DNA sequences, pCtKpnI-1 and pCtKpnI-2, were isolated from Carthamus tinctorius (2n = 2x = 24) and cloned. Both represent tandemly repeated sequences. The pCtKpnI-1 and pCtKpnI-2 clones constitute repeat units of 343-345 bp and 367 bp, respectively, with 63% sequence heterogeneity between the two. Fluorescence in situ hybridization (FISH) was employed on metaphase chromosomes of C. tinctorius using, simultaneously, pCtKpnI-1 and pCtKpnI-2 repeated sequences. The pCtKpnI-1 sequence was found to be exclusively localized at subtelomeric regions on most of the chromosomes. On the other hand, sequence of the pCtKpnI-2 clone was distributed on two nucleolar and one nonnucleolar chromosome pairs. The satellite, and the intervening chromosome segment between the primary and secondary constrictions, in the two nucleolar chromosome pairs were wholly constituted by pCtKpnI-2 repeated sequence. The pCtKpnI-2 repeated sequence, showing partial homology to intergenic spacer (IGS) of 18S-25S ribosomal RNA genes of an Asteraceae taxon (Centaurea stoebe), and the 18S-25S rRNA gene clusters were located at independent, but juxtaposed sites in the nucleolar chromosomes. Variability in the number, size, and location of the two repeated sequences provided identification of most of the chromosomes in the otherwise not too distinctive homologues within the complement. This article reports the start of a molecular cytogenetics program targeting the genome of safflower, a major world oil crop about whose genetics very little is known. PMID:15731214

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

    PubMed

    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. PMID:25576649

  19. Fluorescence in situ hybridization with Alu and L1 polymerase chain reaction probes for rapid characterization of human chromosomes in hybrid cell lines.

    PubMed Central

    Lichter, P; Ledbetter, S A; Ledbetter, D H; Ward, D C

    1990-01-01

    Human-rodent hybrid cell lines have been analyzed with regard to their human DNA content by using various DNA probe sets, derived from the hybrids, for in situ hybridization to normal human metaphase chromosome spreads. Total genomic hybrid DNA was compared with probe sets of hybrid DNA that were highly enriched in human sequences. The latter probes were obtained by amplification through the polymerase chain reaction (PCR) using oligonucleotide primers directed to human specific subsequences of the interspersed repetitive sequences Alu and L1. Previously unidentified chromosomal material within hybrid lines was characterized with speed and precision. It is demonstrated that the complete human complement of hybrid lines can be rapidly assessed by comparing the data obtained with the Alu-PCR products with the results from the L1-PCR products or from the genomic hybrid DNA. This approach using interspersed repetitive sequence-PCR products is simple and fast and also provides an alternative way of generating complex DNA probe sets for the specific delineation of entire chromosomes or subchromosomal regions by in situ hybridization. Images PMID:2395866

  20. Preparations of Meiotic Pachytene Chromosomes and Extended DNA Fibers from Cotton Suitable for Fluorescence In Situ Hybridization

    PubMed Central

    Liu, Fang; Ling, Jian; Wang, Chunying; Li, Shaohui; Zhang, Xiangdi; Wang, Yuhong; Wang, Kunbo

    2012-01-01

    Fluorescence in situ hybridization (FISH) has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in chromosome preparation. The focus of this article was FISH performed not only on cotton pachytene chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs) instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy's solution spreading chromosome was adopted instead of nitrogen removed method to avoid chromosomes losing and fully stretch chromosome. With the above-improved steps, the high-quality well-differentiated pachytene chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene chromosomes preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent oxidation. The probability of success in isolating nuclei for DNA fiber preparation is almost 100% tested with this method in cotton. So a rapid, safe, and efficient method for the preparation of cotton extended DNA fibers suitable for FISH was established. PMID:22442728

  1. Localization of single- and low-copy sequences on tomato synaptonemal complex spreads using fluorescence in situ hybridization (FISH).

    PubMed Central

    Peterson, D G; Lapitan, N L; Stack, S M

    1999-01-01

    Fluorescence in situ hybridization (FISH) is a powerful means by which single- and low-copy DNA sequences can be localized on chromosomes. Compared to the mitotic metaphase chromosomes that are normally used in FISH, synaptonemal complex (SC) spreads (hypotonically spread pachytene chromosomes) have several advantages. SC spreads (1) are comparatively free of debris that can interfere with probe penetration, (2) have relatively decondensed chromatin that is highly accessible to probes, and (3) are about ten times longer than their metaphase counterparts, which permits FISH mapping at higher resolution. To investigate the use of plant SC spreads as substrates for single-copy FISH, we probed spreads of tomato SCs with two single-copy sequences and one low-copy sequence (ca. 14 kb each) that are associated with restriction fragment length polymorphism (RFLP) markers on SC 11. Individual SCs were identified on the basis of relative length, arm ratio, and differential staining patterns after combined propidium iodide (PI) and 4', 6-diamidino-2-phenylindole (DAPI) staining. In this first report of single-copy FISH to SC spreads, the probe sequences were unambiguously mapped on the long arm of tomato SC 11. Coupled with data from earlier studies, we determined the distance in micrometers, the number of base pairs, and the rates of crossing over between these three FISH markers. We also observed that the order of two of the FISH markers is reversed in relation to their order on the molecular linkage map. SC-FISH mapping permits superimposition of markers from molecular linkage maps directly on pachytene chromosomes and thereby contributes to our understanding of the relationship between chromosome structure, gene activity, and recombination. PMID:10224272

  2. Vulvovaginal angiomyofibroblastomas: morphologic, immunohistochemical, and fluorescence in situ hybridization analysis for deletion of 13q14 region.

    PubMed

    Magro, Gaetano; Righi, Alberto; Caltabiano, Rosario; Casorzo, Laura; Michal, Michal

    2014-08-01

    Angiomyofibroblastoma (AMFB) is a benign tumor that belongs to the category of the "stromal tumors of the lower female genital tract," together with cellular angiofibroma and myofibroblastoma. Previous studies have shown overlapping morphologic and immunohistochemical features between these tumors and spindle cell lipoma, mammary-type myofibroblastoma, and vulvovaginal cellular angiofibroma and myofibroblastoma. In addition, typical loss of genetic material from the 13q14 region has been documented in all the above-mentioned tumors, suggesting that they are histogenetically related. We report the clinicopathologic features of 11 new cases of vulvovaginal AMFBs. Histologically, the basic common theme was a proliferation of bland-looking spindle to round-to-epithelioid cells set in an edematous to fibrous stroma, frequently arranged around thin-walled blood vessels. Two cases were composed of a prominent mature fatty component closely admixed with typical areas of AMFB, and thus, they were designated as "lipomatous AMFBs." Notably, 1 case was closely reminiscent of Sertoli cell tumor, sclerosing type, because of its predominant cord-like arrangement. Immunohistochemically, all tumors were diffusely positive for vimentin, whereas desmin and ?-smooth muscle actin were expressed in a minority of cases, suggesting a fibroblastic rather than myofibroblastic differentiation. Most cases of AMFBs coexpressed Bcl-2 protein and CD99. Interestingly, all 5 cases of AMFB with evaluable signals failed to show monoallelic loss of FOXO1 loci (13q14) by fluorescence in situ hybridization. These cytogenetic findings suggest that vulvovaginal AMFB is not genetically related to cellular angiofibroma and myofibroblastoma of the lower female genital tract. PMID:24880711

  3. Trisomy 10p resulting from an inv dup of 10p defined by fluorescence in situ hybridization

    SciTech Connect

    Clement, S.J.; Easterling, T.R.; Leppig, K.A. [Univ. of Washington, Seattle, WA (United States)] [and others

    1994-09-01

    De novo cases of trisomy for the entire short arm of chromosome 10 are infrequently reported and are most commonly the result of translocation of 10p to an acrocentric chromosome. Most reported cases of trisomy 10p are not trisomy for the complete short arm of chromosome 10, but are duplication, deficiency syndromes that result from either inheritance of an unbalanced translocation from a parent possessing a balanced reciprocal translocation, or from a recombinant chromosome derived from a parental pericentric inversion of chromosome 10. Here, we report a case of a de novo trisomy 10p that resulted from an inverted duplication of the entire short arm of chromosome 10. A 42 year old G7,P5,SAB1 woman was referred for amniocentesis because of advanced maternal age. Ultrasound examination at 17 weeks demonstrated a fetus of normal size with no apparent anatomic abnormalities. Cytogenetic evaluation demonstrated one homologue of chromosome 10 had a tandem inverted duplication of the short arm. The fetal karyotype was interpreted to be 46,XX,inv dup (10) (peter-cen::cen-p15::q11-pter). Parental karyotype are normal. Fluorescence in situ hybridization (FISH) using a chromosome 10 paint, chromosome 10 centromere, and all telomere probe, confirmed the inverted duplication involved the entire short arm of chromosome 10. Termination of pregnancy was performed at 20 weeks gestation. Autopsy revealed multiple anomalies including low-set posteriorly rotated ears, cleft of the soft palate, ocular hypertelorism, small upturned nose, agenesis of the gallbladder, sacral hemivertebrae, and abnormal flexion of the thumbs. The fetal karyotype was confirmed by cytogenetic analysis in lung and kidney. This is the second reported case of a de novo tandem duplication of 10p of which we are aware, and the first using FISH technology to characterize the abnormality.

  4. Fluorescence In Situ Hybridization: A Sensitive Method for Trisomy 8 Detection in Bone Marrow Specimens

    Microsoft Academic Search

    Robert B. Jenkins; Michelle M. Le Beau; William J. Kraker; Thomas J. Borell; Paul G. Stalboerger; Elizabeth M. Davis; Lolita Penland; Anthony Fernald; Rafael Espinosa; Daniel J. Schaid; Gordon W. Dewald

    1992-01-01

    RISOMY 8 is a common cytogenetic abnormality T found in the bone marrow (BM) of patients with myeloproliferative disorders (MPD), myelodysplastic syn- dromes (MDS), or acute nonlymphocytic leukemia (ANLL).1-5 Using conventional cytogenetic methods in which 20 to 30 metaphase cells are typically examined, it is possible to find 2 or more metaphases with trisomy 8 in many of these patients.

  5. Evaluation of HER-2/neu status in breast cancer specimens using immunohistochemistry (IHC) & fluorescence in-situ hybridization (FISH) assay

    PubMed Central

    Goud, Kalal Iravathy; Dayakar, Seetha; Vijayalaxmi, Kolanupaka; Babu, Saidam Jangu; Vijay, Anand Reddy P.

    2012-01-01

    Background & objectives: Fluorescence in situ hybridization (FISH) is increasingly being recognized as the most accurate and predictive test for HER2/neu gene amplification and response to therapy in breast cancer. In the present study we investigated HER-2/neu gene amplification by FISH in breast carcinoma tissue specimens and compared the results with that of immunohistochemical (IHC) analysis. Methods: A total of 90 breast carcinoma tissue samples were used for immunohistochemical (IHC) and FISH analysis. IHC was performed by using mouse monoclonal antibody to the intracellular domain of HER-2/neu protein. Each slide was scored in a blinded fashion by two pathologists according to the manufacturer's recommended criteria. FISH analysis was performed on paraffin embedded breast tumour tissue sections. The polysomy for centromere 17 (Spec green signal) was read as green signals less than 4 as moderate polysomy, and more than 4 as highly polysomy. Results: Thirty of the 90 patients had negative results by IHC and FISH. Of the 28 patients with the score of 2+ by IHC, 20 were FISH positive for HER-2/neu gene amplification, three were FISH negative and five patients showed equivocal (1.8-2.2) results by FISH. These five cases were retested for IHC and FISH on different paraffin embedded tissue blocks, and all five were found positive for HER-2/neu gene amplification. Twenty five patients with the score of 3+ by IHC were FISH positive for HER-2/neu gene amplification (>2.2). Seven cases with the score of 3+ by IHC were FISH negative for HER-2/neu gene amplification (>2.2), and showed polysomy of chromosome number 17 high polysomy > 4. Interpretation & conclusions: Our results indicated that HER-2/neu status by FISH should be performed in all cases of breast tumour with a 2+ score by IHC. Cases demonstrating a 3+ score by IHC may be subjected to FISH to rule out polysomy of chromosome 17 which could be falsely interpreted as HER-2/neu overexpression by IHC analysis. There is also a need for establishing a clinically validated cut-off value for HER-2/neu FISH amplification against IHC which may be further compared and calibrated. PMID:22561616

  6. Trisomy 12 in pediatric granulosa-stromal cell tumors. Demonstration by a modified method of fluorescence in situ hybridization on paraffin-embedded material.

    PubMed Central

    Schofield, D. E.; Fletcher, J. A.

    1992-01-01

    The use of fluorescence in situ hybridization (FISH) to detect chromosomal abnormalities has many applications. Use of FISH on archival, paraffin-embedded material has been limited to microscopic sections. We have carried out FISH on preparations of disaggregated nuclei obtained from paraffin-embedded tissue to evaluate chromosome 12 copy number in granulosa-stromal cell neoplasms occurring in infants, children, and adolescents. Trisomy 12 was detected in the majority of cells from three of four juvenile granulosa cell tumors (three ovarian and one testicular) and one malignant granulosa cell tumor. Tetrasomy 12 was observed in a case of ovarian thecoma. Images Figure 1 Figure 2 PMID:1466394

  7. Trisomy 12 in pediatric granulosa-stromal cell tumors. Demonstration by a modified method of fluorescence in situ hybridization on paraffin-embedded material.

    PubMed

    Schofield, D E; Fletcher, J A

    1992-12-01

    The use of fluorescence in situ hybridization (FISH) to detect chromosomal abnormalities has many applications. Use of FISH on archival, paraffin-embedded material has been limited to microscopic sections. We have carried out FISH on preparations of disaggregated nuclei obtained from paraffin-embedded tissue to evaluate chromosome 12 copy number in granulosa-stromal cell neoplasms occurring in infants, children, and adolescents. Trisomy 12 was detected in the majority of cells from three of four juvenile granulosa cell tumors (three ovarian and one testicular) and one malignant granulosa cell tumor. Tetrasomy 12 was observed in a case of ovarian thecoma. PMID:1466394

  8. Novel karyotype in the Ullrich-Turner syndrome - 45,X/46,X,r(X)/46,X,dic(X) - investigated with fluorescence in situ hybridization

    SciTech Connect

    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.

  9. Establishment of a new human pleomorphic malignant fibrous histiocytoma cell line, FU-MFH-2: molecular cytogenetic characterization by multicolor fluorescence in situ hybridization and comparative genomic hybridization

    PubMed Central

    2010-01-01

    Background Pleomorphic malignant fibrous histiocytoma (MFH) is one of the most frequent malignant soft tissue tumors in adults. Despite the considerable amount of research on MFH cell lines, their characterization at a molecular cytogenetic level has not been extensively analyzed. Methods and results We established a new permanent human cell line, FU-MFH-2, from a metastatic pleomorphic MFH of a 72-year-old Japanese man, and applied multicolor fluorescence in situ hybridization (M-FISH), Urovysion™ FISH, and comparative genomic hybridization (CGH) for the characterization of chromosomal aberrations. FU-MFH-2 cells were spindle or polygonal in shape with oval nuclei, and were successfully maintained in vitro for over 80 passages. The histological features of heterotransplanted tumors in severe combined immunodeficiency mice were essentially the same as those of the original tumor. Cytogenetic and M-FISH analyses displayed a hypotriploid karyotype with numerous structural aberrations. Urovysion™ FISH revealed a homozygous deletion of the p16INK4A locus on chromosome band 9p21. CGH analysis showed a high-level amplification of 9q31-q34, gains of 1p12-p34.3, 2p21, 2q11.2-q21, 3p, 4p, 6q22-qter, 8p11.2, 8q11.2-q21.1, 9q21-qter, 11q13, 12q24, 15q21-qter, 16p13, 17, 20, and X, and losses of 1q43-qter, 4q32-qter, 5q14-q23, 7q32-qter, 8p21-pter, 8q23, 9p21-pter, 10p11.2-p13, and 10q11.2-q22. Conclusion The FU-MFH-2 cell line will be a particularly useful model for studying molecular pathogenesis of human pleomorphic MFH. PMID:21092322

  10. 21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...with FISH assays as an aid in the detection, counting and classification of cells based on recognition of cellular color, size, and shape, and in the detection and enumeration of FISH signals in interphase nuclei of formalin-fixed,...

  11. Fluorescence in situ hybridization-based karyotyping of soybean translocation lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean [Glycine max (L.) Merr.] is a major crop species and a target of a substantial investment of genomic and genetic studies; yet, in contrast to other plant species, relatively few chromosomal aberrations have been identified and characterized in soybean. This is due in part to the difficulty ...

  12. Single molecule fluorescent in situ hybridization (smFISH) of C. elegans worms and embryos

    E-print Network

    Ji, Ni

    In C. elegans, the expression pattern of a gene provides important clues to understanding its biological function. To accurately depict endogenous transcriptional activity, a highly sensitive method is required to measure ...

  13. QUANTITATIVE FLUORESCENCE IN SITU HYBRIDIZATION OF AUREOBASIDIUM PULLULANS ON MICROSCOPE SLIDES AND LEAF SURFACES. (R823845)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  14. Rapid method for measuring clastogenic fingerprints using fluorescence in situ hybridization

    DOEpatents

    Lucas, Joe N. (San Ramon, CA)

    2000-01-01

    A method is provided for determining a clastogenic signature of a sample of chromosomes by quantifying a frequency of a first type of chromosome aberration present in the sample; quantifying a frequency of a second, different type of chromosome aberration present in the sample; and comparing the frequency of the first type of chromosome aberration to the frequency of the second type of chromosome aberration. A method is also provided for using that clastogenic signature to identify a clastogenic agent or dosage to which the cells were exposed.

  15. Analysis of amniotic fluid specimens for common chromosome disorders using interphase fluorescence in situ hybridization

    Microsoft Academic Search

    Tariq Moatter; Zahida Khilji; Farzana Murad; Shama Munim

    Objective: The aim of the study was to examine the usage of multi colour FISH technology as an adjunct to con- ventional cytogenetics for the prenatal diagnosis of aneuploidy in interphase nuclei from high risk pregnancies. Methods: Amniotic fluid samples were collected for interphase FISH analysis using DNA probes for chromo- somes 13, 18, 21, X and Y. All the

  16. Partial trisomy 11q involving chromosome 1 detected by fluorescence in situ hybridization

    SciTech Connect

    McCorquodale, M.; Bereziouk, O.; McCorquodale, D.J. [Univ. of Illinois College of Medicine, Chicago, IL (United States)] [and others

    1994-09-01

    Partial trisomy 11q was detected in an infant delivered 3-4 weeks prematurely. The phenotype included slanted palpebral fissures, high arched palate, developmental delay, microcephaly, and cardiac defects, all of which occur in the majority of cases with this syndrome. Other features included a column-shaped skull, preauricular pit, single palmar crease, short, broad great toes, flat occiput, unilateral kidney agenesis, and strabismus. Chromosomes obtained from peripheral blood cells revealed the presence of extra material on the long arm of chromosome 1. The G-banding pattern of this extra material indicated that it might be derived from chromosome 1 or 11. Chromosomal {open_quotes}paints{close_quotes} showed that it was not chromosome 1 material, but was chromosome 11 material extending from band q21 to qter. Partial trisomy 11q arising from translocation of the 11q material to chromosome 2, 3, 4, 5, 6, 9, 10, 13, 17, 21, 22, and X has been reported previously, whereas translocation to chromosome 1 has not. The chromosome to which the 11q material is translocated does not alter the most frequent features of the partial trisomy 11q syndrome, but may influence other less common features.

  17. Mapping of rDNA on the chromosomes of Eleusine species by fluorescence in situ hybridization.

    PubMed

    Bisht, M S; Mukai, Y

    2000-12-01

    Mapping of rDNA sites on the chromosomes of four diploid and two tetraploid species of Eleusine has provided valuable information on genome relationship between the species. Presence of 18S-5.8S-26S rDNA on the largest pair of the chromosomes, location of 5S rDNA at four sites on two pairs of chromosomes and presence of 18S-5.8S-26S and 5S rDNA at same location on one pair of chromosomes have clearly differentiated E. multiflora from rest of the species of Eleusine. The two tetraploid species, E. coracana and E. africana have the same number of 18S-5.8S-26S and 5S rDNA sites and located at similar position on the chromosomes. Diploid species, E. indica, E. floccifolia and E. tristachya have the same 18S-5.8S-26S sites and location on the chromosomes which also resembled with the two pairs of 18S-5.8S-26S rDNA locations in tetraploid species, E. coracana and E. africana. The 5S rDNA sites on chromosomes of E. indica and E. floccifolia were also comparable to the 5S rDNA sites of E. africana and E. coracana. The similarity of the rDNA sites and their location on chromosomes in the three diploid and two polyploid species also supports the view that genome donors to tetraploid species may be from these diploid species. PMID:11280009

  18. Assessment of retrospective dose estimation, with fluorescence in situ hybridization (FISH), of six victims previously exposed to accidental ionizing radiation.

    PubMed

    Liu, Qing-Jie; Lu, Xue; Zhao, Xiao-Tao; Feng, Jiang-Bin; Lü, Yu-Min; Jiang, En-Hai; Zhang, Shu-Lan; Chen, De-Qing; Jia, Ting-Zhen; Liang, Li

    2014-01-01

    The present study aims to evaluate the use of the fluorescence in situ hybridization (FISH) translocation assay for retrospective dose estimation of acute accidental exposure to radiation in the past. Reciprocal translocation analysis by FISH with three whole-chromosome probes was performed on normal peripheral blood samples. Samples were irradiated with 0-5Gy (60)Co ?-rays in vitro, and dose-effect curves were established. FISH-based translocation analyses for six accident victims were then performed, and biological doses were estimated retrospectively by comparison with the dose-effect curves. Reconstructed doses by FISH were compared with estimated doses obtained by analysis of di-centrics performed soon after exposure, or with dose estimates from tooth-enamel electron paramagnetic resonance (EPR) data obtained at the same time as the FISH analysis. Follow-up FISH analyses for an adolescent victim were performed. Results showed that dose-effect curves established in the present study follow a linear-quadratic model, regardless of the background translocation frequency. Estimated doses according to two dose-effect curves for all six victims were similar. FISH dose estimations of three adult victims exposed to accidental radiation less than a decade prior to analysis (3, 6, or 7 years ago) were consistent with those estimated with tooth-enamel EPR measurements or analyses of di-centrics. Estimated doses of two other adult victims exposed to radiation over a decade prior to analysis (16 or 33 years ago) were underestimated and two to three times lower than the values obtained from analysis of di-centrics or tooth-enamel EPR. Follow-up analyses of the adolescent victim showed that doses estimated by FISH analysis decrease rapidly over time. Therefore, the accuracy of dose estimates by FISH is acceptable only when analysis is performed less than 7 years after exposure. Measurements carried out more than a decade after exposure through FISH analysis resulted in underestimation of the biological doses compared with values obtained through analysis of di-centrics and tooth-enamel EPR. PMID:24246721

  19. Comparison of culture, polymerase chain reaction, and fluorescent in situ hybridization for detection of Brachyspira hyodysenteriae and "Brachyspira hampsonii" in pig feces.

    PubMed

    Wilberts, Bailey L; Warneke, Hallie L; Bower, Leslie P; Kinyon, Joann M; Burrough, Eric R

    2015-01-01

    Swine dysentery is characterized by mucohemorrhagic diarrhea and can occur following infection by Brachyspira hyodysenteriae or "Brachyspira hampsonii?". A definitive diagnosis is often based on the isolation of strongly beta-hemolytic spirochetes from selective culture or by the application of species-specific polymerase chain reaction (PCR) assays directly to feces. While culture is highly sensitive, it typically requires 6 or more days to complete, and PCR, although rapid, can be limited by fecal inhibition. Fluorescent in situ hybridization (FISH) has been described in formalin-fixed tissues; however, completion requires approximately 2 days. Because of the time constraints of available assays, a same-day FISH assay was developed to detect B. hyodysenteriae and "B. hampsonii?" in pig feces using previously described oligonucleotide probes Hyo1210 and Hamp1210 for B. hyodysenteriae and "B. hampsonii", respectively. In situ hybridization was simultaneously compared with culture and PCR on feces spiked with progressive dilutions of spirochetes to determine the threshold of detection for each assay at 0 and 48 hr. The PCR assay on fresh feces and FISH on formalin-fixed feces had similar levels of detection. Culture was the most sensitive method, detecting the target spirochetes at least 2 log-dilutions less when compared to other assays 48 hr after sample preparation. Fluorescent in situ hybridization also effectively detected both target species in formalin-fixed feces from inoculated pigs as part of a previous experiment. Accordingly, FISH on formalin-fixed feces from clinically affected pigs can provide same-day identification and preliminary speciation of spirochetes associated with swine dysentery in North America. PMID:25525136

  20. Chromosomal translocation t(X;18) in human synovial sarcomas analyzed by fluorescence in situ hybridization using paraffin-embedded tissue.

    PubMed Central

    Nagao, K.; Ito, H.; Yoshida, H.

    1996-01-01

    Synovial sarcoma is characterized cytogenetically by translocation t(X;18)(p11.2;q11.2). In this study, 28 cases that had been diagnosed initially as synovial sarcoma, including 2 fibrosarcomas, and 1 leiomyosarcoma were collected and examined for translocation t(X;18) on paraffin-embedded tissues by fluorescence in situ hybridization (FISH). Of the synovial sarcomas, 25 showed findings consistent with translocation t(X;18) with an additional copy signal for the total probe of X and 18 chromosomes. The other three cases, as well as the two fibrosarcomas and the leiomyosarcoma, did not show this translocation. One (case 26) of three negative cases was diagnosed finally as leiomyosarcoma and another (case 27) as malignant peripheral nerve sheath tumor from histological and immunohistochemical analysis. Thus, in all, 25 (96%) of 26 synovial sarcomas showed findings consistent with translocation t(X;18). In summary, translocation t(X;18) is a chromosomal aberration specific for synovial sarcoma. The fluorescence in situ hybridization technique can be used even on cells from paraffin-embedded tissues, and is a useful diagnostic aid for synovial sarcoma. Images Figure 1 Figure 2 PMID:8579122

  1. Fluorescence in situ hybridization to interphase cell nuclei in suspension allows flow cytometric analysis of chromosome content and microscopic analysis of nuclear organization

    Microsoft Academic Search

    Barbara Trask; Ger van den Engh; Dan Pinkel; Jim Mullikin; Fred Waldman; Herman van Dekken; Joe Gray

    1988-01-01

    Fluorescence hybridization to interphase nuclei in liquid suspension allows quantification of chromosome-specific DNA sequences using flow cytometry and the analysis of the three-dimensional positions of these sequences in the nucleus using fluorescence microscopy. The three-dimensional structure of nuclei is substantially intact after fluorescence hybridization in suspension, permitting the study of nuclear organization by optical sectioning. Images of the distribution of

  2. Fluorescence in situ hybridization mapping of human chromosome 19: Cytogenetic band location of 540 cosmids and 70 genes or DNA markers

    SciTech Connect

    Trask, B.; Fertitta, A.; Christensen, M.; Bergmann, A.; Copeland, A.; Jong, P. de; Mohrenweiser, H.; Olsen, A.; Carrano, A.; Tynan, K. (Lawrence Livermore National Lab., CA (United States)); Youngblom, J. (California State Univ., Turlock (United States))

    1993-01-01

    We report here the band location of 540 cosmids mapped to chromosome 19. The cosmids were mapped by fluorescence in situ hybridization (FISH) relative to chromosomal bands produced by DAPI/actinomycin staining. The cosmids are distributed throughout the chromosome, with a sampling bias for the q-arm. A detailed analysis of the distribution of three different subtelomeric and 22 pericentromeric chromosome 19 cosmids on other chromosomes is also reported. Colony hybridization identified 142 cosmids that contain sequences representing genes or DNA markers that map to chromosome 19. FISH mapping of these cosmids sublocalizes a total of 70 genes and DNA markers on chromosome 19, revises the previously published map assignments of 2 genes, and narrows the location of over 20 markers. 83 refs., 5 figs., 3 tabs.

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

    PubMed Central

    Neuenschwander, Stefan M.; Salcher, Michaela M.; Pernthaler, Jakob

    2015-01-01

    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 catalyzed 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 labeled 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. PMID:25873914

  4. Analysis of centromere signal patterns in breast cancer cells with chromosomal instability using image cytometry combined with centromere fluorescence in situ hybridization.

    PubMed

    Ito, Hideaki; Oga, Atsunori; Ikemoto, Kenzo; Furuya, Tomoko; Maeda, Noriko; Yamamoto, Shigeru; Kawauchi, Shigeto; Itoh, Hiroshi; Oka, Masaaki; Sasaki, Kohsuke

    2014-09-01

    Fluorescence in situ hybridization (FISH) with centromeric probes is a method used to detect chromosomal instability (CIN), a hallmark of most cancers. However, no studies thus far have investigated the relationship between centromeric FISH signals and the cell cycle in cancer cells. In this study, the chromosome content in each cell cycle phase was evaluated with respect to the number of centromeric FISH signals in two breast cancer cell lines and eight surgically resected breast cancer specimens using image cytometry. Variations in chromosome number were detected at each phase of the cell cycle but were not associated with proliferative capacity in the cell lines. Furthermore, the chromosome doubling frequency differed in each cell line and clinical specimen. These results reveal two aspects of centromeric FISH signal variation in breast cancers that exhibit CIN, and suggest that chromosome doubling is a remarkable occurrence that may increase the heterogeneity of tumors. PMID:25044720

  5. Molecular detection of Helicobacter pylori in a large Mediterranean river, by direct viable count fluorescent in situ hybridization (DVC-FISH).

    PubMed

    Tirodimos, Ilias; Bobos, Mattheos; Kazakos, Evangelos; Haidich, Anna-Bettina; Dardavessis, Theodore; Kostopoulos, Ioannis; Arvanitidou, Malamatenia

    2014-12-01

    Although the precise route and mode of transmission of Helicobacter pylori are still unclear, molecular methods have been applied for the detection of H. pylori in environmental samples. In this study, we used the direct viable count fluorescent in situ hybridization (DVC-FISH) method to detect viable cells of H. pylori in the River Aliakmon, Greece. This is the longest river in Greece, and provides potable water in metropolitan areas. H. pylori showed positive detection for 23 out of 48 water samples (47.9%), while no seasonal variation was found and no correlation was observed between the presence of H. pylori and indicators of fecal contamination. Our findings strengthen the evidence that H. pylori is waterborne while its presence adds to the potential health hazards of the River Aliakmon. PMID:25473996

  6. Comparative mapping of DNA probes derived from the V{sub k} immunoglobulin gene regions on human and great ape chromosomes by fluorescence in situ hybridization

    SciTech Connect

    Arnold, N.; Wienberg, J.; Ermert, K. [Universitaet Muenchen (Germany)] [and others] [Universitaet Muenchen (Germany); and others

    1995-03-01

    Fluorescence in situ hybridization (FISH) of cosmid clones of human V{sub K} gene regions to human and primate chromosomes contributed to the dating of chromosome reorganizations in evolution. A clone from the K locus at 2p11-p12 (cos 106) hybridized to the assumed homologous chromosome bands in the chimpanzees Pan troglodytes (PTR) and P. paniscus (PPA), the Gorilla gorilla (GGO), and the orangutan Pongo Pygmaeus (PPY). Human and both chimpanzees differed from gorilla and orangutan by the mapping of cos 170, a clone derived from chromosome 2cen-q11.2; the transposition of this orphon to the other side of the centromere can, therefore, be dated after the human/chimpanzee and gorilla divergence. Hybridization to homologous bands was also found with a cosmid clone containing a V{sub K}I orphon located on chromosome 1 (cos 115, main signal at 1q31-q32), although the probe is not fully unique. Also, a clone derived from the orphon V{sub K} region on chromosome 22q11 (cos 121) hybridized to the homologous bands in the great apes. This indicates that the orphons on human chromosomes 1 and 22 had been translocated early in primate evolution. 18 refs., 2 figs.

  7. Clinical and cytogenetic findings in seven cases of inverted duplication of 8p with evidence of a telomeric deletion using fluorescence in situ hybridization

    SciTech Connect

    Guo, Wen-Jun; Callif-Daley, F.; Zapata, M.C.; Miller, M.E. [Children`s Medical Center, Dayton, OH (United States)

    1995-09-11

    We report on the clinical and cytogenetic findings in 7 cases of inverted duplication of region 8p11.2-p23. The phenotype of inv dup (8p) compiled from this series and the literature (N = 29) consists of severe mental retardation (100%), minor facial alterations (97%), agenesis of the corpus callosum (80%), hypotonia (66%), orthopedic abnormalities (58%), scoliosis/kyphosis (40%), and congenital heart defect (26%). A telomeric deletion of region 8p23.3-pter was confirmed in 3 of our cases studied using fluorescent in situ hybridization with a telomeric probe for 8p. Thus, these karyotypes are inv dup del(8) (qter{r_arrow} p23.1::p23.1{r_arrow}p11.2:). Our findings suggest that most cases of inv dup(8p) probably have a telomeric deletion. 20 refs., 4 figs., 2 tabs.

  8. Subcuticular bacteria associated with two common New Zealand echinoderms: Characterization using 16S rRNA sequence analysis and fluorescence in situ hybridization.

    PubMed

    Lawrence, Scott A; O'Toole, Ronan; Taylor, Michael W; Davy, Simon K

    2010-02-01

    Many echinoderms contain subcuticular bacteria (SCB), symbionts which reside in the lumen between the host's epidermal cells and outer cuticle. This relationship is common, existing in about 60% of echinoderms studied so far, yet the function of SCB remains largely unknown. In this study, phylogenetic analysis was carried out on 16S rRNA sequences obtained from echinoderm-associated bacteria, resulting in the identification of four species of putative SCB. All four bacteria were identified from the holothurian Stichopus mollis, and two of the four were also found in the asteroid Patiriella sp. Two of these bacteria belong to the Alphaproteobacteria, and two to the Gammaproteobacteria. In addition to phylogenetic analysis, fluorescence in situ hybridization (FISH) assays were carried out on Patiriella sp., S. mollis, and the asteroid Astrostole scabra. Results showed that Patiriella sp. and S. mollis contain SCB, in agreement with the phylogenetic analysis, while SCB were not detected in A. scabra. Of the bacteria detected using FISH, more than 80% were recognized as belonging to the Alphaproteobacteria in both host species. However, in S. mollis about 20% of the detected SCB successfully hybridized with the Gammaproteobacteria-specific probe, whereas bacteria belonging to this class were never observed in Patiriella sp. This is only the second study to characterize SCB by molecular means, and is the first to identify SCB in situ using FISH. PMID:20203257

  9. Comparative Fluorescence in Situ Hybridization Mapping of a 431-kb Arabidopsis thaliana Bacterial Artificial Chromosome Contig Reveals the Role of Chromosomal Duplications in the Expansion of the Brassica rapa Genome

    Microsoft Academic Search

    Scott A. Jackson; Zhukuan Cheng; Ming Li Wang; Howard M. Goodman; Jiming Jiang

    2000-01-01

    Comparative genome studies are important contributors to our understanding of genome evolution. Most comparative genome studies in plants have been based on genetic mapping of homologous DNA loci in different genomes. Large-scale comparative physical mapping has been hindered by the lack of efficient and affordable techniques. We report here the adaptation of fluorescence in situ hybridization (FISH) techniques for comparative

  10. Prenatal diagnosis of trisomy 21 using interphase fluorescence in situ hybridization of post-replicated cells with site-specific cosmid and cosmid contig probes.

    PubMed

    Soloviev, I V; Yurov, Y B; Vorsanova, S G; Fayet, F; Roizes, G; Malet, P

    1995-03-01

    Interphase fluorescence in situ hybridization (FISH) with chromosome 21-specific cosmid clones was used to identify trisomy 21 in cultured and uncultured amniotic cells. Two novel site-specific cosmid clones (regions 21q22 and 21qtel) were compared with a cosmid contig (Zheng et al., 1992). Correct identification of chromosome 21 copy number was made in 65-75 per cent of trisomic cells and in 70-75 per cent of normal disomic cells by using all the tested probes. However, the chromosome 21-specific telomeric probe (cos 17F8) showed the best results due to more intense and clearly visible hybridization. Utilization of a directly fluorophorated telomeric probe using Cy3-dCTP and FluorX-dCTP allows accurate detection of chromosome 21 in a fast 'one-step' FISH procedure on uncultured interphase nuclei. In addition, we compared the efficacy of FISH analysis for the total population of interphase cells and cells in the post-replication (late S, G2) periods of the cell cycle. Selective scoring of cells in the post-replicative period (showing a pair of hybridization signals on each chromatid of the replicated interphase chromosome) increased the number of informative nuclei by up to 95-97 per cent. This approach allows cells with overlapping chromosomes, artificial double hybridization signals on separate chromatids in interphase chromosomes, background hybridization, and polyploid cells to be analysed. Application of directly labelled telomeric cosmid probes and integral analysis of hybridized nuclei in the pre- and post-replication periods of the cell cycle may help to further improve the prenatal detection of trisomy 21. PMID:7784382

  11. Detection of Ralstonia solanacearum, Which Causes Brown Rot of Potato, by Fluorescent In Situ Hybridization with 23S rRNA-Targeted Probes

    PubMed Central

    Wullings, B. A.; Van Beuningen, A. R.; Janse, J. D.; Akkermans, A. D. L.

