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1

Fluorescence In Situ Hybridization  

Microsoft Academic Search

Dr. Seuss’s eloquent “One FISH, two FISH, red FISH, blue FISH” (1) could have been describing one of the most significant advancements in clinical cytogenetics, fluorescence in situ hybridization (FISH). The process, as described by Pinkel et al. in 1988 (2), involved fluorescent detection of probe DNA hybridized to chromosomal target sequences. The overall hybridization was essentially\\u000a the same one

Daynna J. Wolff; Stuart Schwartz

2

Molecular cytogenetics using fluorescence in situ hybridization.  

National Technical Information Service (NTIS)

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...

J. W. Gray W. L. Kuo J. Lucas D. Pinkel H. U. Weier

1990-01-01

3

Molecular cytogenetics using fluorescence in situ hybridization  

SciTech Connect

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.

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

1990-12-07

4

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

Code of Federal Regulations, 2010 CFR

... 2009-04-01 false Automated fluorescence in situ hybridization (FISH) enumeration...Reagents § 866.4700 Automated fluorescence in situ hybridization (FISH) enumeration...Controls Guidance Document: Automated Fluorescence in situ Hybridization (FISH)...

2009-04-01

5

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

Code of Federal Regulations, 2010 CFR

... 2010-04-01 false Automated fluorescence in situ hybridization (FISH) enumeration...Reagents § 866.4700 Automated fluorescence in situ hybridization (FISH) enumeration...Controls Guidance Document: Automated Fluorescence in situ Hybridization (FISH)...

2010-04-01

6

Fluorescence in situ hybridization with comets.  

PubMed

We have adapted the fluorescence in situ hybridization technique to single-cell gel electrophoresis (comet assayed) preparations. Since cells were embedded in agarose, probed regions could be visualized in three dimensions. This system makes it possible to determine the spatial distribution of chromosome-specific DNA sequences at the level of the individual nucleus (nonelectrophoresed) as well as in chromatin fibers of comets (electrostretched chromosomal DNA). This methodology is likely to bring new insights into the field of interphase nuclear ultrastructure. Here, we present the preliminary data obtained with human blood lymphocytes in Gzero after they have been electrophoresed for different times. Chromosome-specific areas (all centromeres, all telomeres, chromosome 7-specific centromere, and long arm of chromosome 3-specific telomere, as well as three segments of the O6-methylguanine-DNA methyltransferase gene) were investigated. Our results are in agreement with the concept that telomeres are in close association with the nuclear membrane and suggest that centromeres are relatively less condensed structures located in the center of the interphase nucleus. PMID:9168819

Santos, S J; Singh, N P; Natarajan, A T

1997-05-01

7

Reutilization of previously hybridized slides for fluorescence in situ hybridization  

SciTech Connect

Application of fluorescence in situ hybridization (FISH) to clinical material is sometimes limited by sample size. In addition, heterogeneity among slides prepared from a single sample may lead to variation in FISH analyses. Reutilization of material for repeated FISH analyses would help to alleviate these problems. We have developed a simple procedure for repeated FISH analyses with directly conjugated probes. Previously hybridized probes are removed by incubation in denaturing solution, and slides can then be rehybridized without residual signals remaining. Several cycles of this procedure allow a full complement of chromosomal loci to be analyzed on the same population of cells. Advantages of this protocol include gaining more cytogenetic information from small samples and eliminating the problem of intratumorvariability. 5 refs., 4 figs.

Epstein, L.; DeVries, S.; Waldman, F.M. [Univ. of California, San Francisco, CA (United States)

1995-12-01

8

Concomitant Oncoprotein Detection with Fluorescence in Situ Hybridization (CODFISH)  

PubMed Central

We sought the validation of a three-color fluorescence-based system that simultaneously profiles Her-2/neu oncogene copy by fluorescence in situ hybridization (FISH) and Her-2/neu encoded protein by the use of a versatile alkaline phosphatase chromogen fast red K in either fluorescence or bright-field mode. Nuclei were counterstained with DAPI. Nineteen infiltrating ductal carcinomas of breast were comprehensively evaluated for Her-2/neu amplification/overexpression by direct and indirect FISH using digoxigenin (DigFISH) and direct fluorescently labeled probes, autoradiographic RNA:RNA in situ hybridization, and immunohistochemistry using monoclonal antibody CB11. CODFISH results correlated well with DigFISH, direct-label FISH, mRNA expression, and oncoprotein expression as assessed with CB11, and enabled simultaneous visualization of gene copy and protein. In addition, qualitative immunohistochemistry may be followed by CODFISH gene copy enumeration to clarify ambiguous cases.

Tubbs, Raymond R.; Pettay, James; Roche, Pat; Stoler, Mark H.; Jenkins, Robert; Myles, Jon; Grogan, Thomas

2000-01-01

9

Supernumerary ring chromosome 17 identified by fluorescent in situ hybridization  

SciTech Connect

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.

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

1997-04-14

10

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

ERIC Educational Resources Information Center

|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…

Baker, William P.; Jones, Carleton Buck

2006-01-01

11

PGD for dystrophin gene deletions using fluorescence in situ hybridization  

Microsoft Academic Search

Duchenne muscular dystrophy and Becker muscular dystrophy (DMD and BMD) are caused by mutations in the dystrophin gene (Xp21). In two-thirds of DMD\\/BMD cases, the mutation is a large deletion of one or several exons. We have established PGD for DMD\\/BMD using interphase fluorescence in situ hybridization (FISH) analysis on single nuclei from blastomeres for the detection of deletions of

H. Malmgren; I. White; S. Johansson; L. Levkov; E. Iwarsson; M. Fridström; Elisabeth Blennow

2006-01-01

12

Labeling meiotic chromosomes in maize with fluorescence in situ hybridization.  

PubMed

Fluorescence in situ hybridization (FISH) can be used to visualize chromosomal features using repetitive or single gene probes above a minimum target size. When applied to meiosis, each chromosome of the karyotypic complement can be identified, which can facilitate an understanding of the interrelationship of different chromosomes during this process. On the other hand, the pachytene stage of early meiosis is characterized by slightly but not strongly condensed chromosomes that permit more detailed analyses of adjacent features than can be achieved with somatic metaphase chromosomes. PMID:23559200

Gao, Zhi; Han, Fangpu; Danilova, Tatiana V; Lamb, Jonathan C; Albert, Patrice S; Birchler, James A

2013-01-01

13

10p Duplication characterized by fluorescence in situ hybridization  

SciTech Connect

We describe a patient with severe failure to thrive, mild-moderate developmental delay, cleft lip and palate, and other anomalies. Routine cytogenetic analysis documented a de novo chromosome rearrangement involving chromosome 4, but the origin of the derived material was unknown. Using chromosome specific painting probes, the karyotype was defined as 46,XY,der(4)t(4;10)(q35;p11.23). Characterization of the dup(10p) by fluorescence in situ hybridization (FISH) analysis provides another example of the usefulness of this technology in identifying small deletions, duplications, or supernumerary marker chromosomes. 19 refs., 4 figs.

Wiktor, A.; Feldman, G.L.; Van Dyke, D.L.; Kratkoczki, P.; Ditmars, D.M. Jr. [Henry Ford Hospital, Detroit, MI (United States)

1994-09-01

14

Fluorescence in situ hybridization for the identification of environmental microbes.  

PubMed

This chapter presents a protocol for the phylogenetic identification of microorganisms in environmental samples (water and sediments) by means of fluorescence in situ hybridization (FISH) with ribosomal RNA-targeted oligonucleotide probes and signal amplification (catalyzed reporter deposition [CARD]). The FISH probes are labeled with the enzyme, horseradish peroxidase (HRP). A subsequent deposition of fluorescently labeled tyramides results in substantially higher signal intensities of target cells than after FISH with probes directly labeled with fluorochromes. Sample preparation and cell permeabilization strategies for various microbial cell wall types are discussed. The custom labeling of tyramides with different fluorochromes is described. A sequential multicolor CARD-FISH protocol is outlined for the simultaneous detection of different phylogenetic groups. PMID:17332640

Pernthaler, Annelie; Pernthaler, Jakob

2007-01-01

15

Spatial Distribution of Sperm-Derived Chromatin in Zygotes Determined by Fluorescence In situ Hybridization.  

National Technical Information Service (NTIS)

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

B. F. Brandriff L. A. Gordon

1992-01-01

16

Rapid prenatal aneuploidy screening by fluorescence in situ hybridization (FISH).  

PubMed

The most common aneuploidies in prenatal diagnostics of the second trimenon are trisomies of chromosomes 13, 18, and 21 and gonosomal abnormalities. To detect these trisomies as quickly as possible after amniocentesis, besides using polymerase chain reaction, fluorescence in situ hybridization (FISH), applying corresponding centromeric or locus-specific probes, is the method of choice. Results of a rapid prenatal aneuploidy screening in uncultured amniocytes by using FISH are available within 24 hr or less. However, care has to be taken against possible pitfalls in connection with the commercially available probe sets and thus interpretation of results in general. Here, we explain how rapid prenatal aneuploidy screening is performed using the Food and Drug Administration-approved Aneu Vysion kit (Abbott/Vysis), and a review is given of drawbacks and opportunities of this method. PMID:18425470

Weise, Anja; Liehr, Thomas

2008-01-01

17

Prenatal diagnosis from cystic hygroma fluid: The value of fluorescence in situ hybridization  

Microsoft Academic Search

Objective: We sought to determine the optimal approach to the prenatal chromosome analysis of cystic hygroma fluid using traditional cytogenetic analysis and fluorescence in situ hybridization. Study Design: A retrospective evaluation of our experience with traditional cytogenetic and fluorescence in situ hybridization analysis on cystic hygroma fluid was performed through a systematic review of the Genzyme Genetics database from January

Alan E. Donnenfeld; David Lockwood; Allen N. Lamb

2001-01-01

18

Flow sorting of marine bacterioplankton after fluorescence in situ hybridization.  

PubMed

We describe an approach to sort cells from coastal North Sea bacterioplankton by flow cytometry after in situ hybridization with rRNA-targeted horseradish peroxidase-labeled oligonucleotide probes and catalyzed fluorescent reporter deposition (CARD-FISH). In a sample from spring 2003 >90% of the cells were detected by CARD-FISH with a bacterial probe (EUB338). Approximately 30% of the microbial assemblage was affiliated with the Cytophaga-Flavobacterium lineage of the Bacteroidetes (CFB group) (probe CF319a), and almost 10% was targeted by a probe for the beta-proteobacteria (probe BET42a). A protocol was optimized to detach cells hybridized with EUB338, BET42a, and CF319a from membrane filters (recovery rate, 70%) and to sort the cells by flow cytometry. The purity of sorted cells was >95%. 16S rRNA gene clone libraries were constructed from hybridized and sorted cells (S-EUB, S-BET, and S-CF libraries) and from unhybridized and unsorted cells (UNHYB library). Sequences related to the CFB group were significantly more frequent in the S-CF library (66%) than in the UNHYB library (13%). No enrichment of beta-proteobacterial sequence types was found in the S-BET library, but novel sequences related to Nitrosospira were found exclusively in this library. These bacteria, together with members of marine clade OM43, represented >90% of the beta-proteobacteria in the water sample, as determined by CARD-FISH with specific probes. This illustrates that a combination of CARD-FISH and flow sorting might be a powerful approach to study the diversity and potentially the activity and the genomes of different bacterial populations in aquatic habitats. PMID:15466568

Sekar, Raju; Fuchs, Bernhard M; Amann, Rudolf; Pernthaler, Jakob

2004-10-01

19

Pallister-Killian syndrome detected by fluorescence in situ hybridization  

SciTech Connect

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.

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

1995-07-03

20

PGD for dystrophin gene deletions using fluorescence in situ hybridization.  

PubMed

Duchenne muscular dystrophy and Becker muscular dystrophy (DMD and BMD) are caused by mutations in the dystrophin gene (Xp21). In two-thirds of DMD/BMD cases, the mutation is a large deletion of one or several exons. We have established PGD for DMD/BMD using interphase fluorescence in situ hybridization (FISH) analysis on single nuclei from blastomeres for the detection of deletions of specific exons in the dystrophin gene. We performed PGD for two carrier females; one had a deletion of exons 45-50 (DMD), and the other had a deletion of exons 45-48 (BMD). An exon 45-specific probe was used in combination with probes for the X and Y centromeres. Using this straightforward approach, we can distinguish affected and unaffected male embryos as well as carrier female and normal female embryos. Three cycles were performed for each patient, which resulted in a pregnancy and the birth of a healthy girl. To the best of our knowledge, this approach for PGD has not been previously reported. The use of interphase FISH is an attractive alternative to sexing or PCR-based mutation detection for PGD patients with known deletions of the dystrophin gene. PMID:16608904

Malmgren, H; White, I; Johansson, S; Levkov, L; Iwarsson, E; Fridström, M; Blennow, Elisabeth

2006-04-11

21

Automated Image Analysis for Quantitative Fluorescence In Situ Hybridization with Environmental Samples  

Microsoft Academic Search

When fluorescence in situ hybridization (FISH) analyses are performed with complex environmental sam- ples, difficulties related to the presence of microbial cell aggregates and nonuniform background fluorescence are often encountered. The objective of this study was to develop a robust and automated quantitative FISH method for complex environmental samples, such as manure and soil. The method and duration of sample

Zhi Zhou; Marie Noelle Pons; Lutgarde Raskin; Julie L. Zilles

2007-01-01

22

Single-step multicolor fluorescence In situ hybridization using semiconductor quantum dot-DNA conjugates  

Microsoft Academic Search

We report a rapid method for the direct multicolor imaging of multiple subnuclear genetic sequences using novel quantum dot-based\\u000a fluorescence in situ hybridization (FISH) probes (QD-FISH). Short DNA oligonucleotides were attached on QDs and used in a single hybridization\\/detection\\u000a step of target sites in situ. QD-FISH probes penetrate both intact interphase nuclei and metaphase chromosomes and showed good targeting of

Laurent A. Bentolila; Shimon Weiss

2006-01-01

23

Fluorescence in situ hybridization for intracellular localization of nifH mRNA.  

PubMed

Few reports on in situ mRNA detection in bacteria have been published, even though a major aim in environmental microbiology is to link function/activity to the identity of the organisms. This study reports a reliable approach for the in situ detection of nifH mRNA using fluorescence hybridization based on a previously described protocol for pmoA. nifH codes for a dinitrogenase reductase, a key enzyme in dinitrogen fixation. nifH mRNA was hybridized with a digoxigenin-labelled polynucleotide probe. The hybrid was detected with an anti-DIG-antibody labelled with horseradish peroxidase. Subsequently, the signal was amplified by catalyzed reporter deposition (CARD) with fluorochrome-labelled tyramides. Furthermore, the imaged organisms were identified using standard fluorescence in situ hybridization of rRNA. Thus, the approach enabled us specifically to link in situ the information from the dinitrogen fixation activity of an organism to its identity. Unexpectedly, the signals derived from nifH mRNA hybridization showed a distinct uneven pattern within the cells. This indicated that the method used could even give insights about the localization of the detected mRNA within the cell, which is a potential use of mRNA fluorescence in situ hybridization (FISH) that has not been reported up to now for bacterial cells. PMID:19217232

Pilhofer, Martin; Pavlekovic, Marko; Lee, Natuschka M; Ludwig, Wolfgang; Schleifer, Karl-Heinz

2009-02-12

24

Comparative genomic hybridization and multiplex-fluorescence in situ hybridization: an appraisal in elderly patients with acute myelogenous leukemia  

Microsoft Academic Search

Comparative genomic hybridization (CGH) and multiplex-fluorescence in situ hybridization (M-FISH) were used to evaluate the presentation karyotype in 15 and 18 patients respectively, aged 560 years, with acute myeloid leukemia (AML). Conventional G-banded analysis was performed in all patients prior to evaluation. Comparative genomic hybridization confirmed the G-banded karyotype fully in 12 patients and partially in two patients. Normal CGH

Christopher D Dalley; Michael J Neat; Nicola J Foot; Margaret Burridge; Loretta Byrne; John A Amess; Ama Z S Rohatiner; Andrew Lister; Bryan D Young; Debra M Lillington

2002-01-01

25

Applications and technical challenges of fluorescence in situ hybridization in stem cell research  

Microsoft Academic Search

Stem cell research, maintenance, and manipulations have advanced significantly in recent years, and we now witness successful clinical applications of stem therapies. However, challenges in regard to karyotypic stability and the ploidy status of stem cell lines have been addressed only marginally. Our approach to develop technology to address these highly relevant issues is based on fluorescence in situ hybridization

Heinz-Ulli G Weier; Lisa W Chu; John P Murnane; Jingly F Weier

2004-01-01

26

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...

27

Detection of aneuploidy in human oocytes and corresponding first polar bodies by fluorescent in situ hybridization  

Microsoft Academic Search

Purpose: The purpose of the study was to investigate the reliability of the fluorescent in situ hybridization (FISH) analysis of the first polar body (IPB) for cytogenetic evaluation of human oocytes as a method of choice in preimplantation diagnosis of chromosomal aneuploidies.

Andrei Dyban; Michael Freidine; Elena Severova; Jeanine Cieslak; Victor Ivakhnenko; Yury Verlinsky

1996-01-01

28

ERBB2 Amplification in Breast Cancer Analyzed by Fluorescence in situ Hybridization  

Microsoft Academic Search

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

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

29

Presence of endophytic bacteria in Vitis vinifera leaves as detected by fluorescence in situ hybridization  

Microsoft Academic Search

Fluorescence in situ hybridization (FISH) in combination with confocal laser scanning microscopy (CLSM) was applied to detect\\u000a and localize bacterial colonies in leaf tissues of Vitis vinifera. Leaves were cleared to minimize the autofluorescence of plant fragments. The use of fluorescently labeled bacterial probe\\u000a EUB338 on discolored grapevine leaf disks allowed the estimation of the spatial distribution of different bacterial

Sandra Lo Piccolo; Valeria Ferraro; Antonio Alfonzo; Luca Settanni; Danilo Ercolini; Santella Burruano; Giancarlo Moschetti

2010-01-01

30

FISH - (Fluoresence In Situ Hybridization)  

NSDL National Science Digital Library

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.

Darryl Leja (National Human Genome Research Institute REV)

2005-04-04

31

Application of fluorescence in situ hybridization (FISH) techniques to fish genetics: a review  

Microsoft Academic Search

The technology is now in place for major advances in the genetics and cytogenetics of fishes at the molecular level. One promising method with broad application is fluorescence in situ hybridization (FISH). Methodologies of FISH and the current and potential uses of these chromosomal techniques in fish genetics are reviewed. Highly repetitive ribosomal genes (rDNAs) and the multicopy genes for

Ruth B. Phillips; Kent M. Reed

1996-01-01

32

Genomic alterations in lung adenocarcinomas detected by multicolor fluorescence in situ hybridization and comparative genomic hybridization.  