    1998-01-01

    During the past few years, Ralstonia (Pseudomonas) solanacearum race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of five R. solanacearum strains and one Ralstonia pickettii strain were PCR amplified, sequenced, and analyzed by sequence alignment. This resulted in the construction of an unrooted tree and supported previous conclusions based on 16S rRNA sequence comparison in which R. solanacearum strains are subdivided into two clusters. Based on the alignments, two specific probes, RSOLA and RSOLB, were designed for R. solanacearum and the closely related Ralstonia syzygii and blood disease bacterium. The specificity of the probes was demonstrated by dot blot hybridization with RNA extracted from 88 bacterial strains. Probe RSOLB was successfully applied in FISH detection with pure cultures and potato tissue samples, showing a strong fluorescent signal. Unexpectedly, probe RSOLA gave a less intense signal with target cells. Potato samples are currently screened by indirect immunofluorescence (IIF). By simultaneously applying IIF and the developed specific FISH, two independent targets for identification of R. solanacearum are combined, resulting in a rapid (1-day), accurate identification of the undesired pathogen. The significance of the method was validated by detecting the pathogen in soil and water samples and root tissue of the weed host Solanum dulcamara (bittersweet) in contaminated areas. PMID:9797321

  12. Detection of Ralstonia solanacearum, which causes brown rot of potato, by fluorescent in situ hybridization with 23S rRNA-targeted probes.

    PubMed

    Wullings, B A; Van Beuningen, A R; Janse, J D; Akkermans, A D

    1998-11-01

    During the past few years, Ralstonia (Pseudomonas) solanacearum race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of five R. solanacearum strains and one Ralstonia pickettii strain were PCR amplified, sequenced, and analyzed by sequence alignment. This resulted in the construction of an unrooted tree and supported previous conclusions based on 16S rRNA sequence comparison in which R. solanacearum strains are subdivided into two clusters. Based on the alignments, two specific probes, RSOLA and RSOLB, were designed for R. solanacearum and the closely related Ralstonia syzygii and blood disease bacterium. The specificity of the probes was demonstrated by dot blot hybridization with RNA extracted from 88 bacterial strains. Probe RSOLB was successfully applied in FISH detection with pure cultures and potato tissue samples, showing a strong fluorescent signal. Unexpectedly, probe RSOLA gave a less intense signal with target cells. Potato samples are currently screened by indirect immunofluorescence (IIF). By simultaneously applying IIF and the developed specific FISH, two independent targets for identification of R. solanacearum are combined, resulting in a rapid (1-day), accurate identification of the undesired pathogen. The significance of the method was validated by detecting the pathogen in soil and water samples and root tissue of the weed host Solanum dulcamara (bittersweet) in contaminated areas. PMID:9797321

  13. Specific detection of Pasteurella multocida in chickens with fowl cholera and in pig lung tissues using fluorescent rRNA in situ hybridization.

    PubMed

    Mbuthia, P G; Christensen, H; Boye, M; Petersen, K M; Bisgaard, M; Nyaga, P N; Olsen, J E

    2001-07-01

    A Pasteurella multocida species-specific oligonucleotide probe, pmhyb449, targeting 16S rRNA was designed and evaluated by whole-cell hybridization against 22 selected reference strains in animal tissues. It differentiated P. multocida from other bacterial species of the families Pasteurellaceae and Enterobacteriaceae and also from divergent species of the order Cytophagales (except biovar 2 strains of Pasteurella avium and Pasteurella canis, which have high 16S rRNA similarity to P. multocida). The potential of the probe for specific identification and differentiation of P. multocida was further detected in formalin-fixed paraffin-embedded lung tissues from experimental fowl cholera in chickens and infections in pigs. In chicken lung tissues P. multocida cells were detected singly, in pairs, as microcolonies, and as massive colonies within air capillaries (septa and lumen), parabronchial septa, and blood vessels (wall and lumen). In pig lung, postmortem-injected P. multocida was detected in the alveoli (lumen and wall), and in both animals the bacterial cells were seen in the bronchi. The results showed that with the oligonucleotide probe pmhyb449, fluorescent in situ hybridization is a suitable and fast method for specific detection of P. multocida in histological formalin-fixed tissues. The test was replicable and reproducible and is recommended as a supplementary test for diagnosis and as a tool in pathogenesis studies of fowl cholera and respiratory tract infections in pigs due to P. multocida. PMID:11427580

  14. Inflammatory myofibroblastic tumor of the posterior mediastinum: an older adult case with anaplastic lymphoma kinase abnormalities determined using immunohistochemistry and fluorescence in situ hybridization.

    PubMed

    Makimoto, Yoshifumi; Nabeshima, Kazuki; Iwasaki, Hiroshi; Ishiguro, Akiko; Miyoshi, Tatsu; Shiraishi, Takeshi; Iwasaki, Akinori; Shirakusa, Takayuki

    2005-04-01

    Inflammatory myofibroblastic tumor (IMT) is a rare neoplasm that usually occurs in children and young adults. Anaplastic lymphoma kinase (ALK) abnormalities in IMT, determined using immunohistochemistry and/or molecular genetic studies, including fluorescence in situ hybridization (FISH), have almost been limited to children and young adults. In elderly cases of IMT, these ALK abnormalities are very rare. We report on a case of IMT arising in the posterior mediastinum of a 59-year-old Japanese man that showed ALK abnormalities determined using immunohistochemistry and FISH, suggesting the neoplastic nature of a subset of IMTs in older patients similar to those in younger ones and the presence of an additional mechanism(s) that allows them to start to grow late. PMID:15778844

  15. Multicolor fluorescent in situ hybridization on post-mortem brain in schizophrenia as an approach for identification of low-level chromosomal aneuploidy in neuropsychiatric diseases.

    PubMed

    Yurov, Y B; Vostrikov, V M; Vorsanova, S G; Monakhov, V V; Iourov, I Y

    2001-12-01

    Fluorescence in situ hybridization (FISH) of DNA-DNA or DNA-RNA using post-mortem brain samples is one approach to study low-level chromosomal aneuploidy and selective expression of specific genes in the brain of patients with neuropsychiatric diseases. We have performed a pilot molecular-cytogenetic analysis of post-mortem brain of schizophrenic patients. Multicolor FISH on two post-mortem brain samples of normal individuals and six schizophrenic individuals (area 10 of cortex) was applied. A set of DNA probes for FISH included: (i) centromeric alphoid DNA probes for chromosomes 7, 8, 13 and 21, 18, X and Y; (ii) classical satellite DNA probes for chromosomes 1 and 16; and (iii) region-specific DNA probes for chromosomes 13, 21 and 22. A statistically significant level of aneuploidy (up to 0.5-4% of neurons) involving chromosomes X and 18 was detected in two post-mortem brains of patients with schizophrenia. These results indicate that low-level chromosomal aneuploidy could be involved in the pathogenesis of schizophrenia. FISH could be applied to extended studies of chromosomal aneuploidy, abnormal patterns of chromosomal organization and functional gene expression in situ in the neurons of the brain in different psychiatric and neurodevelopmental diseases. Schizophrenia and Rett syndrome might be considered as psychiatric diseases of special interest for molecular-cytogenetic analysis as both of them could be associated with mutations in genes involving regulation of neurodevelopmental processes in the brain. PMID:11738870

  16. Ultrasound-guided fine-needle aspiration of a posterior neck dedifferentiated liposarcoma with MDM2 fluorescence in situ hybridization performed on a Pap-stained smear.

    PubMed

    Zreik, Riyam; Soyalp, Krystal; Ruiz, Steve; Ward, Russell; Dobin, Sheila; Chen, Xiangbai; Liu, Lina; Rao, Arundhati

    2015-04-01

    Head and neck liposarcomas, while rare, tend to be subcutaneous and well-differentiated. Dedifferentiated liposarcomas of the head and neck are exceedingly rare in the literature. We present a case of a dedifferentiated liposarcoma arising in the soft tissue of the posterior neck of an 86-year-old man and diagnosed by fine-needle aspiration. Aspirate smears showed a dual population of atypical lipomatous and spindled cells. MDM2 (murine double minute 2) amplification was demonstrated on a Pap-stained smear using fluorescence in situ hybridization (FISH). To the best of our knowledge, this is the first report of MDM2 FISH amplification in a liposarcoma performed on an aspirate smear. Diagn. Cytopathol. 2015;43:320-324. © 2014 Wiley Periodicals, Inc. PMID:25132684

  17. Mosaic vs. nonmosaic trisomy 9: Report of a liveborn infant evaluated by fluorescence in situ hybridization and review of the literature

    SciTech Connect

    Cantu, E.S.; Eicher, D.J.; Shashidhar Pai, G.; Donahue, C.J.; Harley, R.A. [Medical Univ. of South Carolina, Chalreston, SC (United States)

    1996-04-24

    We report on a newborn infant with multiple congenital anomalies and apparent nonmosaic trisomy 9 in the blood (by conventional cytogenetic studies) who died shortly after birth. Clinical observations at birth and autopsy are compared with phenotypes of mosaic and nonmosaic trisomy 9 cases reported previously. Unlike the initial cytogenetic analysis, fluorescence in situ hybridization (FISH) studies of metaphase and interphase blood cells and skin fibroblasts detected the presence of euploid and trisomy 9 cells. These results suggest that earlier reports of trisomy 9, which relied on conventional chromosome analysis of a few metaphase cells and/or only one tissue type, may not have excluded mosaicism, and that trisomy 9 may be viable only in the mosaic state. 39 refs., 3 figs., 2 tabs.

  18. Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy

    Microsoft Academic Search

    Thomas J. Deerinck; Maryalm E. Martone; Varda Lev-Ram; David P. L. Green; Roger Y. Tsien; David L. Spector; Sui Huang; Mark H. Ellisman

    1994-01-01

    A simple method is described for high- resolution light and electron microscopic im- munolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other con- ventional fluorescent compounds. The technique allows

  19. Microfluidic-Based Amplification-Free Bacterial DNA Detection by Dielectrophoretic Concentration and Fluorescent Resonance Energy Transfer Assisted in Situ Hybridization (FRET-ISH) †,‡

    PubMed Central

    Packard, Michelle M.; Shusteff, Maxim; Alocilja, Evangelyn C.

    2012-01-01

    Although real-time PCR (RT-PCR) has become a diagnostic standard for rapid identification of bacterial species, typical methods remain time-intensive due to sample preparation and amplification cycle times. The assay described in this work incorporates on-chip dielectrophoretic capture and concentration of bacterial cells, thermal lysis, cell permeabilization, and nucleic acid denaturation and fluorescence resonance energy transfer assisted in situ hybridization (FRET-ISH) species identification. Combining these techniques leverages the benefits of all of them, allowing identification to be accomplished completely on chip less than thirty minutes after receipt of sample, compared to multiple hours required by traditional RT-PCR and its requisite sample preparation. PMID:25586031

  20. Determination of HER2 Gene Amplification by Fluorescence In situ Hybridization and Concordance with the Clinical Trials Immunohistochemical Assayin Women with Metastatic Breast Cancer Evaluated for Treatment with Trastuzumab

    Microsoft Academic Search

    Noël Dybdal; Grazyna Leiberman; Steven Anderson; Bryan McCune; Alex Bajamonde; Robert L. Cohen; Robert D. Mass; Corsee Sanders

    2005-01-01

    Summary Purpose. To evaluate the concordance between HER2 gene amplification, determined by fluorescence in situ hybridization (FISH), and HER2 protein overexpression assessed by an immunohistochemical (IHC) assay. The IHC protocol used was a research assay, known as the Clinical Trial Assay (CTA), developed to select women with metastatic breast cancer (MBC) for three pivotal clinical trials of trastuzumab therapy.

  1. Numerical and structural genomic aberrations are reliably detectable in tissue microarrays of formalin-fixed paraffin-embedded tumor samples by fluorescence in-situ hybridization.

    PubMed

    Horn, Heike; Bausinger, Julia; Staiger, Annette M; Sohn, Maximilian; Schmelter, Christopher; Gruber, Kim; Kalla, Claudia; Ott, M Michaela; Rosenwald, Andreas; Ott, German

    2014-01-01

    Few data are available regarding the reliability of fluorescence in-situ hybridization (FISH), especially for chromosomal deletions, in high-throughput settings using tissue microarrays (TMAs). We performed a comprehensive FISH study for the detection of chromosomal translocations and deletions in formalin-fixed and paraffin-embedded (FFPE) tumor specimens arranged in TMA format. We analyzed 46 B-cell lymphoma (B-NHL) specimens with known karyotypes for translocations of IGH-, BCL2-, BCL6- and MYC-genes. Locus-specific DNA probes were used for the detection of deletions in chromosome bands 6q21 and 9p21 in 62 follicular lymphomas (FL) and six malignant mesothelioma (MM) samples, respectively. To test for aberrant signals generated by truncation of nuclei following sectioning of FFPE tissue samples, cell line dilutions with 9p21-deletions were embedded into paraffin blocks. The overall TMA hybridization efficiency was 94%. FISH results regarding translocations matched karyotyping data in 93%. As for chromosomal deletions, sectioning artefacts occurred in 17% to 25% of cells, suggesting that the proportion of cells showing deletions should exceed 25% to be reliably detectable. In conclusion, FISH represents a robust tool for the detection of structural as well as numerical aberrations in FFPE tissue samples in a TMA-based high-throughput setting, when rigorous cut-off values and appropriate controls are maintained, and, of note, was superior to quantitative PCR approaches. PMID:24733537

  2. Numerical and Structural Genomic Aberrations Are Reliably Detectable in Tissue Microarrays of Formalin-Fixed Paraffin-Embedded Tumor Samples by Fluorescence In-Situ Hybridization

    PubMed Central

    Horn, Heike; Bausinger, Julia; Staiger, Annette M.; Sohn, Maximilian; Schmelter, Christopher; Gruber, Kim; Kalla, Claudia; Ott, M. Michaela; Rosenwald, Andreas; Ott, German

    2014-01-01

    Few data are available regarding the reliability of fluorescence in-situ hybridization (FISH), especially for chromosomal deletions, in high-throughput settings using tissue microarrays (TMAs). We performed a comprehensive FISH study for the detection of chromosomal translocations and deletions in formalin-fixed and paraffin-embedded (FFPE) tumor specimens arranged in TMA format. We analyzed 46 B-cell lymphoma (B-NHL) specimens with known karyotypes for translocations of IGH-, BCL2-, BCL6- and MYC-genes. Locus-specific DNA probes were used for the detection of deletions in chromosome bands 6q21 and 9p21 in 62 follicular lymphomas (FL) and six malignant mesothelioma (MM) samples, respectively. To test for aberrant signals generated by truncation of nuclei following sectioning of FFPE tissue samples, cell line dilutions with 9p21-deletions were embedded into paraffin blocks. The overall TMA hybridization efficiency was 94%. FISH results regarding translocations matched karyotyping data in 93%. As for chromosomal deletions, sectioning artefacts occurred in 17% to 25% of cells, suggesting that the proportion of cells showing deletions should exceed 25% to be reliably detectable. In conclusion, FISH represents a robust tool for the detection of structural as well as numerical aberrations in FFPE tissue samples in a TMA-based high-throughput setting, when rigorous cut-off values and appropriate controls are maintained, and, of note, was superior to quantitative PCR approaches. PMID:24733537

  3. Fluorescent in situ hybridization shows DIPLOSPOROUS located on one of the NOR chromosomes in apomictic dandelions (Taraxacum) in the absence of a large hemizygous chromosomal region.

    PubMed

    Vašut, Radim J; Vijverberg, Kitty; van Dijk, Peter J; de Jong, Hans

    2014-11-01

    Apomixis in dandelions (Taraxacum: Asteraceae) is encoded by two unlinked dominant loci and a third yet undefined genetic factor: diplosporous omission of meiosis (DIPLOSPOROUS, DIP), parthenogenetic embryo development (PARTHENOGENESIS, PAR), and autonomous endosperm formation, respectively. In this study, we determined the chromosomal position of the DIP locus in Taraxacum by using fluorescent in situ hybridization (FISH) with bacterial artificial chromosomes (BACs) that genetically map within 1.2-0.2 cM of DIP. The BACs showed dispersed fluorescent signals, except for S4-BAC 83 that displayed strong unique signals as well. Under stringent blocking of repeats by C0t-DNA fragments, only a few fluorescent foci restricted to defined chromosome regions remained, including one on the nucleolus organizer region (NOR) chromosomes that contains the 45S rDNAs. FISH with S4-BAC 83 alone and optimal blocking showed discrete foci in the middle of the long arm of one of the NOR chromosomes only in triploid and tetraploid diplosporous dandelions, while signals in sexual diploids were lacking. This agrees with the genetic model of a single dose, dominant DIP allele, absent in sexuals. The length of the DIP region is estimated to cover a region of 1-10 Mb. FISH in various accessions of Taraxacum and the apomictic sister species Chondrilla juncea, confirmed the chromosomal position of DIP within Taraxacum but not outside the genus. Our results endorse that, compared to other model apomictic species, expressing either diplospory or apospory, the genome of Taraxacum shows a more similar and less diverged chromosome structure at the DIP locus. The different levels of allele sequence divergence at apomeiosis loci may reflect different terms of asexual reproduction. The association of apomeiosis loci with repetitiveness, dispersed repeats, and retrotransposons commonly observed in apomictic species may imply a functional role of these shared features in apomictic reproduction, as is discussed. PMID:25760668

  4. Arm-specific telomere dynamics of each individual chromosome in induced pluripotent stem cells revealed by quantitative fluorescence in situ hybridization.

    PubMed

    Terai, Masanori; Izumiyama-Shimomura, Naotaka; Aida, Junko; Ishikawa, Naoshi; Kuroiwa, Mie; Arai, Tomio; Toyoda, Masashi; Nakamura, Ken-ichi; Takubo, Kaiyo

    2014-12-01

    We have reported that telomere fluorescence units (TFUs) of established induced pluripotent stem cells (iPSCs) derived from human amnion (hAM933) and fetal lung fibroblasts (MRC-5) were significantly longer than those of the parental cells, and that the telomere extension rates varied quite significantly among clones without chromosomal instability, although the telomeres of other iPSCs derived from MRC-5 became shorter as the number of passages increased along with chromosomal abnormalities from an early stage. In the present study we attempted to clarify telomere dynamics in each individual chromosomal arm of parental cells and their derived clonal human iPSCs at different numbers of passages using quantitative fluorescence in situ hybridization (Q-FISH). Although no speci?c arm of any particular chromosome appeared to be consistently shorter or longer than most of the other chromosomes in any of the cell strains, telomere elongation in each chromosome of an iPSC appeared to be random and stochastic. However, in terms of the whole genome of any specific cell, the telomeres showed overall elongation associated with iPSC generation. We have thus demonstrated the specific telomere dynamics of each individual chromosomal arm in iPSCs derived from parental cells, and in the parental cells themselves, using Q-FISH. PMID:25217290

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

    PubMed

    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

  6. 5-bp Classical Satellite DNA Loci from Chromosome-1 Instability in Cervical Neoplasia Detected by DNA Breakage Detection/Fluorescence in Situ Hybridization (DBD-FISH)

    PubMed Central

    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

  7. Re-appraisal of the phylogeny and fluorescence in situ hybridization probes for the analysis of the Competibacteraceae in wastewater treatment systems.

    PubMed

    McIlroy, Simon J; Nittami, Tadashi; Kanai, Eri; Fukuda, Junji; Saunders, Aaron M; Nielsen, Per Halkjaer

    2015-04-01

    Members of the family Competibacteraceae are common in wastewater treatment plants (WWTPs) designed for enhanced biological phosphorus removal (EBPR) and are putatively deleterious to the process of P removal. Their ability to accumulate large amounts of polyhydroxyalkanoates is also suggested to be of potential commercial interest for bioplastic production. In this study we have updated the 16S rRNA-based phylogeny of the Competibacter and the Plasticicumulans lineages. The former is delineated by 13 clades including two described genera; 'Ca.?Competibacter' and 'Ca.?Contendobacter'. The oligonucleotide probes used for detection of the family by fluorescence in situ hybridization (FISH) were re-evaluated and designed for coverage of these clades. Surveys of full-scale WWTPs based on 16S rRNA gene amplicon sequencing and FISH analysis indicate that a number of member clades always coexist, with their relative abundances varying substantially between and temporally within plants. The hypothesis that these differences are based on niche partitioning is supported by marked phenotypic differences between clades. An in-depth understanding of the ecology of the family requires further studies of the metabolism of individual clades in situ. The proposed phylogeny and FISH probes will provide the foundation for such studies. PMID:25224028

  8. Evaluation of Peptide Nucleic Acid-Fluorescence In Situ Hybridization for Identification of Clinically Relevant Mycobacteria in Clinical Specimens and Tissue Sections

    PubMed Central

    Lefmann, Michael; Schweickert, Birgitta; Buchholz, Petra; Göbel, Ulf B.; Ulrichs, Timo; Seiler, Peter; Theegarten, Dirk; Moter, Annette

    2006-01-01

    With fluorescently labeled PNA (peptide nucleic acid) probes targeting 16S rRNA, we established a 3-h fluorescence in situ hybridization (FISH) procedure for specific visualization of members of the Mycobacterium tuberculosis complex, M. leprae, M. avium, and M. kansasii. Probe specificity was tested against a panel of 25 Mycobacterium spp. and 10 gram-positive organisms. After validation, probes were used to identify 52 mycobacterial culture isolates. Results were compared to conventional genotypic identification with amplification-based methods. All isolates (M. tuberculosis complex, n = 24; M. avium, n = 7; M. kansasii, n = 1) were correctly identified by FISH. In addition, the technique was used successfully for visualization of mycobacteria in biopsies from infected humans or animals. In conclusion, PNA-FISH is a fast and accurate tool for species-specific identification of culture-grown mycobacteria and for direct visualization of these organisms in tissue sections. It may be used successfully for both research and clinical microbiology. PMID:17021106

  9. Evaluation of peptide nucleic acid-fluorescence in situ hybridization for identification of clinically relevant mycobacteria in clinical specimens and tissue sections.

    PubMed

    Lefmann, Michael; Schweickert, Birgitta; Buchholz, Petra; Göbel, Ulf B; Ulrichs, Timo; Seiler, Peter; Theegarten, Dirk; Moter, Annette

    2006-10-01

    With fluorescently labeled PNA (peptide nucleic acid) probes targeting 16S rRNA, we established a 3-h fluorescence in situ hybridization (FISH) procedure for specific visualization of members of the Mycobacterium tuberculosis complex, M. leprae, M. avium, and M. kansasii. Probe specificity was tested against a panel of 25 Mycobacterium spp. and 10 gram-positive organisms. After validation, probes were used to identify 52 mycobacterial culture isolates. Results were compared to conventional genotypic identification with amplification-based methods. All isolates (M. tuberculosis complex, n = 24; M. avium, n = 7; M. kansasii, n = 1) were correctly identified by FISH. In addition, the technique was used successfully for visualization of mycobacteria in biopsies from infected humans or animals. In conclusion, PNA-FISH is a fast and accurate tool for species-specific identification of culture-grown mycobacteria and for direct visualization of these organisms in tissue sections. It may be used successfully for both research and clinical microbiology. PMID:17021106

  10. Mapping of low-frequency chimeric yeast artificial chromosome libraries from human chromosomes 16 and 21 by fluorescence in situ hybridization and quantitative image analysis

    SciTech Connect

    Marrone, B.L.; Campbell, E.W.; Anzick, S.L.; Shera, K.; Campbell, M.; Yoshida, T.M.; McCormick, M.K.; Deaven, L. (Los Alamos National Lab., NM (United States))

    1994-05-01

    Yeast artificial chromosome (YAC) clones from low-frequency chimeric libraries of human chromosomes 16 and 21 were mapped onto human diploid fibroblast metaphase chromosomes using fluorescence in situ hybridization (FISH) and digital imaging microscopy. YACs mapped onto chromosome 21 were selected to provide subregional location and ordering of known and unknown markers on the long arm of chromosome 21, particularly in the Down syndrome region (q22). YACs mapped onto chromosome 16 were selected to overlap regions spanning chromosome 16 cosmid maps. YAC clones were indirectly labeled with fluorescein, and the total DNA of the chromosome was counterstained with propidium iodide. A single image containing both the FISH signal and the whole chromosome was acquired for each chromosome of interest containing the fluorescent probe signal in a metaphase spread. From the digitized image, the fluorescence intensity profile through the long axis of the chromosome gave the total chromosome length and the probe position. The map position of the probe was expressed as the fractional length (FL) of the total chromosome relative to the end of the short arm (Flpter). From each clone hybridized, 20-40 chromosome images were analyzed. Thirty-eight YACs were mapped onto chromosome 16, and their FLs were distributed along the short and long arms. On chromosome 21, 47 YACs were mapped, including 12 containing known markers. To confirm the order of a dense population of YACs within the Down syndrome region, a two-color mapping strategy was used in which an anonymous YAC was located relative to one or two known markers on the metaphase chromosome. The chromosome FL maps have a 1- to 2-Mb resolution, and the FL measurement of each probe has a typical standard error of 0.5-1 Mb. 14 refs., 3 figs., 3 tabs.

  11. Genetically Abnormal Circulating Cells in Lung Cancer Patients: An Antigen Independent Fluorescence in-situ Hybridization Based Case-Control Study

    PubMed Central

    Katz, Ruth L.; He, Weigong; Khanna, Abha; Fernandez, Ricardo L.; Zaidi, Tanweer M.; Krebs, Matthew; Caraway, Nancy P.; Zhang, Hua-Zhong; Jiang, Feng; Spitz, Margaret R.; Blowers, David P.; Jimenez, Carlos A.; Mehran, Reza J.; Swisher, Stephen G.; Roth, Jack; Morris, Jeffrey S.; Etzel, Carol; El-Zein, Randa

    2010-01-01

    Purpose We performed a study to determine if a fluorescence in-situ hybridization (FISH)-based assay using isolated peripheral blood mononuclear cells (PBMCs) with DNA probes targeting specific sites on chromosomes known to have abnormalities in Non Small Cell Lung Cancer (NSCLC) cases could detect circulating genetically abnormal cells (CACs). Experimental Design We evaluated 59 NSCLC cases with stage I through IV disease and 24 controls. PBMCs and matched tumors were hybridized with 2 two-color (3p22.1/CEP3 and 10q22.3 [SP-A]/CEP10) and 2 four-color (CEP3, CEP7, CEP17, and 9p21.3 [URO]) and (EGFR, c-MYC, 6p11-q11, and 5p15.2 [LAV]) FISH probes. Percentages of cytogenetically abnormal cells (CACs) in peripheral blood and in matched tumor specimens were quantified using an automated fluorescent scanner. Numbers of CACs were calculated based on the percentage of CACs (defined as PBMCs with genetic abnormalities) per mL of blood and expressed per microliter of blood. Results Patients with NSCLC had significantly higher numbers of CACs than did controls. Mean number of CACs ranged from 7.23±1.32/?l for deletions of 10q22.3/CEP10 to 45.52±7.49/?l for deletions of 3p22.1/CEP3. Numbers of CACs with deletions of 3p22.1, 10q22.3, and 9p21.3, and gains of URO, increased significantly from early to advanced stage of disease. Conclusions We have developed a sensitive and quantitative antigen-independent FISH-based test for detecting CACs in peripheral blood of patients with NSCLC which showed a significant correlation with the presence of cancer. If this pilot study can be validated in a larger study, CACs may have a role in the management of patients with NSCLC. PMID:20651054

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

    SciTech Connect

    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.

  13. Monitoring of Saccharomyces cerevisiae, Hanseniaspora uvarum, and Starmerella bacillaris (synonym Candida zemplinina) populations during alcoholic fermentation by fluorescence in situ hybridization.

    PubMed

    Wang, Chunxiao; Esteve-Zarzoso, Braulio; Mas, Albert

    2014-11-17

    Various molecular approaches have been applied as culture-independent techniques to monitor wine fermentations over the last decade. Among them, those based on RNA detection have been widely used for yeast cell detection, assuming that RNA only exists in live cells. Fluorescence in situ hybridization (FISH) targeting intracellular rRNA is considered a promising technique for the investigation of wine ecology. For the present study, we applied the FISH technique in combination with epifluorescence microscopy and flow cytometry to directly quantify populations of Saccharomyces cerevisiae, Hanseniaspora uvarum, and Starmerella bacillaris during alcoholic fermentations. A new specific probe that hybridizes with eight species of Hanseniaspora genus and a second probe specific for Starm. bacillaris were designed, and the conditions for their application to pure cultures, mixed cultures, and wine samples were optimized. Single and mixed fermentations were performed with natural, concentrated must at two different temperatures, 15 °C and 25 °C. The population dynamics revealed that the Sacch. cerevisiae population increased to 10(7)-10(8)cells/ml during all fermentations, whereas H. uvarum and Starm. bacillaris tended to increase in single fermentations but remained at levels similar to their inoculations at 10(6)cells/ml in mixed fermentations. Temperature mainly affected the fermentation duration (slower at the lower temperature) but did not affect the population sizes of the different species. The use of these probes in natural wine fermentations has been validated. PMID:25218463

  14. Simultaneous quantification of active carbon- and nitrogen-fixing communities and estimation of fixation rates using fluorescence in situ hybridization and flow cytometry.

    PubMed

    McInnes, Allison S; Shepard, Alicia K; Raes, Eric J; Waite, Anya M; Quigg, Antonietta

    2014-11-01

    Understanding the interconnectivity of oceanic carbon and nitrogen cycles, specifically carbon and nitrogen fixation, is essential in elucidating the fate and distribution of carbon in the ocean. Traditional techniques measure either organism abundance or biochemical rates. As such, measurements are performed on separate samples and on different time scales. Here, we developed a method to simultaneously quantify organisms while estimating rates of fixation across time and space for both carbon and nitrogen. Tyramide signal amplification fluorescence in situ hybridization (TSA-FISH) of mRNA for functionally specific oligonucleotide probes for rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase; carbon fixation) and nifH (nitrogenase; nitrogen fixation) was combined with flow cytometry to measure abundance and estimate activity. Cultured samples representing a diversity of phytoplankton (cyanobacteria, coccolithophores, chlorophytes, diatoms, and dinoflagellates), as well as environmental samples from the open ocean (Gulf of Mexico, USA, and southeastern Indian Ocean, Australia) and an estuary (Galveston Bay, Texas, USA), were successfully hybridized. Strong correlations between positively tagged community abundance and (14)C/(15)N measurements are presented. We propose that these methods can be used to estimate carbon and nitrogen fixation in environmental communities. The utilization of mRNA TSA-FISH to detect multiple active microbial functions within the same sample will offer increased understanding of important biogeochemical cycles in the ocean. PMID:25172848

  15. Simultaneous Quantification of Active Carbon- and Nitrogen-Fixing Communities and Estimation of Fixation Rates Using Fluorescence In Situ Hybridization and Flow Cytometry

    PubMed Central

    Shepard, Alicia K.; Raes, Eric J.; Waite, Anya M.; Quigg, Antonietta

    2014-01-01

    Understanding the interconnectivity of oceanic carbon and nitrogen cycles, specifically carbon and nitrogen fixation, is essential in elucidating the fate and distribution of carbon in the ocean. Traditional techniques measure either organism abundance or biochemical rates. As such, measurements are performed on separate samples and on different time scales. Here, we developed a method to simultaneously quantify organisms while estimating rates of fixation across time and space for both carbon and nitrogen. Tyramide signal amplification fluorescence in situ hybridization (TSA-FISH) of mRNA for functionally specific oligonucleotide probes for rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase; carbon fixation) and nifH (nitrogenase; nitrogen fixation) was combined with flow cytometry to measure abundance and estimate activity. Cultured samples representing a diversity of phytoplankton (cyanobacteria, coccolithophores, chlorophytes, diatoms, and dinoflagellates), as well as environmental samples from the open ocean (Gulf of Mexico, USA, and southeastern Indian Ocean, Australia) and an estuary (Galveston Bay, Texas, USA), were successfully hybridized. Strong correlations between positively tagged community abundance and 14C/15N measurements are presented. We propose that these methods can be used to estimate carbon and nitrogen fixation in environmental communities. The utilization of mRNA TSA-FISH to detect multiple active microbial functions within the same sample will offer increased understanding of important biogeochemical cycles in the ocean. PMID:25172848

  16. A physical map of 15 loci on human chromosome 5q23-q33 by two-color fluorescence in situ hybridization

    SciTech Connect

    Saltman, D.L.; Dolganov, G.M. (Genelabs Inc. Redwood City, CA (United States)); Warrington, J.A.; Wasmuth, J.J. (Univ. of California, Irvine (United States)); Lovett, M. (Univ. of Texas Southwestern Medical Center, Dallas (United States))

    1993-06-01

    The q23-q33 region of human chromosome 5 encodes a large number of growth factors, growth factor receptors, and hormone/neurotransmitter receptors. This is also the general region into which several disease genes have been mapped, including diastrophic dysplasia, Treacher Collins syndrome, hereditary startle disease, the myeloid disorders that are associated with the 5q-syndrome, autosomal-dominant forms of hereditary deafness, and limb girdle muscular dystrophy. The authors have developed a framework physical map of this region using cosmid clones isolated from the Los Alamos arrayed chromosome 5-specific library. Entry points into this library included 14 probes to genes within this interval and one anonymous polymorphic marker locus. A physical map has been constructed using fluorescence in situ hybridization of these cosmids on metaphase and interphase chromosomes, and this is in good agreement with the radiation hybrid map of the region. The derived order of loci across the region is cen-IL4-IL5-IRF1-IL3-IL9-EGR1-CD14-FGFA-GRL-D5S207-ADRB2-SPARC-RPS14-CSF1R-ADRA1, and the total distance spanned by these loci is approximately 15 Mb. The framework map, genomic clones, and contig expansion within 5q23-q33 should provide valuable resources for the eventual isolation of the clinically relevant loci that reside in this region. 31 refs., 3 figs., 2 tabs.

  17. Fluorescence in situ hybridization as an adjunct tool in the diagnosis of primary and metastatic renal cell carcinoma in fine needle aspiration specimens.

    PubMed

    Kos, Zuzana; Williams, Phillip A; Belanger, Eric C; Mai, Kien T

    2014-12-01

    We investigated the role of fluorescence in situ hybridization (FISH) in the diagnosis of primary renal neoplasms and lesions suspicious for metastatic renal cell carcinoma. Consecutive fine-needle aspiration biopsies (FNAB) of 39 renal masses and 41 metastatic tumours suspicious for renal cell origin were assessed with an immunohistochemical panel for CK7, RCC antigen, CD10, AMACR, PAX8, vimentin, and CD117. In addition, FISH was performed using probes for chromosomes 1p, 3p, 7, 17, X, and Y. A total of 31 of 39 primary renal masses and 33 of 41 metastatic tumors suspicious for renal origin demonstrated typical cytological and immunohistochemical (IHC) features of subtypes of renal neoplasms (40 clear cell renal cell carcinoma (RCC), 20 papillary RCC, and 4 renal oncocytomas). FISH analysis of 15 randomly selected cases each of primary and metastatic lesions revealed chromosomal abnormalities consistent with the diagnosis in 73% of these cases. Of 8 primary renal masses demonstrating atypical microscopic features and noncontributory IHC profiles, FISH was helpful in subtyping 5 (62%) of these lesions (2 clear cell RCC, 1 solid variant of oncocytic papillary RCC, 1 mixed clear cell and papillary RCC, and 1 chromophobe RCC with papillary architecture). Of 8 metastatic tumors clinically suspicious for renal cell origin and supportive, but nondiagnostic IHC, FISH revealed supportive chromosomal changes in 6 (75%) cases. In conclusion FISH analysis on FNAB material, even with limited tissue, may be contributory to the diagnosis and subtyping of RCC in diagnostically challenging biopsies. PMID:24692327

  18. High-resolution cytogenetic mapping of 342 new cosmid markers including 43 RFLP markers on human chromosome 17 by fluorescence in situ hybridization

    SciTech Connect

    Inazawa, Johji; Ariyama, Takeshi; Abe, Tatsuo (Kyoto Prefectual Univ. of Medicine (Japan)); Saito, Hiroko; Nakamura, Yusuke (Cancer Institute, Tokyo (Japan))

    1993-07-01

    The authors have constructed a high-resolution cytogenetic map of human chromosome 17 with 342 cosmid markers, each newly isolated from a cosmid library constructed from a human-mouse hybrid cell line containing a single human chromosome 17. Direct mapping on R- and/or G-banded (pro)metaphase chromosomes by fluorescence in situ hybridization localized these markers throughout the chromosome, although density was highest in the R-band-dominant regions of 17p13, 17p11.2, 17q11.2-q12, 17q21.3, 17q23, and 17q25. By screening some of the cosmid clones, they identified 71 polymorphic systems with 43 markers; 11 of these are VNTRs. As the high-resolution cytogenetic map contains a large number of markers, it can provide useful landmarks for a contig map of chromosome 17. Furthermore, the map will contribute to positional cloning of aberrant genes responsible for inherited diseases such as Miller-Dieker syndrome (MDS), Smith-Magenis syndrome (SMS), and familial early-onset breast cancer, as well as putative tumor suppressor genes on this chromosome. 47 refs., 2 figs., 2 tabs.

  19. Chromosomal Bar Codes Produced by Multicolor Fluorescence In Situ Hybridization with Multiple YAC Clones and Whole Chromosome Painting Probes

    Microsoft Academic Search

    Christoph Lengauer; Michael R. Speicher; Susanne Popp; Anna Jauch; Masafumi Taniwaki; Ramaiah Nagaraja; Harold C. Riethman; Helen Donis-Keller; Michele DUrso; David Schelssinger; Thomas Cremer

    1993-01-01

    Colored chromosome staining patterns, termed chromosomal ‘bar codes’ (CBCs), were obtained on human chromosomes by fluorescence in situhybridization (FISH) with pools of Alu-PCR products from YAC dones containing human DNA inserts ranging from 100 kbp to 1 Mbp. In contrast to conventional G- or R-bands, the chromosomal position, extent, Individual color and relative signal intensity of each ‘bar’ could be

  20. Automated Design of Probes for rRNA-Targeted Fluorescence In Situ Hybridization Reveals the Advantages of Using Dual Probes for Accurate Identification

    PubMed Central

    Yilmaz, L. Safak; Corcoran, Andrew M.; Ökten, Hatice E.; Noguera, Daniel R.

    2014-01-01

    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). PMID:24928876

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

    PubMed

    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). PMID:24928876

  2. Comparison of Vertical Distributions of Prokaryotic Assemblages in the Anoxic Cariaco Basin and Black Sea by Use of Fluorescence In Situ Hybridization

    PubMed Central

    Lin, Xueju; Wakeham, Stuart G.; Putnam, Isabell F.; Astor, Yrene M.; Scranton, Mary I.; Chistoserdov, Andrei Y.; Taylor, Gordon T.

    2006-01-01

    Individual prokaryotic cells from two major anoxic basins, the Cariaco Basin and the Black Sea, were enumerated throughout their water columns using fluorescence in situ hybridization (FISH) with the fluorochrome Cy3 or horseradish peroxidase-modified oligonucleotide probes. For both basins, significant differences in total prokaryotic abundance and phylogenetic composition were observed among oxic, anoxic, and transitional (redoxcline) waters. Epsilon-proteobacteria, Crenarchaeota, and Euryarchaeota were more prevalent in the redoxclines, where previous studies reported high rates of chemoautotrophic production relative to those in waters above and below the redoxclines. Relative abundances of Archaea in both systems varied between 1% and 28% of total prokaryotes, depending on depth. The prokaryotic community composition varied between the two anoxic basins, consistent with distinct geochemical and physical conditions. In the Black Sea, the relative contributions of group I Crenarchaeota (median, 5.5%) to prokaryotic communities were significantly higher (P < 0.001; n = 20) than those of group II Euryarchaeota (median, 2.9%). In contrast, their proportions were nearly equivalent in the Cariaco Basin. Beta-proteobacteria were unexpectedly common throughout the Cariaco Basin's water column, accounting for an average of 47% of 4?,6?-diamidino-2-phenylindole (DAPI)-stained cells. This group was below the detection limit (<1%) in the Black Sea samples. Compositional differences between basins may reflect temporal variability in microbial populations and/or systematic differences in environmental conditions and the populations for which they select. PMID:16597973

  3. Identification of Treponema pedis as the predominant Treponema species in porcine skin ulcers by fluorescence in situ hybridization and high-throughput sequencing.

    PubMed

    Karlsson, Frida; Klitgaard, Kirstine; Jensen, Tim Kåre

    2014-06-25

    Skin lesions often seen in pig production are of great animal welfare concern. To study the potential role of Treponema bacteria in porcine skin ulcers, we investigated the presence and distribution of these organisms in decubital shoulder ulcers (n=51) and ear necroses (n=54) by fluorescence in situ hybridization (FISH) and high-throughput sequencing. In addition, two cases of facial ulcers and five cases of other skin ulcers were included in the study. Samples from all 112 skin lesions and intact skin from pigs without skin ulcers (n=14) were screened by FISH. Three different oligonucleotide probes targeting 16S rRNA were used, specific for domain bacterium, Treponema spp. and species T. pedis. Screening showed that two cases each of facial and other ulcers, 35 (69%) of shoulder ulcers and 32 (59%) of ear necroses were positive for Treponema spp. T. pedis was the unequivocally, predominant species typically constituting more than 90% of the treponemes in a lesion, assessed visually by microscopy. Altogether, T. pedis was demonstrated in 69 of the 71 Treponema spp. positive lesions. We conclude that Treponema spp. are frequently present and abundant in various skin ulcers of pigs. The results from this study point toward an important role of T. pedis as a secondary bacterial infection in porcine skin ulcers, especially in severe and chronic lesions. PMID:24725449

  4. Is monosomy 5 an uncommon aberration? Fluorescence in situ hybridization reveals translocations and deletions in myelodysplastic syndromes or acute myelocytic leukemia.

    PubMed

    Bram, Susanne; Swolin, Birgitta; Rödjer, Stig; Stockelberg, Dick; Ogärd, Inger; Bäck, Hans

    2003-04-15

    Acquired loss of material from chromosome 5 in bone marrow cells is common in myelodysplastic syndromes (MDS) and acute myelocytic leukemia (AML). In this study, we have applied fluorescence in situ hybridization (FISH) analyses with probes for the three regions 5p15.2, 5q31, 5q33-q34, and whole chromosome 5 painting probes (WCP 5) to investigate what further information could be gained regarding the cytogenetic abnormalities of chromosome 5 in 35 patients with MDS or AML. With FISH, a del(5q) was found in all patients except for two. Translocations of material from chromosome 5 were found in 10 patients. Among 16 patients with clones of monosomy 5 seen by cytogenetics, 14 had deletions or translocations. Different breakpoints on chromosome 5 were observed. In conclusion, the extended FISH analyses yielded additional information about chromosome 5 abnormalities in 60% of the patients. Of interest is the finding of a high proportion of translocations and that monosomy 5 occurs less often than is generally believed. PMID:12699885

  5. Diversity of Cultivable Methane-Oxidizing Bacteria in Microsites of a Rice Paddy Field: Investigation by Cultivation Method and Fluorescence in situ Hybridization (FISH)

    PubMed Central

    Dianou, Dayéri; Ueno, Chihoko; Ogiso, Takuya; Kimura, Makoto; Asakawa, Susumu

    2012-01-01

    The diversity of cultivable methane-oxidizing bacteria (MOB) in the rice paddy field ecosystem was investigated by combined culture-dependent and fluorescence in situ hybridization (FISH) techniques. Seven microsites of a Japanese rice paddy field were the focus of the study: floodwater, surface soil, bulk soil, rhizosphere soil, root, basal stem of rice plant, and rice stumps of previous harvest. Based on pmoA gene analysis and transmission electron microscopy (TEM), four type I, and nine type II MOB isolates were obtained from the highest dilution series of enrichment cultures. The type I MOB isolates included a novel species in the genus Methylomonas from floodwater and this is the first type I MOB strain isolated from floodwater of a rice paddy field. In the type I MOB, two isolates from stumps were closely related to Methylomonas spp.; one isolate obtained from rhizosphere soil was most related to Methyloccocus-Methylocaldum-Methylogaea clade. Almost all the type II MOB isolates were related to Methylocystis methanotrophs. FISH confirmed the presence of both types I and II MOB in all the microsites and in the related enrichment cultures. The study reported, for the first time, the diversity of cultivable methanotrophs including a novel species of type I MOB in rice paddy field compartments. Refining growth media and culture conditions, in combination with molecular approaches, will allow us to broaden our knowledge on the MOB community in the rice paddy field ecosystem and consequently to implement strategies for mitigating CH4 emission from this ecosystem. PMID:22446309

  6. Fluorescence in situ hybridization is necessary to detect an association between chromosome aberrations and polycyclic aromatic hydrocarbon exposure in utero and reveals nonrandom chromosome involvement.

    PubMed

    Bocskay, Kirsti A; Orjuela, Manuela A; Tang, Deliang; Liu, Xinhua; Warburton, Dorothy; Perera, Frederica P

    2007-03-01

    Chromosome aberrations are associated with environmental exposures in infants and children. Recently we reported that prenatal exposure to airborne polycyclic aromatic hydrocarbons (PAHs) was significantly (P < 0.01) associated with stable aberration frequencies in cord blood from a subset of 60 newborns from the Columbia Center for Children's Environmental Health Prospective Cohort Study (Bocskay K et al. [ 2005]: Cancer Epidemiol Biomarkers Prev 14:506-511). To determine whether the environmental exposures may be targeting specific chromosomes and to compare various methods for measuring chromosome aberrations, we further evaluated this same subset of subjects composed of African-American and Dominican nonsmoking mother-newborn pairs residing in low-income neighborhoods of New York City, and exposed to varying levels of airborne PAHs. Chromosome aberrations were measured in cord blood lymphocytes, both by whole chromosome probe (WCP) fluorescence in situ hybridization (FISH) and traditional Giemsa-staining. Prenatal exposures were assessed by personal air monitoring. Breaks in chromosomes 1-6, as detected by WCP FISH, were nonrandomly distributed, underscoring the importance of appropriate chromosome probe selection to capture cytogenetic damage in response to exposure. FISH for stable aberrations was found to be a more sensitive method for detecting aberration frequencies associated with environmental exposures, when compared with FISH for unstable aberrations or Giemsa-staining for aberrations. Together, these results suggest that PAHs may be targeting specific chromosomes and highlight the importance of using the more sensitive detection methods to assess risk in populations with low levels of exposure. PMID:17253628

  7. Topoisomerase 2? status in invasive breast carcinoma - comparison of its clinical value according to immunohistochemical and fluorescence in situ hybridization methods of evaluation.

    PubMed

    Olszewski, Wt; Pie?kowski, T; Olszewski, Wp; Mrozkowiak, A; Bauer-Kosi?ska, B; Pia?cik, A; Olszewska, K; Wojnowska, A; Michalski, W

    2014-12-01

    The main purpose of the study was to compare topoisomerase 2? (TOP2A) status in invasive breast carcinomas to the outcome of a therapy containing neoadjuvant treatment with anthracyclines (a combination chemotherapy treatment for breast cancer, namely AC [cyclophosphamide, doxorubicin]). To achieve these goals we created a method of evaluation with criteria based on two methods used in the present study (immunohistochemical [IHC] and fluorescence in situ hybridization [FISH]). The threshold for positive immunohistochemically evaluated status was set for all cases with: nuclear stain intensity score 3+ in 10% or more nuclei and nuclear stain intensity score 2+ in 50% or more nuclei. Our results suggest that TOP2A status may be used as a predictive factor for patient selection for protocols which include anthracyclines as one of the chemotherapeutics. Both methods, IHC and FISH, are suitable for implementation for diagnostic purposes, but IHC positive status measured according to the criteria presented above is the best predictor of longer disease-free survival (DFS) according to our study. Immunohistochemical also gave satisfactory results in all analyzed cases in comparison to only 60% of cases analyzed by FISH. PMID:25693082

  8. Effects of Compost on Colonization of Roots of Plants Grown in Metalliferous Mine Tailings, as Examined by Fluorescence In Situ Hybridization?

    PubMed Central

    Iverson, Sadie L.; Maier, Raina M.

    2009-01-01

    The relationship between compost amendment, plant biomass produced, and bacterial root colonization as measured by fluorescence in situ hybridization was examined following plant growth in mine tailings. Mine tailings can remain devoid of vegetation for decades after deposition due to a combination of factors that include heavy metal toxicity, low pH, poor substrate structure and water-holding capacity, and a severely impacted heterotrophic microbial community. Research has shown that plant establishment, a desired remedial objective to reduce eolian and water erosion of such tailings, is enhanced by organic matter amendment and is correlated with significant increases in rhizosphere populations of neutrophilic heterotrophic bacteria. Results show that for the acidic metalliferous tailings tested in this study, compost amendment was associated with significantly increased bacterial colonization of roots and increased production of plant biomass. In contrast, for a Vinton control soil, increased compost had no effect on root colonization and resulted only in increased plant biomass at high levels of compost amendment. These data suggest that the positive association between compost amendment and root colonization is important in the stressed mine tailings environment where root colonization may enhance both microbial and plant survival and growth. PMID:19047384

  9. Fluorescence in situ hybridization of 12p in germ cell tumors using a bacterial artificial chromosome clone 12p probe on paraffin-embedded tissue: clinical test validation.

    PubMed

    Wehle, Danielle; Yonescu, Raluca; Long, Patricia P; Gala, Nalini; Epstein, Jonathan; Griffin, Constance A

    2008-06-01

    Most germ cell tumors have an isochromosome 12p (detected by metaphase cytogenetics), 12p overrepresentation (detected by fluorescence in situ hybridization [FISH]), or both. Although interphase FISH on paraffin-embedded tissue is a sensitive method of detection of 12p anomalies, use of FISH for clinical diagnostic purposes is not well defined. We describe an interphase FISH assay for detection of increased 12p copy number in germ cell tumors using a bacterial artificial chromosome-derived probe localized to 12p12.1 and a commercially available probe for the centromere of chromosome 12. Twenty-four paraffin-embedded blocks from 14 tumor cases (7 malignant mixed germ cell tumors, 2 dysgerminomas, 4 non-germ cell malignancies arising in germ cell tumors, and 1 mediastinal adenocarcinoma) and 18 normal controls were studied. Negative controls included normal lymph node, lung, and mediastinal tissue. The signals for 12p and 12cen were counted, and the ratio of the averaged signals was calculated; a ratio of 1.3 was considered positive. All germ cell tumors and non-germ cell malignancies arising in germ cell tumors were positive for 12p overrepresentation. All control cases were negative. Because germ cell tumors may metastasize with non-germ cell tumor morphology, interphase FISH may be helpful in distinguishing de novo malignancy from germ cell tumor recurrence in its various forms. PMID:18503827

  10. The use of tetradecanoylphorbol acetate-stimulated peripheral blood cells enhances the prognostic value of interphase fluorescence in situ hybridization in patients with chronic lymphocytic leukemia.

    PubMed

    Delgado, Julio; Aventin, Anna; Briones, Javier; Sanchez, Jana; Nomdedeu, Josep; Sierra, Jorge

    2010-04-01

    Interphase fluorescence in situ hybridization (I-FISH) studies have a remarkable prognostic value in patients with chronic lymphocytic leukemia (CLL). I-FISH studies can be performed either on tetradecanoylphorbol acetate stimulated peripheral blood cells (I-FISH-TPA) or unstimulated peripheral blood mononuclear cells (I-FISH-PBMC). The aim of the study was to evaluate whether this finding was clinically relevant in a group of 235 patients with CLL. Fifty-six patients had both I-FISH-TPA and I-FISH-PBMC results. Compared with uncultured cells, the cytogenetic detection rate rose from 57 to 80% with the use of TPA-stimulated cells (P = 0.014). I-FISH-TPA provided a better prediction of treatment-free survival compared with I-FISH-PBMC (P = 0.031 vs. 0.166). Then, I-FISH-PBMC results from 93 historical patients were compared with 86 recent patients with I-FISH-TPA results. Genomic aberrations were detected in 46 and 67% of patients from the I-FISH-PBMC and I-FISH-TPA cohorts, respectively. The detection rate of 13q deletion as the only aberration increased from 10% with I-FISH-PBMC to 37% with I-FISH-TPA (P = 0.006). In conclusion, I-FISH-TPA increased the detection rate of 13q deletion and had an improved prognostic value compared with I-FISH-PBMC. PMID:20033916

  11. Multiplex fluorescence in situ hybridization and cross species color banding of a case of chronic myeloid leukemia in blastic crisis with a complex Philadelphia translocation.

    PubMed

    Harrison, C J; Gibbons, B; Yang, F; Butler, T; Cheung, K L; Kearney, L; Dirscherl, L; Bray-Ward, P; Gregson, M; Ferguson-Smith, M

    2000-01-15

    Exciting new techniques in molecular cytogenetics--namely, spectral karyotyping, multiplex fluorescence in situ hybridization (M-FISH), and cross species color banding--have been recently developed. An increasing number of reports demonstrate the success of these procedures in providing additional cytogenetic information--identifying marker chromosomes and revealing the presence of previously undetected chromosomal changes. However, these procedures have their limitations, and their absolute sensitivity in the accurate identification of subtle chromosomal abnormalities remains to be established. M-FISH and color banding have been applied to a case of chronic myeloid leukemia with a complex Philadelphia translocation involving chromosomes 9, 17, and 22, which had initially been identified from G-banded chromosome analysis. The abnormalities were confirmed by chromosome "painting" and specific probes. Although M-FISH and color banding revealed no additional cryptic chromosomal changes, this study has clearly demonstrated the success of these multiple color FISH approaches in the accurate characterization of a complex rearrangement with subtle abnormalities. PMID:10640141

  12. Radiation-induced chromosome aberrations in ataxia telangiectasia cells: high frequency of deletions and misrejoining detected by fluorescence in situ hybridization

    NASA Technical Reports Server (NTRS)

    Kawata, Tetsuya; Ito, Hisao; George, Kerry; Wu, Honglu; Uno, Takashi; Isobe, Kouichi; Cucinotta, Francis A.

    2003-01-01

    The mechanisms underlying the hyper-radiosensitivity of AT cells were investigated by analyzing chromosome aberrations in the G(2) and M phases of the cell cycle using a combination of chemically induced premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH) with chromosome painting probes. Confluent cultures of normal fibroblast cells (AG1522) and fibroblast cells derived from an individual with AT (GM02052) were exposed to gamma rays and allowed to repair at 37 degrees C for 24 h. At doses that resulted in 10% survival, GM02052 cells were approximately five times more sensitive to gamma rays than AG1522 cells. For a given dose, GM02052 cells contained a much higher frequency of deletions and misrejoining than AG1522 cells. For both cell types, a good correlation was found between the percentage of aberrant cells and cell survival. The average number of color junctions, which represent the frequency of chromosome misrejoining, was also found to correlate well with survival. However, in a similar surviving population of GM02052 and AG1522 cells, induced by 1 Gy and 6 Gy, respectively, AG1522 cells contained four times more color junctions and half as many deletions as GM02052 cells. These results indicate that both repair deficiency and misrepair may be involved in the hyper-radiosensitivity of AT cells.

  13. Parental exposure to environmental concentrations of diuron leads to aneuploidy in embryos of the Pacific oyster, as evidenced by fluorescent in situ hybridization.

    PubMed

    Barranger, Audrey; Benabdelmouna, Abdellah; Dégremont, Lionel; Burgeot, Thierry; Akcha, Farida

    2015-02-01

    Changes in normal chromosome numbers (i.e. aneuploidy) due to abnormal chromosome segregation may arise either spontaneously or as a result of chemical/radiation exposure, particularly during cell division. Coastal ecosystems are continuously subjected to various contaminants originating from urban, industrial and agricultural activities. Genotoxicity is common to several families of major environmental pollutants, including pesticides, which therefore represent a potential important environmental hazard for marine organisms. A previous study demonstrated the vertical transmission of DNA damage by subjecting oyster genitors to short-term exposure to the herbicide diuron at environmental concentrations during gametogenesis. In this paper, Fluorescent in situ hybridization (FISH) was used to further characterize diuron-induced DNA damage at the chromosomal level. rDNA genes (5S and 18-5.8-28S), previously mapped onto Crassostrea gigas chromosomes 4, 5 and 10, were used as probes on the interphase nuclei of embryo preparations. Our results conclusively show higher aneuploidy (hypo- or hyperdiploidy) level in embryos from diuron-exposed genitors, with damage to the three studied chromosomal regions. This study suggests that sexually developing oysters are vulnerable to diuron exposure, incurring a negative impact on reproductive success and oyster recruitment. PMID:25498420

  14. Heterogeneity of 11q13 region rearrangements in laryngeal squamous cell carcinoma analyzed by microarray platforms and fluorescence in situ hybridization.

    PubMed

    Jarmuz-Szymczak, Ma?gorzata; Pelinska, Kinga; Kostrzewska-Poczekaj, Magdalena; Bembnista, Ewa; Giefing, Maciej; Brauze, Damian; Szaumkessel, Marcin; Marszalek, Andrzej; Janiszewska, Joanna; Kiwerska, Katarzyna; Bartochowska, Anna; Grenman, Reidar; Szyfter, Witold; Szyfter, Krzysztof

    2013-07-01

    We reinvestigated rearrangements occurring in region q13 of chromosome 11 aiming to: (i) describe heterogeneity of the observed structural alterations, (ii) estimate amplicon size and (iii) identify of oncogenes involved in laryngeal cancer progression as potential targets for therapy. The study included 17 cell lines derived from laryngeal cancers and 34 specimens from primary laryngeal tumors. The region 11q13 was analyzed by fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH) and gene expression microarray. Next, quantitative real time PCR was used for chosen genes to confirm results from aCGH and gene expression microarray. The observed pattern of aberrations allows to distinguish three ways, in which gain and amplification involving 11q13 region may occur: formation of a homogeneously staining region; breakpoints in/near 11q13, which lead to the three to sevenfold increase of the copy number of 11q13 region; the presence of additional copies of the whole chromosome 11. The minimal altered region of gain and/or amplification was limited to ~1.8 Mb (chr.11:69,395,184-71,209,568) and comprised mostly 11q13.3 band which contain 12 genes. Five, out of these genes (CCND1, ORAOV1, FADD, PPFIA1, CTTN) had higher expression levels in comparison to healthy controls. Apart from CCND1 gene, which has an established role in pathogenesis of head and neck cancers, CTTN, ORAOV1 and FADD genes appear to be oncogene-candidates in laryngeal cancers, while a function of PPFIA1 requires further studies. PMID:23652995

  15. Human carbonyl reductase (CBR) localized to band 21q22. 1 by high-resolution fluorescence in situ hybridization displays gene dosage effects in trisomy 21 cells

    SciTech Connect

    Lemieux, N. (Universite de Montreal (Canada)); Malfoy, B. (Institut Curie Section de Biologie, Paris (France)); Forrest, G.L. (Beckman Research Institute at the City of Hope, Duarte, CA (United States))

    1993-01-01

    Human carbonyl reductase (CBR) belongs to a group of NADPH-dependent enzymes called aldo-keto reductases. The enzyme can function as an aldo-keto reductase or as a quinone reductase with potential for modulating quinone-mediated oxygen free radicals. The CBR gene was mapped by high-resolution fluorescence in situ hybridization to band 21q22.12, very close to the SOD1 locus at position 2lq22.11. CBR displayed gene dosage effects in trisomy 21 human lymphoblasts at the DNA and mRNA levels. Lymphoblasts with increasing chromosome 21 ploidy also showed increased aldo-keto reductase activity and increased quinone reductase activity. Both aldo-keto reductase activity and quinone reductase activity have been shown to be associated with carbonyl reductase. The location of CBR near SOD1 and the increased enzyme activity and potential for free radical modulation in trisomy 21 cells implicate CBR as a candidate for contributing to the pathology of certain diseases such as Down syndrome and Alzheimer disease. 28 refs., 1 fig., 1 tab.

  16. XY sex reversal in the wood lemming is associated with deletion of Xp21-23 as revealed by chromosome microdissection and fluorescence in situ hybridization.

    PubMed

    Liu, W S; Eriksson, L; Fredga, K

    1998-08-01

    In the wood lemming (Myopus schisticolor), XY sex reversal occurs naturally because of the presence of an X chromosome variant designated X*. The two types of X chromosome, X and X*, can be distinguished by G-banding, and analyses have demonstrated complex rearrangements of the short arm of X*. Here, chromosomal microdissection, degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) and fluorescence in situ hybridization (FISH) techniques have been used to generate and map DNA probes for different parts of the X and X* chromosomes. The results showed that the region of Xp21-23 is deleted from the X* and some of the deleted DNA sequences are homologous to the mouse gamma-satellite. The deletion must be associated with the sex reversal in this species. FISH experiments with dissected probes of X and distal half of Xq provided evidence for presence of homologous sequences between large regions of the X and Y chromosomes, including euchromatic and heterochromatic parts of the sex chromosomes. The findings of this study will be of significance for further cloning of important candidate gene(s) responsible for the XY sex reversal. PMID:9872667

  17. Aneuploidy in 165,330 human sperm; results of two- and three-colour fluorescence in situ hybridization for chromosomes 1, 12, 15, 18, X, and Y

    SciTech Connect

    Spriggs, E.L.; Martin, R.H. [Alberta Childrens Hospital, Alberta (Canada)]|[Univ. of Calgary, Alberta (Canada)

    1994-09-01

    To understand the mechanisms that affect aneuploidy, fluorescence in situ hybridization (FISH), using chromosome-specific centromeric probes, was employed to screen a large population of human sperm for numerical errors. To determine the true rate of disomy for chromosomes 1, 12, 15, 18, two-color FISH was performed, and for the gonosomes, three-color FISH. The use of multiple, differently-colored probes allows one to distingish a true disomic sperm from a diploid cell. For each centromeric probe, a minimum of 10,000 sperm nuclei for each of five donors was scored, giving a total count of 165,330 sperm nuclei. The incidence of disomic sperm for the sex chromosomes was significantly increased as compared to the frequency for the autosomes ({chi}{sup 2}=232.3, p<0.001), confirming the results observed in studies of sperm karyotypes and spontaneous abortions. The disomy frequencies for autosomes 1, 12, 15, and 18 were found to be uniform. Inter-donor heterogeneity for disomy frequencies was found to exist for the sex chromosomes and for chromosomes 1 and 15, suggesting significant variation among normal men.

  18. In situ hybridization to somatic chromosomes in Drosophila.

    PubMed

    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. PMID:21880819

  19. Evaluation of a Fluorescence In Situ Hybridization Assay for Differentiation between Tuberculous and Nontuberculous Mycobacterium Species in Smears of Lowenstein-Jensen and Mycobacteria Growth Indicator Tube Cultures Using Peptide Nucleic Acid Probes

    Microsoft Academic Search

    POONPILAS HONGMANEE; HENRIK STENDER; OLE F. RASMUSSEN

    A new fluorescence in situ hybridization assay based on peptide nucleic acid probes (MTB and NTM probes targeting tuberculous and nontuberculous species, respectively) for the identification of Mycobacterium tuber- culosis complex and differentiation between tuberculous and nontuberculous mycobacteria (NTM) was evalu- ated using Lowenstein-Jensen (LJ) solid cultures from 100 consecutive sputum samples and 50 acid-fast bacillus (AFB)-positive sputum samples as

  20. Resolving Genetic Functions within Microbial Populations: In Situ Analyses Using rRNA and mRNA Stable Isotope Probing Coupled with Single-Cell Raman-Fluorescence In Situ Hybridization ? †

    PubMed Central

    Huang, Wei E.; Ferguson, Andrew; Singer, Andrew C.; Lawson, Kathryn; Thompson, Ian P.; Kalin, Robert M.; Larkin, Michael J.; Bailey, Mark J.; Whiteley, Andrew S.

    2009-01-01

    Prokaryotes represent one-half of the living biomass on Earth, with the vast majority remaining elusive to culture and study within the laboratory. As a result, we lack a basic understanding of the functions that many species perform in the natural world. To address this issue, we developed complementary population and single-cell stable isotope (13C)-linked analyses to determine microbial identity and function in situ. We demonstrated that the use of rRNA/mRNA stable isotope probing (SIP) recovered the key phylogenetic and functional RNAs. This was followed by single-cell physiological analyses of these populations to determine and quantify in situ functions within an aerobic naphthalene-degrading groundwater microbial community. Using these culture-independent approaches, we identified three prokaryote species capable of naphthalene biodegradation within the groundwater system: two taxa were isolated in the laboratory (Pseudomonas fluorescens and Pseudomonas putida), whereas the third eluded culture (an Acidovorax sp.). Using parallel population and single-cell stable isotope technologies, we were able to identify an unculturable Acidovorax sp. which played the key role in naphthalene biodegradation in situ, rather than the culturable naphthalene-biodegrading Pseudomonas sp. isolated from the same groundwater. The Pseudomonas isolates actively degraded naphthalene only at naphthalene concentrations higher than 30 ?M. This study demonstrated that unculturable microorganisms could play important roles in biodegradation in the ecosystem. It also showed that the combined RNA SIP-Raman-fluorescence in situ hybridization approach may be a significant tool in resolving ecology, functionality, and niche specialization within the unculturable fraction of organisms residing in the natural environment. PMID:18997025

  1. Zoo-fluorescence in situ hybridization analysis of human and Indian muntjac karyotypes (Muntiacus muntjak vaginalis) reveals satellite DNA clusters at the margins of conserved syntenic segments.

    PubMed

    Frönicke, L; Scherthan, H

    1997-06-01

    Zoo-fluorescence in situ hybridization (FISH) with human whole chromosome-specific paint probes revealed extensive homoeologies between Indian muntjac (2n=6, 7 female, male) and human karyotypes (2n=46). Forty-two conserved syntenic segments, corresponding to all human chromosomes except the Y chromosome, produced a near-complete coverage of the muntjac complement and revealed margins of interspecific segmental homoeology. To test the hypothesis that interstitial satellite DNA loci, illuminated by a Chinese muntjac C5-satellite probe in Indian muntjac chromosome arms, mark ancestral fusion points (Lin CC, Sasi R, Fan YS, Chen Z-Q (1991) New evidence for tandem chromosome fusions in the karyotypic evolution of the Asian muntjacs. Chromosoma 101: 19-24), we combined Zoo-FISH with C5 satellite mapping. Twenty-six interstitial satellite DNA loci were detected in the haploid Indian muntjac genome and were found to co-localize with the margins of conserved human/Indian muntjac syntenic segments. These results were confirmed by two-colour FISH and are in accordance with the tandem fusion hypothesis for Indian muntjac chromosomes. Furthermore, conserved syntenic segment combinations detected in pig, cattle and Indian muntjac Zoo-FISH maps reveal ancestral artiodactyl chromosomes. PMID:9244453

  2. Chromosomal study of lettuce and its allied species (Lactuca spp., Asteraceae) by means of karyotype analysis and fluorescence in situ hybridization.

    PubMed

    Matoba, Hideyuki; Mizutani, Takayuki; Nagano, Katsuya; Hoshi, Yoshikazu; Uchiyama, Hiroshi

    2007-12-01

    In this study, in addition to the karyotype analysis, the chromosomal distributions of 5 S and 18 S rDNAs, and the Arabidopsis-type (T3AG3) telomeric sequences were detected by means of fluorescence in situ hybridization (FISH) to promote the information of chromosomal organization and evolution in the cultivated lettuce and its wild relatives, L. sativa, L. serriola, L. saligna and L. virosa. The karyotype analysis revealed the dissimilarity between L. virosa and the remaining species. In all four Lactuca species studied, one 5 S rDNA and two 18 S rDNA loci were detected. The simultaneous FISH of 5 S and 18 S rDNAs revealed that both rDNA loci of L. sativa, L. serriola and L. saligna were identical, however, that of L. virosa was different from the other species. These analyses indicate the closer relationships between L. sativa/L. serriola and L. saligna rather than L. virosa. Arabidopsis-type telomeric sequences were detected at both ends of their chromatids of all chromosomes not in the other regions. This observation suggests the lack of telomere-mediated chromosomal rearrangements among the Lactuca chromosomes. PMID:18215246

  3. [Antibiotic resistance rates of Helicobacter pylori isolates and the comparison of E-test and fluorescent in situ hybridization methods for the detection of clarithromycin resistant strains].

    PubMed

    Bakir Ozbey, Saliha; Ozakin, Cüneyt; Keskin, Murat

    2009-04-01

    Helicobacter pylori which is one of the commonly seen chronic bacterial infections in the world, has been demonstrated to have a relationship with chronic gastritis, peptic ulcer disease and gastric cancer. Current management of H. pylori infection involves the use of a proton pump inhibitor (PPI) and any two of amoxicillin, clarithromycin and metronidazole in combination. Antibiotic resistance which is in an increasing trend in H. pylori since the recent years, is the main cause of treatment failure. This study was conducted to determine the susceptibility of 31 H. pylori strains to several antibiotics by using E-test method (AB Biodisk, Sweden) and also to detect clarithromycin resistance by fluorescent in situ hybridization (FISH; SeaFAST, Hungary). The strains were isolated from the gastric biopsy specimens of patients who were admitted to Uluda? University Hospital, Bursa, Turkey with dyspeptic complaints. Clarithromycin, amoxycillin, metronidazole, tetracycline and ciprofloxacin resistance rates were as 41.9%, 3.2%, 41.9%, 3.2% and 45.2%, respectively. Resistance to single antibiotic was detected in 32.2% of the isolates whereas multiresistance was seen in 45.2%. For the hybridization process one probe specific for 16S rRNA and labeled with a fluorescein dye and the other probe specific for the mutations in 23S rRNA and labeled with Cy3 stain were used. Green signalling denoted presence of H. pylori in the specimen and red signalling was associated with clarithromycin resistance. All of the isolates yielded green signalling and the 13 isolates found to be resistant to clarithromycin by E-test, gave red signalling. No difference was detected between the two methods in terms of clarithromycin resistance determination. This was a preliminary study reporting the H. pylori resistance rates in our region, however, further larger scale studies are required for obtaining countrywide data. PMID:19621607

  4. Renal oncocytoma: a comparative clinicopathologic study and fluorescent in-situ hybridization analysis of 73 cases with long-term follow-up

    PubMed Central

    2010-01-01

    Clinical studies have confirmed that renal oncocytoma (RO) is a benign neoplasm with excellent prognosis. In diagnostically challenging cases of renal oncocytic epithelial neoplasms, fluorescent in-situ hybridization (FISH) is increasingly being used and its ability to distinguish RO from chromophobe renal cell carcinoma (ChRCC) has been documented. In this study, we evaluated the differential diagnostic contribution of FISH in cases of RO. Clinicopathologic data and glass slides from 73 patients with RO were reviewed; 20 cases of ChRCC were included for comparison. FISH analysis of formalin-fixed, paraffin-embedded sections was performed using centromeric probes for chromosomes 1, 2, 7 and 17. FISH analysis revealed ROs had frequent loss of signal for chromosome 1 (56%) and 17 (44%). Tumors with more than one loss were common (41%) and 10% cases showed loss of all chromosomes examined. A total of 18% cases did not show any abnormality. Our study shows that chromosomal abnormalities in both ROs and ChRCCs are common with frequent loss of chromosomes 1 and 17. No association was found between overall patient survival and the extent of chromosomal abnormalities. FISH results, even those showing significant chromosomal abnormalities, should not alter the primarily morphology-based diagnosis of RO. PMID:20497539

  5. Characterization of G-banded chromosomes of a female saola (Pseudoryx nghetinhensis,2n = 50) and X chromosome i dentification by means of fluorescent in situ hybridization.