PubMed

We used two molecular cytogenetic techniques, multicolor fluorescence in situ hybridization (M-FISH) and comparative genomic hybridization (CGH), to analyze three established lung adenocarcinoma cell lines (A549, H1650, and SPC-A-1) and primary lung adenocarcinoma samples, to identify common chromosomal aberrations. M-FISH revealed numerous complex chromosomal rearrangements. Chromosomes 5, 6, 11, 12, and 17 were most frequently involved in interchromosomal translocations. CGH revealed regions on 1q, 2p, 3q, 5p, 5q, 7p, 8q, 11q, 12q, 14q, 16p, 17p, 19q, 20q, 21q, and 22q to be commonly overrepresented and regions on 2q, 3p, 4p, 5q, 7q, 8p, 9p, 13q, 14q, and 17p to be underrepresented. The most common gains were found in 16p13 (in 50% of samples), and 16p13 amplification was associated with relatively poor differentiation and late stage. M-FISH and CGH can be a powerful tool in identification of genomic alterations in lung cancer, as well as in diagnosis. The overrepresented regions may harbor potential candidate genes involved in lung adenocarcinoma pathogenesis. PMID:18295661

Shen, Hua; Zhu, Yu; Wu, Yu-Jie; Qiu, Hai-Rong; Shu, Yong-Qian

2008-03-01

33

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

SciTech Connect

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.

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

1995-07-17

34

Fluorescence in situ hybridization of chorionic interphase cells for prenatal screening of Down syndrome  

Microsoft Academic Search

Objective: Our purpose was to determine the usefulness and reliability of fluorescence in situ hybridization on interphase chorionic villi cells in the prenatal diagnosis of Down syndrome. Methods: A total of 336 samples of chorionic villi were analysed by direct chromosome preparation and FISH with a DNA probe specific to chromosome 21. The samples were obtained as part of the

András Tóth; Erika P. Tardy; Krisztina Hajdu; József Bátorfi; József Doszpod; Jenõ Egyed; István Gáti

2001-01-01

35

Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition for the Identification of Marine Bacteria  

Microsoft Academic Search

Fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition (CARD), is currently not generally applicable to heterotrophic bacteria in marine samples. Penetration of the HRP molecule into bacterial cells requires permeabilization procedures that cause high and most probably species-selective cell loss. Here we present an improved protocol for

Annelie Pernthaler; Jakob Pernthaler; Rudolf Amann

2002-01-01

36

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

Microsoft Academic Search

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

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

2003-01-01

37

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

Microsoft Academic Search

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

Ruth B. Phillips

2001-01-01

38

The analysis of one or two blastomeres for PGD using fluorescence in-situ hybridization  

Microsoft Academic Search

BACKGROUND: The analysis of one or two blastomeres for PGD using fluorescence in-situ hybridization (FISH) is debated. The proportion of analysable embryos, false negatives, false positives, sensitivity, specificity, negative pre- dictive value (NPV), positive predictive value (PPV) and efficiency were evaluated when one or two blastomeres were analysed. METHODS: Embryos of patients having PGD for aneuploidy screening were assigned non-randomly

An Michiels; Elvire Van Assche; Inge Liebaers; André Van Steirteghem; Catherine Staessen

39

Rapid Diagnosis of Bacterial Meningitis by Real-Time PCR and Fluorescence In Situ Hybridization  

Microsoft Academic Search

Real-time PCR and fluorescence in situ hybridization (FISH) were evaluated as rapid methods for the diagnosis of bacterial meningitis and compared to standard diagnostic procedures. For PCR, a LightCycler approach was chosen, implementing eubacterial and specific PCR assays for the most relevant bacteria. For FISH, a similar probe set containing eubacterial and specific probes was composed of published and newly

Sven Poppert; Andreas Essig; Barbara Stoehr; Adelinde Steingruber; Beate Wirths; Stefan Juretschko; Udo Reischl; Nele Wellinghausen

2005-01-01

40

Adjunct Prenatal Testing: Patient Decisions Regarding Ethnic Carrier Screening and Fluorescence In Situ Hybridization  

Microsoft Academic Search

Little has been reported regarding how women make decisions about genetic carrier screening for Ashkenazi Jewish genetic disease and cystic fibrosis (CF), and for fluorescent in situ hybridization (FISH) during pregnancy. Thirty-seven women who underwent genetic counseling and prenatal diagnosis were interviewed about their prenatal decision making. Respondents were largely Caucasian (95%), and undergoing prenatal diagnosis because of maternal age

Erica L. Sturm; Kelly E. Ormond

2004-01-01

41

Microbial Community Composition of Wadden Sea Sediments as Revealed by Fluorescence In Situ Hybridization  

Microsoft Academic Search

The microbial community composition of Wadden Sea sediments of the German North Sea coast was investigated by in situ hybridization with group-specific fluorescently labeled, rRNA-targeted oligonucleotides. A large fraction (up to 73%) of the DAPI (4*,6-diamidino-2-phenylindole)-stained cells hybridized with the bacterial probes. Nearly 45% of the total cells could be further identified as belonging to known phyla. Members of the

ENRIC LLOBET-BROSSA; RAMON ROSSELLO ´-MORA; RUDOLF AMANN; Max Planck

1998-01-01

42

Analysis of ovarian borderline tumors using comparative genomic hybridization and fluorescence in situ hybridization.  

PubMed

It is unclear whether ovarian borderline tumors (tumors of low malignant potential) are independent entities or whether they are part of a continuum of tumor progression that culminates in ovarian carcinoma. Little is known about genetic abnormalities in borderline tumors because of the difficulty of growing them in culture for chromosome studies, and because the low ratio of tumor to nontumor cells can interfere with molecular genetic examination. To circumvent these problems, we performed comparative genomic hybridization (CGH) on 10 serous borderline tumors from nine patients, using microdissection to enrich the samples for tumor DNA and reduce contamination from stromal and inflammatory cells. CGH analysis revealed that three of the tumors had detectable chromosomal imbalances, whereas seven were in a balanced state. In those tumors with imbalances, the number of abnormalities ranged from 3-6 per tumor. Additional studies by fluorescence in situ hybridization (FISH) on disaggregated nuclei confirmed the imbalances detected by CGH, revealed one tumor to be hypertriploid, and indicated that the remaining tumors were diploid and in a balanced state. All abnormalities observed in the aneuploid cases are consistent with chromosomal aberrations previously reported for ovarian carinomas, providing further evidence that some borderline tumors are part of a continuum of tumor progression. These results also suggest that there may be different mechanisms leading to borderline tumor formation, including one associated with multiple chromosomal imbalances, and others that do not involve imbalances detectable by CGH. Genes Chromosomes Cancer 25:307-315, 1999. PMID:10398423

Wolf, N G; Abdul-Karim, F W; Farver, C; Schröck, E; du Manoir, S; Schwartz, S

1999-08-01

43

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

SciTech Connect

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.

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

1996-12-30

44

HER2\\/ neu gene amplification status in prostate cancer by fluorescence in situ hybridization  

Microsoft Academic Search

HER-2\\/neu expression has been established as a prognostic factor in breast and other cancers. In prostate cancer (PC), a similar predictive role has been hindered by variable immunohistochemical (IHC) results. The authors studied DNA amplification of the HER-2\\/ neu gene on 4-?m sections obtained from 62 formalin-fixed, paraffin-embedded PCs by fluorescence in situ hybridization (FISH). The results were compared with

Jeffrey S Ross; Christine Sheehan; Alida M Hayner-Buchan; Robert A Ambros; Bhaskar V. S Kallakury; Ronald Kaufman; Hugh A. G Fisher; Patrick J Muraca

1997-01-01

45

Fluorescence in situ hybridization analysis of chromosome aberrations in 60 Chinese patients with multiple myeloma  

Microsoft Academic Search

Conventional cytogenetic analysis is often hampered owing to the low mitotic index of multiple myeloma (MM) cells in bone\\u000a marrow samples of MM. Interphase fluorescence in situ hybridization (I-FISH) analysis combined with magnetic-activated cell\\u000a sorting (MACS) has substantially enhanced the sensitivity of cytogenetic analysis. Here, we used I-FISH to explore the incidence\\u000a of chromosomal abnormalities in 60 Chinese patients with

Xiao GaoChunming; Chunming Li; Run Zhang; Ruifang Yang; Xiaoyan Qu; Hairong Qiu; Jiaren Xu; Hua Lu; Jianyong Li; Lijuan Chen

46

Numerical chromosome alterations in colorectal carcinomas detected by fluorescence in situ hybridization  

Microsoft Academic Search

This study concerns DNA ploidy, numerical changes of chromosomes 7, 8, 10, 17 and 18, and allelic losses at chromosomes 17p13.3 (flanking the p53 gene) and 18q21 (location of the DCC gene) in 31 freshly resected colorectal tumours. Cytological smears were used to determine DNA ploidy by image analysis, and chromosome numbers by fluorescence in situ hybridization (FISH) using chromosome-specific

Akishi Ooi; Isao Nakanishi; C.-D. Huang; M. Mai

1996-01-01

47

Demonstration of a mechanism of aneuploidy in human oocytes using Multifluor fluorescence in situ hybridization  

Microsoft Academic Search

Objective: To evaluate the potential of Multifluor fluorescence in situ hybridization (M-FISH) for karyotyping the human oocyte and first polar body.Design: Prospective case study.Setting: Research laboratories, university hospital.Patient(s): A 33-year-old woman with polycystic ovary syndrome who was undergoing ovarian stimulation and ICSI.Main Outcome Measure(s): Karyotyping of all chromosomes within an oocyte and first polar body, using GV stage oocytes matured

Julie M Clyde; Roger G Gosden; Anthony J Rutherford; Helen M Picton

2001-01-01

48

Efficiency of fluorescence in situ hybridization for bacterial cell identification in temporary river sediments with contrasting water content  

Microsoft Academic Search

We studied the efficiency of two hybridization techniques for the analysis of benthic bacterial community composition under varying sediment water content. Microcosms were set up with sediments from four European temporary rivers. Wet sediments were dried, and dry sediments were artificially rewetted. The percentage of bacterial cells detected by fluorescence in situ hybridization with fluorescently monolabeled probes (FISH) significantly increased

Stefano Fazia; Stefano Amalfitano; Ilaria Pizzetti; Jakob Pernthaler

2007-01-01

49

Increase in Fluorescence Intensity of 16S rRNA In Situ Hybridization in Natural Samples Treated with Chloramphenicol  

Microsoft Academic Search

Despite the numerous advantages of fluorescent in situ hybridization for the identification of single prokaryotic cells with 16S rRNA probes, use of the technique with natural samples, especially those from the marine environment, is still problematic. The low percentage of fluorescently labeled cells constitutes the primary problem for in situ hybridization of natural samples, probably due to low cellular rRNA

Cleber C. Ouverney; Jed A. Fuhrman

1997-01-01

50

An improved fixation technique for fluorescence in situ hybridization for preimplantation genetic diagnosis  

Microsoft Academic Search

Objective: To improve existing preimplantation genetic diagnosis fixation techniques.Design: Prospective randomized in vitro study.Setting: Academic medical center.Patient(s): None.Intervention(s): None.Main Outcome Measure(s): The intensity and clarity of fluorescence in situ hybridization (FISH) signals and the percentage of successfully fixed blastomeres.Result(s): The described fixation technique resulted in 100% fixation and 100% adequate FISH signals. Two conventional techniques resulted in 94% and 87%

Dmitri I Dozortsev; Kevin T McGinnis

2001-01-01

51

Sensitive whole-mount fluorescent in situ hybridization in zebrafish using enhanced tyramide signal amplification.  

PubMed

Whole-mount in situ hybridization is the preferred method for detecting transcript distributions in whole embryos, tissues, and organs. We present here a sensitive fluorescent in situ hybridization method for colocalization analysis of different transcripts in whole embryonic zebrafish brains. The method is based on simultaneous hybridization of differently hapten-labeled RNA probes followed by sequential rounds of horseradish peroxidase (POD)-based transcript detection. Sequential detection involves enhancement of fluorescent signals by tyramide signal amplification (TSA) and effective inactivation of the antibody-POD conjugate prior to the following detection round. We provide a detailed description of embryo preparation, hybridization, antibody detection, POD-TSA reaction, and mounting of embryos for imaging. To achieve high signal intensities, we optimized key steps of the method. This includes improvement of embryo permeability by hydrogen peroxide treatment and efficacy of hybridization and TSA-POD reaction by addition of the viscosity-increasing polymer dextran sulfate. The TSA-POD reaction conditions are further optimized by application of substituted phenol compounds as POD accelerators and use of highly efficient bench-made tyramide substrates. The obtained high signal intensities and cellular resolution of our method allows for co-expression analysis and generation of three-dimensional models. Our protocol is tailored to optimally work in zebrafish embryos, but can surely be modified for application in other species as well. PMID:24048934

Lauter, Gilbert; Söll, Iris; Hauptmann, Giselbert

2014-01-01

52

Fluorescent in situ hybridization applied on samples taken with adhesive tape strips.  

PubMed

Fluorescent in situ hybridization (FISH), applied directly on samples taken with adhesive tape, is proposed as method to detect and identify microorganisms from the surfaces of valuable objects without being destructive. Results of tests carried out in laboratory conditions as well on samples taken from deteriorated surfaces of Roman Catacombs showed the feasibility of FISH when applied on adhesive tape. The potential as well as the limits of the technique were also discussed. PMID:14499996

La Cono, V; Urzì, C

2003-10-01

53

Cot1 banding of human chromosomes using fluorescence in situ hybridization with Cy3 labeling  

Microsoft Academic Search

We developed a new chromosome banding method by in situ hybridization of human Cot-1 DNA as a probe. Clear banding was produced on metaphase chromosomes of lymphoblastoid cells after probe detection with a fluorescent dye Cy3. Comparison with the known banding patterns revelaed a similarity to the R-banding with some significant differences: some centromeric heterochromatin regions show Cot-1 positive bands.

Yimin Wang; Shinsei Minoshima; Nobuyoshi Shimizu

1995-01-01

54

Sequence-Based Design of Single-Copy Genomic DNA Probes for Fluorescence In Situ Hybridization  

Microsoft Academic Search

Chromosomal rearrangements are frequently monitored by fluorescence in situ hybridization (FISH) using large, recombinant DNA probes consisting of contiguous genomic intervals that are often distant from disease loci. We developed smaller, targeted, single-copy probes directly from the human genome sequence. These single-copy FISH (scFISH) probes were designed by computational sequence analysis of ?100-kb genomic sequences. ScFISH probes are produced by

Peter K. Rogan; Patricia M. Cazcarro; Joan H. M. Knoll

2001-01-01

55

Fluorescent In Situ Hybridization to Detect Transgene Integration into Plant Genomes  

NASA Astrophysics Data System (ADS)

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.

Schwarzacher, Trude

56

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

National Technical Information Service (NTIS)

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 th...

J. R. Korenberg

1993-01-01

57

Detecting MicroRNA in Human Cancer Tissues with Fluorescence In Situ Hybridization.  

PubMed

The technique of nucleic acid in situ hybridization is an effective method for identifying the existence and abundance of nucleic acids in tissue sections or cytological preparations. Such a method has the advantage of keeping morphological relationships intact while identifying changes at the molecular level. As a noncoding regulatory RNA, microRNA has been found to intricately control many physiological and pathological conditions. We provide here a representative fluorescence in situ hybridization protocol for microRNA detection, and note commonly used alternatives, and some troubleshooting points. The method described is based on formalin-fixed paraffin-embedded oral cancer tissues but should be broadly applicable to similarly processed tissues of other types of cancer. PMID:24026683

Shi, Zonggao; Johnson, Jeff J; Stack, M Sharon

2013-01-01

58

Application of Pseudomurein Endoisopeptidase to Fluorescence In Situ Hybridization of Methanogens within the Family Methanobacteriaceae?  

PubMed Central

In situ detection of methanogens within the family Methanobacteriaceae is sometimes known to be unsuccessful due to the difficulty in permeability of oligonucleotide probes. Pseudomurein endoisopeptidase (Pei), a lytic enzyme that specifically acts on their cell walls, was applied prior to 16S rRNA-targeting fluorescence in situ hybridization (FISH). For this purpose, pure cultured methanogens within this family, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, and Methanothermobacter thermautotrophicus together with a Methanothermobacter thermautotrophicus-containing syntrophic acetate-oxidizing coculture, endosymbiotic Methanobrevibacter methanogens within an anaerobic ciliate, and an upflow anaerobic sludge blanket (UASB) granule were examined. Even without the Pei treatment, Methanobacterium bryantii and Methanothermobacter thermautotrophicus cells are relatively well hybridized with oligonucleotide probes. However, almost none of the cells of Methanobrevibacter ruminantium, Methanosphaera stadtmanae, cocultured Methanothermobacter thermautotrophicus, and the endosymbiotic methanogens and the cells within UASB granule were hybridized. Pei treatment was able to increase the probe hybridization ratio in every specimen, particularly in the specimen that had shown little hybridization. Interestingly, the hybridizing signal intensity of Methanothermobacter thermautotrophicus cells in coculture with an acetate-oxidizing H2-producing syntroph was significantly improved by Pei pretreatment, whereas the probe was well hybridized with the cells of pure culture of the same strain. We found that the difference is attributed to the differences in cell wall thicknesses between the two culture conditions. These results indicate that Pei treatment is effective for FISH analysis of methanogens that show impermeability to the probe.

Nakamura, Kohei; Terada, Takeshi; Sekiguchi, Yuji; Shinzato, Naoya; Meng, Xian-Ying; Enoki, Miho; Kamagata, Yoichi

2006-01-01

59

Fluorescent in situ hybridization with arbitrarily amplified DNA fragments differentiates carrot (Daucus carota L.) chromosomes.  

PubMed

Carrot (Daucus carota L.) chromosomes are small and poorly differentiated in size and morphology. Here we demonstrate that fluorescent in situ hybridization (FISH) signals derived from arbitrary PCR probes can be used for chromosome identification in carrot. To prepare probes, we searched for nonpolymorphic products abundantly amplified with arbitrary decamer primers in a group of accessions representing carrot genetic diversity. As a result, 13 fragments ranging in size from 517 to 1758 bp were selected, sequenced, and used as probes for fluorescent in situ hybridization. Four of these probes produced clear and reproducible hybridization signals. The sequences showed similarity to a number of carrot BAC-end sequences, indicating their repetitive character. Three of them were similar to internal portions of gypsy and copia LTR retrotransposons previously identified in plants. Hybridization signals for the four probes were observed as dotted tracks on chromosomes, differing in distribution and intensity. Generally, they were present in pericentromeric and (or) interstitial localizations on chromosome arms. The use of the four probes allowed discrimination of chromosome pairs and construction of more detailed karyotypes and idiograms of carrot. PMID:22360760

Nowicka, Anna; Grzebelus, Ewa; Grzebelus, Dariusz

2012-02-23

60

Rapid identification of Candida albicans in blood by combined capillary electrophoresis and fluorescence in situ hybridization.  