    PubMed

    Nguyen, T T; Nguyen, B X; Stranzinger, G

    2005-01-01

    The saola (Pseudoryx nghetinhensis) is a newly discovered large mammal species, belongs to the subfamily Bovinae and is listed as being endangered. Due to the limitation of the material available, no cytogenetic studies have been carried out on this species. In the present study, preliminary cytogenetic analysis was undertaken on cultured female fibroblast cells to characterize the karyotype organization of saola. An examination of 120 Giemsa stained metaphases showed the diploid chromosome number of 2n = 50, including five bi-armed chromosome pairs. The distribution of constitutive heterochromatin in saola was studied. However, the variability in the size of C-bands was not significant on all the homologous chromosomes. The X chromosome pair, corresponding to the largest telocentric chromosomes, was identified by fluorescent in situ hybridization (FISH) using a bacterial artificial chromosome clone (BAC 0577G05, which maps to BTAXq25-->q33). In comparison to the standard karyotype of cattle (ISCNDB 2000), a G-banded ideogram of saola (about 390 band level) was presented. This work, therefore, provided a basic insight into the karyotype organization of this endangered species and will be particularly useful to improve the understanding of differences of genomes between related species. PMID:15905645

  6. Localization of a Female-Specific Marker on the Chromosomes of the Brown Seaweed Saccharina japonica Using Fluorescence In Situ Hybridization

    PubMed Central

    Gu, JunGang; Li, LiHua; Zhou, ZhiGang

    2012-01-01

    Background There is a heteromorphic alternative life in the brown seaweed, Saccharina japonica (Aresch.) C. E. Lane, C. Mayes et G. W. Saunders (?=?Laminaria japonica Aresch.), with macroscopic monoecious sporophytes and microscopic diecious gametophytes. Female gametophytes are genetically different from males. It is very difficult to identify the parent of a sporophyte using only routine cytological techniques due to homomorphic chromosomes. A sex-specific marker is one of the best ways to make this determination. Methodology/Principal Findings To obtain clear images, chromosome preparation was improved using maceration enzymes and fluorochrome 4?, 6-diamidino-2-phenylindole (DAPI). The chromosome number of both male and female haploid gametophytes was 31, and there were 62 chromosomes in diploid sporophytes. Although the female chromosomes ranged from 0.77 µm to 2.61 µm in size and were larger than the corresponding ones in the males (from 0.57 µm to 2.16 µm), there was not a very large X chromosome in the females. Based on the known female-related FRML-494 marker, co-electrophoresis and Southern blot profiles demonstrated that it was inheritable and specific to female gametophytes. Using modified fluorescence in situ hybridization (FISH), this marker could be localized on one unique chromosome of the female gametophytes as well as the sporophytes, whereas no hybridization signal was detected in the male gametophytes. Conclusions/Significance Our data suggest that this marker was a female chromosome-specific DNA sequence. This is the first report of molecular marker localization on algal chromosomes. This research provides evidence for the benefit of using FISH for identifying molecular markers for sex identification, isolation of specific genes linked to this marker in the females, and sex determination of S. japonica gametophytes in the future. PMID:23166593

  7. Clonal profiling of mixed lobular and ductal carcinoma revealed by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization.

    PubMed

    Tajiri, Ryosuke; Inokuchi, Masafumi; Sawada-Kitamura, Seiko; Kawashima, Hiroko; Nakamura, Ritsuko; Oyama, Takeru; Dobashi, Yoh; Ooi, Akishi

    2014-05-01

    A needle biopsy of a mass in the right breast of a 36-year-old woman revealed invasive ductal carcinoma (IDC), and approximately 20% of cancer cells showed unequivocal membranous staining with the HercepTest. After systemic therapy with trastuzumab and paclitaxel followed by FEC (fluorouracil + epirubicin + cyclophosphamide), a right mastectomy was performed. By histological and immunohistochemical examinations, the resected tumor consisted mainly of E-cadherin-negative invasive lobular carcinoma (ILC), and the rest was ERBB2-positive IDC; thus, the diagnosis was mixed ductal and lobular carcinoma. Multiplex ligation-dependent probe amplification and fluorescence in situ hybridization (FISH) analyses revealed that ILC and IDC shared high-level amplification of CCND1 in homogeneously staining regions (HSR) and that IDC had an additional HSR-type amplicon of ERBB2. These findings strongly indicate that IDC and ILC had a common precursor cell with CCND1 amplification. Review of the biopsy specimen with FISH showed IDC with gene amplifications of CCND1 and ERBB2 as a minor component, IDC without amplification of CCND1 or ERBB2 as a major component, and a minute portion of ILC with CCND1 amplification. We speculate that chemotherapy and trastuzumab caused a marked reduction in IDC; however, ILC with CCND1 amplification was resistant to chemotherapy and grew. PMID:24888777

  8. Let them fly or light them up: matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry and fluorescence in situ hybridization (FISH).

    PubMed

    Schweickert, Birgitta; Moter, Annette; Lefmann, Michael; Göbel, Ulf B

    2004-01-01

    This review focuses on clinical bacteriology and by and large does not cover the detection of fungi, viruses or parasites. It discusses two completely different but complementary approaches that may either supplement or replace classic culture-based bacteriology. The latter view may appear provocative in the light of the actual market penetration of molecular genetic testing in clinical bacteriology. Despite its elegance, high specificity and sensitivity, molecular genetic diagnostics has not yet reached the majority of clinical laboratories. The reasons for this are manifold: Many microbiologists and medical technologists are more familiar with classical microbiological methods than with molecular biology techniques. Culture-based methods still represent the work horse of everyday routine. The number of available FDA-approved molecular genetic tests is limited and external quality control is still under development. Finally, it appears difficult to incorporate genetic testing in the routine laboratory setting due to the limited number of samples received or the lack of appropriate resources. However, financial and time constraints, particularly in hospitals as a consequence of budget cuts and reduced length of stay, lead to a demand for significantly shorter turnaround times that cannot be met by culture-dependent diagnosis. As a consequence, smaller laboratories that do not have the technical and personal equipment required for molecular genetic amplification techniques may adopt alternative methods such as fluorescence in situ hybridization (FISH) that combines easy-to-perform molecular hybridization with microscopy, a technique familiar to every microbiologist. FISH is hence one of the technologies presented here. For large hospital or reference laboratories with a high sample volume requiring massive parallel high-throughput testing we discuss matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) of nucleic acids, a technology that has evolved from the post-genome sequencing era, for high-throughput sequence variation analysis (1, 2). PMID:15638841

  9. Dual-color fluorescence in situ hybridization reveals an association of chromosome 8q22 but not 8p21 imbalance with high grade invasive breast carcinoma.

    PubMed

    Walker, Logan C; McDonald, Margaret; Wells, J Elisabeth; Harris, Gavin C; Robinson, Bridget A; Morris, Christine M

    2013-01-01

    We previously reported molecular karyotype analysis of invasive breast tumour core needle biopsies by comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) (Walker et al, Genes Chromosomes Cancer, 2008 May;47(5):405-17). That study identified frequently recurring gains and losses involving chromosome bands 8q22 and 8p21, respectively. Moreover, these data highlighted an association between 8q22 gain and typically aggressive grade 3 tumors. Here we validate and extend our previous investigations through FISH analysis of tumor touch imprints prepared from excised breast tumor specimens. Compared to post-surgical tumor excisions, core needle biopsies are known to be histologically less precise when predicting tumor grade. Therefore investigating these chromosomal aberrations in tumor samples that offer more reliable pathological assessment is likely to give a better overall indication of association. A series of 60 breast tumors were screened for genomic copy number changes at 8q22 and 8p21 by dual-color FISH. Results confirm previous findings that 8p loss (39%) and 8q gain (74%) occur frequently in invasive breast cancer. Both absolute quantification of 8q22 gain across the sample cohort, and a separate relative assessment by 8q22:8p21 copy number ratio, showed that the incidence of 8q22 gain significantly increased with grade (p = 0.004, absolute and p = 0.02, relative). In contrast, no association was found between 8p21 loss and tumor grade. These findings support the notion that 8q22 is a region of interest for invasive breast cancer pathogenesis, potentially harboring one or more genes that, when amplified, precipitate the molecular events that define high tumor grade. PMID:23936250

  10. Nucleic acid in-situ hybridization detection of infectious agents

    NASA Astrophysics Data System (ADS)

    Thompson, Curtis T.

    2000-04-01

    Limitations of traditional culture methods and newer polymerase chain reaction (PCR)-based methods for detection and speciation of infectious agents demonstrate the need for more rapid and better diagnostics. Nucleic acid hybridization is a detection technology that has gained wide acceptance in cancer and prenatal cytogenetics. Using a modification of the nucleic acid hybridization technique known as fluorescence in-situ hybridization, infectious agents can be detected in a variety of specimens with high sensitivity and specificity. The specimens derive from all types of human and animal sources including body fluids, tissue aspirates and biopsy material. Nucleic acid hybridization can be performed in less than one hour. The result can be interpreted either using traditional fluorescence microscopy or automated platforms such as micro arrays. This paper demonstrates proof of concept for nucleic acid hybridization detection of different infectious agents. Interpretation within a cytologic and histologic context is possible with fluorescence microscopic analysis, thereby providing confirmatory evidence of hybridization. With careful probe selection, nucleic acid hybridization promises to be a highly sensitive and specific practical diagnostic alternative to culture, traditional staining methods, immunohistochemistry and complicated nucleic acid amplification tests.

  11. Localization of ZNF164, ZNF146, GGTA1, SOX2, PRLR and EEF2 on homoeologous cattle, sheep and goat chromosomes by fluorescent in situ hybridization and comparison with the human gene map

    Microsoft Academic Search

    H. Hayes; C. Le Chalony; G. Goubin; D. Mercier; E. Payen; C. Bignon; K. Kohno

    1996-01-01

    The six following genes: zinc finger proteins 164 (ZNF164) and 146 (ZNF146), alpha-galactosyltransferase 1 (GGTA1), SRY-related HMG-box 2 (SOX2), prolactin receptor (PRLR) and elongation factor 2 (EEF2) have been localized by fluorescent in situ hybridization respectively on bovine and caprine chromosomes 17, 18, 11, 1, 20 and 7 and on sheep chromosomes 17, 14, 3, 1, 16, and 5. The

  12. Application of ELN cosmid probes using fluorescence in situ hybridization (FISH) towards a clinical diagnostic test for Williams syndrome

    SciTech Connect

    Lowery, M.; Brothman, L.; Leonard, C. [Univ. of Utah Health Center, Salt Lake City, UT (United States)] [and others

    1994-09-01

    Williams syndrome (WS) is characterized by mental deficiency, gregarious personalities, dysmorphic facies, supravalvular aortic stenosis (SVAS), and idiopathic infantile hypercalcemia. Expression of the phenotype is variable. Deletions including an elastin allele (ELN) are thought to be the basis of the connective tissue and vascular abnormalities in WS patients. Patients with WS are hemizygous for ELN, exhibiting a submicroscopic deletion at 7q11.23 detected by FISH. To substantiate the hemizygosity hypothesis and define molecular cytogenetics in patients with familial and sporadic WS, a series of 71 patients were evaluated. EBV-transformed lymphocytes from 48 selected patients were cultured and harvested according to cytogenetic protocol. Cosmids containing ELN were biotinylated and hybridized to metaphase cells by routine procedures. In addition, an alpha-satellite probe for chromosome 7 was included in hybridizations as an internal control. Thirty-one of these 48 patients (65%) showed a deletion in one ELN allele by FISH. Negative patients were shown to be non-affected family members or patients in the {open_quotes}uncertain{close_quotes} category (expressing some, but not all features characteristic of WS). The FISH data were consistent with molecular analyses of ELN deletions. Twenty-three additional patients were referred to confirm or rule out a diagnosis of WS. Nine patients (39%) showed a deletion of ELN by FISH. Correlations between phenotype and FISH results are in progress. These results suggest that a rapid, accurate diagnostic technique for WS using FISH can be implemented in the cytogenetics laboratory as a routine clinical service. Identification of the deletion in patients suspected of having WS will facilitate classification of these patients and improve clinical management.

  13. Concurrent use of flow cytometry and fluorescence in-situ hybridization techniques for detecting faulty meiosis in a human sperm sample.

    PubMed

    Weissenberg, R; Aviram, A; Golan, R; Lewin, L M; Levron, J; Madgar, I; Dor, J; Barkai, G; Goldman, B

    1998-01-01

    Routine semen analysis in an infertile patient revealed severe teratospermia associated with malformation of head and tail in 100% of the sperm cells. Flow cytometry and fluorescence in-situ hybridization (FISH) were shown to supplement routine semen analysis by providing information on the sperm chromatin. Using flow cytometry, propidium iodide-stained spermatozoa from the same sperm sample were compared with a normal reference pool, and with human lymphocytes. The results point to a population of diploid sperm cells rather than to mature haploid spermatozoa. Numerical chromosomal abnormalities of the spermatozoa were subsequently evaluated using FISH. A total of 1000 sperm cells were scored for X and Y chromosomes, and an additional 1128 sperm cells for chromosome 18. Aneuploidy of chromosomes X and Y was revealed in 96.9% of the cells and of chromosome 18 in 90.3% of the cells. Non-disjunction of chromosome X and Y in meiosis I and II occurred in 54.8 and 2.7% of the sperm cells respectively. Non-disjunction in both meiosis I and II occurred in 39.4% of the sperm cells. A normal haploid pattern for chromosomes X and Y was observed in only 3.1%, and for chromosome 18 in 9.7%, of the cells. Using three colour FISH for the sex chromosomes and for chromosome 18, diploidy was demonstrated in 19.4% of 500 sperm cells and aneuploidy in virtually all sperm cells (99.2%). The use of flow cytometry and FISH in cases where genetic and developmental chromatin abnormalities are suspected is a valuable adjunct to other available techniques, and can guide the clinicians to decide which samples are unsuitable for intracytoplasmic injection. PMID:9510012

  14. Genetic and immunophenotypic profile of IGH@ rearrangement detected by fluorescence in situ hybridization in 149 cases of B-cell chronic lymphocytic leukemia.

    PubMed

    Lu, Gary; Kong, Yue; Yue, Changjun

    2010-01-01

    Recent studies have shown a higher frequency of immunoglobulin heavy (IGH@) locus rearrangement in B-cell chronic lymphocytic leukemia (B-CLL) than previously reported. However, association of the IGH@ rearrangement with specific chromosomal abnormalities and immunophenotypic markers in B-CLL is still under further investigation. In this study, we analyzed 149 bone marrow aspirate or peripheral blood specimens from patients diagnosed with B-CLL, evaluated by four different laboratory studies: morphology examination, three- or four-color flow cytometry analysis, conventional cytogenetics, and fluorescence in situ hybridization (FISH) with a dual-color, break-apart IGH@ probe in addition to a B-CLL FISH probe panel for del(11)(q22) ATM, del(13)(q14.3), del(17)(p13) TP53, and +12. An IGH@ rearrangement was found by FISH in 24 cases (16.0%). Of these 24 cases, 16 (67%) contained chromosomal abnormalities, including t(14;19)(q32;q13.2), t(8;14)(q24;q32), and t(14;18)(q32;q21). In addition, a cryptic deletion of the immunoglobulin heavy variable region (IGHV) was revealed. Using 30% as the cutoff for positive CD38 expression, 22 of the 24 cases (92%) were positive for CD38. The present results further confirm that IGH@ rearrangement is not a rare genomic abnormality in B-CLL, and also show both that t(14;19)(q32;q13.2) is the most common cytogenetic change involving IGH@ rearrangement detected by FISH in B-CLL and that IGH@ rearrangement is correlated with CD38 expression. It is appropriate to include an IGH@ probe in the FISH panel for B-CLL diagnosis. PMID:19963136

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

    PubMed Central

    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 and rearrangement of the 11q13 locus. Using specimen (nephrectomy)-histology as the ‘gold standard’, a genomic aberration-based decision tree was generated to classify specimens. The diagnostic potential of the decision tree was assessed by comparing the FISH-based classification and biopsy histology with specimen histology. Results Of the 114 biopsies diagnostic by either method, a higher diagnostic yield was achieved by FISH (92 and 96%) than histology alone (82 and 84%) in the 65 biopsies from SRMs (<4?cm) and 49 from larger masses, respectively. An optimized decision tree was constructed based on aberrations detected in eight chromosomes, by which the maximum concordance of classification achieved by FISH was 79%, irrespective of mass size. In SRMs, the overall sensitivity of diagnosis by FISH compared with histopathology was higher for benign oncocytoma, was similar for the chromophobe renal cell carcinoma subtype, and was lower for clear-cell and papillary subtypes. The diagnostic accuracy of classification of needle biopsy specimens (from SRMs) increased from 80% obtained by histology alone to 94% when combining histology and FISH. Conclusion The present study suggests that a novel FISH assay developed by us has a role to play in assisting in the yield and accuracy of diagnosis of renal cortical neoplasms in needle biopsies in particular, and can help guide the clinical management of patients with SRMs that were non-diagnostic by histology. PMID:24467611

  16. Gene amplification of ESR1 in breast cancers--fact or fiction? A fluorescence in situ hybridization and multiplex ligation-dependent probe amplification study.

    PubMed

    Ooi, Akishi; Inokuchi, Masafumi; Harada, Shinichi; Inazawa, Johji; Tajiri, Ryousuke; Kitamura, Seiko Sawada-; Ikeda, Hiroko; Kawashima, Hiroko; Dobashi, Yoh

    2012-05-01

    Oestrogen receptor-alpha (ER?), encoded by the ESR1 gene located on 6q25, is a nuclear transcription factor. Since it was reported in 2007 that more than 20% of breast cancers show ESR1 gene amplification, there has been considerable controversy about its frequency and clinical significance. We set out to assess the frequency and levels of ESR1 amplification in breast cancers. In a total of 106 breast needle biopsy specimens examined by immunohistochemistry, 78 tumours contained more than 10% ER?-positive cancer cells. In fluorescence in situ hybridization (FISH) analysis with an ESR1-specific probe, variously extended ESR1 signals were found in ER?-expressing cells. Some of these were indistinguishable from large clustered signals generally accepted to mean high-level gene amplification in homogeneously staining regions (HSRs), and could be considered to represent gene amplification. However, with RNase treatment, the 'HSR-like' signals changed to small compact signals, and are thus thought to represent concentrated RNA. FISH using two differently labelled probes corresponding to the non-overlapping 5'- and 3'-end portions of the ESR1 gene on touch smears showed a preserved spatial relationship of the 3' to 5' sequence of ESR1, therefore strongly suggesting that the RNA consisted of primary transcripts. Using touch smears obtained from 51 fresh tumours, precise enumeration of ESR1 signals with a correction by the number of centromere 6 on FISH after RNase A treatment revealed that three tumours (5.9%) had tumour cells with one to three additional copies of ESR1 as predominant subpopulations. This infrequent and low level of gene amplification of ESR1 was also detected as a 'gain' of the gene by analysis with multiplex ligation-dependent probe amplification (MLPA). The consistent results from immunohistochemistry, FISH, and MLPA in the present study settle the long-standing debate concerning gene amplification of ESR1 in breast carcinoma. PMID:22170254

  17. Physical mapping of ribosomal RNA genes in peonies (Paeonia, Paeoniaceae) by fluorescent in situ hybridization: implications for phylogeny and concerted evolution.

    PubMed

    Zhang, D; Sang, T

    1999-05-01

    Physical maps of the 18S-5.8S-26S ribosomal RNA genes (rDNA) were generated by fluorescent in situ hybridization for five diploid Paeonia species, P. delavayi and P. rockii of section Moutan, and P. emodi, P. tenuifolia, and P. veitchii of section Paeonia. Of five pairs of mitotic chromosomes, rDNA loci were mapped near the telomeres of chromosomes 3, 4, and 5 of P. rockii and P. tenuifolia, chromosomes 2, 3, 4, and 5 of P. delavayi, and all five pairs of chromosomes of P. emodi and P. veitchii. Combining this information with the previously obtained rDNA maps of P. brownii and P. californica of section Oneapia, we hypothesized that the most recent common ancestor of extant peony species had three rDNA loci located on chromosomes 3, 4, and 5. Increase in number of rDNA loci occurred later in each of the three sections, and the increase from three to four loci represents a parallel gain of an rDNA locus on chromosome 2 in P. delavayi of section Moutan and P. brownii of section Oneapia. The increase in number of rDNA loci likely resulted from the translocation of rDNA repeats from chromosomes bearing rDNA loci to chromosomes without them; such translocation is probably facilitated by the telomeric location of rDNA loci. For allotetraploid peony species lacking polymorphism in sequences of the internal transcribed spacers (ITS) of rDNA, the rDNAs derived from divergent diploid parents may have been homogenized through concerted evolution among at least six rDNA loci in the allotetraploids. Chromosomal location of rDNA loci has a more substantial impact on the tempo of concerted evolution than the number of loci. PMID:10330077

  18. Reliability Evaluation of Fluorescence In Situ Hybridization (FISH) and G-Banding on Bone Marrow and Peripheral Blood Cells in Chronic Myelogenous Leukemia Patients

    PubMed Central

    Manaflouyan Khajehmarjany, Soheila; Rahmani, Seyed Ali; Chavoshi, Seyed Hadi; Esfahani, Ali; Movassaghpour Akbari, Ali Akbar

    2015-01-01

    Chronic myeloid leukemia (CML) is a myeloproliferative disease. The cytogenetic hallmark of CML is Philadelphia (Ph) chromosome. This study aimed to diagnose suspected CML patients, to monitor CML patients under therapy using cytogenetic and fluorescence in situ hybridization (FISH) techniques to analyze their bone marrow (BM) and peripheral blood (PB) samples, and finally to compare their obtained results for both specimens. This study was conducted during one-year period (2012-2013). The participants were recruited from the Hematology and Oncology Clinic of Shahid Gazi (Emam Reza) Hospital of Tabriz University of Medical Sciences, Tabriz, East Azerbaijan Province, Iran. We analyzed 90 samples from 60 suspected CML patients (30 BM and 60 PB samples). All samples were analyzed using G-banding, 5 samples using dual fusion FISH (DF-FISH) probes, as well as 30 samples using both FISH and G-banding. Among the 90 analyzed samples of 60 patients, 25 (41.66%) were Ph+ using karyotyping, whereas five cases were not analyzable, so FISH was applied and the results confirmed that only two individuals were BCR-ABL+. In the comparison between 25 BM and 25 PB samples using karyotyping, 15 (60%) and 10 (40%) were ph+, respectively. The comparison of FISH and karyotyping on 30 samples showed that 9 (30%) and 8 (26.66%) were Ph+, respectively, and only 18.18% of Ph+ patients showed atypical patterns. In the comparison between BM-cytogenetic and PB- interphase-FISH (I-FISH), BM-cytogenetic was more reliable than PB-I-FISH in detecting Ph. Our data demonstrate that FISH analysis is a rapid, reliable and sensitive technique. The comparison between BM and PB showed that PB can not be replaced by BM, even in detecting by FISH.

  19. Identification of a microdeletion at 7q21.3 with fluorescence in situ hybridization in a patient with split hand/split foot (ectrodactyly)

    SciTech Connect

    Hudgins, L. [Children`s Hospital and Medical Center, Seattle, WA (United States); Massa, H.; Disteche, C. [Univ. of Washington, Seattle, WA (United States)] [and others

    1994-09-01

    Split hand/split foot (SHSF), often referred to as ectrodactyly or lobster claw deformity, is a human developmental disorder characterized by a deep median cleft of the hands and feet, missing digits, and fusion of remaining digits. This anomaly can be seen alone, frequently autosomal dominant, or in association with other abnormalities. One locus for this defect has been localized to chromosome 7q21.3-q22.1. We report a patient with SHSF plus mental retardation, short stature and dysmorphic features who was found to have a microdeletion at this locus detected only with the aid of fluorescence in situ hybridization (FISH). T.H. is a 7 y.o. male who was referred for evaluation of foot anomalies and mild mental retardation. History was remarkable for growth retardation of postnatal onset and hypotonia. Renal ultrasound and audiology evaluation were normal. Physical exam revealed dysplastic ears, micrognathia, long philtrum, high narrow palate, and malformations of the feet consistent with SHSF. Family history was negative for limb abnormalities and mental retardation. A number of patients with SHSF and other anomalies have been found to have deletions involving chromosome 7q21-q22; therefore, high resolution chromosome analysis was performed in T.H. but was inconclusive. Cosmids and yeast artificial chromosomes which we had previously mapped to the SHSF critical region were used as FISH probes and a microdeletion was detected. We were thus able to determine the etiology of this child`s abnormalities and provide accurate genetic counseling, which would not have been possible with standard cytogenetic techniques. This technique also allowed us to further refine the SHSF critical region. This case illustrates the utility of FISH for the rapid identification of suspect microdeletions in SHSF. This approach should also be useful as an expeditious way of defining the critical regions for the location of genes which give rise to other developmental malformations.

  20. Male infertility and copy number variants (CNVs) in the dog: a two-pronged approach using Computer Assisted Sperm Analysis (CASA) and Fluorescent In Situ Hybridization (FISH)

    PubMed Central

    2013-01-01

    Background Infertility affects ~10-15% of couples trying to have children, in which the rate of male fertility problems is approximately at 30-50%. Copy number variations (CNVs) are DNA sequences greater than or equal to 1 kb in length sharing a high level of similarity, and present at a variable number of copies in the genome; in our study, we used the canine species as an animal model to detect CNVs responsible for male infertility. We aim to identify CNVs associated with male infertility in the dog genome with a two-pronged approach: we performed a sperm analysis using the CASA system and a cytogenetic-targeted analysis on genes involved in male gonad development and spermatogenesis with fluorescence in situ hybridization (FISH), using dog-specific clones. This analysis was carried out to evaluate possible correlations between CNVs on targeted genes and spermatogenesis impairments or infertility factors. Results We identified two genomic regions hybridized by BACs CH82-321J09 and CH82-509B23 showing duplication patterns in all samples except for an azoospermic dog. These two regions harbor two important genes for spermatogenesis: DNM2 and TEKT1. The genomic region encompassed by the BAC clone CH82-324I01 showed a single-copy pattern in all samples except for one dog, assessed with low-quality sperm, displaying a marked duplication pattern. This genomic region harbors SOX8, a key gene for testis development. Conclusion We present the first study involving functional and genetic analyses in male infertility. We set up an extremely reliable analysis on dog sperm cells with a highly consistent statistical significance, and we succeeded in conducting FISH experiments on sperm cells using BAC clones as probes. We found copy number differences in infertile compared with fertile dogs for genomic regions encompassing TEKT1, DNM2, and SOX8, suggesting those genes could have a role if deleted or duplicated with respect to the reference copy number in fertility biology. This method is of particular interest in the dog due to the recognized role of this species as an animal model for the study of human genetic diseases and could be useful for other species of economic interest and for endangered animal species. PMID:24373333

  1. Rat karyotyping by fluorescence in situ hybridization (FISH): Localization of oncogene c-raf to 4q42, retinoblastoma antioncogene to 15q12, and mitochondrial D-loop-like sequences to the Y chromosome

    SciTech Connect

    Zullo, S. [Yale Univ. School of Medicine, New Haven, CT (United States)] [Yale Univ. School of Medicine, New Haven, CT (United States); [NIMH Neuroscience Center, Washington, DC (United States); Upender, M. [Yale Univ. School of Medicine, New Haven, CT (United States)] [Yale Univ. School of Medicine, New Haven, CT (United States)

    1995-02-10

    Fluorescence in situ hybridization (FISH) has been used to karyotype the rat genome with long interspersed repetitive elements (LINEs). Two-color FISH experiments were used to localize the rat c-raf oncogene to 4q42 and the rat retinoblastoma anti-oncogene to 15q12. In addition, sequences similar to the rat mitochondrial origin of replication (D-loop-like sequences) have been found to be concentrated among repetitive element sequences on the Y chromosome. This report extends hybridization-based karyotype technology to the rat, thus facilitating the application of FISH to rat gene mapping. 13 refs., 1 fig.

  2. The human gene for xeroderma pigmentosum complementation group G (XPG) maps to 13q33 by fluorescence in situ hybridization

    SciTech Connect

    Samec, S.; Corlet, J.; Scherly, D.; Clarkson, S.G. (Centre Medical Universitaire, Geneva (Switzerland)); Jones, T.A.; Sheer, D. (Imperial Cancer Research Fund, London (United Kingdom)); Wood, R.D. (Clare Hall Labs., Hertfordshire (United Kingdom))

    1994-05-01

    Recently, a human cDNA was isolated that restores normal levels of UV resistance and DNA repair synthesis when expressed in vivo in a lymphoblastoid cell line representing XP group G. The XP-G complementing gene (XPG) generates an mRNA of [approximately]4 kb and encodes a protein (XPGC) with homology to the RAD2 DNA repair protein of Saccharomyces cerevisiae. One hundred twenty nanograms of labeled probe was mixed with 2 [mu]g of human C[sub 0]t-1 DNA as a competitor for repetitive elements. Denaturation, dehydration, hybridization, and washing were performed as described. Probe detection was achieved by incubating metaphase spreads sequentially with 2 [mu]g/ml avidin-Texas Red, 5 [mu]g/ml biotinylated goat anti-avidin antibody, and 2 [mu]g/ml avidin-Texas Red. R-banding was revealed after incubation with fluorescein-labeled anti-BrdU mouse monoclonal antibody. Chromosomes were counter-stained with 0.06 [mu]g/ml DAPI in Citifluor. Analysis of 40 metaphase spreads showed paired signals on both copies of chromosome 13 at band 13q33. No other paired signals were seen consistently on any other chromosome. 9 refs., 1 fig.

  3. Large-scale polymorphism near the ends of several human chromosomes analyzed by using fluorescence in situ hybridization (FISH)

    SciTech Connect

    Trask, B.J.; Friedman, C. [Univ. of Washington, Seattle, WA (United States); Giorgi, D. [CNRS, Montpelier (France)] [and others

    1994-09-01

    We have discovered a large DNA segment that is polymorphically present at the ends of several human chromosomes. The segment, f7501, was originally derived form a human chromosome 19-specific cosmid library. FISH was used to determine the cosmid`s chromosomal distribution on 44 unrelated humans and several closely related primates. The human subjects represent a diversity of reproductively isolated ethnic populations. FISH analysis revealed that sequences highly homologous to the cosmid`s insert are present on both homologs at 3q, 15q,. and 19p in almost all individuals (88, 85, and 87 of 88 homologs, respectively). Other chromosomes sites were labeled much more rarely in the sampled individuals. For example, 56 of the 88 analyzed chromosomes 11 were labeled (18+/+, 6-/-, and 20+/- individuals). In contrast, 2q was labeled on only 1/88 sampled chromosomes. The termini of 2q, 5q, 6p, 6q, 7p, 8p, 9p, 9q, 11p, 12q, 16p, 19q, and 20q and an interstitial site at 2q13-14 were labeled in at least one individual of the set. EcoR1-fragments derived from the cosmid showed the same hybridization pattern as the entire cosmid, indicating that at least 40 kbp is shared by these chromosome ends. Ethnic differences in the allele frequency of these polymorphic variants was observed. For example, signals were observed on 8/10 and 7/10 of the chromosomes 7p and 16q, respectively, derived form Biakan Pygmies, but these sites were infrequently labeled in non-Pygmy human populations (2/68, respectively). This region has undergone significant changes in chromosome location during human evolution. Strong signal was seen on chimpanzee and gorilla chromosome 3, which is homologous to human chromosome 4, a chromosome unlabeled in any of the humans we have analyzed.

  4. Plasma Cell Enrichment Enhances Detection of High-Risk Cytogenomic Abnormalities by Fluorescence In Situ Hybridization and Improves Risk Stratification of Patients With Plasma Cell Neoplasms

    PubMed Central

    Lu, Gary; Muddasani, Ramya; Orlowski, Robert Z.; Abruzzo, Lynne V.; Qazilbash, Muzaffar H.; You, M. James; Wang, Yaping; Zhao, Ming; Chen, Su; Glitza, Isabella Claudia; Medeiros, L. Jeffrey

    2015-01-01

    Context Methods for plasma cell enrichment in bone marrow (BM) specimens can increase the sensitivity of fluorescence in situ hybridization (FISH) for detecting cytogenomic abnormalities, but there is no published report using these methods to evaluate high-risk cytogenomic abnormalities in patients with treated plasma cell neoplasms (PCN) and clinicopathologic data in follow-up. Objective To evaluate the utility of plasma cell enrichment combined with FISH and follow-up data for high-risk cytogenomic abnormalities in post-therapy PCN patients. Design twenty-eight PCN patients with 22 treated were included in this study. Plasma cells were enriched in BM aspirates using a magnetic cell-sorting procedure to select CD138+ cells. Probes were chosen to assess for del(17p13/TP53), del(13q14/RB1), 1q21/CKS1B gain, IgH/FGFR3 and IgH/MAF. Clinicopathologic data were collected during clinical follow-up after plasma cell enrichment. Results Plasma cells in non-enriched specimens ranged from 1%–28% (median, 8%) compared with 28%–96% (median, 73%) in enriched specimens (p<0.0001). In a subset of treated-patients in clinical remission, FISH detected high-risk cytogenomic abnormalities only in plasma cell enriched samples. This approach also detected abnormalities in cases of solitary plasmacytoma and monoclonal gammopathy of undetermined significance. Conclusions Plasma cell enrichment of BM samples increases FISH sensitivity to detect high-risk cytogenomic abnormalities, particularly in treated-patients, and these results, in combination with data from clinical follow-up, can be of value to improve risk stratification and patient management. PMID:23627452

  5. In Situ Analysis of Nitrifying Biofilms as Determined by In Situ Hybridization and the Use of Microelectrodes

    Microsoft Academic Search

    SATOSHI OKABE; HISASHI SATOH; YOSHIMASA WATANABE

    1999-01-01

    We investigated the in situ spatial organization of ammonia-oxidizing and nitrite-oxidizing bacteria in do- mestic wastewater biofilms and autotrophic nitrifying biofilms by using microsensors and fluorescent in situ hybridization (FISH) performed with 16S rRNA-targeted oligonucleotide probes. The combination of these tech- niques made it possible to relate in situ microbial activity directly to the occurrence of nitrifying bacterial populations. In

  6. Fluorescence In Situ Hybridization for MDM2 Amplification as a Routine Ancillary Diagnostic Tool for Suspected Well-Differentiated and Dedifferentiated Liposarcomas: Experience at a Tertiary Center

    PubMed Central

    Wang, Jayson; Swansbury, John; Min, Toon

    2015-01-01

    Background. The assessment of MDM2 gene amplification by fluorescence in situ hybridization (FISH) has become a routine ancillary tool for diagnosing atypical lipomatous tumor (ALT)/well-differentiated liposarcoma and dedifferentiated liposarcoma (WDL/DDL) in specialist sarcoma units. We describe our experience of its utility at our tertiary institute. Methods. All routine histology samples in which MDM2 amplification was assessed with FISH over a 2-year period were included, and FISH results were correlated with clinical and histologic findings. Results. 365 samples from 347 patients had FISH for MDM2 gene amplification. 170 were positive (i.e., showed MDM2 gene amplification), 192 were negative, and 3 were technically unsatisfactory. There were 122 histologically benign cases showing a histology:FISH concordance rate of 92.6%, 142 WDL/DDL (concordance 96.5%), and 34 cases histologically equivocal for WDL (concordance 50%). Of 64 spindle cell/pleomorphic neoplasms (in which DDL was a differential diagnosis), 21.9% showed MDM2 amplification. Of the cases with discrepant histology and FISH, all but 3 had diagnoses amended following FISH results. For discrepancies of benign histology but positive FISH, lesions were on average larger, more frequently in “classical” (intra-abdominal or inguinal) sites for WDL/DDL and more frequently core biopsies. Discrepancies of malignant histology but negative FISH were smaller, less frequently in “classical” sites but again more frequently core biopsies. Conclusions. FISH has a high correlation rate with histology for cases with firm histologic diagnoses of lipoma or WDL/DDL. It is a useful ancillary diagnostic tool in histologically equivocal cases, particularly in WDL lacking significant histologic atypia or DDL without corresponding WDL component, especially in larger tumors, those from intra-abdominal or inguinal sites or core biopsies. There is a significant group of well-differentiated adipocytic neoplasms which are difficult to diagnose on morphology alone, in which FISH for MDM2 amplification is diagnostically contributory.

  7. Karyotypic characterization of the great sturgeon, Huso huso, by multiple staining techniques and fluorescent in situ hybridization

    Microsoft Academic Search

    F. Fontana; J. Tagliavini; L. Congiu; M. Lanfredi; M. Chicca; C. Laurente; R. Rossi

    1998-01-01

    A karyotype analysis by several staining techniques was carried out on the great sturgeon, Huso huso (Linnaeus, 1758). The karyotype (2n?=?118?±?2) was composed of 42 pairs of meta-\\/submetacentric chromosomes and 17 pairs of acrocentrics\\/microchromosomes. Constitutive\\u000a heterochromatin was mainly located at the centromeric regions of the acrocentric chromosomes. The biarmed chromosomes showed\\u000a weak C-bands. Fluorescent staining with GC-specific chromomycin A3 showed

  8. Diagnostic utility of NCOA2 fluorescence in situ hybridization and Stat6 immunohistochemistry staining for soft tissue angiofibroma and morphologically similar fibrovascular tumors.

    PubMed

    Sugita, Shintaro; Aoyama, Tomoyuki; Kondo, Kei; Keira, Yoshiko; Ogino, Jiro; Nakanishi, Katsuya; Kaya, Mitsunori; Emori, Makoto; Tsukahara, Tomohide; Nakajima, Hisaya; Takagi, Masayuki; Hasegawa, Tadashi

    2014-08-01

    Soft tissue angiofibroma (STA), a recently suggested new histologic entity, is a benign fibrovascular soft tissue tumor composed of bland spindle-shaped tumor cells with abundant collagenous to myxoid stroma and branching small vessels. The lesion has a characteristic AHRR-NCOA2 fusion gene derived from chromosomal translocation of t(5;8)(p15;q13). However, morphologically similar tumors containing abundant fibrovascular and myxoid stroma can complicate diagnosis. We designed an original DNA probe for detecting NCOA2 split signals on fluorescence in situ hybridization (FISH) and estimated its utility with 20 fibrovascular tumors: 4 each of STAs, solitary fibrous tumors (SFTs), and cellular angiofibromas and 3 each of low-grade myxofibrosarcomas, myxoid liposarcomas, and low-grade fibromyxoid sarcomas. We also performed FISH for 13q14 deletion and immunohistochemistry (IHC) staining for estrogen receptor, progesterone receptor, retinoblastoma protein, and MUC-4 expression. Furthermore, IHC for Stat6 was conducted in the 20 cases analyzed by FISH and in an additional 26 SFTs. We found moderate to strong nuclear Stat6 expression in all SFTs but no expression in the other tumors. Both estrogen receptor and progesterone receptor expressions were observed in STAs, SFTs, and cellular angiofibromas. Expression of retinoblastoma protein was found in less than 10% of cells in all tumor types except myxoid liposarcoma. The low-grade fibromyxoid sarcomas were strongly positive for MUC-4. All STAs showed NCOA2 split signals on FISH. All tumors, regardless of histologic type, had 13q14 deletion. The NCOA2 FISH technique is a practical method for confirming STA diagnosis. The combination of NCOA2 FISH and Stat6 IHC proved effective for the differential diagnosis of STA, even when using small biopsy specimens. PMID:24856853

  9. Detection of Escherichia coli O157 by Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA-FISH) and Comparison to a Standard Culture Method

    PubMed Central

    Almeida, C.; Sousa, J. M.; Rocha, R.; Cerqueira, L.; Fanning, S.; Azevedo, N. F.

    2013-01-01

    Despite the emergence of non-O157 Shiga toxin-producing Escherichia coli (STEC) infections, E. coli serotype O157 is still the most commonly identified STEC in the world. It causes high morbidity and mortality and has been responsible for a number of outbreaks in many parts of the world. Various methods have been developed to detect this particular serotype, but standard bacteriological methods remain the gold standard. Here, we propose a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the rapid detection of E. coli O157. Testing on 54 representative strains showed that the PNA probe is highly sensitive and specific to E. coli O157. The method then was optimized for detection in food samples. Ground beef and unpasteurized milk samples were artificially contaminated with E. coli O157 concentrations ranging from 1 × 10?2 to 1 × 102 CFU per 25 g or ml of food. Samples were then preenriched and analyzed by both the traditional bacteriological method (ISO 16654:2001) and PNA-FISH. The PNA-FISH method performed well in both types of food matrices with a detection limit of 1 CFU/25 g or ml of food samples. Tests on 60 food samples have shown a specificity value of 100% (95% confidence interval [CI], 82.83 to 100), a sensitivity of 97.22% (95% CI, 83.79 to 99.85%), and an accuracy of 98.33% (CI 95%, 83.41 to 99.91%). Results indicate that PNA-FISH performed as well as the traditional culture methods and can reduce the diagnosis time to 1 day. PMID:23934486

  10. Comparative study of ProEx C immunocytochemistry and UroVysion fluorescent in-situ hybridization assays on urine cytology specimens

    PubMed Central

    Chang, Sue; Smith, Elaine; Levin, Mary; Rao, Jian-Yu; Moatamed, Neda A.

    2015-01-01

    Background: Detection of urothelial carcinoma (UC) by urine cytology can be challenging. Recently, ProEx C has been studied as a marker to improve detection of UC. ProEx C is an assay targeting expression of topoisomerase IIa and minichromosome maintenance protein-2 and is currently utilized to assist in diagnoses of the gynecological specimens. In this study, we compared the utility of ProEx C and UroVysion in urine specimens. Materials and Methods: Twenty-seven urine specimens with UroVysion assay analysis and surgical biopsy follow-up were selected. The smears were stained with ProEx C. ProEx C and UroVysion assay results were separated into two categories based on surgical biopsy follow-up (benign or neoplastic). Surgical biopsy diagnoses were used as the gold standard for comparative evaluation of the two assays. The surgical follow-up was 9 benign, 2 low grade, and 16 high grade UCs. Results: The sensitivity was 88.9% for ProEx C and 55.6% for UroVysion, while the specificity was 77.8% for ProEx C and 44.4% for UroVysion. Positive predictive value was 88.9% for ProEx C and 66.7% for UroVysion. Negative predictive value was 77.8% and 33.3% for ProEx C and UroVysion, respectively. Using the two-tailed paired t-test, P value of 0.033 was obtained when ProEx C stain was compared with the UroVysion assay. Conclusion: ProEx C immunocytochemistry has a more favorable performance than fluorescent in-situ hybridization with a significant difference between the two assays using paired two-tail t-test (P = 0.0033). PMID:25685171

  11. Targeting the Treponemal Microbiome of Digital Dermatitis Infections by High-Resolution Phylogenetic Analyses and Comparison with Fluorescent In Situ Hybridization

    PubMed Central

    Foix Bretó, Antoni; Boye, Mette; Jensen, Tim K.

    2013-01-01

    Modern pyrosequencing technology allows for a more comprehensive approach than traditional Sanger sequencing for elucidating the etiology of bovine digital dermatitis. We sought to describe the composition and diversity of treponemes in digital dermatitis lesions by using deep sequencing of the V3 and V4 hypervariable regions of the 16S rRNA gene coupled with species-level taxonomic identification. Treponema-specific 16S rRNA gene PCRs and pyrosequencing were performed on biopsy specimens originating from 10 different Catalan dairy herds (n = 36) with digital dermatitis, and this analysis yielded 75,297 sequences. We identified 20 different taxa, including a potentially novel phylotype that displayed 95% sequence identity to members of the Treponema denticola/Treponema pedis-like cluster. Species frequencies and abundances that were determined by pyrosequencing analysis were highly correlated with the results of fluorescent in situ hybridization using phylotype-specific oligonucleotide probes. In a limited number of animals from a single geographic region, we detected most of the Treponema phylotypes that were described in previous investigations of digital dermatitis. Additionally, we identified a number of phylotypes that mapped to oral treponemes of humans and dogs that had not been reported for digital dermatitis lesions. The results presented here support previous observations of a polytreponemal etiology of infections, with Treponema phagedenis-like, Treponema medium/Treponema vincentii-like, and T. denticola/T. pedis-like phylotypes being highly associated with disease. Using this new approach, it has become feasible to study large herds and their surrounding environments, which might provide a basis for a better understanding of the pathogenesis of this disease. PMID:23658264

  12. Open L-lactic acid fermentation of food refuse using thermophilic Bacillus coagulans and fluorescence in situ hybridization analysis of microflora.

    PubMed

    Sakai, Kenji; Ezaki, Yutaka

    2006-06-01

    In the production of commercially useful poly-L-lactic acid plastic from biomass wastes, a feasible fermentation process to produce optically active L-lactic acid would be required. Here, model kitchen refuse (MKR) was inoculated with Bacillus coagulans NBRC12583 under nonsterilized openculture conditions. At temperatures below 45 degrees C, a racemic mixture of D- and L-lactic acids was accumulated, whereas only L-lactic acid was selectively accumulated by incubation at 50-65 degrees C. At 45 degrees C, the results of fermentation could not be consistently reproduced. To analyze microflora in this type of mixed culture system, whole-cell fluorescence in situ hybridization (FISH) using 16S rRNA-targeted oligonucleotide probes for B. coagulans, Bcoa191, and LAC722(L), a group-specific probe for a wide range of mesophilic lactic acid bacteria was applied. The dominancy of mesophilic lactic acid bacteria at lower temperatures, and that of B. coagulans at higher temperatures were confirmed. By using a saccharified liquid of collected kitchen refuse, 86 g/l of L-lactic acid was accumulated under nonsterile conditions by a 5-d incubation at 55 degrees C, pH 6.5, with 53% carbon yield and 97% optical purity. To conclude, high temperature open lactic acid fermentation is a simple and promising method for producing high-grade L-lactic acid from biomass waste, and FISH analysis of such mixed-culture systems is helpful for monitoring the microflora in these cultures. PMID:16935246

  13. Genomic In Situ Hybridization (GISH) as a Tool to Identify Chromosomes of Parental Species in Sunflower Interspecific Hybrids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Interspecific hybridization has been widely used to transfer genes from wild species into cultivated sunflower. Fluorescent genomic in situ hybridization (GISH) has been used to identify alien chromosomes or segments in other crops, but an equivalent technique for sunflower is lacking. The objective...

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

    SciTech Connect

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

    1987-05-01

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

  15. Phylogenetic Analysis and Fluorescence In Situ Hybridization Detection of Archaeal and Bacterial Endosymbionts in the Anaerobic Ciliate Trimyema Compressum

    Microsoft Academic Search

    Naoya Shinzato; Ichiro Watanabe; Xian-Ying Meng; Yuji Sekiguchi; Hideyuki Tamaki; Toru Matsui; Yoichi Kamagata

    2007-01-01

    The anaerobic free-living ciliate, Trimyema compressum, is known to harbor both methanogenic archaeal and bacterial symbionts in the cytoplasm. To clarify their phylogenetic belongings,\\u000a a full-cycle rRNA approach was applied to this symbiosis. Phylogenetic analysis showed that the methanogenic symbiont was\\u000a related to Methanobrevibacter arboriphilicus, which was distantly related to symbionts found in other Trimyema species. This result suggested that

  16. Fluorescence in situ hybridization-based detection of Salmonella spp. and Listeria monocytogenes in complex food matrices

    Microsoft Academic Search

    Bledar Bisha

    2009-01-01

    Current methods for detection of Salmonella spp. and Listeria monocytogenes in food are culture-based methods and require performing numerous steps, between preenrichment, enrichment, selective plating, identification, and confirmation. Conducting these procedures can take several days; they require extensive manual labor and large amounts of media and reagents which can increase the cost of the testing. Molecular-based rapid high throughput methods

  17. Fluorescence in situ hybridization probes targeting members of the phylum Candidatus?Saccharibacteria falsely target Eikelboom type 1851 filaments and other Chloroflexi members.

    PubMed

    Nittami, Tadashi; Speirs, Lachlan B M; Fukuda, Junji; Watanabe, Masatoshi; Seviour, Robert J

    2014-12-01

    The FISH probe TM7-305 is thought to target the filamentous Eikelboom morphotype 0041 as a member of the Candidatus ‘Saccharibacteria’ (formerly TM7) phylum. However, with activated sludge samples in both Japan and Australia, this probe hybridized consistently with filamentous bacteria fitting the description of the morphotype 1851, which also responded positively to the CHL1851 FISH probe designed to target Chloroflexi members of this morphotype. 16S rRNA clone libraries from samples containing type 1851 TM7-305-positive filaments yielded Chloroflexi clones with high sequence similarity to Kouleothrix aurantiaca. These contained a variant TM7-305 probe target site possessing weakly destabilizing mismatches insufficient to prevent probe hybridization. Furthermore, the TM7-905 FISH probe, designed to target members of the entire Candidatus ‘Saccharibacteria’ phylum, also hybridized with the filament morphotypes 0041/0675, which responded also to the phylum level Chloroflexi probes. Many Chloroflexi sequences have only a single base mismatch to the TM7-905 probe target sequence. When competitor probes for both the TM7-305 and TM7-905 Chloroflexi non-target sites were applied, no fluorescent signal was seen in any of the filamentous organisms also hybridizing with the aforementioned Chloroflexi probes. These data indicate that these competitor probes must be included in hybridizations when both the TM7-905 and TM7-305 FISH probes are applied, to minimize potential false positive FISH results. PMID:25756114

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

    PubMed Central

    Robertson, Kelly L.; Vora, Gary J.

    2012-01-01

    Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6. Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5. Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2). PMID:22258228

  19. FLUORESCENT IN SITU DETECTION OF ENCEPHALITOZOON HELLEM SPORES WITH A 6-CARBOXYFLUORESCEIN-LABELED RNA-TARGETED OLIGONUCLEOTIDE PROBE

    EPA Science Inventory

    A fluorescent in situ hybridization assay has been developed for the detection of the human-pathogenic microsporidian, Encephalitozoon hellem, in water samples using epifluorescence microscopy. The assay employs a 19-nucleotide species-specific 6-carboxyfluorescein-labeled oligo...

  20. Spatial Preservation of Nuclear Chromatin Architecture during Three-Dimensional Fluorescence in Situ Hybridization (3D-FISH)

    Microsoft Academic Search

    Irina Solovei; Antonio Cavallo; Lothar Schermelleh; Françoise Jaunin; Catia Scasselati; Dusan Cmarko; Christoph Cremer; Stanislav Fakan; Thomas Cremer

    2002-01-01

    3D-FISH has become a major tool for studying the higher order chromatin organization in the cell nucleus. It is not clear, however, to what extent chromatin arrangement in the nucleus after fixation and 3D-FISH still reflects the order in living cells. To study this question, we compared higher order chromatin arrangements in living cells with those found after the 3D-FISH

  1. Cytomolecular characterization of rRNA gene sequences among Citrullus species and subspecies using fluorescent in situ hybridization (FISH) technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In previous studies, many DNA markers showed strong preferential (non-Mendelian) segregation in F2 and BC1 genetic populations derived from crosses between wild type watermelon [C. lanatus (Thunb.) Matsum. et Nakai subsp. lanatus var. citroides (Bailey) Mansf. ex Greb.] (CLC) and watermelon cultivar...

  2. Miller-Dieker syndrome associated with duplication of 17p13.3 confirmed by fluorescence in situ hybridization (FISH)

    SciTech Connect

    Li, S.; Tuck-Muller, C.M.; Martinez, J.E. [Univ. of South Alabama, Mobile, AL (United States)] [and others

    1994-09-01

    Miller-Dieker syndrome is characterized by profound mental retardation, craniofacial abnormalities, and lissencephaly (smooth brain). Microscopic or submicroscopic deletions of the 17p13.3 region have been reported in Miller-Dieker patients. We report a patient with this syndrome in whom a duplication of the 17p13.3 region was detected by FISH. The 9-year-old female proband was referred because of features of Miller-Dieker syndrome: microcephaly, profound psychomotor retardation, seizures, characteristic facies, and lissencephaly shown by MRI studies. High-resolution G-banding failed to demonstrate an abnormality in chromosome 17. However, FISH analysis with the DNA probe (Oncor No. 5101) specific for Miller-Dieker region of chromosome 17p13.3 demonstrated duplication of this segment instead of the classic deletion. We know of no other report of Miller-Dieker syndrome associated with duplication of 17p13.3. The family study revealed normal chromosomes in both parents by cytogenetic and FISH analysis. Our investigation suggests that duplications, as well as deletions, of the 17p13.3 region are associated with the Miller-Dieker syndrome. The presence of deletions or duplications of the same chromosomal region in patients with features of Miller-Dieker syndrome suggests that its pathogenesis may be due to gene dosage effects.

  3. Human cDNA mapping using fluorescence in situ hybridization. Final progress report, April 1, 1994--July 31, 1997

    SciTech Connect

    Korenberg, J.R.

    1997-12-31

    The ultimate goal of this research is to generate and apply novel technologies to speed completion and integration of the human genome map and sequence with biomedical problems. To do this, techniques were developed and genome-wide resources generated. This includes a genome-wide Mapped and Integrated BAC/PAC Resource that has been used for gene finding, map completion and anchoring, breakpoint definition and sequencing. In the last period of the grant, the Human Mapped BAC/PAC Resource was also applied to determine regions of human variation and to develop a novel paradigm of primate evolution through to humans. Further, in order to more rapidly evaluate animal models of human disease, a BAC Map of the mouse was generated in collaboration with the MTI Genome Center, Dr. Bruce Birren.

  4. Differentiation of Candida albicans and Candida dubliniensis by Fluorescent In Situ Hybridization with Peptide Nucleic Acid Probes

    Microsoft Academic Search

    KENNETH OLIVEIRA; GERHARD HAASE; CLETUS KURTZMAN; JENS JO; HENRIK STENDER; Boston Probes

    2001-01-01

    The recent discovery of Candida dubliniensis as a separate species that traditionally has been identified as Candida albicans has led to the development of a variety of biochemical and molecular methods for the differentiation of these two pathogenic yeasts. rRNA sequences are well-established phylogenetic markers, and probes targeting species-specific rRNA sequences have been used in diagnostic assays for the detection

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

    PubMed Central

    Tinawi-Aljundi, Rima; King, Lauren; Knuth, Shannon T; Gildea, Michael; Ng, Carrie; Kahl, Josh; Dion, Jacqueline; Young, Chris; Schervish, Edward W; Frontera, J Rene; Hafron, Jason; Kernen, Kenneth M; Di Loreto, Robert; Aurich-Costa, Joan

    2015-01-01

    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% sensitivity for high-grade tumors (95% CI: 92.5%–100%) and 87.5% sensitivity (95% CI: 68.8%–95.5%) for low-grade tumors. All the clinical parameters for the OligoFISH panel were higher than the UroVysion test’s published performance. We found significantly higher clinical sensitivity and NPV at initial diagnosis and significantly higher specificity and PPV for recurrence. Conclusion The OligoFISH probe panel is a fast, easy, and reproducible test for bladder cancer diagnosis and monitoring, with excellent clinical performance and utility.

  6. Novel, rapid DNA-based on-chip bacterial identification system combining dielectrophoresis and amplification-free fluorescent resonance energy transfer assisted in-situ hybridization (FRET-ISH)

    NASA Astrophysics Data System (ADS)

    Packard, Michelle M.; Shusteff, Maxim; Alocilja, Evangelyn

    2011-10-01

    Although real-time PCR (RT-PCR) has become a diagnostic standard for rapid identification of bacterial species, typical methods remain time-intensive due to sample preparation and amplification cycle times. The assay described in this work incorporates on-chip dielectrophoretic capture and concentration of bacterial cells, thermal lysis, cell permeabilization, and nucleic acid denaturation and fluorescence resonance energy transfer assisted in-situ hybridization (FRET-ISH) species identification. Identification is achieved completely on chip in less than thirty minutes from receipt of sample compared to multiple hours required by traditional RT-PCR and its requisite sample preparation.

  7. Le diagnostic anténatal de la trisomie 21 par l'hybridation in situ en fluorescence (FISH): à propos des premiers tests réalisés au Maroc

    PubMed Central

    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 une faible quantité de liquide amniotique. Elle offre aux couples l'avantage de prendre, dans des délais raisonnables, la décision qui leur convient concernant la poursuite ou non de la grossesse. Elle permet souvent, avec un résultat normal, de rassurer rapidement les femmes enceintes trop angoissées. PMID:23330029

  8. Assignment of the tyrosinase-related protein-2 gene (TYRP2) to human chromosome 13q31-q32 by fluorescence in situ hybridization: Extended synteny with mouse chromosome 14

    SciTech Connect

    Sturm, R.A. (Univ. of Queensland, Brisbane (Australia)); Baker, E.; Sutherland, G.R. (Centre for Medical Genetics, North Adelaide (Australia))

    1994-05-01

    A recombinant human genomic liver DNA [lambda]-phage library was screened with the insert of the pHuTRP-2 cDNA clone to isolate a series of bacteriophage with inserts spanning the human TYRP2 gene. One of the [lambda]-phage clones ([lambda]HuT-YRP2-7) containing a 2-kb HindIII fragment with the 5[prime] exon sequence of the cDNA as determined by sequence analysis was used for the gene localization study. DNA prepared from the phage by Qiagen chromatography was nick-translated with biotin-14-dATP and hybridized in situ at a final concentration of 5 ng/[mu]l to metaphases from two normal males. The fluorescence in situ hybridization method was modified from that previously described in that chromosomes were stained before analysis with both propidium iodide as counterstain and DAPI for chromosome identification. Twenty metaphases from the first normal male were examined for fluorescent signal. All of these metaphases showed signal on one or both chromatids of chromosome 13 in the region 13q31-q33; 88% of this signal was at the interface of bands 13q31-q32. There was a total of four nonspecific background dots observed in these 20 metaphases. A similar result was obtained from hybridization of the probe to 20 metaphases from the second normal male (data not shown). This region has also been shown to contain the propionyl coenzyme A carboxylase [alpha]-chain gene by in situ hybridization. The localization of the TYRP2 locus to human chromosome 13q31-q32 extends the syntenic region of chromosome 13 with mouse chromosome 14. 15 refs., 1 fig.

  9. In situ identification of bacteria in drinking water and adjoining biofilms by hybridization with 16S and 23S rRNA-directed fluorescent oligonucleotide probes.

    PubMed Central

    Manz, W; Szewzyk, U; Ericsson, P; Amann, R; Schleifer, K H; Stenström, T A

    1993-01-01

    Free-water-phase and surface-associated microorganisms from drinking water were detected and roughly identified by hybridization with fluorescence-labeled oligonucleotide probes complementary to regions of 16S and 23S rRNA characteristic for the domains Bacteria, Archaea, and Eucarya and the beta and gamma subclasses of Proteobacteria. Samples of glass-attached biofilms and plankton were taken from a Robbins device installed in a water distribution system. More than 70% of the surface-associated cells and less than 40% of the planktonic cells visualized by 4',6-diamidino-2-phenylindole staining bound detectable amounts of rRNA-targeted probes. These findings are an indication for higher average rRNA content and consequently higher physiological activity of the attached microbial cells compared with the free-living cells. All detectable cells hybridized with the bacterial probe, whereas no Archaea and no Eucarya cells could be detected. Simultaneous hybridization with probes specific for the beta and gamma subclasses of Proteobacteria revealed that microcolonies already consisted of mixed populations in early stages with fewer than 50 cells. These observations provide further evidence that the coexistence and interaction of bacteria in drinking water biofilms may be an integral part of their growth and survival strategies. Images PMID:8357261

  10. Detection of in-situ hybridization to human metaphase chromosomes by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Okamoto, Naoaki; Ishikawa, Mitsuru

    2000-04-01

    Detection of in situ hybridization to human metaphase chromosomes provides important information about gene mappings and about analysis of chromosomal disorders. We applied atomic force microscopy (AFM) to the detection of in situ hybridization to get better resolution as compared to light microscopy. Chromosomes were spread over a glass substrate and hybridized with DNA probes labeled with biotin or digoxigenin. The hybridized probes were reacted with streptavidin or anti-digoxigenin antibody, both of which were conjugated with 5-nm gold colloidal particles. We missed direct detection of the conjugated gold colloidal particles by micro-meter scale AFM scanning , but obtained clear topographic difference between the site of hybridization and the chromosome arm with the help of silver enhancement. We thus clearly detected the in situ hybridization using chromosome painting probes, alpha satellite probes, and locus specific gene probes by AFM. The in situ hybridization to DNA fiber was also detected by AFM. The detection of in situ hybridization by AFM has advantages over fluorescence in situ hybridization: no reduction of signal intensity under light irradiation. Application of AFM to the detection of in situ hybridization will be a useful method to analyze chromosomes.

  11. Identification of new winter wheat - winter barley addition lines (6HS and 7H) using fluorescence in situ hybridization and the stability of the whole 'Martonvásári 9 kr1' - 'Igri' addition set.

    PubMed

    Szakács, E; Molnár-Láng, M

    2010-01-01

    A previous paper reported the development of disomic addition lines (2H, 3H, 4H, and 1HS isochromosomic) from hybrids between the winter wheat 'Martonvásári 9 kr1' and the two-rowed winter barley cultivar 'Igri'. The present paper describes the isolation of two new additions, the 7H disomic and 6HS ditelosomic additions, using fluorescence in situ hybridization with the repetitive DNA probes Afa-family and HvT01. The identification of the barley chromosomes in the wheat genome was confirmed with simple sequence repeat markers. The morphological characterization of the new addition lines is also discussed. Studies of the genetic stability of the whole set (2H, 3H, 4H, 7H, 1HS iso, 6HS) of 'Martonvásári 9 kr1' - 'Igri' additions revealed that the most stable disomic additions are 2H and 3H and the most unstable line is the 1HS isochromosomic addition. PMID:20130747

  12. Detection of {open_quotes}cryptic{close_quotes}karyotypic rearrangements in closely related primate species by fluorescence in situ hybridization (FISH) using human subtelomeric DNA probes

    SciTech Connect

    Youngblom, J.J. [California State University-Stanislaus, Turlock, CA (United States); Trask, B.J.; Friedman, C. [Univ. of Washington, Seattle, WA (United States)] [and others

    1994-09-01

    Specific human subtelomeric DNA probes were used to reveal cryptic chromosomal rearrangements that cannot be detected by conventional high resolution cytogenetic techniques, or by chromosomal in situ suppression hybridization using whole chromosome paint analysis. Two cosmids containing different subtelomeric DNA sequences were derived from human chromosome 19 and designated as 7501 and 16432. Cosmid 7501 was hybridized to chromosomes from humans, chimpanzee, gorilla and orangutan. In humans, 7501 consistently labeled chromosomes 3q, 15q, and 19p. Additional chromosomes were labeled in different individuals, indicating a polymorphic distribution of this sequence in the human genome. In contrast, 7501 consistently and strongly labeled only the q arm terminus of chromosome 3 in both chimp and gorilla. The identification of the chromosome was made by two-color FISH analysis using human chromosome 4-specific paint and homologous to human chromosome 4. None of the human subjects showed labeling of chromosome 4 with 7501. This finding suggests that in the course of human evolution, subsequent to the divergence of humans and African apes, a cryptic translocation occurred between the ancestral human chromosome 4 and one or more of the other human chromosomes that now contain this DNA segment. In orangutan, 7501 labeled a single acrocentric chromosome pair, a distinctly different chromosome than that labeled in chimp and gorilla. Comparison of chromosome sites labeled with cosmid 16432 showed the distribution of signals on chromosome 1q arm is the same for humans and chimp, but different in the gorilla. Humans and chimps show distinct labeling on sites 1q terminus and 1q41-42. In gorilla, there is instead a large cluster of intense signal near the terminus of 1q that clearly does not extend all the way to the terminus. A paracentric inversion or an unequal cross-over event may account for the observed difference between these species.

  13. The oxytocin receptor gene (OXTR) localizes to human chromosome 3p25 by fluorescence in situ hybridization and PCR analysis of somatic cell hybrids

    SciTech Connect

    Simmons, C.F. Jr.; Clancy, T.E.; Quan, R. [Children`s Hospital, Boston, MA (United States)] [and others] [Children`s Hospital, Boston, MA (United States); and others

    1995-04-10

    The human oxytocin receptor regulates parturition and myometrial contractility, breast milk let-down, and reproductive behaviors in the mammalian central nervous system. Kimura et al. recently identified a human oxytocin receptor cDNA by means of expression cloning from a human myometrial cDNA library. To elucidate further the molecular mechanisms that regulate oxytocin receptor gene expression and to define the expected Mendelian inheritance of possible human disease states, we must determine the number of genes, their localization, and their organization and structure. We summarize below our data indicating that the human oxytocin receptor gene is localized to 3p25 and exists as a single copy in the haploid genome. 9 refs., 2 figs.

  14. Assignment of the human skeletal muscle alpha actin gene (ACTA1) to 1q42 by fluorescence in situ hybridisation

    Microsoft Academic Search

    P. A. Akkari; H. J. Eyre; S. D. Wilton; D. F. Callen; C. Meredith; L. Kedes; N. G. Laing

    1994-01-01

    The human skeletal muscle alpha actin gene (ACTAl) has previously been localized to 1p21?qter using somatic cell hybrids and a specific probe from the 3’ untranslated region of the gene. Using fluorescence in situ hybridization the localization has been confirmed and the ACTAl gene precisely mapped to 1q42.Copyright © 1994 S. Karger AG, Basel

  15. Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rRNA-targeted probes and its application in fecal samples

    Microsoft Academic Search

    P. S. Langendijk-Geneveaux; Frits Schut; Gijsbert J. Jansen; Gerwin C. Raangs; Ger R. Kamphuis; Michael H. F. Wilkinson; Gjalt W. Welling

    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 V8 of the 16S rRNA contained sequences unique to this genus and proved applicable as target sites for oligodeoxynucleotide probes. Determination of the genus specificity of the oligonucleotides was performed by whole-cell hybridization with fluorescein

  16. Characterization of the temporal persistence of chromosomal abnormalities in the semen of Hodkin`s disease patients after treatment with NOVP chemotherapy using multi-chromosome fluorescence in situ hybridization

    SciTech Connect

    Cassel, M.J.; Robbins, W.A.; Wyrobek, A.J. [Lawrence Livermore National Laboratory, CA (United States); Meistrich, M.L. [Univ. of Texas, Houston, TX (United States)

    1994-12-31

    Three-chromosome fluorescence in situ hybridization (FISH) was applied to sperm of men with Hodgkin`s disease to measure the persistence of chromosomally abnormal sperm within the time interval of 3 to 33 months after the end of treatment. NOVP chemotherapy includes the agents novantrone, oncovin, vinblastine, and prednisone, two of which are spindle poisons expected to induce aneuploidy. Semen samples were evaluated for the frequencies of fluorescence phenotypes representing hyperhaploidy, hypohaploidy, and genomic duplications using DNA probes specific for repetitive sequences on chromosomes X,Y, and 8. Using this procedure, NOVP was previously shown to induce chromosomally abnormal sperm in treated patients. In a longitudinal assessment of 11 semen samples from 2 men, frequencies of abnormal sperm appeared to return to pre-treatment levels at {approximately}6 months after the end of treatment and remained at these levels up to 33 months after the end of treatment. However, pre-treatment frequencies of chromosomally abnormal cells in Hodgkin`s patients were elevated above those found in normal healthy men. Additional patients are being evaluated to determine how long after therapy Hodgkin`s disease patients remain at increased risk for producing chromosomally abnormal sperm.

  17. Fluorescence Photooxidation with Eosin: A Method for High Resolution Immunolocalization and In Situ

    E-print Network

    Tsien, Roger Y.

    Fluorescence Photooxidation with Eosin: A Method for High Resolution Immunolocalization and In Situ of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin

  18. In situ sensitive fluorescence imaging of neurons cultured on a plasmonic dish using fluorescence microscopy.

    PubMed

    Tawa, Keiko; Yasui, Chikara; Hosokawa, Chie; Aota, Hiroyuki; Nishii, Junji

    2014-11-26

    A plasmonic dish was fabricated as a novel cell-culture dish for in situ sensitive imaging applications, in which the cover glass of a glass-bottomed dish was replaced by a grating substrate coated with a film of silver. Neuronal cells were successfully cultured over a period of more than 2 weeks in the plasmonic dish. The fluorescence images of their cells including dendrites were simply observed in situ using a conventional fluorescence microscope. The fluorescence from neuronal cells growing along the dish surface was enhanced using the surface plasmon resonance field. Under an epi-fluorescence microscope and employing a donut-type pinhole, the fluorescence intensity of the neuron dendrites was found to be enhanced efficiently by an order of magnitude compared with that using a conventional glass-bottomed dish. In a transmitted-light fluorescence microscope, the surface-selective fluorescence image of a fine dendrite growing along the dish surface was observed; therefore, the spatial resolution was improved compared with the epi-fluorescence image of the identical dendrite. PMID:25321614

  19. In situ Measurements of Phytoplankton Fluorescence Using Low Cost Electronics

    PubMed Central

    Leeuw, Thomas; Boss, Emmanuel S.; Wright, Dana L.

    2013-01-01

    Chlorophyll a fluorometry has long been used as a method to study phytoplankton in the ocean. In situ fluorometry is used frequently in oceanography to provide depth-resolved estimates of phytoplankton biomass. However, the high price of commercially manufactured in situ fluorometers has made them unavailable to some individuals and institutions. Presented here is an investigation into building an in situ fluorometer using low cost electronics. The goal was to construct an easily reproducible in situ fluorometer from simple and widely available electronic components. The simplicity and modest cost of the sensor makes it valuable to students and professionals alike. Open source sharing of architecture and software will allow students to reconstruct and customize the sensor on a small budget. Research applications that require numerous in situ fluorometers or expendable fluorometers can also benefit from this study. The sensor costs US$150.00 and can be constructed with little to no previous experience. The sensor uses a blue LED to excite chlorophyll a and measures fluorescence using a silicon photodiode. The sensor is controlled by an Arduino microcontroller that also serves as a data logger. PMID:23783738

  20. Heterogeneity and meiotic behaviour of B and sex chromosomes, banding patterns and localization of (TTAGGG)n sequences by fluorescence in situ hybridization in the neotropical water rat Nectomys (Rodentia, Cricetidae).

    PubMed

    de Jesus Silva, M J; Yonenaga-Yassuda, Y

    1998-09-01

    A cytogenetic study using fluorescence in situ hybridization (FISH) of telomere probes, CBG, GTG and RBG banding patterns and synaptonemal complex data was carried out in 41 specimens of Nectomys from three Brazilian states: Pernambuco, Mato Grosso and São Paulo. The specimens presented 2n = 52, 53, 56 and 57, and the differences in chromosome number were due to the presence of three different supernumeraries and also to the occurrence of tandem fusions. The tandem fusions involved chromosome pairs 3 + 11 and 5 + 24 from karyotype with 2n = 56 that originated pairs 1 and 4 in specimens with 2n = 52. Sex chromosome polymorphism was also detected, and the X presented three different morphologies, which could be explained by heterochromatin addition/deletion. FISH results revealed interstitial telomeric bands (ITBs) in a submetacentric B, but no ITB was detected in the chromosomes originated by tandem fusion. The supernumeraries presented a remarkable heterogeneity of size and morphology, constitutive heterochromatin pattern and localization of telomeric sequences. Synaptonemal complex by light and electron microscopy showed the supernumerary as an autopaired univalent. The sex chromosome pairing in meiotic cells involved the heterochromatic short arm of the X chromosome and the short arm of the Y chromosome. PMID:9865784

  1. GeneCARD-FISH: Detection of tceA and vcrA reductive dehalogenase genes in Dehalococcoides mccartyi by fluorescence in situ hybridization.

    PubMed

    Matturro, B; Rossetti, S

    2015-03-01

    Due to the direct involvement in the biodegradation of chlorinated solvents, reductive dehalogenase genes (RDase) are considered biomarkers of the metabolic potential of different strains of Dehalococcoides mccartyi (Dhc). This is known to be the only microbe able to completely reduce toxic chlorinated solvents to harmless ethene. In the last years, several Molecular Biological Tools (MBTs) have been developed to optimize the detectability of Dhc cells and/or the RDase genes, with particular attention to the most important indicators of ethene formation, namely tceA and vcrA genes. Despite qPCR has been indicated as the MBT of choice, the use of CARD-FISH recently demonstrated to provide a more accurate quantification of Dhc cells in a wide concentration range, overcoming the drawbacks of loosing nucleic acids during the preparation of the sample associated with qPCR. CARD-FISH assays usually target 16S rRNA and up to date no protocol able to discriminate different Dhc strains by detecting RDase genes has been developed. This study reports the first evidence of in situ detection of tceA and vcrA genes into Dhc cells by applying a new procedure named geneCARD-FISH. Dhc strains carrying tceA and vcrA genes were identified and quantified in a PCE-to-ethene dechlorinating microbial enrichment and overall they represented 58.63%±2.45% and 40.46%±1.86% of the total Dhc cells, respectively. These values were markedly higher than those obtained by qPCR, which strongly underestimated the actual concentration of vcrA gene (0.08%±0.01% of Dhc 16S rRNA gene copies). The assay was successfully applied also for the analysis of environmental samples and remarkably strengthens the biomonitoring activities at field scale by providing the specific in situ discrimination of Dhc cells carrying the key-RDase genes. PMID:25595619

  2. Evaluation of the genotoxic activity of paclitaxel by the in vitro micronucleus test in combination with fluorescent in situ hybridization of a DNA centromeric probe and the alkaline single cell gel electrophoresis technique (comet assay) in human T-lymphocytes.