PubMed

A CE method based on whole-cell molecular labeling via fluorescence in situ hybridization was developed for the detection of Candida albicans in whole blood. Removal of potentially interfering red blood cells (RBC) with a simple hypotonic/detergent lysis step enabled us to detect and quantitate contaminating C. albicans cells at concentrations that were orders of magnitude lower than background RBC counts ( approximately 7.0 x 10(9) RBC/mL). In the presence of the lysed blood matrix, yeast cells aggregated without the use of a blocking plug to stack the cells. Short (15 min) hybridizations yielded bright Candida-specific fluorescence in situ hybridization signals, enabling us to detect as few as a single injected cell. The peak area response of the stacked Candida cells showed a strong linear correlation with cell concentrations determined by plate counts, up to approximately 10(7) CFU/mL (or approximately 1 x 10(4) injected cells). This rapid and quantitative method for detecting Candida in blood may have advantageous applications in both human and veterinary diagnostics. PMID:20665522

Lantz, Andrew W; Bisha, Bledar; Tong, Man-Yung; Nelson, Ryan E; Brehm-Stecher, Byron F; Armstrong, Daniel W

2010-08-01

61

[Use of photo-anchoring of DNA probes for fluorescent in situ hybridization].  

PubMed

A possibility was investigated to use photo-crosslinking DNA probes for fluorescent in situ hybridization (FISH). DNA probes were modified by incorporating photonucleotides in these, containing a photoreactive group (tetrafluorobenzazid) and capable of making covalent bonds with the examined DNA, when irradiated in 300-330 nm region. The photonucleotide was incorporated into the probe either by nick-translation, or upon elongation of the hybridized probe by the Kljonow fragment. It has been shown that the DNA probe, cross-linking to a chromosome as a result of covalent bonds, is not removed from the place of hybridization under consequent denaturating washing, which makes it possible to carry out the following DNA hybridization with selective conservation of signals obtained due to previous hybridization. This peculiarity of photo-linking DNA probes makes it possible to use them for the two-step DNA hybridization. To demonstrate this, preparations of human chromosomes were investigated. On the first step, chromosomal DNA was hybridized by means of DNA probe having nucleotide sequences of centromeric regions of chromosomes 13 and 21, the probe being linked to chromosomal DNA by the photonucleotide. Following the denaturation treatment of the preparation, and after the second chromosomal DNA hybridization with cosmid DNA, containing chromosome 13 DNA nucleotide sequence, the signal in chromosome 13 centromeric region was retained to serve a marker of this chromosome, thus fascilitating its easier identification following the hybridization of its DNA with cosmic DNA. The denaturation stability of photo-crosslinking probes opens some new possibilities in technology of DNA in situ hybridization. PMID:9821246

Nasedkina, T V; Mal'kov, R B; Fedorova, L I; Godovikova, T S; Kolpashchikov, D M; Poletaev, A I

1998-01-01

62

Fluorescence in situ hybridization on single cells. (Sex determination and chromosome rearrangements).  

PubMed

Fluorescence in situ hybridization (FISH) is the technique of choice for preimplantation genetic diagnosis (PGD) selection of female embryos in families with X-linked disease, for which there is no mutation-specific test. FISH with target-specific DNA probes is also the primary technique used for PGD detection of chromosome imbalance associated with Robertsonian translocations, reciprocal translocations, inversions, and other chromosome rearrangements, because the DNA probes, labeled with different fluorochromes or haptens, detect the copy number of their target loci. The methods described outline strategies for PGD for sex determination and chromosome rearrangements. These methods are assessment of reproductive risks, the selection of suitable probes for interphase FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring using directly labeled and indirectly labeled probes. PMID:17876073

Scriven, Paul N; Ogilvie, Caroline Mackie

2007-01-01

63

Crystal ball: fluorescence in situ hybridization in the age of super-resolution microscopy.  

PubMed

Super-resolution microscopy encompasses a suite of cutting edge microscopy methods able to surpass the resolution limits of light microscopy. The recent commercial availability of super-resolution microscopy is advancing many fields of biology. In this crystal ball forward look, we briefly examine the perspectives of combining super-resolution microscopy and fluorescence in situ hybridization (FISH). We strongly believe, based on first evidence presented here, that using super-resolution microscopy in environmental microbiology has the potential to reshape the way we analyze the results obtained with FISH, by improving both the localization and quantification of target molecules. PMID:23140662

Moraru, Cristina; Amann, Rudolf

2012-11-07

64

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

PubMed

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

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

2007-11-29

65

Fluorescence In situ hybridization (FISH) of the tetrathionate reductase (ttr) gene in marine sulfur-reducing bacteria  

Microsoft Academic Search

Fluorescence in situ hybridization (FISH) has been used in numerous ways to identify the presence or absence of complementary nucleic acid sequences in a cell. Nucleotide probes are labeled with fluorescent molecules and designed to target genes or any complementary nucleic acid sequence of interest. In microbial ecology, FISH is used for the identification of microorganisms based on their 16s

Sean D Jarvis

2011-01-01

66

Three-dimensional acrylamide fluorescence in situ hybridization for plant cells.  

PubMed

Plant meiosis involves complex and dynamic processes that occur within the space inside the nucleus. Direct inspection of meiotic chromosomes by fluorescence microscopy has been used to investigate many of these processes. In particular, optical sectioning microscopy of fluorescence in situ hybridization (FISH)-stained nuclei provides three-dimensional spatial information about the organization and distribution of specific sequences and chromosomal loci within the nucleus. Here we provide a fully detailed three-dimensional (3D) acrylamide FISH method for the analysis of plant meiotic nuclei. Several examples illustrate the versatility of this technique for the investigation of meiotic telomere dynamics in maize, Arabidopsis, and oat. Additional examples of 3D FISH include chromosome painting in a maize chromosome-addition line of oat and telomere FISH with maize nuclei from plants expressing a fluorescently tagged fusion protein, histone H2B-mCherry. PMID:23559202

Howe, Elizabeth S; Murphy, Shaun P; Bass, Hank W

2013-01-01

67

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

Microsoft Academic Search

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

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

1999-01-01

68

Single-mRNA counting using fluorescent in situ hybridization in budding yeast.  

PubMed

Fluorescent in situ hybridization (FISH) allows the quantification of single mRNAs in budding yeast using fluorescently labeled single-stranded DNA probes, a wide-field epifluorescence microscope and a spot-detection algorithm. Fixed yeast cells are attached to coverslips and hybridized with a mixture of FISH probes, each conjugated to several fluorescent dyes. Images of cells are acquired in 3D and maximally projected for single-molecule analysis. Diffraction-limited labeled mRNAs are observed as bright fluorescent spots and can be quantified using a spot-detection algorithm. FISH preserves the spatial distribution of cellular RNA distribution within the cell and the stochastic fluctuations in individual cells that can lead to phenotypic differences within a clonal population. This information, however, is lost if the RNA content is measured on a population of cells by using reverse transcriptase PCR, microarrays or high-throughput sequencing. The FISH procedure and image acquisition described here can be completed in 3 d. PMID:22301778

Trcek, Tatjana; Chao, Jeffrey A; Larson, Daniel R; Park, Hye Yoon; Zenklusen, Daniel; Shenoy, Shailesh M; Singer, Robert H

2012-02-02

69

Mapping of Regions of Physical Deletion on Chromosome 16q in Prostate Cancer Cells by Fluorescence in Situ Hybridization (FISH)  

Microsoft Academic Search

Recent evidence suggests that a tumor suppressor gene important in the progression of prostatic carcinoma may reside on chromosome 16q. The exact location and identity of this gene are unknown. We used fluorescence in situ hybridization in a novel manner to define more clearly the location of this gene. Region-specific chromosome 16 cosmid contig probes were hybridized directly to interphase

Michael L. Cher; Takashi Ito; Noel Weidner; Peter R. Carroll; Ronald H. Jensen

1995-01-01

70

Physical mapping of chromosome 17 cosmids by fluorescence in situ hybridization and digital image analysis  

SciTech Connect

The authors used fluorescence in situ hybridization and digital image analysis to localize cosmids along human chromosome 17. Seventy-one cosmids were selected at random from a chromosome 17 library constructed from a partial Sau3AI digest of flow-sorted chromosomes from a mouse-human hybrid cell line. Sixty-three of these (89%) gave a signal only on chromosome 17. The 40 cosmids producing the most distinct hybridization signals in metaphase and interphase cells were precisely mapped using digital image analysis. An additional 20 cosmids, previously mapped by linkage analysis, were also mapped. The order of these probes determined by metaphase mapping was consistent with the order determined by linkage analysis. 13 refs., 1 fig., 1 tab.

Kallioniemi, O.P.; Kallioniemi, A.; Sudar, D.; Pinkel, D.; Gray, J. (Univ. of California, San Francisco, CA (United States)); Mascio, L. (Lawrence Livermore National Lab., CA (United States)); Deaven, L. (Los Alamos National Lab., NM (United States))

1994-03-01

71

Prenatal exclusion of subtelomeric deletion 1p by fluorescent in situ hybridization  

Microsoft Academic Search

Background  Subtelomeric deletion 1p is difficult to detect from banded karyotypes. Recent developments in the field of molecular cytogenetics\\u000a have made it possible for submicroscopic rearrangements within chromosomes to be detected using fluorescence in situ hybridization\\u000a (FISH) techniques.\\u000a \\u000a \\u000a \\u000a Materials and methods  We describe prenatal FISH testing of subtelomeric 1p deletion in a fetus of a mother whose previous child had subtelomeric\\u000a 1p

Vorapong Phupong; Verayuth Praphanphoj; Vorasuk Shotelersuk

2007-01-01

72

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

PubMed Central

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. Images Figure 3 Figure 2 p434-a Figure 1

Blennow, E; Anneren, G; Bui, T H; Berggren, E; Asadi, E; Nordenskjold, M

1993-01-01

73

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

PubMed Central

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.

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

74

Combined fluorescence in situ hybridization and PRINS for the analysis of the Dystrophin gene.  

PubMed

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy are caused in most cases by deletions of the DMD gene. These rearrangements are detectable in affected boys and men by a simple multiplex polymerase chain reaction approach. However, this technique is not able to disclose DMD deletions in heterozygous female carriers, and different approaches must be used in these cases. Here, we describe an approach based on the combined use of primed in situ labeling and fluorescence in situ hybridization techniques for the detection of single DMD exons in fixed metaphase chromosomes and interphase nuclei of both male and female subjects, suggesting the usefulness of this tool in the detection of small intragenic deletions of the DMD gene. PMID:16861757

Stuppia, Liborio; La Sala, Dario; Cinti, Caterina

2006-01-01

75

Direct eye visualization of Cy5 fluorescence for immunocytochemistry and in situ hybridization.  

PubMed

Cyanine 5.18 (or Cy5) is a fluorochrome emitting in the long-red/far-red range, usually regarded as unsuitable for direct observation by the human eye. We describe here the optimization of a direct visualization approach to Cy5 labeling, based on a standard fluorescence microscope with mercury light excitation and applicable to both immunocytochemistry and fluorescent in situ hybridization. Crucial factors were (a) an excitation path in the microscope not absorbing light in the orange-red range, up to 640 nm, (b) a 588-640-nm excitation filter range, distinctly below the excitation optimum for Cy5, (c) a 650-700-nm emission filter range, transmitting the low-wavelength portion of Cy5 emission, and (d) high-efficiency filter set components allowing a narrow gap between excitation and emission ranges without visible cross-talk of excitation light in the emission path. PMID:10681398

Ferri, G L; Isola, J; Berger, P; Giro, G

2000-03-01

76

Two-color fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems  

PubMed Central

Background Whole-mount in situ hybridization (WISH) is extensively used to characterize gene expression patterns in developing and adult brain and other tissues. To obtain an idea whether a novel gene might be involved in specification of a distinct brain subdivision, nucleus or neuronal lineage, it is often useful to correlate its expression with that of a known regional or neuronal marker gene. Two-color fluorescent in situ hybridization (FISH) can be used to compare different transcript distributions at cellular resolution. Conventional two-color FISH protocols require two separate rounds of horseradish peroxidase (POD)-based transcript detection, which involves tyramide signal amplification (TSA) and inactivation of the first applied antibody-enzyme conjugate before the second detection round. Results We show here that the alkaline phosphatase (AP) substrates Fast Red and Fast Blue can be used for chromogenic as well as fluorescent visualization of transcripts. To achieve high signal intensities we optimized embryo permeabilization properties by hydrogen peroxide treatment and hybridization conditions by application of the viscosity-increasing polymer dextran sulfate. The obtained signal enhancement allowed us to develop a sensitive two-color FISH protocol by combining AP and POD reporter systems. We show that the combination of AP-Fast Blue and POD-TSA-carboxyfluorescein (FAM) detection provides a powerful tool for simultaneous fluorescent visualization of two different transcripts in the zebrafish brain. The application of different detection systems allowed for a one-step antibody detection procedure for visualization of transcripts, which significantly reduced working steps and hands-on time shortening the protocol by one day. Inactivation of the first applied reporter enzyme became unnecessary, so that false-positive detection of co-localization by insufficient inactivation, a problem of conventional two-color FISH, could be eliminated. Conclusion Since POD activity is rather quickly quenched by substrate excess, less abundant transcripts can often not be efficiently visualized even when applying TSA. The use of AP-Fast Blue fluorescent detection may provide a helpful alternative for fluorescent transcript visualization, as the AP reaction can proceed for extended times with a high signal-to-noise ratio. Our protocol thus provides a novel alternative for comparison of two different gene expression patterns in the embryonic zebrafish brain at a cellular level. The principles of our method were developed for use in zebrafish but may be easily included in whole-mount FISH protocols of other model organisms.

2011-01-01

77

Fluorescence in situ hybridization applications for super-resolution 3D structured illumination microscopy.  

PubMed

Fluorescence in situ hybridization on three-dimensionally preserved cells (3D-FISH) is an efficient tool to analyze the subcellular localization and spatial arrangement of targeted DNA sequences and RNA transcripts at the single cell level. 3D reconstructions from serial optical sections obtained by confocal laser scanning microscopy (CLSM) have long been considered the gold standard for 3D-FISH analyses. Recent super-resolution techniques circumvent the diffraction-limit of optical resolution and have defined a new state-of-the-art in bioimaging. Three-dimensional structured illumination microscopy (3D-SIM) represents one of these technologies. Notably, 3D-SIM renders an eightfold improved volumetric resolution over conventional imaging, and allows the simultaneous visualization of differently labeled target structures. These features make this approach highly attractive for the analysis of spatial relations and substructures of nuclear targets that escape detection by conventional light microscopy. Here, we focus on the application of 3D-SIM for the visualization of subnuclear 3D-FISH preparations. In comparison with conventional fluorescence microscopy, the quality of 3D-SIM data is dependent to a much greater extent on the optimal sample preparation, labeling and acquisition conditions. We describe typical problems encountered with super-resolution imaging of in situ hybridizations in mammalian tissue culture cells and provide optimized DNA-/(RNA)-FISH protocols including combinations with immunofluorescence staining (Immuno-FISH) and DNA replication labeling using click chemistry. PMID:23086869

Markaki, Yolanda; Smeets, Daniel; Cremer, Marion; Schermelleh, Lothar

2013-01-01

78

Analysis of restriction enzyme-induced chromosomal aberrations by fluorescence in situ hybridization  

SciTech Connect

Fluorescence in situ hybridization and Giemsa staining of metaphase chromosomes were used to determine the relative frequencies of symmetric exchange aberrations (translocations) and asymmetric exchange aberrations (rings, dicentrics, and polycentrics) after exposure of human lymphoblastoid cells to restriction enzymes or X-rays. The yield of symmetric exchanges was determined with the use of chromosome-specific probes for human chromosomes 2 or 4, which were hybridized to metaphase chromosomes from cells exposed to the enzymes Pvull, Sacl, or Xbal or 3 or 5 Gy of X-rays. The yield of asymmetric exchanges was determined in Giemsa-stained metaphase chromosomes from the same enzyme-treated or irradiated cell population. About 1.5- to 3-fold more symmetric than asymmetric exchanges were induced after restriction enzyme treatment. However, after X-ray treatment the yield of dicentrics relative to the yield of reciprocal translocations was close to the expected 1:1 ratio. 28 refs., 5 figs., 2 tabs.

Columna, E.A.; Giaccia, A.J.; Evans, J.W.; Yates, B.L.; Morgan, W.F. (Univ. of California, San Francisco, CA (United States) Stanford Medical School, CA (United States))

1993-01-01

79

Combined RNA/DNA Fluorescence In Situ Hybridization on Whole-Mount Drosophila Ovaries.  

PubMed

DNA FISH (fluorescent in situ hybridization) analysis reveals the chromosomal location of the gene of interest. RNA in situ hybridization is used to examine the amounts and cell location of transcripts. This method is commonly used to describe the localization of processed transcripts in different tissues or cell lines. Gene activation studies are often aimed at determining the mechanism of this activation (transcriptional or posttranscriptional). Elucidation of the mechanism of piRNA-mediated silencing of genomic repeats is at the cutting edge of small RNA research. The RNA/DNA FISH technique is a powerful method for assessing transcriptional changes at any particular genomic locus. Colocalization of the RNA and DNA FISH signals allows a determination of the accumulation of nascent transcripts at the transcribed genomic locus. This would be suggest that this gene is activated at the transcriptional (or co-transcriptional) level. Moreover, this method allows for the identification of transcriptional derepression of a distinct copy (copies) among a genomic repeat family. Here, a RNA/DNA FISH protocol is presented for the simultaneous detection of RNA and DNA in situ on whole-mount Drosophila ovaries using tyramide signal amplification. With subsequent immunostaining of chromatin components, this protocol can be easily extended for studying the interdependence between chromatin changes at genomic loci and their transcriptional activity. PMID:24178564

Shpiz, Sergey; Lavrov, Sergey; Kalmykova, Alla

2014-01-01

80

Quantitative fluorescence in situ hybridization of microbial communities in the rumens of cattle fed different diets.  

PubMed

At present there is little quantitative information on the identity and composition of bacterial populations in the rumen microbial community. Quantitative fluorescence in situ hybridization using newly designed oligonucleotide probes was applied to identify the microbial populations in liquid and solid fractions of rumen digesta from cows fed barley silage or grass hay diets with or without flaxseed. Bacteroidetes, Firmicutes, and Proteobacteria were abundant in both fractions, constituting 31.8 to 87.3% of the total cell numbers. They belong mainly to the order Bacteroidales (0.1 to 19.2%), hybridizing with probe BAC1080; the families Lachnospiraceae (9.3 to 25.5%) and Ruminococcaceae (5.5 to 23.8%), hybridizing with LAC435 and RUM831, respectively; and the classes Deltaproteobacteria (5.8 to 28.3%) and Gammaproteobacteria (1.2 to 8.2%). All were more abundant in the rumen communities of cows fed diets containing silage (75.2 to 87.3%) than in those of cows fed diets containing hay (31.8 to 49.5%). The addition of flaxseed reduced their abundance in the rumens of cows fed silage-based diets (to 45.2 to 58.7%) but did not change markedly their abundance in the rumens of cows fed hay-based diets (31.8 to 49.5%). Fibrolytic species, including Fibrobacter succinogenes and Ruminococcus spp., and archaeal methanogens accounted for only a small proportion (0.4 to 2.1% and 0.2 to 0.6%, respectively) of total cell numbers. Depending on diet, between 37.0 and 91.6% of microbial cells specifically hybridized with the probes used in this study, allowing them to be identified in situ. The identities of other microbial populations (8.4 to 63.0%) remain unknown. PMID:20802069

Kong, Yunhong; He, Maolong; McAlister, Tim; Seviour, Robert; Forster, Robert

2010-08-27

81

Detection of the oyster parasite Bonamia ostreae by fluorescent in situ hybridization.  