    PubMed

    Digue, L; Orsière, T; De Méo, M; Mattéi, M G; Depetris, D; Duffaud, F; Favre, R; Botta, A

    1999-01-01

    Paclitaxel is a recent chemotherapeutic agent that inhibits tubulin depolymerization in tumoral cells. Despite its increasing use against various human cancers, the genotoxicity of paclitaxel has never been studied on normal human cells. The in vitro genotoxic effects of the drug were evaluated with two complementary mutagenesis tests on human T-lymphocytes: (1) the cytokinesis-blocked micronuclei assay (CBMN) in combination with fluorescent in situ hybridization (FISH) of nonspecific centromeric probes and (2) the comet assay performed in three ways: on stimulated lymphocytes as in the CBMN, and on freshly isolated lymphocytes at both 4 and 37 degrees C. A slight cytotoxicity of 2.5 to 10 nM paclitaxel was found in the CBMN and a significant increase in the binucleated micronucleated cell rates was observed, with a concentration-dependent manner. In the FISH analysis, more than 85% of the micronuclei (MN) were centromere positive, and a ratio of 72. 2 to 78.6% of these MN contained more than one centromere. Moreover, at 10 nM of paclitaxel, 35.6% of the cells are multimicronucleated lymphocytes. Unexpectedly, paclitaxel induced single-strand breaks on proliferating lymphocytes at 5 and 7.5 nM but not in resting cells, even at 5 to 15 microM. These in vitro results showed that (1) paclitaxel does not present any direct DNA action in resting cells, (2) DNA damage detected in stimulated lymphocytes may be linked either to a high frequency of cells in the S-phase cell cycle or to a direct DNA damaging effect on replicating cells, and (3) paclitaxel is a strong in vitro aneugenic drug on human normal cells, at clinically relevant concentrations. PMID:10618175

  3. Interstitial deletion of chromosome 1q [del(1)(q24q25.3)] identified by fluorescence in situ hybridization and gene dosage analysis of apolipoprotein A-II, coagulation factor V, and antithrombin III

    SciTech Connect

    Takano, Takako; Yamanouchi, Yasuko; Mori, Yosuke [Teikyo Univ. School of Medicine, Tokyo (Japan)] [and others] [Teikyo Univ. School of Medicine, Tokyo (Japan); and others

    1997-01-20

    We report on a 12-month-old Japanese boy with an interstitial deletion of the long-arm of chromosome 1 and meningomyelocele, hydrocephalus, anal atresia, atrial septal defect, left renal agenesis, bilateral cryptorchidism, talipes equinovarus, low birth weight, growth/developmental retardation, and many minor anomalies. By conventional GTG-banding, his karyotype was first interpreted as 46,XY,de1(1)(q23q24), but it was corrected as 46,XY.ish del(1)(q24q25.3) by fluorescence in situ hybridization using 11 known cosmid clones as probes. His serum levels of apolipoprotein A-II (gene symbol: APOA2, previously assigned to 1q21-q23) and coagulation factor V (F5, 1q21-q25) were normal, while serum concentration and activity of antithrombin III (AT3, 1q23-q25.1) was low. The results indicated that localization of APOA2 and F5 are proximal to the deleted region and AT3 is located within the deletion extent in the patient. 16 refs., 4 figs.

  4. Detection of mixed chimerism and leukemic relapse after allogeneic bone marrow transplantation in subpopulations of leucocytes by fluorescent in situ hybridization in combination with the simultaneous immunophenotypic analysis of interphase cells.

    PubMed

    Kögler, G; Wolf, H H; Heyll, A; Arkesteijn, G; Wernet, P

    1995-01-01

    Serial blood and marrow specimens from eight adult recipients of sex-mismatched transplants (BMT) for chronic myeloid leukemia (CML, n = 3), Ewing sarcoma (n = 1), acute myeloid leukemia (AML) in second remission (n = 1), acute lymphatic leukemia (ALL, n = 1) and multiple myeloma (n = 2) were analyzed by the simultaneous immunophenotypic CD3, CD4, CD8, CD20, CD34, CD10 and genotypic analysis (for X and Y chromosomes). This combined technique of moAb/APAAP staining for cell surface and cytoplasmic antigens and fluorescence in situ hybridization (FISH) for the detection of sex chromosomes allowed the qualitative and quantitative evaluation of mixed chimerism and/or relapse. Using the same slides for moAb/APAAP and FISH allowed the simultaneous identification of the cell lineage, the lymphocyte subpopulation and the genotype (XX or YX) in every blood or BM specimen analyzed. A mixed chimerism in the T cell (CD4, CD8+: median 26% host cells, range 5-44%) and in the myelomonocytic cell population (CD14+ median 16% host cells, range 5-50%) was observed at day +7 after BMT. By days +14 to +18 this mixed chimerism was reduced to 18% host T cells (range 5-50%) and 7% host myelomonocytic cells (range 0-20%). Beyond days +21 to +28 a stable donor chimerism for T cells, myelomonocytic cells and granulocytes was observed in seven of eight patients. Still 0.5-1% host cells of different lineages were detectable in five from the eight patients at later time points (> day + 100). In three patients with CML these cells were CD13 or CD13, CD34 positive and in one was CD4, CD8 positive.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7742754

  5. Applications of Strand-Specific in situ Hybridization

    SciTech Connect

    Goodwin, E.H.; Meyne, J.; Bailey, S.M.; Quigley, D.; Smith, L.; Tennyson, R.

    1997-01-01

    This is the final report of a three-year, Laboratory-Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). Fluorescence in situ hybridization (FISH) is used to determine the location of specific DNA sequences on chromosomes. It is an effective tool in genomic mapping and is finding increasing use in medical diagnosis. A ''strand-specific'' version of FISH has been developed in the Life Sciences Division of LANL. The new procedure, named CO-FISH, reveals not only location but also the 5'-to-3'direction of a target sequence, such as the sense strand of a gene. This project was designed to investigate applications of the new technique. Strand-specific FISH was found to be useful and informative for genomic mapping of repetitive DNA sequences. The method provide a valuable new tool for investigating the mechanisms of aneuploidy inducing agents and the cytogenetic phenomena called lateral asymmetry. Finally, using strand-specific FISH, the authors were able to detect certain types of chromosome aberrations (isochromosomes, inversions and Robertsonian translocations) that can be difficult to observe with standard techniques.

  6. Cytogenetic Analysis Using Quantitative, High-Sensitivity, Fluorescence Hybridization

    Microsoft Academic Search

    D. Pinkel; T. Straume; J. W. Gray

    1986-01-01

    This report describes the use of fluorescence in situ hybridization for chromosome classification and detection of chromosome aberrations. Biotin-labeled DNA was hybridized to target chromosomes and subsequently rendered fluorescent by successive treatments with fluorescein-labeled avidin and biotinylated anti-avidin antibody. Human chromosomes in human-hamster hybrid cell lines were intensely and uniformly stained in metaphase spreads and interphase nuclei when human genomic

  7. A small supernumerary marker chromosome X identified by in situ hybridization.

    PubMed

    Silahtaroglu, A N; Hacihanefioglu, S; Yilmaz, S; Tarkan, Y; Cenani, A; Tümer, Z

    1995-05-01

    Cytogenetic analysis of a girl with moderate mental retardation and dysmorphic features revealed a 46,XX/47,XX,+mar karyotype. Fluorescence in situ hybridization using chromosome specific alpha satellite probes showed that the supernumerary marker originated from the X chromosome. To our knowledge, this is the first reported case of a female patient mosaic for a supernumerary small marker chromosome derived from X, and hence mosaic for trisomy of the pericentric region of the X chromosome. PMID:7554355

  8. Comparative Cytogenetics Analysis of Chlamys farreri, Patinopecten yessoensis, and Argopecten irradians with C0t-1 DNA by Fluorescence In Situ Hybridization

    PubMed Central

    Hu, Li-Ping; Shang, Wen-Cong; Sun, Yan; Wang, Shan; Ren, Xiao-Liang; Huang, Xiao-Ting; Bao, Zhen-Min

    2011-01-01

    The chromosomes of Chlamys farreri, Patinopecten yessoensis, and Argopecten irradians were studied by FISH using C. farreri C0t-1 DNA probes. The results showed that C0t-1 DNA signals spread on all chromosomes in the three scallops, whereas signal density and intensity were different strikingly. Clustering brighter signals presented in the centromeric and telomeric regions of most C. farreri chromosomes, and in the centromeric or pericentromeric regions of several P. yessoensis chromosomes. Comparative analysis of the mapping indicated a relatively higher homology in the repetitive DNA sequences of the genome between C. farreri and P. yessoensis than that between C. farreri and A. irradians. In addition, FISH showed that the distribution of C0t-1 DNA clustering signals in C. farreri displayed completely similar signal bands between homologous chromosomes. Based on the C0t-1 DNA fluorescent bands, a more exact karyotype of C. farreri has been obtained. In this study, the comparative analysis based on C0t-1 DNA provides a new insight into the chromosomal reconstructions during the evolution process, and it is helpful for understanding an important source of genomic diversity, species relationships, and genome evolution. PMID:21845202

  9. Chromosome mapping of 18S rDNA and 5S rDNA by dual-color fluorescence in situ hybridization in the half-smooth tongue sole (Cynoglossus semilaevis).

    PubMed

    Jiang, L; Jiang, J; Liu, J; Yuan, J; Chen, Y; Zhang, Q; Wang, X

    2014-01-01

    Half-smooth tongue sole (Cynoglossus semilaevis) is an important aquaculture flatfish in China. Cytogenetic analysis has revealed that its sex determination system is female heterogametic (ZZ/ZW). The W chromosome is morphologically larger and has been considered evolutionarily younger than any other chromosome in the set. However, the genetic origin and evolution process of this neo-chromosome remains unclear. In this study, 2 tandem arrays of rRNA genes were chosen to address this question. Both the major rDNA (18S rDNA) and the minor rDNA (5S rDNA) were located on the C. semilaevis chromosomes by fluorescence in situ hybridization (FISH). Six 18S rDNA signals were observed on the centromeric regions of 3 pairs of autosomes in both males and females. In females, there was an additional 18S rDNA signal mapping to the telomeric region of the W chromosome long arm. With respect to the 5S rDNA, 12 signals were mapped to the centromeric regions of six pairs of autosomes. Two-color FISH further confirmed that the two pairs of the 5S rDNA signals were correspondingly located at the same positions of the same autosomes as those of the 18S rDNA signals. These results allowed us to speculate about the evolution process of the W chromosome. Chromosome fusions and repetitive sequence accumulations might have occurred in C. semilaevis. The synteny and non-synteny of C. semilaevis 18S rDNA and 5S rDNA might imply the original and evolutionary characteristics of this species. These findings will facilitate studies on karyotype evolution of the order Pleuronectiformes. PMID:25526196

  10. Comparison of a multiplex reverse transcriptase-polymerase chain reaction for BCR-ABL to fluorescence in situ hybridization, Southern blotting, and conventional cytogenetics in the monitoring of patients with Ph1-positive leukemias.

    PubMed

    Colleoni, G W; Jhanwar, S C; Ladanyi, M; Chen, B

    2000-12-01

    A multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) assay for both major forms of BCR-ABL was compared with fluorescence in situ hybridization (FISH), karyotyping, and Southern blotting for disease monitoring in 37 follow-up bone marrow samples from 32 patients with Ph1-positive leukemia. Of these 37 samples, 33 were from patients with chronic myeloid leukemia (CML) (26 post allogeneic bone marrow transplantation [AlloBMT] and seven during interferon-alpha therapy) and 4 from Ph1-positive acute lymphoblastic leukemia (ALL) patients (1 post AlloBMT and 3 post high dose chemotherapy). For the 27 samples studied after AlloBMT (26 CML and 1 Ph1-positive ALL) the time after transplantation ranged from 1 to 107 months (median 47.5 months). In 8 (22%) of the 37 samples there were discrepant results among methods. The discrepancy rates relative to other techniques were: karyotyping 17% (5 of 29), Southern blotting 18% (6 of 33), multiplex RT-PCR 8% (3 of 37), and FISH 8% (3 of 37). Therefore, the relative accuracy of each method for disease monitoring in Ph1-positive leukemia was: 83% (24 of 29) for karyotyping, 82% (27 of 33) for Southern blotting, 92% (34 of 37) for FISH, and 92% (34 of 37) for multiplex RT-PCR. This multiplex RT-PCR assay appears equivalent to FISH in terms of accuracy, simplicity, and turnaround time and both are superior to Southern blot and conventional cytogenetics in the laboratory monitoring of Ph1-positive leukemias. PMID:11129444

  11. The detection of HIV by in situ hybridization

    SciTech Connect

    Shapshak, P.; Sun, N.C.; Resnick, L.; Hsu, M.Y.; Tourtellotte, W.W.; Schmid, P.; Conrad, A.; Fiala, M.; Imagawa, D.T. (Mount Sinai Medical Center, Miami Beach, FL (USA))

    1990-03-01

    A simplified method of in situ hybridization is described for the detection of HIV targets. This standardized method can be applied to sections of formalin-fixed paraffin-embedded tissues, cell blocks, and smears of cultured cells using {sup 3}H- or {sup 35}S-labeled DNA and {sup 35}S-labeled RNA probes. In order to use elevated stringencies in the hybridization and wash steps, tissue sections and cells are covalently bonded to silanated glass slides without their subsequent loss from the slides. Routine hematoxylin and eosin or Romanovsky's stains enable the identification of the cells detected by in situ hybridization. HIV-infected neuroblastoma and lymphoid cell lines, lymph nodes, bone marrow, kidney, as well as brain tissue from patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex are used to demonstrate the generality of the procedure. The standardized method described widens the ease and applicability of in situ hybridization in the investigation of pathologic tissues with the use of diverse specimens and probes.

  12. Nanogold In Situ Hybridization for Phylogenetic Identification in Geologic Samples Using Environmental Scanning Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Ehrhardt, C.; Haymon, R.; Sievert, S.; Holden, P.

    2006-12-01

    Collecting phylogenetic information simultaneously with mineral textures and associations for geomicrobiological studies has always been a challenge. Recently a new type of nucleotide reporter system has been developed that utilizes small particles of nanogold (1.4 nm) covalently attached to oligonucelotide probes. Due to the small size and electron density of these nanogold reporter molecules, this in situ hybridization technique allows for the phylogenetic identification of microbial targets with a scanning electron microscope. Here we present new applications of the nanogold hybridization technique for pure cultures and natural microbial communities in a range of geologic samples including sand grains, basalt chips incubated on deep sea hydrothermal vents, and gypsum crusts sampled from a saline lake. While we do observe nonspecific binding of nanogold probes to minerals and organic compounds in geologic matrices, this can be distinguished from positive hybridization events with a spatial variety analysis. To assess the potential of nanogold hybridizations for quantitative assessments of microbial communities, fluorescent in situ hybridizations (FISH) were performed on all samples and compared to cell counts generated from nanogold hybridizations.

  13. ENHANCING THE IN VITRO AND IN VIVO DETECTION OF ANEUPLOIDY BY FLUORESCENCE IN SITU HYBRIDIZATION WITH THE USE OF BROMODEOXYURIDINE AS A PROLIFERATION MARKER. (R826408)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  14. DETECTION OF HYPERDIPLOIDY IN RAT INTERPHASE HEPATOCYTES FOLLOWING TREATMENT IN VIVO WITH VINBLASTINE SULFATE USING FLUORESCENCE IN SITU HYBRIDIZATION (FISH). (R826408)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  15. Duplication of 10q confirmed by DNA in situ hybridization

    SciTech Connect

    Johnson, V.P.; Sutliff, W.C. [Univ. of South Dakota School of Medicine, Vermillion, SD (United States)

    1994-08-15

    Partial duplication of 10q is a recognizable clinical entity. In most of the reported cases, the trisomic segment is identified by a balanced translocation state in a parent. Verification remains a problem in de novo cases. However, the recent availability of whole chromosome probes allows for confirmatory diagnosis of suspected cases. We describe a case of de novo duplication (10q) with verification using DNA is situ hybridization. 9 refs., 5 figs.

  16. Allelic Loss at SMAD4 in Polyps from Juvenile Polyposis Patients and Use of Fluorescence in Situ Hybridization to Demonstrate Clonal Origin of the Epithelium

    Microsoft Academic Search

    Kelly Woodford-Richens; Jill Williamson; Stephen Bevan; Joanne Young; Barbara Leggett; Ian Frayling; Yi Thway; Shirley Hodgson; Jin Cheon Kim; Takeo Iwama; Marco Novelli; Denise Sheer; Richard Poulsom; Nicholas Wright; Richard Houlston; Ian Tomlinson

    Juvenile polyposis syndrome (JPS; Online Mendelian Inheritance in Man2 174900) is a rare Mendelian disorder in which individuals have typical hamartomatous polyps within the gastrointestinal tract. The stromal element of the polyps has classically been thought to be the proliferative component, although epithelial malignancies (largely gastro- intestinal cancers) occur more frequently than expected in JPS patients. Germ-line mutations in SMAD4

  17. Incidence of diffuse large B-cell lymphoma of germinal center B-cell origin in whole diffuse large B-cell lymphoma: tissue fluorescence in situ hybridization using t(14;18) compared with immunohistochemistry.

    PubMed

    Hirose, Yuko; Masaki, Yasufumi; Karasawa, Hiromi; Shimoyama, Kumiko; Fukushima, Toshihiro; Kawabata, Hiroshi; Ogawa, Noriyoshi; Wano, Yuji; Ozaki, Mamoru

    2005-01-01

    Diffuse large B-cell lymphoma (DLBCL) can be divided into prognostically important categories such as germinal center B (GCB)-like and non-GCB-like groups. The t(14;18)(q32;q21) translocation defines a unique subset of DLBCL cases with a GCB gene expression profile. Two-color fluorescence in situ hybridization (FISH) analysis was applied to detect t(14;18) (q32;q21) in the nuclei of paraffin-embedded tissue sections from 61 patients with de novo DLBCL. Nine (15%) of 61 cases had a positive pattern. Fifty-seven cases were subclassified in an immunohistochemical study with anti-CD10, anti-bcl-6, and anti-MUM1 antibodies. In this classification, 21 cases (37%) were placed in the GCB group, and 36 (63%) were placed in the non-GCB group. There was a discrepancy between t(14;18) occurrence and bcl-2 protein expression. Bcl-2 protein expression was positive in 40 (67%) of 60 cases. The expression of bcl-2 protein in the GCB and non-GCB groups was not significantly different: 15 (71%) of 21 cases in the GCB group and 24 (67%) of 36 cases in the non-GCB group tested positive. We found no difference between the FISH-positive and FISH-negative groups in overall survival time (P = .6019, log-rank test). The overall survival rates of GCB and non-GCB groups did not differ significantly by immunohistochemical classification (P = .5399, log-rank test). Overall survival was significantly longer in the group with a low International Prognostic Index (IPI) score than in the group with a high IPI score (P = .0002, log-rank test). Our results suggest that immunohistochemical study and cytogenetic study with t(14;18) FISH cannot predict the clinical outcomes of DLBCL patients. A study with a larger number of patients may show a difference in clinical outcomes between FISH-positive and FISH-negative groups and between GCB and non-GCB groups. PMID:15717689

  18. DETECTION OF HYPERDIPLOIDY IN BLADDER EPITHELIAL CELLS OF RATS TREATED WITH ORTHO-PHENYLPHENOL USING FLUORESCENCE IN SITU HYBRIDIZATION. (R826408)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  19. Interspecific hybridisation in rhinoceroses: Confirmation of a Black × White rhinoceros hybrid by karyotype, fluorescence in situ hybridisation (FISH) and microsatellite analysis

    Microsoft Academic Search

    T. J. Robinson; V. Trifonov; I. Espie; E. H. Harley

    2005-01-01

    Black and white rhinoceroses are among the most charismatic megaherbivores and have become flagship species for international conservation. They are often subject to intense management that includes being compressed unnaturally in space and density. We present chromosomal and microsatellite evidence to substantiate the first recorded instance of interspecific hybridisation between them. The data suggest that the genetic integrity of the

  20. Variations of Bacterial Populations in Human Feces Measured by Fluorescent In Situ Hybridization with Group-Specific 16S rRNA-Targeted Oligonucleotide Probes

    Microsoft Academic Search

    ALISON H. FRANKS; HERMIE J. M. HARMSEN; GERWIN C. RAANGS; GIJSBERT J. JANSEN; FRITS SCHUT; GJALT W. WELLING

    1998-01-01

    Six 16S rRNA-targeted oligonucleotide probes were designed, validated, and used to quantify predominant groups of anaerobic bacteria in human fecal samples. A set of two probes was specific for species of the Bacteroides fragilis group and the species Bacteroides distasonis. Two others were designed to detect species of the Clostridium histolyticum and the Clostridium lituseburense groups. Another probe was designed

  1. Improved Enumeration of Lactic Acid Bacteria in Mesophilic Dairy Starter Cultures by Using Multiplex Quantitative Real-Time PCR and Flow Cytometry-Fluorescence In Situ Hybridization

    Microsoft Academic Search

    Udo Friedrich; Jan Lenke

    2006-01-01

    Nucleic acid-based assays were developed to enumerate members of the three taxa Lactococcus lactis subsp. cremoris, L. lactis subsp. lactis, and Leuconostoc spp. in mesophilic starter cultures. To our knowledge the present is the first study to present a multiplex quantitative PCR (qPCR) strategy for the relative enumeration of bacteria. The multiplex qPCR strategy was designed to quantify the target

  2. Application of spent sulfidic caustics for autotrophic denitrification in a MLE process and their microbial characteristics by fluorescence in situ hybridization

    Microsoft Academic Search

    Jeung-Jin Park; So-Ra Park; Dong-Jin Ju; Jeong-Keun An; Im-Gyu Byun; Tae-Joo Park

    2008-01-01

    Spent sulfidic caustics (SSCs) produced from petrochemical plants contain a high concentration of hydrogen sulfide and alkalinity,\\u000a and some organic matter. Most of the SSCs are incinerated with the auxiliary fuel causing secondary pollution problems. The\\u000a reuse of this waste is becoming increasingly important in terms of economical and environmental viewpoints. To denitrify wastewater\\u000a with a low COD\\/N ratio, additional

  3. Deletion of 13q14 remains an independent adverse prognostic variable in multiple myeloma despite its frequent detection by interphase fluorescence in situ hybridization

    Microsoft Academic Search

    Niklas Zojer; Robert Konigsberg; Jutta Ackermann; Elke Fritz; Susanne Dallinger; Elisabeth Kromer; Hannes Kaufmann; Lucia Riedl; Heinz Gisslinger; Susanne Schreiber; Renate Heinz; Heinz Ludwig; Heinz Huber; Johannes Drach

    2000-01-01

    have stage III disease (P 5 .022), higher serum levels of b2-microglobulin (P 5 .059), and a higher percentage of bone marrow plasma cells (P 5 .085) than pa- tients with a normal 13q14 status on FISH analysis. In patients with a deletion of 13q14, myeloma cell proliferation (Ki-67) was markedly increased (22.0% 6 6.9% compared with 15.6% 6 8.2%

  4. Quantitative Use of Fluorescent In Situ Hybridization To Examine Relationships between Mycolic AcidContaining Actinomycetes and Foaming in Activated Sludge Plants

    Microsoft Academic Search

    RUSSELL J. DAVENPORT; THOMAS P. CURTIS; MICHAEL GOODFELLOW; FIONA M. STAINSBY; MARC BINGLEY

    2000-01-01

    The formation of viscous foams on aeration basins and secondary clarifiers of activated sludge plants is a common and widespread problem. Foam formation is often attributed to the presence of mycolic acid- containing actinomycetes (mycolata). In order to examine the relationship between the number of mycolata and foam, we developed a group-specific probe targeting the 16S rRNA of the mycolata,

  5. Apparent mosaicism for del(17)(p11.2) ruled out by fluorescence in situ hybridization in a Smith-Magenis syndrome patient

    Microsoft Academic Search

    Ramesh C. Juyal; L. G. Shaffer; J. R. Lupski; F. Greenberg; A. Baldini; P. I. Patel

    1995-01-01

    Smith-Magenis syndrome (SMS) is a multiple congenital anomalies\\/mental retardation syndrome typically associated with a deletion of band p11.2 of human chromosome 17. Finucane et al. reported a 14-year-old boy with mild physical and behavior manifestations of SMS. No evidence for deletion was initially evident in 20 peripheral blood lymphocytes examined at 850 band level of resolution. Examination of metaphase chromosomes

  6. Analysis of HER2 status in breast carcinoma by fully automated HER2 fluorescence in situ hybridization (FISH): comparison of two immunohistochemical tests and manual FISH.

    PubMed

    Yoon, Nara; Do, In-Gu; Cho, Eun Yoon

    2014-09-01

    Easy and accurate HER2 testing is essential when considering the prognostic and predictive significance of HER2 in breast cancer. The use of a fully automated, quantitative FISH assay would be helpful to detect HER2 amplification in breast cancer tissue specimens with reduced inter-laboratory variability. We compared the concordance of HER2 status as assessed by an automated FISH staining system to manual FISH testing. Using 60 formalin-fixed paraffin-embedded breast carcinoma specimens, we assessed HER2 immunoexpression with two antibodies (DAKO HercepTest and CB11). In addition, HER2 status was evaluated with automated FISH using the Leica FISH System for BOND and a manual FISH using the Abbott PathVysion DNA Probe Kit. All but one specimen were successfully stained using both FISH methods. When the data were divided into two groups according to HER2/CEP17 ratio, positive and negative, the results from both the automated and manual FISH techniques were identical for all 59 evaluable specimens. The HER2 and CEP17 copy numbers and HER2/CEP17 ratio showed great agreement between both FISH methods. The automated FISH technique was interpretable with signal intensity similar to those of the manual FISH technique. In contrast with manual FISH, the automated FISH technique showed well-preserved architecture due to low membrane digestion. HER2 immunohistochemistry and FISH results showed substantial significant agreement (? = 1.0, p < 0.001). HER2 status can be reliably determined using a fully automated HER2 FISH system with high concordance to the well-established manual FISH method. Because of stable signal intensity and high staining quality, the automated FISH technique may be more appropriate than manual FISH for routine applications. PMID:24372629

  7. Apparent mosaicism for del(17)(p11.2) ruled out by fluorescence in situ hybridization in a Smith-Magenis syndrome patient

    SciTech Connect

    Juyal, R.C.; Shaffer, L.G.; Lupski, J.R.; Greenberg, F.; Baldini, A.; Patel, P.I. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1995-11-20

    Smith-Magenis syndrome (SMS) is a multiple congenital anomalies/mental retardation syndrome typically associated with a deletion of band p11.2 of human chromosome 17. Finucane et al. reported a 14-year-old boy with mild physical and behavior manifestations of SMS. No evidence for deletion was initially evident in 20 peripheral blood lymphocytes examined at 850 band level of resolution. Examination of metaphase chromosomes of skin fibroblasts showed a deletion of 17p11.2 in 25/25 cells examined which was consistent with the patient`s clinical manifestations of SMS. Subsequent examination of 25 cells from peripheral blood cultures indicated that 11% of cells harbored a deletion at 17p11.2, thus suggesting a mosaicism for the deletion. A third study of 20 peripheral blood lymphocytes examined at 550-850 band length resolution in a different laboratory, indicated that 13 cells had no apparent deletion, 4 cells had an apparent deletion and 3 cells were questionable. 7 refs.

  8. Localization of multiple human dihydrodiol dehydrogenase (DDH1 and DDH2) and chlordecone reductase (CHDR) genes in chromosome 10 by the polymerase chain reaction and fluorescence in situ hybridization

    SciTech Connect

    Khanna, M.; Qin, K.N.; Belkin, S. [Cornell Univ. Medical College, New York, NY (United States)] [and others] [Cornell Univ. Medical College, New York, NY (United States); and others

    1995-01-20

    Multiple human dihydrodiol dehydrogenases and human chlordecone reductase belong to the aldo-keto reductase superfamily. These two enzymes are involved in the metabolism of xenobiotics, such as polycyclic aromatic hydrocarbons and pesticides. Recently we have isolated three closely related genes encoding two dihydrodiol dehydrogenases (DDH1 and DDH2) and the chlordecone reductase (CHDR). Mapping of the location of the genes was performed using the polymerase chain reaction using gene-specific primers to amplify gene sequences in human/hamster hybrid DNA. All three genes were found to be located on chromosome 10. In situ hybridization using a lambda clone as the probe further confirmed regional localization at 10p14-p15. 13 refs., 2 figs.

  9. Assignment of the human neuropeptide Y gene to chromosome 7p15.1 by nonisotopic in situ hybridization

    SciTech Connect

    Baker, E.; Sutherland, G.R. [Women`s and Children`s Hospital, Adelaide (Australia)] [Women`s and Children`s Hospital, Adelaide (Australia); Hort, Y.J. [Garvan Inst. of Medical Research, Sydney (Australia)] [and others] [Garvan Inst. of Medical Research, Sydney (Australia); and others

    1995-03-01

    A 15-kb genomic fragment containing the whole human NPY gene in the vector {lambda}GEM-11 was nick-translated with biotin-14-dATP and hybridized in situ at a final concentration of 5 ng/{mu}l to metaphases from two males. The fluorescence in situ hybridization method was modified from that previously described in that chromosomes were stained before analysis with both propidium iodide (as counterstain) and DAPI (for chromosome identification). Images of metaphase preparations were captured by a CCD camera and computer enhanced.

  10. Accurate Identification of ALK Positive Lung Carcinoma Patients: Novel FDA-Cleared Automated Fluorescence In Situ Hybridization Scanning System and Ultrasensitive Immunohistochemistry

    PubMed Central

    Conde, Esther; Suárez-Gauthier, Ana; Benito, Amparo; Garrido, Pilar; García-Campelo, Rosario; Biscuola, Michele; Paz-Ares, Luis; Hardisson, David; de Castro, Javier; Camacho, M. Carmen; Rodriguez-Abreu, Delvys; Abdulkader, Ihab; Ramirez, Josep; Reguart, Noemí; Salido, Marta; Pijuán, Lara; Arriola, Edurne; Sanz, Julián; Folgueras, Victoria; Villanueva, Noemí; Gómez-Román, Javier; Hidalgo, Manuel; López-Ríos, Fernando

    2014-01-01

    Background Based on the excellent results of the clinical trials with ALK-inhibitors, the importance of accurately identifying ALK positive lung cancer has never been greater. However, there are increasing number of recent publications addressing discordances between FISH and IHC. The controversy is further fuelled by the different regulatory approvals. This situation prompted us to investigate two ALK IHC antibodies (using a novel ultrasensitive detection-amplification kit) and an automated ALK FISH scanning system (FDA-cleared) in a series of non-small cell lung cancer tumor samples. Methods Forty-seven ALK FISH-positive and 56 ALK FISH-negative NSCLC samples were studied. All specimens were screened for ALK expression by two IHC antibodies (clone 5A4 from Novocastra and clone D5F3 from Ventana) and for ALK rearrangement by FISH (Vysis ALK FISH break-apart kit), which was automatically captured and scored by using Bioview's automated scanning system. Results All positive cases with the IHC antibodies were FISH-positive. There was only one IHC-negative case with both antibodies which showed a FISH-positive result. The overall sensitivity and specificity of the IHC in comparison with FISH were 98% and 100%, respectively. Conclusions The specificity of these ultrasensitive IHC assays may obviate the need for FISH confirmation in positive IHC cases. However, the likelihood of false negative IHC results strengthens the case for FISH testing, at least in some situations. PMID:25248157

  11. Fibre Optic Probe For In Situ Fluorescence Measurements

    NASA Astrophysics Data System (ADS)

    King, P. R.; Driver, I.; Dawson, J. B.; Ellis, D. J.; Feather, J. W.

    1988-06-01

    A single fibre fluoroprobe using excitation from an argon ion laser has been constructed. The probe has been used to measure fluorescence from a dye in aqueous solution and simultaneously to detect Raman scattering from the water. It has been shown that the measured fluorescence decreases as scatterer is added to the solution but that the ratio of fluorescence to Raman signal is constant for a given dye concentration. This ratio did not remain constant when an absorbing ink was added.

  12. Molecular Diversity, Cultivation, and Improved Detection by Fluorescent In Situ Hybridization of a Dominant Group of Human Gut Bacteria Related to Roseburia spp. or Eubacterium rectale

    Microsoft Academic Search

    Rustam I. Aminov; Alan W. Walker; Sylvia H. Duncan; Hermie J. M. Harmsen; Gjalt W. Welling; Harry J. Flint

    2006-01-01

    Phylogenetic analysis was used to compare 16S rRNA sequences from 19 cultured human gut strains of Roseburia and Eubacterium rectale with 356 related sequences derived from clone libraries. The cultured strains were found to represent five of the six phylotypes identified. A new oligonucleotide probe, Rrec584, and the previous group probe Rint623, when used in conjunction with a new helper

  13. Analysis and direct quantification of Saccharomyces cerevisiae and Hanseniaspora guilliermondii populations during alcoholic fermentation by fluorescence in situ hybridization, flow cytometry and quantitative PCR

    Microsoft Academic Search

    Margarida Monteiro; Braulio Esteve-Zarzoso; Helena Albergaria; Albert Mas

    2011-01-01

    Traditionally, it was assumed that non-Saccharomyces (NS) yeasts could only survive in the early stages of alcoholic fermentations. However, recent studies applying culture-independent methods have shown that NS populations persist throughout the fermentation process. The aim of the present work was to analyze and quantify Saccharomyces cerevisiae (Sc) and Hanseniaspora guilliermondii (Hg) populations during alcoholic fermentations by plating and culture-independent

  14. Biomimetic, hybrid and in situ composites

    Microsoft Academic Search

    J. E. Mark; P. D. Calvert

    1994-01-01

    Structural biological materials have mechanical properties that would be attractive if they could be duplicated in manufactured polymers, composites and ceramics. Hard tissues are reviewed here with an emphasis on the process by which they form and the resulting microstructures. In parallel the authors summarize synthetic efforts to produce similar structures and the properties which have been achieved.

  15. Establishment of the genomic in situ hybridization (GISH) technique for analysis in interspecific hybrids of Passiflora.

    PubMed

    Melo, C A F; Silva, G S; Souza, M M

    2015-01-01

    The genomic in situ hybridization (GISH) technique was applied to Passiflora interspecific F1 HD13-133 hybrids (Passiflora sublanceolata x Passiflora foetida) and HD15-101 (Passiflora gardineri x Passiflora gibertii), and the backcrossed hybrids (BC1) HD18-106 and HD18-113 (Passiflora sublanceolata x HD13-133). GISH was performed using genomic probes prepared with the DNA from the paternal genitor, whereas the maternal DNA was used as blocking DNA and employed at various concentrations (20X, 40X, 60X, and 100X) in relation to the probe concentration. At the same time, GISH was applied with the use of simultaneous probes from both genomes, paternal and maternal, that were detected with avidin-FITC and anti-digoxigenin-rhodamine, respectively. Both methodologies allowed the distinguishing of the maternal and paternal genomes, thus confirming the hybrid nature of all the analyzed genotypes. Furthermore, the presence of recombinant chromosomes in BC1 hybrids revealed the occurrence of meiotic recombination in HD13 hybrids. This application of the GISH technique is an important step towards genomic analyses of Passiflora hybrids: it can broaden the phylogenetic and evolutionary studies of the genus and, at the same time, contribute to breeding programs. PMID:25867365

  16. Hybrid-type temperature sensor for in situ measurement

    SciTech Connect

    Iuchi, Tohru; Hiraka, Kensuke [Sensor Photonics Research Center, Toyo University, 2100 Kujirai, Kawagoe, Saitama 350-8585 (Japan)

    2006-11-15

    A hybrid-type surface temperature sensor combines the contact and noncontact methods, which allows us to overcome the shortcomings of both methods. The hybrid-type surface thermometer is composed mainly of two components: a metal film sheet that makes contact with an object and a radiometer that is used to detect the radiance of the rear surface of the metal film, which is actually a modified radiation thermometer. Temperature measurement using the hybrid-type thermometer with a several tens micrometer thick Hastelloy sheet, a highly heat and corrosion resistant alloy, is possible with a systematic error of -0.5 K and random errors of {+-}0.5 K, in the temperature range from 900 to 1000 K. This thermometer provides a useful means for calibration of in situ temperature measurement in various processes, especially in the silicon semiconductor industry. This article introduces the basic idea of the hybrid-type surface sensor, presents experimental results and discussions, and finally describes some applications.

  17. In situ measurements of colloid transport and retention using synchrotron X-ray fluorescence

    E-print Network

    Walter, M.Todd

    In situ measurements of colloid transport and retention using synchrotron X-ray fluorescence David] The physics regarding the retention and mobilization of colloids in saturated and unsaturated conditions remains poorly understood, partially because of the inability to measure colloid concentrations in situ

  18. Use of somatic cell hybrids and fluorescence in situ hybridization to localize the functional serum amyloid A (SAA) genes to chromosome 11p15.4-p15.1 and the entire SAA superfamily to chromosome 11p15

    SciTech Connect

    Watson, G.; Woo, P. [Clinical Research Centre, Middlesex (United Kingdom)] [Clinical Research Centre, Middlesex (United Kingdom); See, C.G. [Univ. College London (United Kingdom)] [Univ. College London (United Kingdom)

    1994-10-01

    The serum amyloid A (SAA) superfamily consists of two acute phase genes, SAA1 and SAA2; a pseudogene, SAA3; and a constitutively expressed gene, SAA4. The SAA proteins, which are found associated with high-density lipoprotein, are believed to have an essential function. Chronic infection, inflammation, or trauma causes very high levels of the acute phase SAA proteins. This may result in the potentially fatal condition, amyloidosis, in which amyloid fibrils are deposited in the essential organs. Somatic cell hybrids have been used by several groups to map one or more of the SAA genes to chromosome 11p. We used FISH analysis and PCR amplification of DNA from 17 somatic cell hybrids carrying all or part of chromosome 11 as their only human component to systematically fine map the chromosomal location of the entire SAA superfamily. We demonstrate by these methods that the location of the entire SAA superfamily is at 11p15. Furthermore, we have demonstrated that SAA1, SAA2, and SAA4, i.e., all of the functional genes of the superfamily, map within this region to chromosome 11p15.4-p15.1. 20 refs., 2 figs.

  19. 1Nonradioactive In Situ Hybridization in Atherosclerotic Tissue.

    PubMed

    Apostolopoulos, J

    2001-01-01

    In situ hybridization (ISH) is a powerful and important technique that allows the detection and microscopic localization of nucleic acids within the specific cell, tissue, or chromosome of interest. In addition, it offers increased sensitivity over traditional filter hybridization, since low-copy mRNA molecules in individual cells can be detected. At the time the ISH technique was developed by Pardue and Gall (1), there were restrictions in it since radioisotopes were the only labels for nucleic acids available and autoradiographic film was the only detection system. Current molecular biological cloning techniques have now enabled most researchers to prepare almost any specific probe of choice and, more importantly, modern nonradioactive labels with colorimetric detection have removed all the limitations and restrictions of radioactive labels. The principal advantages of nonradioactive hybridization compared with isotopic hybridization are increased speed, greater resolution, lower costs, and reduced radioactive exposure. Furthermore, it allows the opportunity for combining different labels in one ISH experiment. The procedures behind ISH localization of DNA or RNA are very similar and may be summarized in five areas: (1) sample and glass slide preparation, including fixation, mounting, and ISH pretreatment, (2) probe preparation/labeling, (3) hybridization, (4) probe removal/washing, and (5) detection. Nonradioactive probe labeling itself can be divided into two methods, i.e., direct and indirect. This chapter describes the preparation of atherosclerotic tissue for ISH, indirect labeling of probes with digoxigenin (DIG), and the detection protocols suitable for this type of tissue. The DIG labeling method was developed by Kessler (2) and is based on the steroid digoxigenin, which is isolated from Digitalis purpurea and D. lanata. The DIG molecule is linked to the C-5 position of uridine (UTP, dUTP, or ddUTP) via a spacer arm. The DIG-labeled nucleotides can be incorporated easily into nucleic acid probes by DNA polymerases such as DNA polymerase I,Taq DNA polymerase, T7 DNA polymerase, RNA polymerases, and terminal transferase. These various enzymes therefore allow DIG labeling by random priming, nick translation, PCR, 3'-end labeling/tailing, and in vitro transcription. Following hybridization, DIG-probes may be detected with high-affinity specific anti-DIG antibodies (3). These antibodies are conjugated with alkaline phosphatase, peroxidase, fluoroscein, rhodamine, AMCA (amino-methylcoumarin-acetic acid), or colloidal gold (for electron microscopy) enabling a very versatile detection system. This system can be made even more versatile and sensitive by using unconjugated anti-DIG followed by conjugated secondary antibodies. A detection sensitivity of about 0.1 pg (as determined by Southern blot) can be achieved with combinations of anti-DIG-alkaline phospatase and NBT or BCIP. In this chapter, I describe a protocol that we developed for nonradioactive in situ hybridization of atherosclerotic tissue for the detection of interleukin 8, tissue factor, and tissue factor pathway inhibitor in both frozen and paraffin-embedded tissue (4-7). PMID:21340943

  20. Genome discrimination by in situ hybridization in Icelandic species of Elymus and Elytrigia (Poaceae: Triticeae).

    PubMed

    Orgaard, M; Anamthawat-Jónsson, K

    2001-04-01

    The genome constitution of Icelandic Elymus caninus, E. alaskanus, and Elytrigia repens was examined by fluorescence in situ hybridization using genomic DNA and selected cloned sequences as probes. Genomic in situ hybridization (GISH) of Hordeum brachyantherum ssp. californicum (diploid, H genome) probe confirmed the presence of an H genome in the two tetraploid Elymus species and identified its presence in the hexaploid Elytrigia repens. The H chromosomes were painted uniformly except for some chromosomes of Elytrigia repens which showed extended unlabelled pericentromeric and subterminal regions. A mixture of genomic DNA from H. marinum ssp. marinum (diploid, Xa genome) and H. murinum ssp. leporinum (tetraploid, Xu genome) did not hybridize to chromosomes of the Elymus species or Elytrigia repens, confirming that these genomes were different from the H genome. The St genomic probe from Pseudoroegneria spicata (diploid) did not discriminate between the genomes of the Elymus species, whereas it produced dispersed and spotty hybridization signals most likely on the two St genomes of Elytrigia repens. Chromosomes of the two genera Elymus and Elytrigia showed different patterns of hybridization with clones pTa71 and pAes41, while clones pTa1 and pSc119.2 hybridized only to Elytrigia chromosomes. Based on FISH with these genomic and cloned probes, the two Elymus species are genomically similar, but they are evidently different from Elytrigia repens. Therefore the genomes of Icelandic Elymus caninus and E. alaskanus remain as StH, whereas the genomes of Elytrigia repens are proposed as XXH. PMID:11341738

  1. In Situ Measurement of Bioluminescence and Fluorescence in an Integrated

    E-print Network

    Sinskey, Anthony J.

    INTRODUCTION Light emission from luminescent and fluorescent bacteria (and more recently, yeast) created to act InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20708 Abstract: Reporter strains of bacteria a significant impact in many areas of biology, including toxicity assays for environ- mental pollutants

  2. The Use of Whole-Mount "in Situ" Hybridization to Illustrate Gene Expression Regulation

    ERIC Educational Resources Information Center

    Llamusí, Beatriz; Muñoz-Soriano, Verónica; Paricio, Nuria; Artero, Rubén

    2014-01-01

    "In situ" hybridization is a widely used technique for studying gene expression. Here, we describe two experiments addressed to postgraduate genetics students in which the effect of transcription factors on gene expression is analyzed in "Drosophila embryos of different genotypes by whole-mount in situ hybridization. In one of the…

  3. Strong HER2\\/neu protein overexpression by immunohistochemistry often does not predict oncogene amplification by fluorescence in situ hybridization 1 1 This manuscript consists of original material not previously accepted for publication by another journal. The manuscript does not include use of any investigational or “off-label” products. The authors do not have a financial or other relationship with any of the commercial enterprises whose products or services may be discussed in this original article

    Microsoft Academic Search

    Lauren Hammock; Melinda Lewis; Carol Phillips; Cynthia Cohen

    2003-01-01

    Breast cancer patients with HER-2\\/neu oncogene amplification by fluorescence in situ hybridization (FISH) have been shown to have a better response to trastuzumab (Herceptin) therapy than those showing HER-2\\/neu protein overexpression only. Many centers currently perform FISH only on tumors showing 2+ HER-2\\/neu positivity by immunohistochemistry (IHC), with the assumption that 3+ positivity virtually equates with amplification. Results of FISH

  4. Solar cells Improved Hybrid Solar Cells via in situ UV Polymerization

    E-print Network

    Sibener, Steven

    Solar cells Improved Hybrid Solar Cells via in situ UV Polymerization Sanja Tepavcevic, Seth B-enhanced solar energy conversion. By using this simple in situ UV polymerization method that couples mobility of the photoactive layer can be enhanced. 1. Introduction Hybrid solar cells have been developed

  5. Whole-Mount In Situ Hybridization of Hawaiian Bobtail Squid (Euprymna scolopes) Embryos with DIG-Labeled

    E-print Network

    McFall-Ngai, Margaret

    Protocol Whole-Mount In Situ Hybridization of Hawaiian Bobtail Squid (Euprymna scolopes) Embryos for preparing RNA probes for whole-mount in situ hybridization in Hawaiian bobtail squid (Euprymna scolopes The probe prepared in this protocol is suitable for use in Whole-Mount In Situ Hybridization of Hawaiian

  6. Fetal t(5p;21q) misdiagnosed as monosomy 21: A plea for in situ hybridization studies

    SciTech Connect

    Gill, P.; Uhrich, S.; Cheng, E.; Disteche, C. [Univ. of Washington Medical Center, Seattle, WA (United States)

    1994-10-01

    We report a case of 45,XY,-5,-21,+der (5)t(5;21) (p13 or p14;q11.2 or q21) that was prenatally misdiagnosed as complete monosomy 21 and terminated at 24 weeks of gestation. Subsequent fluorescence in situ hybridization studies with a chromosome 21 painting probe documented the cryptic unbalanced translocation. 17 refs., 2 figs., 1 tab.

  7. In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea

    PubMed Central

    2013-01-01

    Background The freshwater planarian Schmidtea mediterranea has emerged as a powerful model for studies of regenerative, stem cell, and germ cell biology. Whole-mount in situ hybridization (WISH) and whole-mount fluorescent in situ hybridization (FISH) are critical methods for determining gene expression patterns in planarians. While expression patterns for a number of genes have been elucidated using established protocols, determining the expression patterns for particularly low-abundance transcripts remains a challenge. Results We show here that a short bleaching step in formamide dramatically enhances signal intensity of WISH and FISH. To further improve signal sensitivity we optimized blocking conditions for multiple anti-hapten antibodies, developed a copper sulfate quenching step that virtually eliminates autofluorescence, and enhanced signal intensity through iterative rounds of tyramide signal amplification. For FISH on regenerating planarians, we employed a heat-induced antigen retrieval step that provides a better balance between permeabilization of mature tissues and preservation of regenerating tissues. We also show that azide most effectively quenches peroxidase activity between rounds of development for multicolor FISH experiments. Finally, we apply these modifications to elucidate the expression patterns of a few low-abundance transcripts. Conclusion The modifications we present here provide significant improvements in signal intensity and signal sensitivity for WISH and FISH in planarians. Additionally, these modifications might be of widespread utility for whole-mount FISH in other model organisms. PMID:23497040

  8. Digoxigenin-Labeled In Situ Hybridization for the Detection of Streptococcus suis DNA in Polyserositis and a Comparison with Biotinylated In Situ Hybridization

    PubMed Central

    KANG, Ikjae; SEO, Hwi Won; PARK, Changhoon; OH, Yeonsu; LEE, Jeehoon; YOU, Ok Heui; KIM, Sung-Hoon; GOTTSCHALK, Marcelo; CHAE, Chanhee

    2013-01-01

    ABSTRACT The objective of this study was to develop digoxigenin-labeled in situ hybridization (ISH) for the detection of Streptococcus suis in naturally infected pigs with polyserositis and to compare it with biotinylated ISH. Digoxigenin-labeled hybridization signals for S. suis were observed in cells that had infiltrated the fibrous polyserositis and microcolonies in the blood vessels. Mock hybridization showed no hybridization signals for endogenous digoxigenin. Biotinylated hybridization signals for S. suis were observed in cells that had infiltrated the fibrous polyserositis. However, similar hybridization signals were also observed in the fibrous inflammatory area using mock hybridization for endogenous biotin. The present study demonstrated that digoxigenin-labeled ISH is a valuable diagnostic tool for specific detection of S. suis in polyserositic tissues without nonspecific reactions compared with biotinylated ISH. PMID:23985415

  9. [Physical localization of ribosomal genes and chromosome DAPI banding by in situ hybridization in Medicago sativa L].

    PubMed

    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 . PMID:16520314

  10. HER2 assessment by silver in situ hybridization: where are we now?

    PubMed

    Sanguedolce, Francesca; Bufo, Pantaleo

    2015-03-01

    HER2 testing in breast and gastric cancer is critical not only as a prognostic tool but also as a predictive marker for response to the humanized monoclonal antibody trastuzumab. Currently, HER2 status is assessed on histological and cytological specimens by conventional validated methods such as immunohistochemistry and FISH, while bright-field in situ hybridization techniques, such as silver in situ hybridization and chromogenic in situ hybridization, may offer performance benefits over FISH. The major points are first, technical issues, advantages and disadvantages relevant to each methods, and their clinical implications and second, the well-known genetic heterogeneity of HER2, and the occurrence of polysomy of chromosome 17. This review aims to summarize the growing body of literature on the accuracy of bright-field in situ techniques, notably silver in situ hybridization, in assessing HER2 status, and to discuss the role of such methods in pathology practice. PMID:25578771

  11. In situ ultraviolet fluorescence probe for detection of volatile organic hydrocarbons in groundwater

    SciTech Connect

    Morlock, C.R. [Morlock Environmental, Inc., Lebanon, NH (United States)

    1995-12-31

    The authors have developed a miniature ultraviolet (UV) fluorescence device for measuring volatile organic compound (VOC) concentrations in ground water. The device consists of a mercury vapor lamp, excitation filter, sample chamber, emission filter and a miniature photomultiplier tube. Filters are chosen for optimal fluorescence signals from BTEX compounds. The prototype device is a benchtop model where a water sample is introduced into a sample chamber. Fluorescence is measured for the water/contaminant mix. This is followed by an air purge of the water to eliminate all volatile compounds. A second fluorescence measurement is taken after the purge. Any detected fluorescent after the purge results from system noise or fluorescence of substances other than VOCs. By rationing the two fluorescence measurements the authors arrive at a number which is proportional to VOC concentration. The results indicated that the authors can detect BTEX compounds in the sub-ppm range. The authors are currently designing an instrument to be lowered into a bore hole for continuous depth profiling and in situ measurement. They believe that it will be possible to build a device suitable for installation within a cone penetrometer or other sounding device. The intended use of the instrument is for rapid in situ assessment of VOCs and for permanent installation in a well for long term monitoring. The cost of all components that are part of this device is less than $2000.

  12. Understanding early organogenesis using a simplified in situ hybridization protocol in Xenopus.

    PubMed

    Deimling, Steven J; Halabi, Rami R; Grover, Stephanie A; Wang, Jean H; Drysdale, Thomas A

    2015-01-01

    Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems. PMID:25651461

  13. Detection of deletions and cryptic translocations in Miller-Dieker syndrome by in situ hybridization.

    PubMed Central

    Kuwano, A; Ledbetter, S A; Dobyns, W B; Emanuel, B S; Ledbetter, D H

    1991-01-01

    Fluorescence in situ hybridization (FISH) using two cosmid probes (41A and P13) from the Miller-Dieker syndrome (MDS) critical region in 17p13.3 was performed in a blinded comparison of three MDS patients with submicroscopic deletions and in four normal relatives used as controls. The controls showed both chromosome 17 homologues labeled in 85%-95% of cells, while each patient showed only one homologue labeled in 75%-80% of cells. Two MDS patients with cryptic translocations were also studied. In one case, a patient and her mother had the same der(17) (p+), but the reciprocal product of the translocation could not be identified in the mother by G-banding (i.e., it was a "half-cryptic" translocation). FISH revealed a 3q;17p translocation. The other case involved a patient with apparently normal karyotype. Because a large molecular deletion was found, a translocation involving two G-negative telomeres (i.e., a "full-cryptic" translocation) was postulated. FISH studies on her father and normal brother showed an 8q;17p translocation. These studies demonstrate that in situ hybridization is an efficient method for deletion detection in Miller-Dieker syndrome. More important, parental studies by FISH on patients demonstrating molecular deletions and a normal karyotype may identify cryptic translocation events, which cannot be detected by other molecular genetic strategies. Similar in situ strategies for deletion detection can be developed for other microdeletion syndromes, such as Prader-Willi/Angelman syndrome or DiGeorge syndrome. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:1897521

  14. Next-Generation in Situ Hybridization Chain Reaction: Higher Gain, Lower Cost, Greater Durability

    PubMed Central

    2014-01-01

    Hybridization chain reaction (HCR) provides multiplexed, isothermal, enzyme-free, molecular signal amplification in diverse settings. Within intact vertebrate embryos, where signal-to-background is at a premium, HCR in situ amplification enables simultaneous mapping of multiple target mRNAs, addressing a longstanding challenge in the biological sciences. With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The properties of HCR lead to straightforward multiplexing, deep sample penetration, high signal-to-background, and sharp subcellular signal localization within fixed whole-mount zebrafish embryos, a standard model system for the study of vertebrate development. However, RNA reagents are expensive and vulnerable to enzymatic degradation. Moreover, the stringent hybridization conditions used to destabilize nonspecific hairpin binding also reduce the energetic driving force for HCR polymerization, creating a trade-off between minimization of background and maximization of signal. Here, we eliminate this trade-off by demonstrating that low background levels can be achieved using permissive in situ amplification conditions (0% formamide, room temperature) and engineer next-generation DNA HCR amplifiers that maximize the free energy benefit per polymerization step while preserving the kinetic trapping property that underlies conditional polymerization, dramatically increasing signal gain, reducing reagent cost, and improving reagent durability. PMID:24712299

  15. Next-generation in situ hybridization chain reaction: higher gain, lower cost, greater durability.

    PubMed

    Choi, Harry M T; Beck, Victor A; Pierce, Niles A

    2014-05-27

    Hybridization chain reaction (HCR) provides multiplexed, isothermal, enzyme-free, molecular signal amplification in diverse settings. Within intact vertebrate embryos, where signal-to-background is at a premium, HCR in situ amplification enables simultaneous mapping of multiple target mRNAs, addressing a longstanding challenge in the biological sciences. With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The properties of HCR lead to straightforward multiplexing, deep sample penetration, high signal-to-background, and sharp subcellular signal localization within fixed whole-mount zebrafish embryos, a standard model system for the study of vertebrate development. However, RNA reagents are expensive and vulnerable to enzymatic degradation. Moreover, the stringent hybridization conditions used to destabilize nonspecific hairpin binding also reduce the energetic driving force for HCR polymerization, creating a trade-off between minimization of background and maximization of signal. Here, we eliminate this trade-off by demonstrating that low background levels can be achieved using permissive in situ amplification conditions (0% formamide, room temperature) and engineer next-generation DNA HCR amplifiers that maximize the free energy benefit per polymerization step while preserving the kinetic trapping property that underlies conditional polymerization, dramatically increasing signal gain, reducing reagent cost, and improving reagent durability. PMID:24712299

  16. Genomic Origin and Organization of the Allopolyploid Primula egaliksensis Investigated by in situ Hybridization

    PubMed Central

    Guggisberg, Alessia; Baroux, Célia; Grossniklaus, Ueli; Conti, Elena

    2008-01-01

    Background and Aims Earlier studies have suggested that the tetraploid Primula egaliksensis (2n = 40) originated from hybridization between the diploids P. mistassinica (2n = 18) and P. nutans (2n = 22), which were hypothesized to be the maternal and paternal parent, respectively. The present paper is aimed at verifying the hybrid nature of P. egaliksensis using cytogenetic tools, and to investigate the extent to which the parental genomes have undergone genomic reorganization. Methods Genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) with ribosomal DNA (rDNA) probes, together with sequencing of the internal transcribed spacer (ITS) region of the rDNA, were used to identify the origin of P. egaliksensis and to explore its genomic organization, particularly at rDNA loci. Key Results GISH showed that P. egaliksensis inherited all chromosomes from P. mistassinica and P. nutans and did not reveal major intergenomic rearrangements between the parental genomes (e.g. interchromosomal translocations). However, karyological comparisons and FISH experiments suggested small-scale rearrangements, particularly at rDNA sites. Primula egaliksensis lacked the ITS-bearing heterochromatic knobs characteristic of the maternal parent P. mistassinica and maintained only the rDNA loci of P. nutans. These results corroborated sequence data indicating that most ITS sequences of P. egaliksensis were of the paternal repeat type. Conclusions The lack of major rearrangements may be a consequence of the considerable genetic divergence between the putative parents, while the rapid elimination of the ITS repeats from the maternal progenitor may be explained by the subterminal location of ITS loci or a potential role of nucleolar dominance in chromosome stabilization. These small-scale rearrangements may be indicative of genome diploidization, but further investigations are needed to confirm this assumption. PMID:18308718

  17. Hybrid assemblies of fluorescent nanocrystals and membrane proteins in liposomes.

    PubMed

    De Leo, Vincenzo; Catucci, Lucia; Falqui, Andrea; Marotta, Roberto; Striccoli, Marinella; Agostiano, Angela; Comparelli, Roberto; Milano, Francesco

    2014-02-18

    Because of the growing potential of nanoparticles in biological and medical applications, tuning and directing their properties toward a high compatibility with the aqueous biological milieu is of remarkable relevance. Moreover, the capability to combine nanocrystals (NCs) with biomolecules, such as proteins, offers great opportunities to design hybrid systems for both nanobiotechnology and biomedical technology. Here we report on the application of the micelle-to-vesicle transition (MVT) method for incorporation of hydrophobic, red-emitting CdSe@ZnS NCs into the bilayer of liposomes. This method enabled the construction of a novel hybrid proteo-NC-liposome containing, as model membrane protein, the photosynthetic reaction center (RC) of Rhodobacter sphaeroides. Electron microscopy confirmed the insertion of NCs within the lipid bilayer without significantly altering the structure of the unilamellar vesicles. The resulting aqueous NC-liposome suspensions showed low turbidity and kept unaltered the wavelengths of absorbance and emission peaks of the native NCs. A relative NC fluorescence quantum yield up to 8% was preserved after their incorporation in liposomes. Interestingly, in proteo-NC-liposomes, RC is not denatured by Cd-based NCs, retaining its structural and functional integrity as shown by absorption spectra and flash-induced charge recombination kinetics. The outlined strategy can be extended in principle to any suitably sized hydrophobic NC with similar surface chemistry and to any integral protein complex. Furthermore, the proposed approach could be used in nanomedicine for the realization of theranostic systems and provides new, interesting perspectives for understanding the interactions between integral membrane proteins and nanoparticles, i.e., in nanotoxicology studies. PMID:24460372

  18. Regulatory pathway analysis by high-throughput in situ hybridization

    SciTech Connect

    Visel, Axel; Carson, James P.; Oldekamp, Judit; Warnecke, Marei; Jakubcakova, Vladimira; Zhou, Xunlei; Shaw, Chad; Alvarez-Bolado, Gonzalo; Eichele, Gregor

    2007-10-01

    Automated in situ hybridization (ISH) permits construction of comprehensive atlases of gene expression patterns in mammals. When web-accessible, such atlases become searchable digital expression maps of individual genes and offer an entryway to elucidate genetic interactions and signaling pathways. An atlas housing ~1,000 spatial gene expression patterns of the mid-gestation mouse embryo was generated. Patterns were textually annotated using a controlled vocabulary comprising 90 anatomical features. Hierarchical clustering of annotations was carried out using distance scores calculated from the similarity between pairs of patterns across all anatomical structures. This ordered hundreds of complex expression patterns into a matrix that reflected the embryonic architecture and the relatedness of patterns of expression. Clustering yielded twelve distinct groups of expression pattern. Because of similarity of expression patterns within a group, members of this group may be components of regulatory cascades. We focused on one group, which is composed of 83 genes, including Pax6, an evolutionary conserved transcriptional master mediator of the development. Using functional studies, ISH on Pax6-deficient embryos and Pax6 binding site identification and validation by means of electromobility shift assays, we identified numerous genes that are transcriptionally regulated by Pax6. Hence cluster analysis of annotated gene expression patterns obtained by robotic ISH is an entryway for identification of components of signaling cascades in mammals.

  19. In situ DNA PCR and RNA hybridization detection of herpes simplex virus sequences in trigeminal gangliaof latently infected mice

    Microsoft Academic Search

    Anand Mehta; John Maggioncalda; Omar Bagasra; Seshamma Thikkavarapu; Pamujula Saikumari; Tibor Valyi-Nagy; Nigel W. Fraser; Timothy M. Block

    1995-01-01

    The presence of herpes simplex virus (HSV-1) DNA in the trigeminal ganglia of latently infected mice was detected byan in situ DNA polymerase chain reaction (PCR), which includes a DNA:DNA hybridization step (indirect in situ PCR). These results were compared to the number of neurons possessing the HSV-1 latency associated transcript (LAT), as detected by in situ RNA hybridization with

  20. A capillary-based probe for in situ detection of enhanced fluorescence signals

    NASA Astrophysics Data System (ADS)

    Long, F.; Xiao, R.; Zhu, A. N.; Shi, H. C.; Wang, S. Q.

    2013-07-01

    A simple, compact, and high sensitivity capillary-based probe for the in situ detection of fluorescence signals with high sensitivity is demonstrated. A home-made single–multi-mode fiber coupler that is coaxially aligned with the capillary-based probe provides for the transmission of excitation light and the collection and transmission of fluorescence. We propose a conceptually straightforward theoretical model to optimize the factors affecting the fluorescence-capture capability of the capillary-based probe. The fluorescence signal detected by fiber-optic spectroscopy non-linearly increases with the length of the capillary-based probe. In addition, the thicker the capillary tube wall is, the less the fluorescence signals determined are. The performance of the proposed probe is evaluated experimentally by measuring the fluorescence spectra of Cy5.5 dye and blue-green algae. The experimental results show that the proposed probe provides more than a ten-fold increase in fluorescence signal compared with direct measurements by a flat-tipped multi-mode fiber probe. The advantages of the capillary-based probe, which include its simple and compact structure, excellent light collection efficiency, requirement of small sample volume, and recoverability of samples, allow its wide application to in situ detection in the medical, forensic, biological, geological, and environmental fields with high sensitivity.

  1. Analysis of chromosome segregation during mammalian meiosis using combined immunofluorescence and fluorescence in situ hubridization

    SciTech Connect

    Hunt, P.A.; Embury, P.B.; Mroz, K.M. [Case Western Univ., Cleveland, OH (United States)] [and others

    1994-09-01

    Meiotic non-disjunction is thought to occur in 10-20% of all human oocytes, making this the most common genetic abnormality in our species. Aberrant recombination has been implicated in the genesis of these errors; however, direct studies of the meiotic process have been hampered by the lack of material and appropriate technology. We have developed a technique for the evaluation of meiosis in intact mammalian oocytes that combines immunofluorescence and fluorescence in situ hybridization (FISH). This allows for simultaneous, 3-dimensional visualization of the meiotic spindle, the alignment of the chromosomes on the spindle, and the placement of specific chromosomes. We have used this technology to follow meiotic progression in oocytes from XO female mice to evaluate the behavior of an unsynapsed chromosome during mammalian meiosis. Perturbations in chromosome behavior are evident early in meiosis: during the formation of the first meiotic spindle, the univalent X chromosome is properly positioned. With the onset of anaphase, the single X chromosome most commonly segregates as an intact chromosome, although equational segregation of the X chromatids is seen in a significant minority (approximately 20%) of oocytes. These observations demonstrate that failure of pairing/recombination can result in segregation of sister chromatids at meiosis I. This has obvious implications for human non-disjunction, much of which is thought to be due to recombination deficiencies; accordingly, we are now extending our studies to include analyses of human oocytes.

  2. Investigation of polymer electrolyte membrane chemical degradation and degradation mitigation using in situ fluorescence spectroscopy

    PubMed Central

    Prabhakaran, Venkateshkumar; Arges, Christopher G.; Ramani, Vijay

    2012-01-01

    A fluorescent molecular probe, 6-carboxy fluorescein, was used in conjunction with in situ fluorescence spectroscopy to facilitate real-time monitoring of degradation inducing reactive oxygen species within the polymer electrolyte membrane (PEM) of an operating PEM fuel cell. The key requirements of suitable molecular probes for in situ monitoring of ROS are presented. The utility of using free radical scavengers such as CeO2 nanoparticles to mitigate reactive oxygen species induced PEM degradation was demonstrated. The addition of CeO2 to uncatalyzed membranes resulted in close to 100% capture of ROS generated in situ within the PEM for a period of about 7 h and the incorporation of CeO2 into the catalyzed membrane provided an eightfold reduction in ROS generation rate. PMID:22219367

  3. In Situ Hybridization for the Precise Localization of Transcripts in Plants

    PubMed Central

    Javelle, Marie; Marco, Cristina F.; Timmermans, Marja

    2011-01-01

    With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14. PMID:22143276

  4. Elucidating Structure and Function In Vivo With Hybrid Fluorescence and Magnetic Resonance Imaging

    Microsoft Academic Search

    Mark Niedre; Vasilis Ntziachristos

    2008-01-01

    While the mathematics, physics, and technology behind magnetic resonance (MR) and fluorescence image formation are distinctively different, the two modalities have significant complementary features to impart strong preclinical and clinical application synergies. Traditionally, hybrid MR and fluorescence imaging implied the use of a system where optical and MR signals can be concurrently acquired. In this case, the common geometry allows

  5. Simultaneous capture and in situ analysis of circulating tumor cells using multiple hybrid nanoparticles.

    PubMed

    Lee, Hun Joo; Cho, Hyeon-Yeol; Oh, Jin Ho; Namkoong, Kak; Lee, Jeong Gun; Park, Jong-Myeon; Lee, Soo Suk; Huh, Nam; Choi, Jeong-Woo

    2013-09-15

    Using hybrid nanoparticles (HNPs), we demonstrate simultaneous capture, in situ protein expression analysis, and cellular phenotype identification of circulating tumor cells (CTCs). Each HNP consists of three parts: (i) antibodies that bind specifically to a known biomarker for CTCs, (ii) a quantum dot that emits fluorescence signals, and (iii) biotinylated DNA that allows capture and release of CTC-HNP complex to an in-house developed capture & recovery chip (CRC). To evaluate our approach, cells representative of different breast cancer subtypes (MCF-7: luminal; SK-BR-3: HER2; and MDA-MB-231: basal-like) were captured onto CRC and expressions of EpCAM, HER2, and EGFR were detected concurrently. The average capture efficiency of CTCs was 87.5% with identification accuracy of 92.4%. Subsequently, by cleaving the DNA portion with restriction enzymes, captured cells were released at efficiencies of 86.1%. Further studies showed that these recovered cells are viable and can proliferate in vitro. Using HNPs, it is possible to count, analyze in situ protein expression, and culture CTCs, all from the same set of cells, enabling a wide range of molecular- and cellular-based studies using CTCs. PMID:23628845

  6. Portable fluorescence spectroscopy platform for Huanglongbing (HLB) citrus disease in situ detection

    NASA Astrophysics Data System (ADS)

    Mota, Alessandro D.; Rossi, Giuliano; de Castro, Guilherme Cunha; Ortega, Tiago A.; de Castro N., Jarbas C.

    2014-02-01

    In this work, the development of a portable fluorescence spectroscopy platform for Huanglongbing (HLB) citrus disease in situ detection is presented. The equipment consists of an excitation blue LED light source, a commercial miniature spectrometer and embedded software. Measurements of healthy, HLB-symptomatic and HLB-asymptomatic citrus leafs were performed. Leafs were excited with the blue LED and their fluorescence spectra collected. Embedded electronics and software were responsible for the spectrum processing and classification via partial least squares regression. Global success rates above 80% and 100% distinction of healthy and HLB-symptomatic leafs were obtained.

  7. A Hybrid Approach of Using Wavelets and Fuzzy Clustering for Classifying Multispectral Florescence In Situ Hybridization Images

    PubMed Central

    Dandpat, Ashok Kumar

    2006-01-01

    Multicolor or multiplex fluorescence in situ hybridization (M-FISH) imaging is a recently developed molecular cytogenetic diagnosis technique for rapid visualization of genomic aberrations at the chromosomal level. By the simultaneous use of all 24 human chromosome painting probes, M-FISH imaging facilitates precise identification of complex chromosomal rearrangements that are responsible for cancers and genetic diseases. The current approaches, however, cannot have the precision sufficient for clinical use. The reliability of the technique depends primarily on the accurate pixel-wise classification, that is, assigning each pixel into one of the 24 classes of chromosomes based on its six-channel spectral representations. In the paper we introduce a novel approach to improve the accuracy of pixel-wise classification. The approach is based on the combination of fuzzy clustering and wavelet normalization. Two wavelet-based algorithms are used to reduce redundancies and to correct misalignments between multichannel FISH images. In comparison with conventional algorithms, the wavelet-based approaches offer more advantages such as the adaptive feature selection and accurate image registration. The algorithms have been tested on images from normal cells, showing the improvement in classification accuracy. The increased accuracy of pixel-wise classification will improve the reliability of the M-FISH imaging technique in identifying subtle and cryptic chromosomal abnormalities for cancer diagnosis and genetic disorder research. PMID:23165039

  8. Modulation of tyrosine hydroxylase gene expression in the central nervous system visualized by in situ hybridization

    SciTech Connect

    Berod, A.; Biguet, N.F.; Dumas, S.; Bloch, B.; Mallet, J.

    1987-03-01

    cDNA probe was used for in situ hybridization studies on histological sections through the locus coeruleus, substantia nigra, and the ventral tegmental area of the rat brain. Experimental conditions were established that yielded no background and no signal when pBR322 was used as control probe. Using the tyrosine hydroxylase probe, the authors ascertained the specificity of the labeling over catecholaminergic cells by denervation experiments and comparison of the hybridization pattern with that of immunoreactivity. The use of /sup 35/S-labeled probe enabled the hybridization signal to be resolved at the cellular level. A single injection of reserpine into the rat led to an increase of the intensity of the autoradiographic signal over the locus coeruleus area, confirming an RNA gel blot analysis. The potential of in situ hybridization to analyze patterns of modulation of gene activity as a result of nervous activity is discussed.

  9. Amplification of chromosomal DNA in situ

    DOEpatents

    Christian, Allen T. (Tracy, CA); Coleman, Matthew A. (Livermore, CA); Tucker, James D. (Livermore, CA)

    2002-01-01

    Amplification of chromosomal DNA in situ to increase the amount of DNA associated with a chromosome or chromosome region is described. The amplification of chromosomal DNA in situ provides for the synthesis of Fluorescence in situ Hybridization (FISH) painting probes from single dissected chromosome fragments, the production of cDNA libraries from low copy mRNAs and improved in Comparative Genomic Hybridization (CGH) procedures.

  10. Rate of in situ Shattercane x Sorghum Hybridization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sorghum (Sorghum bicolor subsp. bicolor) can interbreed with its close weedy relative shattercane (S. bicolor subsp. drummondii). The introduction of traits from sorghum into a shattercane population could contribute to the invasiveness of the wild shattercane population. An in situ experiment was...

  11. miRNA in situ hybridization in circulating tumor cells - MishCTC.

    PubMed

    Ortega, Francisco G; Lorente, Jose A; Garcia Puche, Jose L; Ruiz, Maria P; Sanchez-Martin, Rosario M; de Miguel-Pérez, Diego; Diaz-Mochon, Juan J; Serrano, Maria J

    2015-01-01

    Circulating tumor cells (CTCs) must be phenotypically and genetically characterized before they can be utilized in clinical applications. Here, we present the first protocol for the detection of miRNAs in CTCs using in situ hybridization (ISH) combined with immunomagnetic selection based on cytokeratin (CK) expression and immunocytochemistry. Locked-Nucleic Acid (LNA) probes associated with an enzyme-labeled fluorescence (ELF) signal amplification approach were used to detect miRNA-21 in CTCs. This protocol was optimized using both epithelial tumor (MDA-MB468) and epithelial non-tumor (MCF-10A) cell lines, and miRNA-21 was selected as the target miRNA because of its known role as an onco-miRNA. Hematopoietic cells do not express miRNA-21; thus, miRNA-21 is an ideal marker for detecting CTCs. Peripheral blood samples were taken from 25 cancer patients and these samples were analyzed using our developed protocol. Of the 25 samples, 11 contained CTCs. For all 11 CTC-positive samples, the isolated CTCs expressed both CK and miRNA-21. Finally, the protocol was applied to monitor miRNA-21 expression in epithelial to mesenchymal transition (EMT)-induced MCF-7 cells, an epithelial tumor cell line. CK expression was lost in these cells, whereas miRNA-21 was still expressed, suggesting that miRNA-21 might be a good marker for detecting CTCs with an EMT phenotype. PMID:25777797

  12. miRNA in situ hybridization in circulating tumor cells - MishCTC

    PubMed Central

    Ortega, Francisco G.; Lorente, Jose A.; Garcia Puche, Jose L.; Ruiz, Maria P.; Sanchez-Martin, Rosario M.; de Miguel-Pérez, Diego; Diaz-Mochon, Juan J.; Serrano, Maria J.