PubMed

Bonamia ostreae is an economically significant protistan parasite of the flat oyster Ostrea edulis in Europe and North America. Management of this parasite depends partly upon its reliable identification in wild and aquacultured oyster populations, but B. ostreae is small and difficult to detect by traditional microscopic methods. We designed a fluorescent in situ hybridization (FISH) assay to sensitively detect B. ostreae in standard histopathological sections of B. ostreae-infected oysters using fluorescently labeled DNA oligonucleotide probes. Hybridization using a cocktail of 3 presumptively B. ostreae-specific, fluorescein iso(thio)cyanate (FITC)-labeled oligonucleotides produced an unambiguous staining pattern of small green rings inside infected oyster hemocytes that was easily distinguished from host tissue background. This pattern is diagnostic for B. ostreae. A negative control cocktail of oligonucleotides containing 2 mismatches relative to target sequences, on the other hand, failed to hybridize at all. B. ostreae-specific probes did not cross-react with a related protist, Haplosporidium nelsoni. PMID:13677511

Carnegie, Ryan B; Barber, Bruce J; Distel, Daniel L

2003-08-01

82

Whole-slide imaging is a robust alternative to traditional fluorescent microscopy for fluorescence in situ hybridization imaging using break-apart DNA probes.  

PubMed

Fluorescence in situ hybridization is an indispensable technique used in routine pathology and for theranostic purposes. Because fluorescence in situ hybridization techniques require sophisticated microscopic workstations and long procedures of image acquisition with sometimes subjective and poorly reproducible results, we decided to test a whole-slide imaging system as an alternative approach. In this study, we used the latest generation of Pannoramic 250 Flash digital microscopes (P250 Flash digital microscopes; 3DHISTECH, Budapest, Hungary) to digitize fluorescence in situ hybridization slides of diffuse large B cells lymphoma cases for detecting MYC rearrangement. The P250 Flash digital microscope was found to be precise with better definition of split signals in cells containing MYC rearrangement with fewer truncated signals as compared to traditional fluorescence microscopy. This digital technique is easier thanks to the preview function, which allows almost immediate identification of the tumor area, and the panning and zooming functionalities as well as a shorter acquisition time. Moreover, fluorescence in situ hybridization analyses using the digital technique appeared to be more reproducible between pathologists. Finally, the digital technique also allowed prolonged conservation of photos. In conclusion, whole-slide imaging technologies represent rapid, robust, and highly sensitive methods for interpreting fluorescence in situ hybridization slides with break-apart probes. In addition, these techniques offer an easier way to interpret the signals and allow definitive storage of the images for pathology expert networks or e-learning databases. PMID:23517924

Laurent, Camille; Guérin, Maxime; Frenois, François-Xavier; Thuries, Valérie; Jalabert, Laurence; Brousset, Pierre; Valmary-Degano, Séverine

2013-03-18

83

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

PubMed Central

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

Hsu, Joanne H.; Zeng, Hui; Lemke, Kalistyn H.; Polyzos, Aris A.; Weier, Jingly F.; Wang, Mei; Lawin-O'Brien, Anna R.; Weier, Heinz-Ulrich G.; O'Brien, Benjamin

2013-01-01

84

Fluorescence in situ hybridization techniques for the rapid detection of genetic prognostic factors in neuroblastoma  

PubMed Central

Neuroblastoma is the commonest extracranial solid tumour in children. There are a number of molecular genetic features known which are of prognostic importance and which are used to direct therapy. Identification and targeting of high-risk individuals with intensive therapeutic regimens may allow an improvement in survival rates. The most powerful biological parameters associated with prognosis in this malignancy are chromosomal changes, especially MYCN amplification, deletion of chromosome 1p and aneuploidy. Rapid characterization of these aberrations at the time of diagnosis is paramount if stratification according to risk group is to be achieved. This paper describes the rapid detection of del(1p), MYCN amplification and trisomy using interphase fluorescence in situ hybridization on imprints from fresh tumour biopsies. The results are related to those obtained by standard molecular methods and karyotyping. © 2000 Cancer Research Campaign

Taylor, C P F; Bown, N P; McGuckin, A G; Lunec, J; Malcolm, A J; Pearson, A D J; Sheer, D

2000-01-01

85

Rapid identification of pathogens in blood culture with fluorescent in situ hybridization (FISH).  

PubMed

Rapid identification of pathogens in bloodstream infections is of utmost importance to improve survival of septic patients. Fluorescent in situ hybridization (FISH) accelerates the identification of most frequent bacterial and yeast pathogens of sepsis. In this study, 210 positive blood cultures were tested with FISH method and the results were evaluated comparing to the traditional cultivation based results. Overall agreement between FISH and conventional identification was 91.4%, with better results for Gram-negative bacteria than for Gram-positives (100% and 89.5%, respectively). FISH results were obtained within 1 hour. FISH may serve as a useful tool to supplement traditional microbiological methods for rapid, provisional identification of sepsis pathogens. PMID:20870594

Horváth, Andrea; Kristóf, Katalin; Konkoly-Thege, Marianne; Nagy, K

2010-09-01

86

Chromosome orientation fluorescence in situ hybridization (CO-FISH) to study sister chromatid segregation in vivo  

PubMed Central

Previously, assays for sister chromatid segregation patterns relied on incorporation of BrdU and indirect methods to infer segregation patterns after two cell divisions. Here we describe a method to differentially label sister chromatids of murine cells and directly assay sister chromatid segregation patterns following one cell division in vitro and in vivo by adaptation of the well-established CO-FISH (chromosome orientation fluorescent in situ hybridization) technique. 5-bromo-2?-deoxyuridine (BrdU) is incorporated into newly-formed DNA strands, followed by photolysis and exonuclease digestion to create single-stranded sister chromatids containing parental template DNA only. Such single-stranded sister chromatids are differentially labeled using unidirectional probes to major satellite sequences coupled to fluorescent markers. Differentially-labeled sister chromatids in post-mitotic cells are visualized using fluorescence microscopy and sister chromatid segregation patterns can be directly assayed after one cell division. This procedure requires four days for in vivo mouse tissues, and two days for in vitro cultured cells.

Falconer, Ester; Chavez, Elizabeth; Henderson, Alexander; Lansdorp, Peter M.

2013-01-01

87

Detection of genetic alterations in micrometastatic cells in bone marrow of cancer patients by fluorescence in situ hybridization  

Microsoft Academic Search

Detection of micrometastatic tumor cells in bone marrow of cancer patients has been shown to be of prognostic significance. To further characterize these cells, we combined antibody labeling and fluorescence in situ hybridization (FISH). For detection of numerical changes of chromosome 17, nine patients with proven breast cancer whose bone marrow contained epithelial tumor cells were evaluated. Epithelial cells were

Peter Müller; Dorothea Weckermann; Gert Riethmüller; Günter Schlimok

1996-01-01

88

Frequent TMPRSS2-ERG rearrangement in prostatic small cell carcinoma detected by fluorescence in situ hybridization: the superiority of fluorescence in situ hybridization over ERG immunohistochemistry.  

PubMed

Small cell carcinoma of the prostate is both morphologically and immunohistochemically similar to small cell carcinoma of other organs such as the urinary bladder or lung. TMPRSS2-ERG gene fusion appears to be a highly specific alteration in prostatic carcinoma that is frequently shared by small cell carcinoma. In adenocarcinoma, immunohistochemistry for the ERG protein product has been reported to correlate well with the presence of the gene fusion, although in prostatic small cell carcinoma, this relationship is not completely understood. We evaluated 54 cases of small cell carcinoma of the prostate and compared TMPRSS2-ERG gene fusion status by fluorescence in situ hybridization (FISH) to immunohistochemical staining with antibody to ERG. Of 54 cases of prostatic small cell carcinoma, 26 (48%) were positive for TMPRSS2-ERG gene fusion by FISH and 12 (22%) showed overexpression of ERG protein by immunohistochemistry. Of the 26 cases positive by FISH, 11 were also positive for ERG protein by immunohistochemistry. One tumor was positive by immunohistochemistry but negative by FISH. Urinary bladder small cell carcinoma (n = 25) showed negative results by both methods; however, 2 of 14 small cell carcinomas of other organs (lung, head, and neck) showed positive immunohistochemistry but negative FISH. Positive staining for ERG by immunohistochemistry is present in a subset of prostatic small cell carcinomas and correlates with the presence of TMPRSS2-ERG gene fusion. Therefore, it may be useful in confirming prostatic origin when molecular testing is not accessible. However, sensitivity and specificity of ERG immunohistochemistry in small cell carcinoma are decreased compared to FISH. PMID:23850495

Schelling, Lindsay A; Williamson, Sean R; Zhang, Shaobo; Yao, Jorge L; Wang, Mingsheng; Huang, Jiaoti; Montironi, Rodolfo; Lopez-Beltran, Antonio; Emerson, Robert E; Idrees, Muhammad T; Osunkoya, Adeboye O; Man, Yan-Gao; Maclennan, Gregory T; Baldridge, Lee Ann; Compérat, Eva; Cheng, Liang

2013-07-12

89

In situ hybridization  

SciTech Connect

These proceedings collect papers on genetic aspects of neurobiology. Topics include: gene expression in mammalian cells, in situ hybridization with RNA probes, mRNA in brain cells, neuropeptide gene expression, genetic mapping, and localization of peptide hormone gene expression.

Valentino, K.L.; Eberwine, J.H.; Barchas, J.D.

1987-01-01

90

Detection of aneuploidy in human spermatozoa using fluorescence in situ hybridization (FISH).  

PubMed

Fluorescence in situ hybridization (FISH) with chromosome specific alpha-satellite DNA probes was used to estimate the rates of aneuploidy of chromosomes 1, 17, 18, X and Y in human ejaculated sperm. Sperm samples were collected from six donors, and biotinylated DNA probes, D1Z5, D17Z1, D18Z1, DXZ1 and DYZ3 were hybridized to interphase sperm which had been pretreated with dithiothreitol to expand their nuclei. A minimum of 3,000 sperm per donor were analyzed. The hybridization efficiency was 99.68% for all the five probes. The frequencies of aneuploidy for chromosomes 1, 17 and 18 were 0.65%, 0.66%, and 0.61%, respectively. For XX- and YY-sperm the frequencies were 0.28% and 0.27%, respectively. To estimate the diploidy and disomy rates, a mixture of D17Z1 and D18Z1 were used as probes, and the frequency of diploid sperm was calculated to be 0.27%. After subtraction of the diploidy rate, the disomy rates for chromosomes 1, 17, and 18 were estimated to be 0.38%, 0.39% and 0.33%, respectively. The proportion of X- and Y-bearing sperm were 49.90% and 49.66%, consistent with an expected 1:1 ratio. PMID:9088108

Hu, H; Miharu, N; Mizunoe, T; Nakaoka, Y; Okamoto, E; Ohama, K

1996-12-01

91

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

Microsoft Academic Search

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

Duongruitai Nicomrat; Warren A. Dick; Olli H. Tuovinen

2006-01-01

92

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

PubMed Central

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.

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

2011-01-01

93

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

Code of Federal Regulations, 2013 CFR

...AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunology Laboratory Equipment and Reagents § 866.4700 Automated fluorescence in situ...

2013-04-01

94

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

PubMed

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

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

2013-07-27

95

Automated segmentation and analysis of fluorescent in situ hybridization (FISH) signals in interphase nuclei of pap-smear specimens  

Microsoft Academic Search

Interphase fluorescence in situ hybridization (FISH) technology is a potential and promising molecular imaging tool, which can be applied to screen and detect cervical cancer. However, manual FISH detection method is a subjective, tedious, and time-consuming process that results in a large inter-reader variability and possible detection error (in particular for heterogeneous cases). Automatic FISH image analysis aims to potentially

Xingwei Wang; Bin Zheng; Shibo Li; Roy R. Zhang; Yuhua Li; John J. Mulvihill; Wei R. Chen; Hong Liu

2009-01-01

96

Cot1 banding of human chromosomes using fluorescence in situ hybridization with Cy3 labeling  

Microsoft Academic Search

Summary We developed a new chromosome banding method byin situ hybridization of human Cot-1 DNA as a probe. Clear banding was produced on metaphase chromosomes of lymphoblastoid cells after probe detection with a fluorescent dye Cy3. Comparison with the known banding patterns revelaed a similarity to the R-banding with some significant differences: some centromeric heterochromatin regions show Cot-1 positive bands.

Yimin Wang; Shinsei Minoshima; Nobuyoshi Shimizu

1995-01-01

97

Potential use of repeated fluorescence in situ hybridization in the same human blastomeres for preimplantation genetic diagnosis  

Microsoft Academic Search

Objective: To assess the feasibility of repeated fluorescence in situ hybridization (FISH) procedures in the same nucleus of a human blastomere.Design: Three consecutive FISH procedures were performed in the same human blastomere by using direct label fluorescence CEP and WCP probes (Vysis).Setting: Hospital-based private IVF program.Patient(s): Twenty-eight infertile couples who underwent conventional IVF in our center.Intervention(s): Embryos from oocytes with

Jiaen Liu; Yieh-Loong Tsai; Xue-Zhong Zheng; Theodore A Baramki; Ricardo A Yazigi; Eugene Katz

1998-01-01

98

Rapid Identification and Enumeration of Antibiotic Resistant Bacteria in Urban Canals by Microcolony-Fluorescence in Situ Hybridization  

Microsoft Academic Search

The abundance and phylogenetic composition of antibiotic resistant bacteria in canals of metropolitan Bangkok, Thailand, were investigated using a microcolony method and fluorescence in situ hybridization (FISH). Cells were directly trapped from aquatic samples onto polycarbonate membranes and incubated for 24 hr on selective agar containing antibiotics. Individual antibiotic resistant bacterial microcolonies samples were classified on the filter using FISH

Takehiko Kenzaka; Nobuyasu Yamaguchi; Fuangfa Utrarachkij; Orasa Suthienkul; Masao Nasu

2006-01-01

99

Analyzing mRNA expression using single mRNA resolution fluorescent in situ hybridization.  

PubMed

As the product of transcription and the blueprint for translation, mRNA is the main intermediate product of the gene expression pathway. The ability to accurately determine mRNA levels is, therefore, a major requirement when studying gene expression. mRNA is also a target of different regulatory steps, occurring in different subcellular compartments. To understand the different steps of gene expression regulation, it is therefore essential to analyze mRNA in the context of a single cell, maintaining spatial information. Here, we describe a stepwise protocol for fluorescent in situ hybridization (FISH) that allows detection of individual mRNAs in single yeast cells. This method allows quantitative analysis of mRNA expression in single cells, permitting "absolute" quantification by simply counting mRNAs. It further allows us to study many aspects of mRNA metabolism, from transcription to processing, localization, and mRNA degradation. PMID:20946829

Zenklusen, Daniel; Singer, Robert H

2010-03-01

100

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

Microsoft Academic Search

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

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

1999-01-01

101

An integrated microfluidic chip for chromosome enumeration using fluorescence in situ hybridization.  

PubMed

Fluorescence in situ hybridization (FISH) is a powerful technique for probing the genetic content of individual cells at the chromosomal scale. Conventional FISH techniques provide a sensitive diagnostic tool for the detection of chromosomal alterations on a cell-by-cell basis; however, the cost-per-test in terms of reagent and highly qualified labour has prevented its wide-spread utilization in clinical settings. Here, we address the inefficient use of labour with the first integrated and automated on-chip FISH implementation, one that requires only minutes of setup time from the technician. Our microfluidic chip has lowered the reagent use by 20-fold, decreased the labour time by 10-fold, and substantially reduced the amount of support equipment needed. We believe this cost-effective platform will make sensitive FISH techniques more accessible for routine clinical usage. PMID:19023479

Sieben, Vincent J; Debes-Marun, Carina S; Pilarski, Linda M; Backhouse, Christopher J

2008-10-23

102

Improvements in cytological preparations for fluorescent in situ hybridization in Passiflora.  

PubMed

Cytological preparations for the fluorescent in situ hybridization (FISH) technique require cytoplasm-free metaphases, with well-spread chromosomes, for the localization of DNA sequences and chromosome mapping. We tested various procedures for FISH analysis of Passiflora cacaoensis, P. gardneri and hybrid F? progeny of P. gardneri x P. gibertii. Two treatments with four enzymes and three incubation times were compared. The material was treated with 1.0 M HCl before enzymatic digestion. The following criteria were used to determine the quality of the metaphases: a) lack or presence of cytoplasm; b) well-spread chromosomes or with overlap; c) complete or incomplete chromosome number (2n). The enzyme Pectinex(®) SP ULTRA gave the best performance, with the shortest incubation time. The best results were observed after 30 min of incubation; more than 70% of the metaphases did not have large amounts of cytoplasm or overlapping chromosomes, and about 75% maintained the chromosome number. FISH was carried out using a 45S rDNA probe (pTa71) labeled with biotin and detected with fluorescein isothiocyanate. Sites with strong staining and without nonspecific signals were observed. Our methodological adaptations allowed the preparation of metaphase slides of high quality for the FISH technique, with less time required for the preparation of samples. PMID:21053178

Souza, M M; Urdampilleta, J D; Forni-Martins, E R

2010-11-03

103

Visualization of ribosomal DNA loci in spore interphasic nuclei of glomalean fungi by fluorescence in situ hybridization  

Microsoft Academic Search

Fluorescence in situ hybridization (FISH) was applied to interphasic nuclei isolated from spores of four species of AM fungi\\u000a : Scutellospora castanea, Glomus mosseae, Glomus intraradices and Gigaspora rosea. Ribosomal DNA loci were visualized using digoxigenin-labeled 25?S rDNA probes obtained by nested PCR. Several hybridization\\u000a sites were detected per nucleus and an internuclear variability was observed in the number of

Sophie Trouvelot; Diederik van Tuinen; Mohamed Hijri; V. Gianinazzi-Pearson

1999-01-01

104

A physical map of chromosome 20 established using fluorescence in situ hybridization and digital image analysis  

SciTech Connect

The physical locations of 46 cosmid clones and 21 P1 clones were determined along the chromosome 20 axis relative to the p terminus (FLpter) using fluorescence in situ hybridization (FISH) and digital image microscopy. The cosmid clones were selected from the chromosomally enriched library LA20NC01. Nine P1 clones were selected from a pooled DuPont genomic library using PCR with primer pairs selected to amplify genetically mapped sequence-tagged sites. This information was used to relate the physical map to the genetic map. Twelve P1 clones were selected from the same library using PCR primer pairs that amplified known genes. Two of these, E2F and BCLX, had not been mapped previously. 14 refs., 1 fig., 2 tabs.

Stokke, T.; Collins, C.; Kuo, W.L. [Univ. of California, San Francisco, CA (United States)] [and others

1995-03-01

105

Chromosomal loci of 50 human keratinocyte cDNAs assigned by fluorescence in situ hybridization  

SciTech Connect

The chromosomal loci of expressed genes provide useful information for a candidate gene approach to the genes responsible for genetic diseases. A large set of randomly isolated cDNAs catalogued by partial sequencing can serve as a resource for accessing and isolating these disease genes. Using fluorescence in situ hybridization, we examined the chromosomal loci of 217 human keratinocyte-derived cDNAs, with independent novel sequence tags at the 3{prime} end region. Among them, we determined the loci of 50 cDNAs. Single-pass sequencing of these from the 5{prime} ends indicated that 39 cDNAs still can be produced for new genes. These cDNAs with identified chromosomal loci are powerful tools that can be used to help elucidate the genes responsible for hereditary skin disorders. 42 refs., 3 figs., 2 tabs.

Morishima, Yohich; Ariyama, Takeshi; Yamanishi, Kiyofumi [Uyoto Prefectural Univ. of Medicine (Japan)] [and others

1995-07-20

106

[Detection of maize centromeric repeats in the relatives of maize using fluorescence in situ hybridization].  

PubMed

In order to analyze the conservation of maize centromeric satellite DNA (CentC) and centromeric retrotransposon (CRM) in the subspecies and relatives of Zea mays, dual fluorescence in situ hybridization (FISH) was used to detect the existence and distribution of the above two repetitive sequences in Zea mays ssp. mexicana, Z. diploperennis, Z. perennis, Tripsacum dactyloides, Coix lacryma-jobi, and Sorghum bicolor. In Z. mays ssp. mexicana, Z. diploperennis, and Z. perennis, both CentC and CRM probes produced strong or relatively strong signals in the centromeric regions of all chromosomes. There was an obvious variation in the intensity of hybridization signals on different chromosomes, indicating that different centromeres have different amounts of CentC and CRM sequences. In some centromeres, the intensity of CentC signals differed from that of CRM signals and was free from overlapping. In T. dactyloides, only weak CentC and CRM signals were detected in the centromeric regions of most chromosomes, while in C. lacryma-jobi and S. bicolor only relatively strong or strong CRM signals primarily located in the centromeric regions were detected. This result indicates that CentC is highly conserved among the subspecies of Z. mays and the species of Zea, and has high conservation in Tripsacum, a genus that is most closely related to Zea, and CRM is conserved among the species of grass family either closely or distantly related to Zea. PMID:20233704

She, Chao-Wen; Jiang, Xiang-Hui; Song, Yun-Chun; Liu, Wei

2010-03-01

107

Fluorescence in-situ hybridization and dermoscopy in the assessment of controversial melanocytic tumors.  

PubMed

Although the 'gold standard' for melanoma diagnosis remains histopathological analysis, presently dermoscopists play a significant role in the diagnostic process. However, even a combined approach may not allow a clear-cut judgment on equivocal melanocytic lesions. Fluorescence in-situ hybridization (FISH) can offer assistance in the evaluation of chromosome abnormalities associated with malignancies, and its role is emerging in melanoma diagnosis. The aim of this study was to evaluate the diagnostic role of the FISH in the assessment of controversial lesions, defined as those lesions showing discrepancies between dermatoscopic and histological evaluations. Twenty clinically and histologically ambiguous melanocytic lesions were selected. After the first histopathologic diagnosis, a second pathologist examined the specimens in a blinded review for a second opinion and to identify the most suitable areas to hybridize using probes specific to RREB1, MYB, and CCND1 genes and the centromere of chromosome 6. The first histopathological evaluation led to the diagnosis of melanoma in seven cases, whereas the second identified eight cases of malignant melanoma and was in agreement with the first in 65% of cases and with dermoscopy in 40% of cases. Cytogenetic abnormalities detected by FISH are markers of malignancy that can be useful in the characterization of difficult-to-diagnose melanocytic tumors, when the dermatologist and the pathologist have a different opinions. PMID:24077512

Ponti, Giovanni; Ruini, Cristel; Massi, Daniela; Pellacani, Giovanni; Tomasi, Aldo; Paglierani, Milena; Loschi, Pietro; Seidenari, Stefania

2013-12-01

108

Rapid prenatal diagnosis of chromosomal aneuploidies by fluorescence in situ hybridization: Clinical experience with 4,500 specimens  

Microsoft Academic Search

Detection of chromosome aneuploidies in uncultured amniocytes is possible using fluorescence in situ hybridization (FISH). The authors herein describe the results of the first clinical program which utilized FISH for the rapid detection of chromosome aneuploidies in uncultured amniocytes. FISH was performed on physician request, as an adjunct to cytogenetics in 4,500 patients. Region-specific DNA probes to chromosomes 13, 18,

B. E. Ward; S. L. Gersen; M. P. Carelli; N. M. McGuire; W. R. Dackowski; K. W. Klinger; M. Weinstein; C. Sandlin

1993-01-01

109

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

SciTech Connect

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.

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

1994-09-01

110

An Approach for Quantitative Assessment of Fluorescence In Situ Hybridization (FISH) Signals for Applied Human Molecular Cytogenetics  

Microsoft Academic Search

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

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

2005-01-01

111

FISHIS: Fluorescence In Situ Hybridization in Suspension and Chromosome Flow Sorting Made Easy  

PubMed Central

The large size and complex polyploid nature of many genomes has often hampered genomics development, as is the case for several plants of high agronomic value. Isolating single chromosomes or chromosome arms via flow sorting offers a clue to resolve such complexity by focusing sequencing to a discrete and self-consistent part of the whole genome. The occurrence of sufficient differences in the size and or base-pair composition of the individual chromosomes, which is uncommon in plants, is critical for the success of flow sorting. We overcome this limitation by developing a robust method for labeling isolated chromosomes, named Fluorescent In situ Hybridization In suspension (FISHIS). FISHIS employs fluorescently labeled synthetic repetitive DNA probes, which are hybridized, in a wash-less procedure, to chromosomes in suspension following DNA alkaline denaturation. All typical A, B and D genomes of wheat, as well as individual chromosomes from pasta (T. durum L.) and bread (T. aestivum L.) wheat, were flow-sorted, after FISHIS, at high purity. For the first time in eukaryotes, each individual chromosome of a diploid organism, Dasypyrum villosum (L.) Candargy, was flow-sorted regardless of its size or base-pair related content. FISHIS-based chromosome sorting is a powerful and innovative flow cytogenetic tool which can develop new genomic resources from each plant species, where microsatellite DNA probes are available and high quality chromosome suspensions could be produced. The joining of FISHIS labeling and flow sorting with the Next Generation Sequencing methodology will enforce genomics for more species, and by this mightier chromosome approach it will be possible to increase our knowledge about structure, evolution and function of plant genome to be used for crop improvement. It is also anticipated that this technique could contribute to analyze and sort animal chromosomes with peculiar cytogenetic abnormalities, such as copy number variations or cytogenetic aberrations.

Giorgi, Debora; Farina, Anna; Grosso, Valentina; Gennaro, Andrea; Ceoloni, Carla; Lucretti, Sergio

2013-01-01

112

A Molecular Cytogenetic Map of Sorghum Chromosome 1: Fluorescence in Situ Hybridization Analysis With Mapped Bacterial Artificial Chromosomes  

Microsoft Academic Search

We used structural genomic resources for Sorghum bicolor (L.) Moench to target and develop multiple molecular cytogenetic probes that would provide extensive coverage for a specific chromosome of sorghum. Bacterial artificial chromosome (BAC) clones containing molecular markers mapped across sorghum linkage group A were labeled as probes for fluorescence in situ hybridization (FISH). Signals from single-, dual-, and multiprobe BAC-FISH

M. N. Islam-Faridi; K. L. Childs; P. E. Klein; G. Hodnett; M. A. Menz; R. R. Klein; W. L. Rooney; J. E. Mullet; D. M. Stelly; H. J. Price

113

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

PubMed Central

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.

Wellinghausen, Nele; Wirths, Beate; Poppert, Sven

2006-01-01

114

Rapid Exclusion of Chromosomal Aneuploidies by Fluorescence in situ Hybridization prior to Fetal Surgery for Obstructive Uropathy – A Case Report  

Microsoft Academic Search

Ultrasound of a fetus at 17 weeks gestation revealed posterior urethral valve syndrome with anhydramnios. Fluorescence in situ hybridization (FISH) to detect aneuploidies of chromosomes 13, 18, 21 X and Y was performed on transitional cells from the fetal bladder obtained at percutaneous vesicocentesis, followed by conventional cytogenetics. Fetal urine was chosen due to unavailability of amniotic fluid for karyotypic

Daniel W. Skupski; Keith A. Eddleman; Nancy Zellers; Brian E. Ward

1994-01-01

115

Counting and Size Classification of Active Soil Bacteria by Fluorescence In Situ Hybridization with an rRNA Oligonucleotide Probe  

Microsoft Academic Search

A fluorescence in situ hybridization (FISH) technique based on binding of a rhodamine-labelled oligonu- cleotide probe to 16S rRNA was used to estimate the numbers of ribosome-rich bacteria in soil samples. Such bacteria, which have high cellular rRNA contents, were assumed to be active (and growing) in the soil. Hybridization to an rRNA probe, EUB338, for the domain Bacteria was

HENRIK CHRISTENSEN; MICHAEL HANSEN; JAN SØRENSEN

1999-01-01

116

The study on improving fluorescence microscopy image effects of genomic in situ hybridization  

Microsoft Academic Search

Fluorescence microscopy is an important optical microscopic observation apparatus used for the research of cytobiology and cytogenetics. By using microscopic photography, digital CCD imaging instrument and other devices, researchers can observe and capture images of FISH, GISH, and Green fluorescent protein (GFP), etc. In practice, however, in order to get good experimental results with a clear background and distinctive hybridization

Min Qu; Yanming Zhang

2010-01-01

117

In-solution fluorescence in situ hybridization and fluorescence-activated cell sorting for single cell and population genome recovery.  

PubMed

Over the past decade, technological advances in whole genome amplification, microfluidics, flow sorting, and high-throughput sequencing have led to the development of single-cell genomics. Single-cell genomic approaches are typically applied to anonymous microbial cells with only morphology providing clues to their identity. However, targeted separation of microorganisms based on phylogenetic markers, such as the 16S rRNA gene, is beginning to emerge in the single-cell genomics field. Here, we describe an in-solution fluorescence in situ hybridization (FISH) protocol which can be combined with fluorescence-activated cell sorting (FACS) for separation of single cells or populations of interest from environmental samples. Sequencing of DNA obtained from sorted cells can be used for the recovery of draft quality genomes, and when performed in parallel with deep metagenomics, can be used to validate and further scaffold metagenomic assemblies. We illustrate in this chapter the feasibility of this FISH-FACS approach by describing the targeted recovery of a novel anaerobic methanotrophic archaeon. PMID:24060113

Haroon, Mohamed F; Skennerton, Connor T; Steen, Jason A; Lachner, Nancy; Hugenholtz, Philip; Tyson, Gene W

2013-01-01

118

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

Microsoft Academic Search

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

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

119

DNA mimics for the rapid identification of microorganisms by fluorescence in situ hybridization (FISH).  

PubMed

Fluorescence in situ hybridization (FISH) is a well-established technique that is used for a variety of purposes, ranging from pathogen detection in clinical diagnostics to the determination of chromosomal stability in stem cell research. The key step of FISH involves the detection of a nucleic acid region and as such, DNA molecules have typically been used to probe for the sequences of interest. However, since the turn of the century, an increasing number of laboratories have started to move on to the more robust DNA mimics methods, most notably peptide and locked nucleic acids (PNA and LNA). In this review, we will cover the state-of-the-art of the different DNA mimics in regard to their application as efficient markers for the presence of individual microbial cells, and consider their potential advantages and pitfalls. Available PNA probes are then reassessed in terms of sensitivity and specificity using rRNA databases. In addition, we also attempt to predict the applicability of DNA mimics in well-known techniques attempting to detect in situ low number of copies of specific nucleic acid sequences such as catalyzed reporter deposition (CARD) and recognition of individual genes (RING) FISH. PMID:19325728

Cerqueira, Laura; Azevedo, Nuno F; Almeida, Carina; Jardim, Tatiana; Keevil, Charles William; Vieira, Maria J

2008-10-20

120

Diagnosis of Hydatidiform Moles by Polymorphic Deletion Probe Fluorescence in Situ Hybridization  

PubMed Central

Because products of conception often contain maternal and villous tissues, the determination of maternal and villous genotypes based on genetic polymorphisms can help discern maternal and paternal chromosomal contribution and aid in the diagnosis of hydatidiform moles. Polymorphic deletion probe (PDP) fluorescence in situ hybridization (FISH) probes based on copy number variants are highly polymorphic and allow in situ determination of genetic identity. By using three informative PDPs on chromosomes 2p, 4q, and 8p, we compared maternal with villous genotypes and determined the ploidy of villous tissue. PDP FISH was performed on 13 complete moles, 13 partial moles, 13 nonmolar abortions, and an equivocal hydropic abortion. PDP FISH permitted definitive diagnosis of complete moles in five of 13 cases for which maternal and villous genotypes were mutually exclusive. A complete mole was highly suspected when all three PDP loci showed homozygous villous genotypes. The diagnosis of a complete mole by PDP FISH yielded a theoretical test sensitivity of 87.5%, specificity of 91.8%, an observed test sensitivity of 100%, and specificity of 92.3%. Triploidy was observed in all partial moles, in which diandric triploidy was confirmed in six cases. In the equivocal hydropic abortion, PDP FISH combined with p57 immunofluorescence revealed placental androgenetic/biparental mosaicism. PDP FISH can be used in clinical practice and research studies to subclassify hydatidiform moles and evaluate unusual products of conception.

Chiang, Sarah; Fazlollahi, Ladan; Nguyen, Anhthu; Betensky, Rebecca A.; Roberts, Drucilla J.; Iafrate, A. John

2011-01-01

121

DNA Mimics for the Rapid Identification of Microorganisms by Fluorescence in situ Hybridization (FISH)  

PubMed Central

Fluorescence in situ hybridization (FISH) is a well-established technique that is used for a variety of purposes, ranging from pathogen detection in clinical diagnostics to the determination of chromosomal stability in stem cell research. The key step of FISH involves the detection of a nucleic acid region and as such, DNA molecules have typically been used to probe for the sequences of interest. However, since the turn of the century, an increasing number of laboratories have started to move on to the more robust DNA mimics methods, most notably peptide and locked nucleic acids (PNA and LNA). In this review, we will cover the state-of-the-art of the different DNA mimics in regard to their application as efficient markers for the presence of individual microbial cells, and consider their potential advantages and pitfalls. Available PNA probes are then reassessed in terms of sensitivity and specificity using rRNA databases. In addition, we also attempt to predict the applicability of DNA mimics in well-known techniques attempting to detect in situ low number of copies of specific nucleic acid sequences such as catalyzed reporter deposition (CARD) and recognition of individual genes (RING) FISH.

Cerqueira, Laura; Azevedo, Nuno F.; Almeida, Carina; Jardim, Tatiana; Keevil, Charles William; Vieira, Maria J.

2008-01-01

122

Assignment of the human ?-tropomyosin gene TPM4 to band 19p13.1 by fluorescence in situ hybridization  

Microsoft Academic Search

Sequence-tagged sites (STSs) were developed for the human ?-tropomyosin gene TPM4. One STS was used to amplify DNA from somatic cell hybrids to localize TPM4 to chromosome 19. The other, a product from a long-range PCR, was used directly as a probe to refine the localization of TPM4 to 19p13.1 by fluorescence in situ hybridization to metaphase chromosome spreads.Copyright ©

S. D. Wilton; L. Lim; S. D. Dorosz; H. C. Gunn; H. J. Eyre; D. F. Callen; N. G. Laing

1996-01-01

123

An improved protocol for quantification of freshwater Actinobacteria by fluorescence in situ hybridization.  

PubMed

We tested a previously described protocol for fluorescence in situ hybridization of marine bacterioplankton with horseradish peroxidase-labeled rRNA-targeted oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) in plankton samples from different lakes. The fraction of Bacteria detected by CARD-FISH was significantly lower than after FISH with fluorescently monolabeled probes. In particular, the abundances of aquatic Actinobacteria were significantly underestimated. We thus developed a combined fixation and permeabilization protocol for CARD-FISH of freshwater samples. Enzymatic pretreatment of fixed cells was optimized for the controlled digestion of gram-positive cell walls without causing overall cell loss. Incubations with high concentrations of lysozyme (10 mg ml(-1)) followed by achromopeptidase (60 U ml(-1)) successfully permeabilized cell walls of Actinobacteria for subsequent CARD-FISH both in enrichment cultures and environmental samples. Between 72 and >99% (mean, 86%) of all Bacteria could be visualized with the improved assay in surface waters of four lakes. For freshwater samples, our method is thus superior to the CARD-FISH protocol for marine Bacteria (mean, 55%) and to FISH with directly fluorochrome labeled probes (mean, 67%). Actinobacterial abundances in the studied systems, as detected by the optimized protocol, ranged from 32 to >55% (mean, 45%). Our findings confirm that members of this lineage are among the numerically most important Bacteria of freshwater picoplankton. PMID:12732568

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

2003-05-01

124

Analysis of DNA Damage and Repair by Comet Fluorescence In Situ Hybridization (Comet-FISH).  

PubMed

A useful tool in the detection of overall and region-specific DNA damage is the Comet-FISH technique. This method combines two well-established methods, the Comet assay (single cell gel electrophoresis), which makes it possible to detect and quantify DNA damage at the single cell level, and FISH (fluorescence in situ hybridization), a technique that allows the specific detection of selected DNA sequences. The influence of specific substances such as water pollutants or food ingredients on individual cells can be measured with the alkaline version of the Comet assay, which involves the embedding of cells in agarose on microscopic slides, lysis of cells, and separation of DNA via electrophoresis. In damaged cells a "comet tail" is formed by fractured DNA migrating from the nucleus (head of the comet) in the electric field.The damaged DNA (DNA strand breaks) correlates with the percentage of DNA in the tail. In combination with the FISH method, DNA damage or repair capacity in single cells can be measured using labelled probes, which hybridize to specific DNA sequences of interest. This protocol exemplarily provides a description of the Comet-FISH technique for the detection of DNA damage using hydrogen peroxide as a genotoxic model substance. PMID:24162978

Glei, Michael; Schlörmann, Wiebke

2014-01-01

125

Faster identification of pathogens in positive blood cultures by fluorescence in situ hybridization in routine practice.  