    2015-01-01

    Circulating tumor cells (CTCs) must be phenotypically and genetically characterized before they can be utilized in clinical applications. Here, we present the first protocol for the detection of miRNAs in CTCs using in situ hybridization (ISH) combined with immunomagnetic selection based on cytokeratin (CK) expression and immunocytochemistry. Locked-Nucleic Acid (LNA) probes associated with an enzyme-labeled fluorescence (ELF) signal amplification approach were used to detect miRNA-21 in CTCs. This protocol was optimized using both epithelial tumor (MDA-MB468) and epithelial non-tumor (MCF-10A) cell lines, and miRNA-21 was selected as the target miRNA because of its known role as an onco-miRNA. Hematopoietic cells do not express miRNA-21; thus, miRNA-21 is an ideal marker for detecting CTCs. Peripheral blood samples were taken from 25 cancer patients and these samples were analyzed using our developed protocol. Of the 25 samples, 11 contained CTCs. For all 11 CTC-positive samples, the isolated CTCs expressed both CK and miRNA-21. Finally, the protocol was applied to monitor miRNA-21 expression in epithelial to mesenchymal transition (EMT)-induced MCF-7 cells, an epithelial tumor cell line. CK expression was lost in these cells, whereas miRNA-21 was still expressed, suggesting that miRNA-21 might be a good marker for detecting CTCs with an EMT phenotype. PMID:25777797

  13. FLUORESCENT IN SITU DETECTION OF ENCEPHALITOZOON HELLEM SPORES WITH A 6-CARBOXYFLUORESCEIN-LABELED RIBOSOMAL RNA-TARGETED OLIGONUCLEOTIDE PROBE

    EPA Science Inventory

    A fluroescent in situ hybridization assay has been developed for the detection of the human-pathogenic microsporidian, Encephalitozoon hellem in water samples using epifluorescence microscopy. The assay employs a 19-nucleotide species-specific 6-carboxyfluorescein-labeled oligonu...

  14. Genomic in situ hybridization to identify alien chromosomes and chromosome segments in wheat

    Microsoft Academic Search

    T. Schwarzacher; K. Anamthawat-Jónsson; G. E. Harrison; A. K. M. R. Islam; J. Z. Jia; I. P. King; A. R. Leitch; T. E. Miller; S. M. Reader; W. J. Rogers; M. Shi; J. S. Heslop-Harrison

    1992-01-01

    Genomic in situ hybridization was used to identify alien chromatin in chromosome spreads of wheat, Triticum aestivum L., lines incorporating chromosomes from Leymus multicaulis (Kar. and Kir.) Tzvelev and Thinopyrum bessarabicum (Savul. and Rayss) Löve, and chromosome arms from Hordeum chilense Roem. and Schult, H. vulgare L. and Secale cereale L. Total genomic DNA from the introgressed alien species was

  15. Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues.

    PubMed

    Lee, Je Hyuk; Daugharthy, Evan R; Scheiman, Jonathan; Kalhor, Reza; Ferrante, Thomas C; Terry, Richard; Turczyk, Brian M; Yang, Joyce L; Lee, Ho Suk; Aach, John; Zhang, Kun; Church, George M

    2015-03-01

    RNA-sequencing (RNA-seq) measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. In contrast, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq, our method enriches for context-specific transcripts over housekeeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d. PMID:25675209

  16. Towards in situ fluorescence spectroscopy and microscopy investigations of asphaltene precipitation kinetics.

    PubMed

    Franco, Juliana C; Gonçalves, Grasiele; Souza, Monique S; Rosa, Samantha B C; Thiegue, Larissa M; Atvars, Teresa D Z; Rosa, Paulo T V; Nome, René A

    2013-12-16

    We perform a spectroscopic analysis of asphaltene in solution and in crude oil with the goal of designing an optical probe of asphaltene precipitation inside high-pressure cells. Quantitative analysis of steady-state spectroscopic data is employed to identify fluorescence and Raman contributions to the observed signals. Time-resolved fluorescence spectroscopy indicates that fluorescence lifetime can be used as a spectroscopic probe of asphaltene in crude oil. Quantitative confocal laser-scanning microscopy studies of asphaltene in n-heptane are used to calculate particle-size distributions as a function of time, both at the sample surface and asphaltene interior. The resulting precipitation kinetics is well described by stochastic numerical simulations of diffusion-limited aggregation. Based on these results, we present the design and construction of an apparatus to optically probe the in situ precipitation of asphaltene suitable for studies inside high pressure cells. Design considerations include the use of a spatial light modulator for aberration correction in microscopy measurements, together with the design of epi-fluorescence spectrometer, both fiber-based and for remote sensing fluorescence spectroscopy. PMID:24514660

  17. 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

    SciTech Connect

    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.

  18. In situ measurements of colloid transport and retention using synchrotron X-ray fluorescence

    NASA Astrophysics Data System (ADS)

    Dicarlo, David A.; Zevi, Yuniati; Dathe, Annette; Giri, Shree; Gao, Bin; Steenhuis, Tammo S.

    2006-12-01

    The physics regarding the retention and mobilization of colloids in saturated and unsaturated conditions remains poorly understood, partially because of the inability to measure colloid concentrations in situ. In this study, we attached Cd+2 ions to clay colloids and used synchrotron X rays to cause the Cd to fluoresce. By measuring the fluorescence and attenuation of the X rays we obtained simultaneous in situ water saturations and colloid concentrations on timescales of tens of seconds. We used this technique to study the transport of colloids consisting of Na and Ca Montmorillonite clays through a preferential flow path in uniform well-sorted sand. This flow path had both saturated and unsaturated zones that travel downward with time. We found that the Na colloids showed little retention in the sand, while the Ca colloids were retarded with respect to the wetting front. By comparing the results to those obtained by infiltrations with a Cd solute we find that the retention of the colloids seen in the unsaturated portion of the column was no greater than that seen in the saturated portion. We discussed the advantages and limitations of this X-ray fluorescence technique and the implications for colloid transport.

  19. Synergetic spin crossover and fluorescence in one-dimensional hybrid complexes.

    PubMed

    Wang, Chun-Feng; Li, Ren-Fu; Chen, Xue-Yuan; Wei, Rong-Jia; Zheng, Lan-Sun; Tao, Jun

    2015-01-26

    Hybrid materials integrated with a variety of physical properties, such as spin crossover (SCO) and fluorescence, may show synergetic effects that find applications in many fields. Herein we demonstrate a promising post-synthetic approach to achieve such materials by grafting fluorophores (1-pyrenecarboxaldehyde and Rhodamine?B) on one-dimensional SCO Fe(II) structures. The resulting hybrid materials display expected one-step SCO behavior and fluorescent properties, in particular showing a coupling between the transition temperature of SCO and the temperature where the fluorescent intensity reverses. Consequently, synergetic effect between SCO and fluorescence is incorporated into materials despite different fluorophores. This study provides an effective strategy for the design and development of novel magnetic and optical materials. PMID:25504738

  20. Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries

    Microsoft Academic Search

    P. Lichter; T. Cremer; J. Borden; L. Manuelidis; D. C. Ward

    1988-01-01

    Summary A method of in situ hybridization for visualizing individual human chromosomes from pter to qter, both in metaphase spreads and interphase nuclei, is reported. DNA inserts from a single chromosomal library are labeled with biotin and partially preannealed with a titrated amount of total human genomic DNA prior to hybridization with cellular or chromosomal preparations. The cross-hybridization of repetitive

  1. Edited by Oliver Hobert. Last revised May 24, 2012. Published December 13, 2012. This chapter should be cited as: Ji N. and van Oudenaarden A. Single molecule fluorescent in situ hybridization (smFISH) of C. elegans worms and embryos (December 13, 2012),

    E-print Network

    van Oudenaarden, Alexander

    stages. Conventional RNA in situ hybridization methods using hapten- (biotin or digoxygenin) labeled RNA differs from conventional approaches by using many short (about 20 base pairs long) oligonucleotide probes Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided

  2. A laser induced fluorescence-based instrument for in-situ measurements of atmospheric formaldehyde.

    PubMed

    Hottle, John R; Huisman, Andrew J; DiGangi, Joshua P; Kammrath, Aster; Galloway, Melissa M; Coens, Katherine L; Keutsch, Frank N

    2009-02-01

    Direct, in situ detection of gas phase formaldehyde (HCHO) via laser induced fluorescence in a White-type multipass cell is demonstrated with a (3sigma) limit of detection of approximately 0.051 parts per billion by volume in a 1 s sampling time. Calibration is performed in two ways: using permeation tubes and with air bubbled through an aqueous solution of HCHO. The concentration of HCHO output from the bubbler is measured by cavity ring-down spectroscopy. Measurement of ambient HCHO is carried out at the University of Wisconsin, Madison for a period of several days. PMID:19245018

  3. Coexistence of t(15;17) and t(15;16;17) detected by fluorescence in situ hybridization in a patient with acute promyelocytic leukemia: A case report and literature review

    PubMed Central

    ZHANG, RUI; KIM, YOUNG-MI; WANG, XIANFU; LI, YAN; PANG, HUI; LEE, JI-YUN; LI, SHIBO

    2014-01-01

    Acute promyelocytic leukemia (APL) is characterized by the t(15;17)(q22;q21), which results in the fusion of the promyelocytic leukemia (PML) gene at 15q22 with the retinoic acid ?-receptor (RARA) gene at 17q21. The current study presents the case of a 54-year-old female with APL carrying the atypical PML/RARA fusion signal due to a novel complex variant translocation t(15;16;17)(q22;q24;q21), as well as the classical PML/RARA fusion signal. Subsequent array comparative genomic hybridization revealed somatic, cryptic deletions on 3p25.3, 8q23.1 and 12p13.2-p13.1, and a duplication on 8q11.2; however, no genetic material loss or gain was observed in the breakpoint regions of chromosomes 15, 16 or 17. To the best of our knowledge, this is the first report of the coexistence of two abnormal clones, one classical and one variant, presenting simultaneously in addition to cryptic chromosome segmental imbalances in an adult APL patient. PMID:25120648

  4. Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer.

    PubMed Central

    Tsuji, A; Sato, Y; Hirano, M; Suga, T; Koshimoto, H; Taguchi, T; Ohsuka, S

    2001-01-01

    We previously showed that a specific kind of mRNA (c-fos) was detected in a living cell under a microscope by introducing two fluorescently labeled oligodeoxynucleotides, each labeled with donor or acceptor, into the cytoplasm, making them hybridize to adjacent locations on c-fos mRNA, and taking images of fluorescence resonance energy transfer (FRET) (A. Tsuji, H. Koshimoto, Y. Sato, M. Hirano. Y. Sei-Iida, S. Kondo, and K. Ishibashi, 2000, Biophys. J. 78:3260-3274). On the formed hybrid, the distance between donor and acceptor becomes close and FRET occurs. To observe small numbers of mRNA in living cells using this method, it is required that FRET fluorescence of hybrid must be distinguished from fluorescence of excess amounts of non-hybridizing probes and from cell autofluorescence. To meet these requirements, we developed a time-resolved method using acceptor fluorescence decays. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay with direct excitation. A combination of Bodipy493/503 and Cy5 was selected as donor and acceptor. When the formed hybrid had a configuration where the target RNA has no single-strand part between the two fluorophores, the acceptor fluorescence of hybrid had a sufficiently longer delay to detect fluorescence of hybrid in the presence of excess amounts of non-hybridizing probes. Spatial separation of 10-12 bases between two fluorophores on the hybrid is also required. The decay is also much slower than cell autofluorescence, and smaller numbers of hybrid were detected with less interference of cell autofluorescence in the cytoplasm of living cells under a time-resolved fluorescence microscope with a time-gated function equipped camera. The present method will be useful when observing induced expressions of mRNA in living cells. PMID:11423432

  5. Femtosecond optical tweezers for in-situ control of two-photon fluorescence.

    PubMed

    Agate, B; Brown, C; Sibbett, W; Dholakia, K

    2004-06-28

    We perform a comparison of optical tweezing using continuous wave (cw) and femtosecond lasers. Measurement of the relative Q-values in the femtosecond and cw regimes shows that femtosecond optical tweezers are just as effective as cw optical tweezers. We also demonstrate simultaneous optical tweezing and in-situ control of two-photon fluorescence (at 400nm) from dye-doped polymer microspheres. By switching the 800 nm tweezing laser source between femtosecond and cw regimes, we turned the fluorescent signal from the tweezed particle on and off while maintaining an equivalent tweezing action. Femtosecond lasers can thus be used for optical tweezing and simultaneously utilized to induce nonlinear multi-photon processes such as two-photon excitation or even photoporation. PMID:19483818

  6. In-Situ Silver Acetylide Silver Nitrate Explosive Deposition Measurements Using X-Ray Fluorescence.

    SciTech Connect

    Covert, Timothy T.

    2014-09-01

    The Light Initiated High Explosive facility utilized a spray deposited coating of silver acetylide - silver nitrate explosive to impart a mechanical shock into targets of interest. A diagnostic was required to measure the explosive deposition in - situ. An X - ray fluorescence spectrometer was deployed at the facility. A measurement methodology was developed to measure the explosive quantity with sufficient accuracy. Through the use of a tin reference material under the silver based explosive, a field calibration relationship has been developed with a standard deviation of 3.2 % . The effect of the inserted tin material into the experiment configuration has been explored.

  7. Whole-Mount In Situ Hybridization of Hawaiian Bobtail Squid (Euprymna scolopes) Embryos with DIG-Labeled

    E-print Network

    Ruby, Edward G.

    Protocol Whole-Mount In Situ Hybridization of Hawaiian Bobtail Squid (Euprymna scolopes) Embryos in preparing Hawaiian bobtail squid (Euprymna scolopes) embryos, hybridizing a DIG-labeled riboprobe in whole Hybridization of Hawaiian Bobtail Squid (Euprymna scolopes) Embryos with DIG- Labeled Riboprobes: I. DNA

  8. ELF{trademark}: A new fluorogenic alkaline phosphatase substrate for nonisotopic mRNA in situ hybridization

    SciTech Connect

    Singer, V.; Paragas, V.; Zhang, Y.Z. [Molecular Probes, Inc., Eugene, OR (United States)

    1994-09-01

    Molecular Probes` researchers have developed a novel alkaline phosphatase substrate that gives rise to a bright yellow-green fluorescent precipitate at the site of enzymatic activity. We refer to this process as ELF, for Enzyme-Labeled Fluorescence. We found that the ELF substrate is an ideal tool for mRNA in situ hybridization. Not only are ELF signals much brighter than those generated using conventional fluorophores, resulting in film exposure times approximately one-fortieth as long as those needed for fluorescein-labeled probes or secondary detection reagents, but the ELF signal is also many-fold more photostable. The ELF signal develops in seconds to minutes, in contrast to radioactive detection, which requires days to weeks for adequate signal development. In addition, because the ELF precipitate has a Stokes shift of greater than 100 nm, we found that ELF signals can be easily distinguished from sample autofluorescence and from signals arising from other fluorescent probes or counterstrains. Also, unlike signals from chromogenic enzyme substrates, the fluorescent ELF signals are easily distinguished from pigmented tissues, even in zebrafish whole-mount embryos.

  9. Cytomegalovirus hepatitis confirmed by in situ hybridization in 3 immunocompetent infants.

    PubMed

    Tajiri, H; Kozaiwa, K; Tanaka-Taya, K; Tada, K; Takeshima, T; Yamanishi, K; Okada, S

    2001-01-01

    We present 3 cases of immunocompetent infants with CMV infection who showed prolonged liver dysfunction. In all cases the CMV genome was detectable in hepatocytes using the in situ hybridization method. Combination therapy with ganciclovir (GCV) and hyperimmune gammaglobulin (HGG) was instituted in 2 cases and successfully suppressed the replication of CMV, with sustained improvement in liver function. In 1 of these cases, signals for CMV DNA were undetectable in the liver 12 months after termination of combination therapy. These results help to confirm the etiology of CMV for persistent hepatitis in immunocompetent infants using the in situ hybridization method and also show the efficacy of combination therapy with a virostatic agent, GCV, and an immune-modulating agent, HGG. PMID:11728056

  10. Localization of parathyroid hormone-related protein in osteoclasts by in situ hybridization and immunohistochemistry

    Microsoft Academic Search

    V. Kartsogiannis; N. Udagawa; K. W. Ng; T. J. Martin; J. M. Moseley; H. Zhou

    1998-01-01

    Using immunohistology with two specific antisera raised against N-terminal parathyroid hormone-related protein (PTHrP) and in situ hybridization (riboprobe to common coding exon), evidence is provided for the expression of PTHrP by mouse, rabbit, and human osteoclasts derived from several in vitro and in vivo sources. In cocultures of mouse bone marrow and calvarial cells treated with 1,25-dihydroxyvitamin D3, the generated

  11. In situ synthesis of photocatalytically active hybrids consisting of bacterial nanocellulose and anatase nanoparticles.

    PubMed

    Wesarg, Falko; Schlott, Franziska; Grabow, Janet; Kurland, Heinz-Dieter; Heßler, Nadine; Kralisch, Dana; Müller, Frank A

    2012-09-18

    Bacterial nanocellulose (BNC) is an extraordinary biopolymer with a wide range of potential technical applications. The high specific surface area and the interconnected pore system of the nanofibrillar BNC network suggest applications as a carrier of catalysts. The present paper describes an in situ modification route for the preparation of a hybrid material consisting of BNC and photocatalytically active anatase (TiO(2)) nanoparticles (NPs). The influence of different NP concentrations on the BNC biosynthesis and the resulting supramolecular structure of the hybrids was investigated. It was found that the number of colony forming units (CFUs) and the consumption of glucose during biosynthesis remained unaffected compared to unmodified BNC. During the formation of the BNC network, the NPs were incorporated in the whole volume of the accruing hybrid. Their distribution within the hybrid material is affected by the anisotropic structure of BNC. The photocatalytic activity (PCA) of the BNC-TiO(2) hybrids was determined by methanol conversion (MC) under UV irradiation. These tests demonstrated that the NPs retained their PCA after incorporation into the BNC carrier structure. The PCA of the hybrid material depends on the amount of incorporated NPs. No alteration of the photocatalyst's efficiency was found during repeated PCA tests. In conclusion, the in situ integration of photocatalytically active NPs into BNC represents an attractive possibility to extend its fields of application to porous filtering media for drinking water purification and air cleaning. PMID:22925063

  12. Mapping of the gene encoding the melanocortin-1 ([alpha]-melanocyte stimulating hormone) receptor (MC1R) to human chromosome 16q24. 3 by fluorescence in situ hybridization

    SciTech Connect

    Gantz, I.; Yamada, Tadataka; Tashiro, Takao; Konda, Yoshitaka; Shimoto, Yoshimasa; Miwa, Hiroto; Trent, J.M. (Univ. of Michigan Medical Center, Ann Arbor, MI (United States))

    1994-01-15

    [alpha]-Melanocyte stimulating hormone ([alpha]-MSH), a hormone originally named for its ability to regulate pigmentation of melanocytes, is a 13-amino-acid post-translational product of the pro-opiomelanocortin (POMC) gene. [alpha]-MSH and the other products of POMC processing, which share the core heptapeptide amino acid sequence Met-Glu (Gly)-His-Phe-Arg-Trp-Gly (Asp), the adrenocorticotropic hormone (ACTH), [beta]-MSH, and [gamma]-MSH, are collectively referred to as melanocortins. While best known for their effects on the melanocyte (pigmentation) and adrenal cortical cells (steroidogenesis), melanocortins have been postulated to function in diverse activities, including enhancement of learning and memory, control of the cardiovascular system, analgesia, thermoregulation, immunomodulation, parturition, and neurotrophism. To identify the chromosomal band encoding the human melanocortin-1 receptor gene, 1 [mu]g of an EMBL clone coding region of the human MC1R and approximately 15 kb of surrounding DNA was labeled with biotin and hybridized to human metaphase chromosomes as previously described. The results indicate that the human MC1R gene is localized to 16q24.3. 15 refs., 1 fig.

  13. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization.

    PubMed Central

    Pinkel, D; Straume, T; Gray, J W

    1986-01-01

    This report describes the use of fluorescence in situ hybridization for chromosome classification and detection of chromosome aberrations. Biotin-labeled DNA was hybridized to target chromosomes and subsequently rendered fluorescent by successive treatments with fluorescein-labeled avidin and biotinylated anti-avidin antibody. Human chromosomes in human-hamster hybrid cell lines were intensely and uniformly stained in metaphase spreads and interphase nuclei when human genomic DNA was used as a probe. Interspecies translocations were detected easily at metaphase. The human-specific fluorescence intensity from cell nuclei and chromosomes was proportional to the amount of target human DNA. Human Y chromosomes were fluorescently stained in metaphase and interphase nuclei by using a 0.8-kilobase DNA probe specific for the Y chromosome. Cells from males were 40 times brighter than those from females. Both Y chromosomal domains were visible in most interphase nuclei of XYY amniocytes. Human 28S ribosomal RNA genes on metaphase chromosomes were distinctly stained by using a 1.5-kilobase DNA probe. Images PMID:3458254

  14. In situ measurement of bioluminescence and fluorescence in an integrated microbioreactor.

    PubMed

    Zanzotto, Andrea; Boccazzi, Paolo; Gorret, Nathalie; Van Dyk, Tina K; Sinskey, Anthony J; Jensen, Klavs F

    2006-01-01

    Reporter strains of bacteria that emit light or a fluorescent marker in response to specific conditions in their environment are having a significant impact in many areas of biology, including toxicity assays for environmental pollutants, chemical detection, and gene expression profiling. We have demonstrated methods for in situ measurements of bioluminescence and fluorescence from bacterial cultures grown in 50 microL instrumented microbioreactors. Results from microbioreactors were compared to results obtained from conventional 500 mL batch bioreactors and shake flasks. Experiments were conducted with reporter strains of Escherichia coli in which luxCDABE or gfp was fused to a promoter that was either expressed constitutively, or that responded to oxygen limitation. With these reporter strains, we have demonstrated the ability to obtain information on growth conditions within the microbioreactor. We have also shown that the large aspect ratio of the microbioreactor provides a unique advantage over measurements in larger bioreactors by reducing the inner filter effect in on-line measurements and eliminating the need for error-prone off-line dilutions. In addition, continuous on-line monitoring of genes in real-time, when expanded to include entire reporter libraries, could potentially provide a true dynamic picture of cellular gene expression from which the kinetics of gene expression can be untangled and elucidated. PMID:16187336

  15. In situ hybridization of oxytocin messenger RNA: macroscopic distribution and quantitation in rat hypothalamic cell groups

    SciTech Connect

    Burbach, J.P.; Voorhuis, T.A.; van Tol, H.H.; Ivell, R.

    1987-05-29

    Oxytocin mRNA was detected in the rat hypothalamus by in situ hybridization to a single stranded /sup 35/S-labelled DNA probe and the distribution of oxytocin mRNA-containing cell groups was studied at the macroscopic level. Specificity of hybridization was confirmed by comparison to vasopressin mRNA hybridization in parallel tissue sections. Cell groups containing oxytocin mRNA were confined to a set of hypothalamic cell groups, i.c. the supraoptic, paraventricular, anterior commissural nuclei, nucleus circularis and scattered hypothalamic islets. These cell groups displayed similar densities of autoradiographic signals indicating that the oxytocin gene is expressed at approximately the same average level at these various sites.

  16. In Situ Immune Infrared Fluorescent Staining for Detection and Quantification of Bluetongue Virus in Cullicoides Insect Cell Culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bluetongue virus (BTV) is transmitted to sheep, cattle and other ruminants by Culicoides spp. of biting midges. Cell lines have been developed from C. sonorensis; however, techniques for directly detecting and quantifying virus in these insect cells are lacking. In situ immune infrared fluorescent s...

  17. Effects of stress treatments on the detection of Salmonella typhimurium by in situ hybridization

    Microsoft Academic Search

    Tim Tolker-Nielsen; Marianne Halberg Larsen; Henriette Kyed; Søren Molin

    1997-01-01

    In order to assess the usefulness of quantitative in situ rRNA hybridization as an indicator of the physiological state of bacteria, we have used this method to measure the cellular contents of 16 S and 23 S rRNA in Salmonella typhimurium subjected to a number of different stress treatments. The contents of rRNA in S. typhimurium decreased when the bacteria

  18. In Situ Dimerization of Multiple Wild Type and Mutant Zinc Transporters in Live Cells Using Bimolecular Fluorescence Complementation

    PubMed Central

    Lasry, Inbal; Golan, Yarden; Berman, Bluma; Amram, Noy; Glaser, Fabian; Assaraf, Yehuda G.

    2014-01-01

    Zinc transporters (ZnTs) facilitate zinc efflux and zinc compartmentalization, thereby playing a key role in multiple physiological processes and pathological disorders, presumed to be modulated by transporter dimerization. We recently proposed that ZnT2 homodimerization is the underlying basis for the dominant negative effect of a novel heterozygous G87R mutation identified in women producing zinc-deficient milk. To provide direct visual evidence for the in situ dimerization and function of multiple normal and mutant ZnTs, we applied here the bimolecular fluorescence complementation (BiFC) technique, which enables direct visualization of specific protein-protein interactions. BiFC is based upon reconstitution of an intact fluorescent protein including YFP when its two complementary, non-fluorescent N- and C-terminal fragments (termed YN and YC) are brought together by a pair of specifically interacting proteins. Homodimerization of ZnT1, -2, -3, -4, and -7 was revealed by high subcellular fluorescence observed upon co-transfection of non-fluorescent ZnT-YC and ZnT-YN; this homodimer fluorescence localized in the characteristic compartments of each ZnT. The validity of the BiFC assay in ZnT dimerization was further corroborated when high fluorescence was obtained upon co-transfection of ZnT5-YC and ZnT6-YN, which are known to form heterodimers. We further show that BiFC recapitulated the pathogenic role that ZnT mutations play in transient neonatal zinc deficiency. Zinquin, a fluorescent zinc probe applied along with BiFC, revealed the in situ functionality of ZnT dimers. Hence, the current BiFC-Zinquin technique provides the first in situ evidence for the dimerization and function of wild type and mutant ZnTs in live cells. PMID:24451381

  19. In situ dimerization of multiple wild type and mutant zinc transporters in live cells using bimolecular fluorescence complementation.

    PubMed

    Lasry, Inbal; Golan, Yarden; Berman, Bluma; Amram, Noy; Glaser, Fabian; Assaraf, Yehuda G

    2014-03-14

    Zinc transporters (ZnTs) facilitate zinc efflux and zinc compartmentalization, thereby playing a key role in multiple physiological processes and pathological disorders, presumed to be modulated by transporter dimerization. We recently proposed that ZnT2 homodimerization is the underlying basis for the dominant negative effect of a novel heterozygous G87R mutation identified in women producing zinc-deficient milk. To provide direct visual evidence for the in situ dimerization and function of multiple normal and mutant ZnTs, we applied here the bimolecular fluorescence complementation (BiFC) technique, which enables direct visualization of specific protein-protein interactions. BiFC is based upon reconstitution of an intact fluorescent protein including YFP when its two complementary, non-fluorescent N- and C-terminal fragments (termed YN and YC) are brought together by a pair of specifically interacting proteins. Homodimerization of ZnT1, -2, -3, -4, and -7 was revealed by high subcellular fluorescence observed upon co-transfection of non-fluorescent ZnT-YC and ZnT-YN; this homodimer fluorescence localized in the characteristic compartments of each ZnT. The validity of the BiFC assay in ZnT dimerization was further corroborated when high fluorescence was obtained upon co-transfection of ZnT5-YC and ZnT6-YN, which are known to form heterodimers. We further show that BiFC recapitulated the pathogenic role that ZnT mutations play in transient neonatal zinc deficiency. Zinquin, a fluorescent zinc probe applied along with BiFC, revealed the in situ functionality of ZnT dimers. Hence, the current BiFC-Zinquin technique provides the first in situ evidence for the dimerization and function of wild type and mutant ZnTs in live cells. PMID:24451381

  20. In situ nanofabrication of hybrid PEG-dendritic-inorganic nanoparticles and preliminary evaluation of their biocompatibility.

    PubMed

    Sousa-Herves, Ana; Sánchez Espinel, Christian; Fahmi, Amir; González-Fernández, África; Fernandez-Megia, Eduardo

    2015-02-19

    An in situ template fabrication of inorganic nanoparticles using carboxylated PEG-dendritic block copolymers of the GATG family is described as a function of the dendritic block generation, the metal (Au, CdSe) and metal molar ratio. The biocompatibility of the generated nanoparticles analysed in terms of their aggregation in physiological media, cytotoxicity and uptake by macrophages relates to the PEG density of the surface of the hybrids. PMID:25530028

  1. Localization of single copy DNA sequences on G-banded human chromosomes by in situ hybridization

    Microsoft Academic Search

    Mary E. Harper; Grady F. Saunders

    1981-01-01

    Recombinant lambda bacteriophage clone H3 containing a human DNA segment of 14.9 kb present in one or two copies per haploid genome was isolated. In situ hybridization to human metaphase chromosomes of the 3H-labeled cloned DNA resulted in highly significant labeling (53% of cells) of band p36 of chromosome 1, such that 22% of all chromosomal grains were located on

  2. Design of Hybrid Steam-In Situ Combustion Bitumen Recovery Processes

    SciTech Connect

    Yang Xiaomeng; Gates, Ian D. [University of Calgary, Department of Chemical and Petroleum Engineering (Canada)], E-mail: ian.gates@ucalgary.ca

    2009-09-15

    Given enormous capital costs, operating expenses, flue gas emissions, water treatment and handling costs of thermal in situ bitumen recovery processes, improving the overall efficiency by lowering energy requirements, environmental impact, and costs of these production techniques is a priority. Steam-assisted gravity drainage (SAGD) is the most widely used in situ recovery technique in Athabasca reservoirs. Steam generation is done on surface and consequently, because of heat losses, the energy efficiency of SAGD can never be ideal with respect to the energy delivered to the sandface. An alternative to surface steam generation is in situ combustion (ISC) where heat is generated within the formation through injection of oxygen at a sufficiently high pressure to initiate combustion of bitumen. In this manner, the heat from the combustion reactions can be used directly to mobilize the bitumen. As an alternative, the heat can be used to generate steam within the formation which then is the agent to move heat in the reservoir. In this research, alternative hybrid techniques with simultaneous and sequential steam-oxygen injection processes are examined to maximize the thermal efficiency of the recovery process. These hybrid processes have the advantage that during ISC, steam is generated within the reservoir from injected and formation water and as a product of oxidation. This implies that ex situ steam generation requirements are reduced and if there is in situ storage of combustion gases, that overall gas emissions are reduced. In this research, detailed reservoir simulations are done to examine the dynamics of hybrid processes to enable design of these processes. The results reveal that hybrid processes can lower emitted carbon dioxide-to-oil ratio by about 46%, decrease the consumed natural gas-to-oil ratio by about 73%, reduce the cumulative energy-to-oil ratio by between 40% and 70% compared to conventional SAGD, and drop water consumption per unit oil produced. However, oil recovery is between 25% and 40% below that of SAGD. Design of successful hybrid steam-oxygen processes must take into account the balance between injected steam and amount of injected oxygen and combustion gas products that dilute injected and in situ-generated steam in the depletion chamber by lowering its partial pressure, and thus its saturation temperature which in turn impacts production rates and recovery.

  3. Rapid identification of marker chromosomes by in situ hybridization under different stringency conditions.

    PubMed

    Vorsanova, S G; Yurov, Y B; Soloviev, I V; Demidova, I A; Malet, P

    1994-10-01

    Six metacentric non-satellite chromosome markers and 4 satellite markers of unknown origin were discovered by routine cytogenetic analysis. These markers were then investigated by isotopic and nonisotopic (FISH) in situ hybridization. In order to determine the origin of small marker chromosomes, a special protocol involving sequential application of defined alphoid and 'classical' satellite DNA probes with chromosome specificity was used. In situ hybridization under low stringency conditions has been performed with DNA probes specific for 4 groups of chromosomes. After preliminary analysis and determination of the possible origin of these marker chromosomes, DNA probes with high chromosome specificity were used under high stringency conditions. Marker chromosomes were found to be derivatives of chromosomes 7, 9 (3 cases), 13, 14 or 22, 21 (2 cases), X and Y. PMID:7848878

  4. Analysis of microRNA expression by in situ hybridization with RNA oligonucleotide probes

    PubMed Central

    Thompson, Robert C.; Deo, Monika; Turner, David L.

    2007-01-01

    In situ hybridization is an important tool for analyzing gene expression and developing hypotheses about gene functions. The discovery of hundreds of microRNA (miRNA) genes in animals has provided new challenges for analyzing gene expression and functions. The small size of the mature miRNAs (?20-24 nucleotides in length) presents difficulties for conventional in situ hybridization methods. However, we have developed a modified in situ hybridization method for detection of mammalian miRNAs in tissue sections, based upon the use of RNA oligonucleotide probes in combination with highly specific wash conditions. Here we present detailed procedures for detection of miRNAs in tissue sections or cultured cells. The methods described can utilize either nonradioactive hapten-conjugated probes that are detected by enzyme-coupled antibodies, or radioactively labeled probes that are detected by autoradiography. The ability to visualize miRNA expression patterns in tissue sections provides an additional tool for the analyses of miRNA expression and function. In addition, the use of radioactively labeled probes should facilitate quantitative analyses of changes in miRNA gene expression. PMID:17889803

  5. High-resolution in situ hybridization to whole-mount zebrafish embryos

    Microsoft Academic Search

    Christine Thisse; Bernard Thisse

    2007-01-01

    The in situ hybridization (ISH) technique allows the sites of expression of particular genes to be detected. This protocol describes ISH of digoxigenin-labeled antisense RNA probes to whole-mount zebrafish embryos. In our method, PCR-amplified sequence of a gene of interest is used as a template for the synthesis of an antisense RNA probe, which is labeled with digoxigenin-linked nucleotides. Embryos

  6. Development of a combined portable x-ray fluorescence and Raman spectrometer for in situ analysis

    NASA Astrophysics Data System (ADS)

    Guerra, M.; Longelin, S.; Pessanha, S.; Manso, M.; Carvalho, M. L.

    2014-06-01

    In this work, we have built a portable X-ray fluorescence (XRF) spectrometer in a planar configuration coupled to a Raman head and a digital optical microscope, for in situ analysis. Several geometries for the XRF apparatus and digital microscope are possible in order to overcome spatial constraints and provide better measurement conditions. With this combined spectrometer, we are now able to perform XRF and Raman measurements in the same point without the need for sample collection, which can be crucial when dealing with cultural heritage objects, as well as forensic analysis. We show the capabilities of the spectrometer by measuring several standard reference materials, as well as other samples usually encountered in cultural heritage, geological, as well as biomedical studies.

  7. Ultrastructural in situ hybridization to nascent transcripts of highly transcribed rRNA genes in chromatin spreads

    Microsoft Academic Search

    Marina M. O'Reilly; Sarah L. French; Martha L. Sikes; Oscar L. Miller Jr

    1994-01-01

    The amplified rRNA genes of amphibian oocytes were used as a model system for the development of an in situ hybridization technique to label nascent transcripts in dispersed chromatin. A biotinylated complementary RNA probe was hybridized to nascent transcripts from dispersed nucleoli, and detected by a two step antibody technique utilizing colloidal gold as an electron dense marker. A specific

  8. Quantitative methods for genome-scale analysis of in situ hybridization and correlation with microarray data

    PubMed Central

    Lee, Chang-Kyu; Sunkin, Susan M; Kuan, Chihchau; Thompson, Carol L; Pathak, Sayan; Ng, Lydia; Lau, Chris; Fischer, Shanna; Mortrud, Marty; Slaughterbeck, Cliff; Jones, Allan; Lein, Ed; Hawrylycz, Michael

    2008-01-01

    With the emergence of genome-wide colorimetric in situ hybridization (ISH) data sets such as the Allen Brain Atlas, it is important to understand the relationship between this gene expression modality and those derived from more quantitative based technologies. This study introduces a novel method for standardized relative quantification of colorimetric ISH signal that enables a large-scale cross-platform expression level comparison of ISH with two publicly available microarray brain data sources. PMID:18234097

  9. In Situ Hybridization Combined With Antibody Staining On Tissue Sections

    E-print Network

    Jessell,