PubMed

Rapid identification of microorganisms in blood cultures is required to optimize empirical treatment at an early stage. Fluorescence in situ hybridization (FISH) can reduce the time to identification of microorganisms in growth-positive blood cultures. In this study, we evaluated the performance, time to identification, and potential clinical benefits of FISH compared to those of conventional culture methods in routine practice. After Gram staining, blood culture fluids were simultaneously further identified with FISH and with conventional culture methods. Results and points in time of FISH and culture identification (provisional and final identifications) were collected and compared. For 91% of microorganisms, the genus or family name was identified, and for 79%, the species name could be attributed. The sensitivity and specificity of the individual probes exceeded 95%, except for the Enterobacteriaceae probe (sensitivity, 89%). Cross-hybridization was obtained with the Klebsiella pneumoniae probe for Klebsiella oxytoca. The time gains of FISH and final culture identification were more than 18 h for bacteria and 42 h for yeasts. With FISH, Staphylococcus aureus was differentiated from coagulase-negative staphylococci 1.4 h faster than by provisional identification (P < 0.001). In conclusion, FISH allows rapid and reliable identification of the majority of microorganisms in growth-positive blood cultures. The substantial time gain of identification with FISH may allow same-day adjustment of antimicrobial therapy, and FISH is especially useful if no provisional identification is obtained. With further extension of the number of probes and a reduction in turnaround time, FISH will become a very useful diagnostic tool in the diagnosis of bloodstream infections. PMID:16390958

Peters, Remco P H; Savelkoul, Paul H M; Simoons-Smit, Alberdina M; Danner, Sven A; Vandenbroucke-Grauls, Christina M J E; van Agtmael, Michiel A

2006-01-01

126

Method for multiplex cellular detection of mRNAs using quantum dot fluorescent in situ hybridization  

PubMed Central

The photostability and narrow emission spectra of non-organic quantum dot fluorophores (QDs) make them desirable candidates for fluorescent in situ hybridization (FISH) to study the expression of specific mRNA transcripts. We developed a novel method for direct QD labeling of modified oligonucleotide probes through streptavidin and biotin interactions, as well as protocols for their use in multiple-label FISH. We validated this technique in mouse brainstem sections. The subcellular localization of the vesicular monoamine transporter (Vmat2) mRNA corresponds when using probes labeled with two different QDs in the same hybridization. We developed protocols for combined direct QD FISH and QD immunohistochemical labeling within the same neurons as well as for simultaneous study of the subcellular distribution of multiple mRNA targets. We demonstrated increased sensitivity of FISH using QDs in comparison with organic fluorophores. These techniques gave excellent histological results both for multiplex FISH and combined FISH and immunohistochemistry. This approach can facilitate the ultrasensitive simultaneous study of multiple mRNA and protein markers in tissue culture and histological section.

Chan, PokMan; Yuen, Tony; Ruf, Frederique; Gonzalez-Maeso, Javier; Sealfon, Stuart C.

2005-01-01

127

Gastric choriocarcinoma shows characteristics of adenocarcinoma and gestational choriocarcinoma: a comparative genomic hybridization and fluorescence in situ hybridization study.  

PubMed

The authors report two cases of the rare primary gastric choriocarcinoma. These tumors showed an overwhelming predominance of cytotrophoblast- and syncytiotrophoblast-like tumor cells that were positive for beta-human chorionic gonadotrophin, with small foci of glandular differentiation. Beta-human chorionic gonadotrophin was also detected serologically in one patient. Comparative genomic hybridization study was performed on one specimen. Copy number gains of chromosomes 12, 17, 20, 22, and X, together with losses on 18q, were the major findings. Except for the gain of chromosome 12, which is known to be uncommon in primary gastric adenocarcinoma but frequently associated with choriocarcinoma, the remaining genomic imbalances were among the most common comparative genomic hybridization findings reported in primary gastric adenocarcinoma. Fluorescence in situ hybridization on paraffin sections of both specimens confirmed the presence of polysomy 17 and trisomy 12. These results suggest that primary gastric choriocarcinoma genetically possesses characteristics of both adenocarcinoma and gestational choriocarcinoma. The authors believe this is the first interphase cytogenetics study on this rare tumor, and that the results support the theory that gastric choriocarcinoma arises from alternate differentiation pathways of adenocarcinoma. PMID:11552718

Liu, A Y; Chan, W Y; Ng, E K; Zhang, X; Li, B C; Chow, J H; Chung, S C

2001-09-01

128

Multicolor fluorescence in situ hybridization and comparative genomic hybridization reveal molecular events in lung adenocarcinomas and squamous cell lung carcinomas.  

PubMed

We have used the molecular cytogenetic techniques of multicolor fluorescence in situ hybridization (M-FISH) and comparative genomic hybridization (CGH) to analyze two established lung cancer cell lines (A549, H520), 80 primary lung adenocarcinoma samples and 80 squamous cell lung carcinoma samples in order to identify common chromosomal aberrations. M-FISH revealed numerous complex chromosomal rearrangements. Chromosomes 5, 6, 11, 12, and 17 were most frequently involved in interchromosomal translocations. CGH revealed regions on 1q, 2p, 3q, 5p, 5q, 7p, 8q, 11q, 12q, 14q, 16p, 17p, 19q, 20q, 21q and 22q to be commonly over-represented and regions on 2q, 3p, 4p, 5q, 7q, 8p, 9p, 13q, 14q, and 17p to be under-represented. In lung adenocarcinomas the most common gains were found in 16p13 (50%); while in squamous cell lung carcinomas the common gains were found in 17q21 (45%) and these alterations were observed to be associated with their specific pathological subtype. In conclusion, the present study contributes to the molecular biological characterization in lung adenocarcinomas and squamous cell lung carcinomas and through evaluation of molecular events to the recently emergent focus on novel markers for lung cancer treatment. PMID:18848758

Shen, Hua; Gao, Wen; Wu, Yu-jie; Qiu, Hai-rong; Shu, Yong-qian

2008-09-16

129

Feature representation and signal classification in fluorescence in-situ hybridization image analysis  

Microsoft Academic Search

Fast and accurate analysis of fluorescence in-situ hy- bridization (FISH) images for signal counting will depend mainly upon two components: a classifier to discriminate between artifacts and valid signals of several fluorophores (colors), and well discrim- inating features to represent the signals. Our previous work has focused on the first component. To investigate the second compo- nent, we evaluate candidate

Boaz Lerner; William F. Clocksin; Seema Dhanjal; Maj A. Hultén; Christopher M. Bishop

2001-01-01

130

Application of fluorescence in situ hybridization technique to detect simazine-degrading bacteria in soil samples.  

PubMed

We propose a new approach to evaluate the natural attenuation capacity of soil by using fluorescence in situ hybridization (FISH). A specific oligonucleotide probe AtzB1 was designed based on the sequence data of the atzB gene involved in the hydrolytic deamination of s-triazines; this gene, located in a multiple copy plasmid was detected by the optimized FISH protocol. Two agricultural soils (Lodi and Henares) with a history of simazine treatments, and two natural soils (Soto and Monza), without previous exposure to simazine, were studied. AtzB1 probe-target cells were found only in the agricultural soils and, in a greater percentage, in the Lodi soil, compared to the Henares one. Moreover, the greatest percentage of AtzB1 probe-target cells in Lodi was accompanied by a greater mineralization rate, compared to the Henares soil. The FISH method used in this study was suitable for the detection of simazine-degrading bacteria and could be a useful indicator of the potential of soil bioremediation. PMID:18082866

Martín, Margarita; Gibello, Alicia; Lobo, Carmen; Nande, Mar; Garbi, Carlos; Fajardo, Carmen; Barra-Caracciolo, Anna; Grenni, Paola; Martínez-Iñigo, M José

2007-12-20

131

Higher axial-resolution and sensitivity pachytene fluorescence in situ hybridization protocol in tetraploid cotton.  

PubMed

Fluorescence in situ hybridization (FISH) based on pachytene chromosomes has become an important cytogenetic tool to construct high axial-resolution and sensitivity cytogenetic maps. However, the application of this technique in cotton has lagged behind due to difficulties in chromosome preparation. To date, successful FISH based on cotton pachytene chromosomes has not been reported. In this study, the first protocol developed for pachytene chromosome preparation in tetraploid cotton is presented. This protocol yielded chromosome spreads suitable for large and small DNA probe FISH labeling. Two important parameters, axial-resolution and sensitivity, of FISH on mitotic metaphase and pachytene chromosomes were systematically analyzed. The results demonstrated that DNA targets separated by 0.6 cM and low-copy targets as small as 3-kb were resolved and detected, respectively, in pachytene FISH. The application of our FISH protocol will continue to improve and provide a point of departure for constructing an integrated high axial-resolution cytogenetic map in cotton. PMID:19844799

Wang, Kai; Yang, Zaijie; Shu, Changshen; Hu, Jing; Lin, Qiuyun; Zhang, Wenpan; Guo, Wangzhen; Zhang, Tianzhen

2009-10-21

132

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

PubMed Central

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 used to analyze prostate tissues from patients with prostate cancer and BPH. P. acnes infection of the prostate gland was demonstrated in prostatic tissue in 5 of 10 randomly selected prostate cancer patients. FISH analysis and confocal laser microscopy imaging revealed intracellular localization and stromal biofilm-like aggregates as common forms of P. acnes infection in prostate tissues from both prostate cancer and BPH patients. A sequential analysis of prostate tissue from individual patients suggested that P. acnes can persist for up to 6 years in the prostate gland. These results indicate that P. acnes can establish a persistent infection in the prostate gland. Further study is needed to clarify the link between this bacterium and prostatic inflammation which may contribute to the development of BPH and prostate cancer.

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

2007-01-01

133

Gene amplification in myeloid leukemias elucidated by fluorescence in situ hybridization.  

PubMed

Gene amplification in hematologic malignancies is uncommon. When karyotyping leukemia cells, gene amplification is generally seen as double-minute (dmin) chromosomes and homogeneously staining regions (hsr). One of the more commonly amplified regions is MYC at 8q24.21, but amplification of MLL at 11q23 and regions on 9p, 19q, and elsewhere on 11q have been reported. Increased copy number of these genes has been associated with poor prognosis. Over an 11-year period, we identified 31 cases of possible gene amplification, 27 of which had enough sample material for further investigations. A total of 17 cases had dmin only, 13 cases had hsr only, and 1 case had both dmin and hsr in the karyotype. Fluorescence in situ hybridization (FISH) analysis identified amplification of MYC in 12 cases, all on dmin, and amplification of MLL in eight cases, all on hsr. Regions other than MYC and MLL were amplified in eight cases and, using multicolor FISH and multicolor banding, we identified a number of novel regions of amplification: 13q11 approximately q12.1, 15q26.1 approximately q26.3, and 17q12. We also identified one case where two different chromosomal regions were simultaneously amplified in the same cell line. PMID:19602463

Rayeroux, Kathleen C; Campbell, Lynda J

2009-08-01

134

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

PubMed

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 the study. A total of 450,580 spermatozoa were studied from 21 subjects (9 fertile, 12 infertile). Significant differences were observed in the disomy rates between chromosomes with the highest frequency observed for chromosome 16 (0.17%) and the lowest for the Y chromosome (0.10%). No differences were observed between fertile and infertile subjects for either diploidy or disomy. Total disomy rates for chromosomes 1, 16, X and Y ranged from 0.34% to 0.84% among infertile subjects, and 0.32% to 0.61% among fertile subjects. Our data suggest that generalized aneuploidy in sperm is not a major contributor to unexplained infertility. PMID:8168824

Miharu, N; Best, R G; Young, S R

1994-05-01

135

Silver-Enhanced In Situ Hybridization as an Alternative to Fluorescence In Situ Hybridization for Assaying HER2 Amplification in Clinical Breast Cancer  

PubMed Central

Purpose Valid determination of HER2 status is a prerequisite to establish an adequate treatment strategy for breast cancer patients, regardless of the disease stage. The goal of this study was to examine the feasibility of the newly developed silver-enhanced in situ hybridization (SISH) technique as an alternative to fluorescence in situ hybridization (FISH) for HER2 assay in primary invasive breast cancer. Methods FISH and SISH for HER2 amplification were performed using tissue microarray. Both methods were used in 257 consecutive primary breast cancers. Results HER2 amplification was observed in 62 (23.1%) of a total of 257 breast cancers based on SISH. Of the 257 breast cancers measured using both methods, the results of the two methods were consistent in 248 (concordance, 96.5%; kappa=0.903). When we compared HER2 amplification in the primary tumor with the metastatic lymph nodes of the same patients, HER2 amplification was observed in nine cases (14.0%) out of 64 cases in which HER2 was not amplified in the primary tumors. In contrast, HER2 status was completely preserved in metastatic lymph nodes showing HER2 amplification in the primary tumor. Conclusion These results indicate that SISH can be a feasible alternative to FISH in the clinical setting. In node-positive breast cancer, confirmation of the HER2 status of the metastatic lymph nodes appears to be mandatory, regardless of the HER2 status of the primary tumors.

Han, Sehwan; Kim, Jung-Yeon; Kim, Hyun-Jung; Kwon, Ji Eun; Gwak, Geumhee

2011-01-01

136

Detection of chromosomal abnormalities of chromosome 12 in uterine leiomyoma using fluorescence in situ hybridization.  

PubMed

Fifty uterine leiomyomas were examined using conventional cytogenetic method and fluorescence in situ hybridization (FISH) for detection of chromosomal abnormalities of chromosome 12. Of the 50 tumors, nine were examined using FISH on the non-cultured samples. Two (4.0%) of 50 tumor samples examined showed chromosomal abnormalities of chromosome 12 by the conventional cytogenetic analysis. For FISH, the whole-chromosome painting probe and D12Z3 probe specific for the centromeric region were used. Of the 50 cultured samples, 10 showed structural aberrations and four showed numerical aberrations of chromosome 12 by FISH analysis. Of the nine non-cultured samples, four showed structural abnormalities of chromosome 12, all of which also showed structural abnormalities of chromosome 12 on the cultured samples. These results indicate that chromosomal abnormalities of chromosome 12 are important in the biology of at least some types of uterine leiomyoma, and that FISH is a useful complement to the conventional cytogenetic analysis in the study of solid tumors. PMID:8914635

Hayashi, S; Miharu, N; Okamoto, E; Samura, O; Hara, T; Ohama, K

1996-03-01

137

Image cytometry-based detection of aneuploidy by fluorescence in situ hybridization in suspension.  

PubMed

Cytogenetic abnormalities are important diagnostic and prognostic criteria for hematologic malignancies. Karyotyping and fluorescence in situ hybridization (FISH) are the conventional methods by which these abnormalities are detected. The sensitivity of these microscopy-based methods is limited by the abundance of the abnormal cells in the samples and therefore these analyses are commonly not applicable to minimal residual disease (MRD) stages. A flow cytometry-based imaging approach was developed to detect chromosomal abnormalities following FISH in suspension (FISH-IS), which enables the automated analysis of several log-magnitude higher number of cells compared with the microscopy-based approaches. This study demonstrates the applicability of FISH-IS for detecting numerical chromosome aberrations, establishes accuracy, and sensitivity of detection compared with conventional FISH, and feasibility to study procured clinical samples of acute myeloid leukemia (AML). Male and female healthy donor peripheral blood mononuclear cells hybridized with combinations of chromosome enumeration probes (CEP) 8, X, and Y served as models for disomy, monosomy, and trisomy. The sensitivity of detection of monosomies and trisomies amongst 20,000 analyzed cells was determined to be 1% with a high level of precision. A high correlation (R(2) = 0.99) with conventional FISH analysis was found based on the parallel analysis of diagnostic samples procured from 10 AML patients with trisomy 8 (+8). Additionally, FISH-IS analysis of samples procured at the time of clinical remission demonstrated the presence of residual +8 cells indicating that this approach may be used to detect MRD and associated chromosomal defects. PMID:22837074

Minderman, Hans; Humphrey, Kristen; Arcadi, Jane K; Wierzbicki, Andrzej; Maguire, Orla; Wang, Eunice S; Block, AnneMarie W; Sait, Sheila N J; George, Thaddeus C; Wallace, Paul K

2012-07-26

138

Metallographic in situ hybridization.  

PubMed

Metallographic methods, in which a target is visualized using a probe or antibody that deposits metal selectively at its binding site, offers many advantages for bright-field in situ hybridization (ISH) detection as well as for other labeling and detection methods. Autometallographically enhanced gold labeling procedures have demonstrated higher sensitivity than conventional enzyme chromogens. Enzyme metallography, a novel procedure in which an enzymatic probe is used to deposit metal directly from solution, has been used to develop bright-field ISH methods for HER2 gene determination in breast cancer and other biopsy specimens. It provides the highest level of sensitivity and resolution, both for visualizing endogenous gene copies in nonamplified tissues and for resolving multiple gene copies to allow copy enumeration in amplified tissues without the need for oil immersion or fluorescence optics. An automated enzyme metallography procedure, silver ISH, has been developed for use in slide-staining instruments. Metallographic staining also provides excellent results for immunohistochemistry and may be combined with other staining procedures for the simultaneous detection of more than one gene or combinations of genes and proteins. PMID:17640553

Powell, Richard D; Pettay, James D; Powell, William C; Roche, Patrick C; Grogan, Thomas M; Hainfeld, James F; Tubbs, Raymond R

2007-08-01

139

Quantitative in situ hybridization  

Microsoft Academic Search

In situ hybridization (ISH) methods make it possible to localize specific RNA and DNA sequences in cells and tissues and are important tools for studies of cytogenetics, microbiology, gene regulation, plasticity, and differentiation. The basic prerequisite for ISH involves synthesis of a labeled DNA or RNA sequence (probe) that is complementary to the target sequence to be investigated. The probe

Lars-Inge Larsson

1997-01-01

140

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

Microsoft Academic Search

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

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

1994-01-01

141

Interchromosomal effects for chromosome 21 in carriers of structural chromosome reorganizations determined by fluorescence in situ hybridization on sperm nuclei  

Microsoft Academic Search

We have used dual color fluorescence in situ hybridization (FISH) on decondensed sperm heads from four carriers of structural chromosome reorganizations, viz. t(3;15), t(Y;7), t(13;22) and inv(9), to assess the possible existence of an interchromosomal effect (ICE) on the segregation of chromosome 21. In the carriers of t(Y;7), t(13;22) and inv(9), all results were within the limits described in controls.

Joan Blanco; Josep Egozcue; Francesca Vidal

2000-01-01

142

A Broad Amplification Pattern at 3q in Squamous Cell Lung Cancer—A Fluorescence In Situ Hybridization Study  

Microsoft Academic Search

Frequent DNA copy number gain at 3q, with minimal overlapping area at 3q24-qter, has previously been reported in squamous cell carcinoma of the lung (SQCC), implicating the importance of genes at 3q in the tumorigenesis of SQCC. To further characterize the gain of DNA sequences at 3q, we performed interphase fluorescence in situ hybridization (FISH) analysis on 16 paraffin-embedded SQCC

Eeva Kettunen; Wa'el El-Rifai; Anna-Maria Björkqvist; Henrik Wolff; Antti Karjalainen; Sisko Anttila; Karin Mattson; Kirsti Husgafvel-Pursiainen; Sakari Knuutila

2000-01-01

143

Combined cytogenetic testing and fluorescence in situ hybridization analysis in the study of chronic lymphocytic leukemia and multiple myeloma  

Microsoft Academic Search

We investigated the usefulness of fluorescence in situ hybridization (FISH) panels when testing for chromosomal aberrations of known prognostic significance in chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). Our CLL panel included probes for 11q, 12 centromere, 13q, 14, and 17p. Karyotype and FISH were abnormal in 13 of 60 (21.6%) cases, two (3.3%) abnormal by karyotype alone, and

Anne Wiktor; Daniel L. Van Dyke

2004-01-01

144

Fluorescence in Situ Hybridization Markers for Prediction of Cervical Lymph Node Metastases  

PubMed Central

The presence of lymph node metastases is associated with poor prognosis in early stage cervical cancer. As of yet, no molecular markers predicting lymph node metastases have been identified. We examined single genetic markers and a composite marker, comprised of three fluorescence in situ hybridization (FISH) probes targeting the genes LAMP3, PROX1, and PRKAA1, in pretreatment cervical biopsies from 16 lymph node positive cases and 15 lymph node negative controls from women with stage IB and IIA cervical cancer. In addition, we determined clonal patterns by including CCND1 to compare the clonal constitution of primary tumors and associated lymph node metastases. The composite FISH marker allowed for classification of patients into those with and without lymph node metastases with a sensitivity and specificity of 75% and 87%, respectively (P = 0.001). The positive predictive value and negative predictive value were 86% and 76%, respectively. Clonal patterns varied among the tumors. In many cases, changes between the primary tumor and lymph node metastases in the most common clones may indicate that certain clones have a growth advantage for establishing metastases in lymph nodes. We conclude that the composite FISH marker may be useful for determining risk for subsequent development of lymph node metastases in patients with cervical cancer.

Wangsa, Darawalee; Heselmeyer-Haddad, Kerstin; Ried, Patricia; Eriksson, Elina; Schaffer, Alejandro A.; Morrison, Larry E.; Luo, Juhua; Auer, Gert; Munck-Wikland, Eva; Ried, Thomas; Lundqvist, Elisabeth Avall

2009-01-01

145

Bacterioplankton Compositions of Lakes and Oceans: a First Comparison Based on Fluorescence In Situ Hybridization  

PubMed Central

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes was used to investigate the phylogenetic composition of bacterioplankton communities in several freshwater and marine samples. An average of about 50% of the cells were detected by probes for the domains Bacteria and Archaea, and of these, about half could be identified at the subdomain level with a set of group-specific probes. Beta subclass proteobacteria constituted a dominant fraction in freshwater systems, accounting for 16% (range, 3 to 32%) of the cells, although they were essentially absent in the marine samples examined. Members of the Cytophaga-Flavobacterium cluster were the most abundant group detected in the marine systems, accounting for 18% (range, 2 to 72%) of the 4?,6-diamidino-2-phenylindole (DAPI) counts, and they were also important in freshwater systems (7%, range 0 to 18%). Furthermore, members of the alpha and gamma subclasses of Proteobacteria as well as members of the Planctomycetales were detected in both freshwater and marine water in abundances <7%.

Glockner, Frank Oliver; Fuchs, Bernhard M.; Amann, Rudolf

1999-01-01

146

Detection of fetal erythroid cells from maternal blood using fluorescence in situ hybridization and liquid culture.  

PubMed

Fetal nucleated erythrocytes circulating in maternal blood are a potential source of fetal DNA for noninvasive prenatal genetic diagnosis. However, the estimated ratio of fetal to maternal cells is extremely small. In order to enrich these cells, we performed direct culture using a two-phase liquid system. Mononuclear cells were obtained from maternal blood samples at 8-10(+3) weeks of gestation and cultured in the first phase. After 4-5 days, the nonadherent cells were harvested and recultured with erythropoietin in the second phase for another 3-5 days. We examined cellular morphology, and counted the number of benzidine- positive cells and the percentage of glycophorin A/CD71 positive erythroid cells. We also did Kleihauer-Betke stain for Hb F, polymerase chain reaction (PCR) for SRY/DYZ1, chromosome analysis, and fluorescence in situ hybridization (FISH). The number of total erythroid cells reached about 0.1 x 10(6)-1.0 x 10(6)/mL with a purity of 84.0-97.3%. Hb F stain showed total erythroid cells of approximately 0.4 x 10(4)-9.8 x 10(4)/mL. Male DNA was detected in one case by PCR. In this case, the XY karyotype was confirmed by FISH and amniocentesis. This approach provides enriched source of fetal cells for further prenatal genetic analysis without complicated separation or sorting procedures. PMID:11306738

Han, J Y; Kim, K H; Park, J I; Kim, I H; Je, G H

2001-04-01

147

Identification of tissue contamination by polymorphic deletion probe fluorescence in situ hybridization.  

PubMed

Potential sources of error in surgical pathology include specimen misidentification, unidentified tissue, and tissue contamination of paraffin blocks and slides. Current molecular approaches to characterize unidentified or misidentified tissue include fluorescence in situ hybridization identification of sex chromosomes (XY FISH) and microsatellite analysis. Polymorphic deletion probe (PDP) FISH, a novel FISH assay based on copy number variants, can distinguish between cells and tissues from 2 individuals in situ, independent of gender. Using a panel of 3 PDPs, we compared the genotypes of potential tissue contaminants (n=19) and unidentified tissues (n=6) with patient tissues to determine the utility of PDP FISH in resolving specimen identity. XY FISH was added to increase the informative potential of the assay, and microsatellite analysis was used as a gold standard to confirm PDP FISH results. PDP FISH distinguished between putative contaminants and patient tissues in 13 of 14 cases and indicated a high likelihood of 2 tissues originating from the same source in 11 of 11 cases. The assay has a sensitivity and specificity of 86% [6/7, exact 95% confidence interval (CI): 42%, 97%] and 100% (9/9, exact 1-sided 97.5% CI: 68%, 100%), respectively, and a positive predictive value and negative predictive value of 100% (6/6, exact 1-sided 97.5% CI: 54%, 100%) and 90% (9/10, exact 95% CI: 55%, 98%), respectively. PDP FISH is an accurate and practical molecular assay for the genetic characterization of potential tissue contaminants and unidentified tissues, especially in the setting of small sample size, and permits concomitant assessment of morphology. PMID:22982889

Chiang, Sarah; Yip, Stephen; Betensky, Rebecca A; Batten, Julie M; Misdraji, Joseph; John Iafrate, A

2012-10-01

148

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

NASA Astrophysics Data System (ADS)

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.

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

2012-05-01

149

A new approach to screen transgenic offspring using fluorescence in situ hybridization  

SciTech Connect

In order to identify the presence of vector, specifically Lacl or LacZ, in putative transgenic mice rapidly and reliably, we developed a new method which utilizes fluorescence in situ hybridization (FISH). Transgenic mouse models are being used with increasing frequency for mutational and toxicological studies. By applying our method, an investigator can reliably determine the presence and the number of integration sites of a transgenic vector in numerous samples with less effort compared to conventional methods. This approach involves gazing a tail with a scalpel to obtain a blood smear on a microscope slide. After fixing the slide in 3:1 methanol: acetic acid, typical FISH analysis using biotinylated lambda DNA as the probe in performed. Our method eliminates the need for DNA extraction, blotting, and PCR, and yields results from a large number of individually identifiable cells from each animal. This assay is more accurate, reliable and easier to perform than conventional schemes presently used for screening transgenic animals. A particular advantage of this assay is the ability to discriminate between animals that are heterozygous and homozygous, something that eludes the PCR-based methods and Southern blotting. We have successfully analyzed over 95 samples with this method. Based on these results, we believe our system is more sensitive and accurate than conventional means of screening.

Swiger, R.R.; Tucker, J.D. [Lawrence Livermore National Laboratory, CA (United States); Heddle, J.A. [York Univ., Ontario (Canada)

1995-11-01

150

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

PubMed

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. PMID:22612116

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

2012-05-01

151

Subregional localization of 20 single-copy loci to chromosome 6 by fluorescence in situ hybridization  

SciTech Connect

Although 338 genetic loci and 1 or more candidate tumor suppressor genes have been assigned to chromosome 6, the physical and genetic map of this chromosome is at a very preliminary stage. In this study, the authors have performed subregional localization of 20 single-copy DNA sequences previously assigned to chromosome 6 using the fluorescence in situ hybridization technique. One locus, D6S6, was localized at 6p12. Two loci, D6S160 and D6S32, were localized in proximal 6q, at 6q12 and 6q23.3, respectively. The remaining loci were clustered in two regions, 4 at 6q23.5-q25 (D6S33, D6S43, D6S85, MYB) and 13 at 6q26-q27 (D6S2, D6S21, D6S22, D6S25, D6S37, D6S39, D6S44, D6S48, D6S132, D6S133, D6S148, D6S170, D6S201). These data will aid in the eventual development of the physical map of chromosome 6. 30 refs., 2 figs., 1 tab.

Rao, P.H.; Murty, V.V.V.S.; Chaganti, R.S.K. [Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Gaidano, G.; Hauptschein, R.; Dalla-Favera, R. [Columbia Univ., New York, NY (United States)

1993-05-01

152

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

Microsoft Academic Search

BackgroundOur 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 FindingsWe employ peptide nucleic acid fluorescence in situ hybridization (PNA FISH) methods to

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

2011-01-01

153

Catalyzed reporter deposition-fluorescent in situ hybridization (CARD-FISH) detection of Dehalococcoides.  

PubMed

Members of the genus Dehalococcoides are well-known for their capacity to reductively dechlorinate chlorinated organic pollutants. The availability of quantitative and sensitive detection methods is of major interest for research on the ecology of those environmentally important micro-organisms. In this paper we describe the development of a Catalyzed Reporter Deposition-Fluorescent In Situ Hybridization (CARD-FISH) for detection of Dehalococcoides cells in enrichment cultures using two oligonucleotide sequences which target two different lineages of Dehalococcoides as probes. Both sequences were previously applied in conventional FISH as probes. Conjugation of the probe to horseradish peroxidase (HRP) did not change the specificity of the probes and bright fluorescent signals were obtained. Despite the use of higher concentrations of probe and the application of longer exposure times in the conventional FISH procedure, CARD-FISH resulted in more intense signals. The CARD-FISH method was applied to a vinyl chloride (VC)-reductively-dechlorinating enrichment culture. Only the probe targeting the CBDB1 lineage of Dehalococcoides reacted with the sample which was in agreement with previous nucleic acid based analysis. The culture consisted of 51%+/-8% of Dehalococcoides cells. Furthermore, the CARD-FISH probes for detecting Dehalococcoides were combined with FISH probes for simultaneous detection of either Bacteria or Archaea which should allow rapid insight into the relative dynamics of the different members of dechlorinating communities as a response to environmental changes. Overall, CARD-FISH proved to be a rapid, reliable and convenient method to detect and quantify Dehalococcoides cells. PMID:18410973

Dijk, J A; Breugelmans, P; Philips, J; Haest, P J; Smolders, E; Springael, D

2008-02-11

154

Intratumoral heterogeneity of her-2/neu in invasive mammary carcinomas using fluorescence in-situ hybridization and tissue microarray.  

PubMed

Fluorescence in-situ hybridization is increasingly being used to determine HER-2/neu status in patients with breast carcinoma. The possibility that intratumoral heterogeneity of HER-2/neu gene amplification may potentially contribute to inaccurate assessment of HER-2/neu status was investigated in routine cases of invasive mammary carcinomas. From 169 representative formalin-fixed, paraffin-embedded blocks of invasive duct mammary carcinomas with grade 3 architecture, 48 cases were analyzed by fluorescence in-situ hybridization and 59 analyses were performed. Intratumoral heterogeneity for HER-2/neu gene amplification was demonstrated in only 5 (16%) of 31 cases where morphologically similar areas of a single tumor were analyzed. We conclude from this study that intratumoral heterogeneity of HER-2/neu gene amplification is a demonstrable but relatively uncommon occurrence. For invasive mammary carcinomas, the accurate assessment of HER-2/neu status by fluorescence in-situ hybridization analysis is not significantly confounded by intratumoral heterogeneity of HER-2/neu gene amplification in individual tumors. PMID:17041191

Shin, Sandra J; Hyjek, Elizabeth; Early, Erin; Knowles, Daniel M

2006-10-01

155

Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria.  

PubMed

Fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition (CARD), is currently not generally applicable to heterotrophic bacteria in marine samples. Penetration of the HRP molecule into bacterial cells requires permeabilization procedures that cause high and most probably species-selective cell loss. Here we present an improved protocol for CARD-FISH of marine planktonic and benthic microbial assemblages. After concentration of samples onto membrane filters and subsequent embedding of filters in low-gelling-point agarose, no decrease in bacterial cell numbers was observed during 90 min of lysozyme incubation (10 mg ml(-1) at 37 degrees C). The detection rates of coastal North Sea bacterioplankton by CARD-FISH with a general bacterial probe (EUB338-HRP) were significantly higher (mean, 94% of total cell counts; range, 85 to 100%) than that with a monolabeled probe (EUB338-mono; mean, 48%; range, 19 to 66%). Virtually no unspecific staining was observed after CARD-FISH with an antisense EUB338-HRP. Members of the marine SAR86 clade were undetectable by FISH with a monolabeled probe; however, a substantial population was visualized by CARD-FISH (mean, 7%; range, 3 to 13%). Detection rates of EUB338-HRP in Wadden Sea sediments (mean, 81%; range, 53 to 100%) were almost twice as high as the detection rates of EUB338-mono (mean, 44%; range, 25 to 71%). The enhanced fluorescence intensities and signal-to-background ratios make CARD-FISH superior to FISH with directly labeled oligonucleotides for the staining of bacteria with low rRNA content in the marine environment. PMID:12039771

Pernthaler, Annelie; Pernthaler, Jakob; Amann, Rudolf

2002-06-01

156

The Bin1 gene localizes to human chromosome 2q14 by PCR analysis of somatic cell hybrids and fluorescence in situ hybridization  

SciTech Connect

This report describes the localization of the box-dependent myc-interacting protein-1 (Bin1) gene to human chromosome 2q14 using fluorescence in situ hybridization and polymerase chain reaction. Loss of Bin1 expression is commonly found in human carcinomas. 5 refs., 1 fig.

Negorev, D.; Simon, D. [Medical College of Pennyslvania and Hahnemann Univ., Philadelphia, PA (United States); Riethman, H. [Wistar Inst., Philadelphia, PA (United States)] [and others

1996-04-15

157

Microfluidic fluorescence in situ hybridization and flow cytometry (uFlowFISH)  

PubMed Central

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.

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

2011-01-01

158

Development of automated brightfield double In Situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) for breast carcinomas and an assay performance comparison to manual dual color HER2 fluorescence In Situ hybridization (FISH)  

Microsoft Academic Search

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) fluorescence in situ hybridization (FISH) is a quantitative assay for selecting breast cancer patients for trastuzumab therapy. However, current HER2 FISH procedures are labor intensive, manual methods that require skilled technologists and specialized fluorescence microscopy. Furthermore, FISH slides cannot be archived for long term storage and review. Our objective was to develop

Hiroaki Nitta; Beatrice Hauss-Wegrzyniak; Megan Lehrkamp; Adrian E Murillo; Fabien Gaire; Michael Farrell; Eric Walk; Frederique Penault-Llorca; Masafumi Kurosumi; Manfred Dietel; Lin Wang; Margaret Loftus; James Pettay; Raymond R Tubbs; Thomas M Grogan

2008-01-01

159

Genotyping the factor VIII intron 22 inversion locus using fluorescent in situ hybridization  

Microsoft Academic Search

The factor VIII intron 22 inversion is the most common cause of hemophilia A, accounting for approximately 40% of all severe cases of the disease. Southern hybridization and multiplex long distance PCR are the most commonly used techniques to detect the inversion in a diagnostic setting, although both have significant limitations. Here we describe our experience establishing a multicolor fluorescent

Campbell R. Sheen; Margaret A. McDonald; Peter M. George; Mark P. Smith; Christine M. Morris

2011-01-01

160

Clinical Evaluation of a Multi-target Fluorescent in Situ Hybridization Assay for Detection of Bladder Cancer  

Microsoft Academic Search

PurposeThe UroVysion fluorescence in situ hybridization assay (UroVysion Bladder Cancer Recurrence Kit, Vysis, Inc., Downers Grove, Illinois) is a multi-target assay that detects aneuploidy of chromosomes 3, 7 and 17, and loss of the 9p21 band in exfoliated cells in urine from patients with transitional cell carcinoma. We performed 2 multicenter trials. In 1 trial we compared the sensitivity of

MICHAEL F. SAROSDY; PAUL SCHELLHAMMER; GARY BOKINSKY; PAUL KAHN; ROBERTO CHAO; LAWRENCE YORE; JOSEPH ZADRA; DANIEL BURZON; GERALD OSHER; JULIA A. BRIDGE; STEVEN ANDERSON; SONNY L. JOHANSSON; MICHAEL LIEBER; MARK SOLOWAY; KERRY FLOM

2002-01-01

161

An extended fluorescence in situ hybridization approach for the cytogenetic study of cholangiocarcinoma on endoscopic retrograde cholangiopancreatography brushing cytology preparations.  

PubMed

The cytological diagnosis of cholangiocarcinoma has been significantly aided by applying a 4-probe fluorescence in situ hybridization system on endoscopic retrograde cholangiopancreatography brushing smears, aiming mainly at the detection of hyperdiploidy. However, this approach adds little to our understanding of the genetic background of the disease. With the prospect of obtaining additional data on chromosomal aberrations, we have extended the fluorescence in situ hybridization study, with the application of 4 independent 2-probe systems in 35 patients with documented cholangiocarcinoma. Fluorescence in situ hybridization assays were performed on endoscopic retrograde cholangiopancreatography brushing smears, with probes for the 7q31, 11q13 (CCND1), 17p53 (TP53), and 9p21 (INK4 locus) bands, together with the respective centromeric probe. Hyperdiploidy, involving at least 2 of the 4 chromosomes targeted, was found in 31 patients. 17p13 deletion was detected in 3, and 9p21 deletion, in 5 of the hyperdiploid cases, with the 2 aberrations concurrent in 1. CCND1 amplification was found in 1 case as the sole abnormality and in another together with hyperdiploidy, but in apparently unrelated clones. This work indicates that interphase fluorescence in situ hybridization is a practical and useful tool for the cytogenetic study of cholangiocarcinoma on endoscopic retrograde cholangiopancreatography brushing smears, which is often the only available tissue specimen of the tumor. Apart from hyperdiploidy, it provides additional data on the genetic profile of cholangiocarcinoma, especially regarding structural chromosomal aberrations and clonal diversity. This line of investigation may prove useful in the delineation of oncogenesis and the interpretation of the diverse clinical features of the disease. PMID:23845469

Vasilieva, Larisa E; Papadhimitriou, Stefanos I; Alexopoulou, Alexandra; Pavlidis, Dimitris; Kostopoulos, Ioannis; Georgiakaki, Maria; Xinopoulos, Dimitrios; Romanos, Andreas; Dourakis, Spyridon P

2013-07-08

162

Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization Allows for Enrichment-Independent Detection of Microcolony-Forming Soil Bacteria  

Microsoft Academic Search

Advances in the growth of hitherto unculturable soil bacteria have emphasized the requirement for rapid bacterial identification methods. Due to the slow-growing strategy of microcolony-forming soil bacteria, successful fluorescence in situ hybridization (FISH) requires an rRNA enrichment step for visualization. In this study, catalyzed reporter deposition (CARD)-FISH was employed as an alternative method to rRNA enhancement and was found to

Belinda C. Ferrari; Niina Tujula; Kate Stoner; Staffan Kjelleberg

2006-01-01

163

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

SciTech Connect

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.

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

1995-08-10

164

Fluorescence in situ hybridization, a diagnostic aid in ambiguous melanocytic tumors: European study of 113 cases.  

PubMed

Some melanocytic tumors are ambiguous, so the reproducible histopathological diagnosis of benign or malignant lesion is difficult. This study evaluated the contribution of fluorescence in situ hybridization (FISH) first in 43 non-equivocal melanomas and nevi, and then in 113 ambiguous melanocytic tumors selected by expert pathologists from six different European institutions. We included two groups of ambiguous tumors: patients without recurrence (5-year minimal follow-up) and with metastases. An independent triple-blind histopathological review was performed to classify tumors as 'favor benign' (A-) or 'favor malignant' (A+). A four-color probe set targeting 6p25, 6q23, 11q13 and CEP6 was used for FISH. In the 43 non-equivocal melanomas and nevi, sensitivity was 85% and specificity 90%. Ninety out of 95 ambiguous melanocytic tumors included were FISH interpretable (67 FISH negative and 23 FISH positive). Of the 90 patients, 69 presented no recurrence and 21/90 exhibited metastases. These ambiguous tumors were mostly spitzoid tumors (45/90). Histopathological reviewers classified these tumors as favor malignant (49/90) and favor benign (32/90), whereas nine cases had a discordant diagnosis. By comparison with outcome, the sensitivity and specificity of histopathological review were 95 and 52%, and the sensitivity and specificity of FISH were 43 and 80%. Compared with histopathological review, the sensitivity and specificity of FISH were 34.5 and 91%. Interestingly, by combining the histopathological diagnosis with FISH results, the diagnosis was optimized, especially by increasing specificity (76% instead of 52% for expert diagnosis alone) and by improving sensitivity compared with FISH alone (90 vs 43% for FISH result alone). The value of this FISH test is to add a reproducible demonstration of malignancy to the histopathological diagnosis, especially in doubtful/ambiguous melanocytic tumors. A positive FISH test reinforces the diagnosis of melanoma, allowing such tumors (particularly thick tumors) to be managed as melanomas. PMID:21151100

Vergier, Beatrice; Prochazkova-Carlotti, Martina; de la Fouchardière, Arnaud; Cerroni, Lorenzo; Massi, Daniela; De Giorgi, Vincenzo; Bailly, Christiane; Wesselmann, Ulrich; Karlseladze, Apollon; Avril, Marie-Francoise; Jouary, Thomas; Merlio, Jean-Philippe

2010-12-10

165

Visualization of the mycelia of wood-rotting fungi by fluorescence in situ hybridization using a peptide nucleic acid probe.  

PubMed

White rot fungus, Phanerochaete chrysosporium, and brown rot fungus, Postia placenta, grown on agar plates, were visualized by fluorescence in situ hybridization (FISH) using a peptide nucleic acid (PNA) probe. Mycelia grown on wood chips were also clearly detected by PNA-FISH following blocking treatment. To the best of our knowledge, this is the first report on the visualization of fungi in wood by FISH. PMID:23391931

Nakada, Yuji; Nakaba, Satoshi; Matsunaga, Hiroshi; Funada, Ryo; Yoshida, Makoto

2013-02-07

166

Characterization of an unbalanced de novo rearrangement by microsatellite polymorphism typing and by fluorescent in situ hybridization  

Microsoft Academic Search

Unbalanced de novo rearrangements, difficult to characterize by conventional cytogenetic techniques, may be elucidated by molecular approaches. By dinucleotide repeat polymorphism typing and fluorescence in situ hybridization (FISH), we have defined the composition of an unbalanced de novo translocation (46,XX,15p+) in a child with multiple congenital anomalies. Use of a microsatellite repeat D5S208 (localized to 5p15) and polymerase chain reaction

Jian Zhao; Patricia L. Gordon; R. S. Jr. Wilroy; Paula R. Martens; Jack Tarleton; Lee P. Shulman; Joe Leigh Simpson; Sherman Elias; Avirachan T. Tharapel

1995-01-01

167

Cellular localization of oriC during the cell cycle of Escherichia coli as analyzed by fluorescent in situ hybridization  

Microsoft Academic Search

The origin of replication of Escherichia coli, oriC, has been labeled by fluorescent in situ hybridization (FISH). The E. coli K12 strain was grown under steady state conditions with a doubling time of 79 min at 28 °C. Under these growth conditions DNA replication starts in the previous cell cycle at -33 min. At birth cells possess two origins which are visible as

Marco Roos; Anton B. M. van Geel; Mirjam E. G. Aarsman; Jacques T. M. Veuskens; Conrad L. Woldringh; Nanne Nanninga

1999-01-01

168

Simple Adhesive-Tape-Based Sampling of Tomato Surfaces Combined with Rapid Fluorescence In Situ Hybridization for Salmonella Detection?  

PubMed Central

A simple adhesive-tape-based method for sampling of tomato surfaces was combined with fluorescence in situ hybridization for rapid culture-independent detection of Salmonella strains. Tapes could also be placed face-down on selective agar for on-tape enrichment of captured Salmonella cells. Overlay of cell-charged tapes with small volumes of liquid enrichment media enabled subsequent detection of tape-captured Salmonella via flow cytometry.

Bisha, Bledar; Brehm-Stecher, Byron F.

2009-01-01

169

Application of fluorescence in situ hybridization to detect residual leukemic cells with 9;22 and 15;17 translocations  

Microsoft Academic Search

We performed fluorescence in situ hybridization (FISH) upon 9;22 and 15;17 translocation-positive bone marrow cells to monitor the clinical course of 46 patients with chronic myelocytic leukemia (CML) and nine with acute promyelocytic leukemia (AML M3) who received chemotherapy and\\/or bone marrow transplantation (BMT). M-BCR-ABL and PML-RAR? probes were used to detect translocations of t(9;22) and t(15;17), respectively. Signals from

K Tanaka; M Arif; M Eguchi; TS Kumaravel; R Ueda; R Ohno; K Iwato; T Kyo; H Dohy; N Kamada

1997-01-01

170

Assignment of the human organic anion transporting polypeptide (OATP) gene to chromosome 12p12 by fluorescence in situ hybridization  

Microsoft Academic Search

Background\\/Aims: The organic anion transporting polypeptide (OATP) of human liver mediates the basolateral hepatocellular uptake of numerous cholephilic anions and steroidal compounds. The aim of this study was to clone the human OATP gene and to map its chromosomal localization by fluorescence in situ hybridization.Methods: A polymerase chain reaction-amplified fragment of the human OATP gene was used to isolate a

Gerd-Achim Kullak-Ublick; Ulrich Beuers; Peter J. Meier; Horst Domdey; Gustav Paumgartner

1996-01-01

171

Detection of Gallibacterium spp. in Chickens by Fluorescent 16S rRNA In Situ Hybridization  

PubMed Central

Gallibacterium has recently been included as a new genus of the family Pasteurellaceae Pohl 1981, which encompasses bacteria previously reported as Pasteurella anatis, “Actinobacillus salpingitidis,” and avian Pasteurella haemolytica-like organisms. So far, identification has exclusively relied on phenotypic characterization. We present a method based on a cyanine dye 3.18-labeled in situ hybridization probe targeting 16S rRNA to allow specific detection of bacteria belonging to the genus Gallibacterium. The probe, GAN850, showed no cross-reactivity to 25 other poultry-associated bacterial species, including members of the families Pasteurellaceae, Enterobacteriaceae, and Flavobacteriaceae, when cross-reactivities were evaluated by whole-cell hybridization. The probe was further evaluated by hybridization to formalin-fixed spleen and liver tissues from experimentally infected chickens, in which it proved to be useful for the detection of Gallibacterium. Additionally, determination of the spatial distribution and the host cell affiliation of Gallibacterium at various times during the infection process was possible. In conclusion, the in situ hybridization technique described may be of use as a diagnostic tool as well as for studies to elucidate the pathogenesis of Gallibacterium infections in chickens.

Bojesen, Anders Miki; Christensen, Henrik; Nielsen, Ole Lerberg; Olsen, John Elmerdahl; Bisgaard, Magne

2003-01-01

172

Detection of Gallibacterium spp. in chickens by fluorescent 16S rRNA in situ hybridization.  

PubMed

Gallibacterium has recently been included as a new genus of the family Pasteurellaceae Pohl 1981, which encompasses bacteria previously reported as Pasteurella anatis, "Actinobacillus salpingitidis," and avian Pasteurella haemolytica-like organisms. So far, identification has exclusively relied on phenotypic characterization. We present a method based on a cyanine dye 3.18-labeled in situ hybridization probe targeting 16S rRNA to allow specific detection of bacteria belonging to the genus Gallibacterium. The probe, GAN850, showed no cross-reactivity to 25 other poultry-associated bacterial species, including members of the families Pasteurellaceae, Enterobacteriaceae, and Flavobacteriaceae, when cross-reactivities were evaluated by whole-cell hybridization. The probe was further evaluated by hybridization to formalin-fixed spleen and liver tissues from experimentally infected chickens, in which it proved to be useful for the detection of Gallibacterium. Additionally, determination of the spatial distribution and the host cell affiliation of Gallibacterium at various times during the infection process was possible. In conclusion, the in situ hybridization technique described may be of use as a diagnostic tool as well as for studies to elucidate the pathogenesis of Gallibacterium infections in chickens. PMID:14605154

Bojesen, Anders Miki; Christensen, Henrik; Nielsen, Ole Lerberg; Olsen, John Elmerdahl; Bisgaard, Magne

2003-11-01

173

Human cDNA mapping using a high-resolution R-banding technique and fluorescence in situ hybridization.  

PubMed

High-resolution fluorescence in situ hybridization (FISH) is now an essential element in both human gene mapping and clinical cytogenetics. To facilitate its application, a series of techniques have been developed using FISH to map DNA probes in the size range of 1-1,000 kb directly on R-banded human chromosomes. Distinctive reverse (R) banding is achieved by staining with chromomycin A3 and distamycin A following in situ hybridization. The use of such counterstains enables simultaneous viewing of both the fluorescent R-bands and in situ hybridization signals by either standard photomicroscopy or an automated image-acquisition system. This method is rapid and reproducibly reveals bands at the 350-700 stage. Further, specific methods for chromosome preparation, hybridization, and signal production have been developed and applied in combination with R-banding. These methods are used for precise chromosomal localization of DNA sequences in sizes ranging from that of cDNA (> 1 kb) through bacterial artificial chromosomes (100-150 kb) to yeast artificial chromosomes (> or = 1 Mb). These techniques provide high-resolution methods for rapid mapping of human genes, expanding the applications of FISH techniques in basic research and clinical analysis. PMID:7698011

Korenberg, J R; Chen, X N

1995-01-01

174

Sample preparations are important for fluorescence in situ hybridization of cells biopsied from preimplantation embryos.  

PubMed

Summary Fluorescence in situ hybridization (FISH) is a cytogenetic technology used to detect chromosomal abnormalities in preimplantation human embryos. However, its efficiency is not stable due to improper sample preparation. The present study was designed to modify the current sample preparation technique and then to evaluate its efficiency in human preimplantation genetic diagnosis (PGD). Day 3 cleavage embryos as well as day 5 and 6 blastocysts were biopsied by mechanical aspiration method. In the present study, two methods were used for sample preparation of the biopsied cells. Method I was the traditional method, in which each blastomere was placed in a hypotonic solution for 5 min and then fixed on glass slides. The slides were kept at room temperature before the FISH procedures. Method II was a modified method, in which all blastomeres were placed individually in hypotonic solution drops covered by oil for at least 5 min and then fixed on slides with 0.1% Tween/HCl. After fixation, the slides were kept at -20°C for at least 30 min before the FISH procedures. The two methods were compared in terms of time consumption and proportions of blastomeres with FISH signals. In total, 329 blastomeres from day 3 embryos were fixed by Method I with an average fixation time of 8-10 min for each blastomere. By contrast, with Method II, 362 blastomeres were fixed and the average time was 3-4 min for each blastomere. After FISH, more nuclei had signals with Method II (97.2%) than with Method I (86.9%). All cells that were biopsied from blastocysts and prepared with Method II had FISH signals. However, Method I was not suitable for the fixation of multiple cells biopsied from blastocysts as cells were not traceable during the fixation. The present study indicates that proper sample preparation is critical for obtaining FISH signals in cells biopsied from preimplantation human embryos; hence these modifications can increase the efficiency of human PGD. PMID:23331574

Li, Lifei; Zhang, Xuehong; Wang, Weihua

2013-01-18

175

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

PubMed

A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the species D. bruxellensis and that the new method is able to identify Brettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity. PMID:11157265

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

2001-02-01

176

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

PubMed Central

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

Stender, Henrik; Kurtzman, Cletus; Hyldig-Nielsen, Jens J.; S?rensen, Ditte; Broomer, Adam; Oliveira, Kenneth; Perry-O'Keefe, Heather; Sage, Andrew; Young, Barbara; Coull, James

2001-01-01

177

Analysis of follicular lymphoma by dual-color fluorescence in situ hybridization.  

PubMed

Follicular lymphoma (FL) is divided into two groups: one with the t(14;18)(q32;q21) and the other without this translocation but with other chromosomal abnormalities including t(3q27) break. The majority of FLs in Western countries have the former chromosomal changes with characteristic clinical features and low histological grades. The goal of this study was to investigate the characteristics of Korean FLs with regard to the underlying molecular defects. Sixty-one cases of FL were evaluated from two centers in Korea by immunostaining for CD10, bcl-2, and bcl-6 proteins. Fluorescence in situ hybridization was performed to detect the t(14;18) and t(3q27) break. Cases with FL grade 3 accounted for 57% of all 61 cases. The t(14;18) was detected in 43.9% of the cases studied, and the frequency was 80, 50, 34.8, and 9.1% from grade 1 through grade 3b. The t(3q27) was detected in 16.1% of cases. Cases without a t(14;18) were mainly histological grade 3 (P < 0.001) and had a tendency to have the t(3q27) and bcl-2 amplification. The incidence of t(14;18)-positive low-grade FL was found to be lower in Korea than in Western countries. The increased frequency of t(14;18)-negative grade 3 FL attributes to the lower incidence of t(14;18) in Korean FL. PMID:18040714

Lee, Dakeun; Seo, Jinwon; Oh, YoungLyun; Kim, Jinman; Ko, YoungHyeh

2007-11-27

178

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

PubMed

Triclosan is considered a ubiquitous pollutant and can be detected in a wide range of environmental samples. Triclosan removal by wastewater treatment plants has been largely attributed to biodegradation processes; however, very little is known about the micro-organisms involved. In this study, DNA-based stable isotope probing (DNA-SIP) combined with microautoradiography-fluorescence in situ hybridization (MAR-FISH) was applied to identify active triclosan degraders in an enrichment culture inoculated with activated sludge. Clone library sequences of 16S rRNA genes derived from the heavy DNA fractions of enrichment culture incubated with (13)C-labelled triclosan showed a predominant enrichment of a single bacterial clade most closely related to the betaproteobacterial genus Methylobacillus. To verify that members of the genus Methylobacillus were actively utilizing triclosan, a specific probe targeting the Methylobacillus group was designed and applied to the enrichment culture incubated with (14)C-labelled triclosan for MAR-FISH. The MAR-FISH results confirmed a positive uptake of carbon from (14)C-labelled triclosan by the Methylobacillus. The high representation of Methylobacillus in the (13)C-labelled DNA clone library and its observed utilization of (14)C-labelled triclosan by MAR-FISH reveal that these micro-organisms are the primary consumers of triclosan in the enrichment culture. The results from this study show that the combination of SIP and MAR-FISH can shed light on the networks of uncultured micro-organisms involved in degradation of organic micro-pollutants. PMID:22956759

Lolas, Ihab Bishara; Chen, Xijuan; Bester, Kai; Nielsen, Jeppe Lund

2012-09-06

179

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

PubMed

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

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

2008-01-01

180

Fluorescent In Situ Hybridization: A New Tool for the Direct Identification and Detection of F. psychrophilum  

PubMed Central

F. psychrophilum is the causative agent of Bacterial Cold Water Disease (BCW) and Rainbow Trout Fry Syndrome (RTFS). To date, diagnosis relies mainly on direct microscopy or cultural methods. Direct microscopy is fast but not very reliable, whereas cultural methods are reliable but time-consuming and labor-intensive. So far fluorescent in situ hybridization (FISH) has not been used in the diagnosis of flavobacteriosis but it has the potential to rapidly and specifically detect F. psychrophilum in infected tissues. Outbreaks in fish farms, caused by pathogenic strains of Flavobacterium species, are increasingly frequent and there is a need for reliable and cost-effective techniques to rapidly diagnose flavobacterioses. This study is aimed at developing a FISH that could be used for the diagnosis of F. psychrophilum infections in fish. We constructed a generic probe for the genus Flavobacterium (“Pan-Flavo”) and two specific probes targeting F. psychrophilum based on 16S rRNA gene sequences. We tested their specificity and sensitivity on pure cultures of different Flavobacterium and other aquatic bacterial species. After assessing their sensitivity and specificity, we established their limit of detection and tested the probes on infected fresh tissues (spleen and skin) and on paraffin-embedded tissues. The results showed high sensitivity and specificity of the probes (100% and 91% for the Pan-Flavo probe and 100% and 97% for the F. psychrophilum probe, respectively). FISH was able to detect F. psychrophilum in infected fish tissues, thus the findings from this study indicate this technique is suitable as a fast and reliable method for the detection of Flavobacterium spp. and F. psychrophilum.

Strepparava, Nicole; Wahli, Thomas; Segner, Helmut; Polli, Bruno; Petrini, Orlando

2012-01-01

181

Automated enumeration of groups of marine picoplankton after fluorescence in situ hybridization.  

PubMed

We describe here an automated system for the counting of multiple samples of double-stained microbial cells on sections of membrane filters. The application integrates an epifluorescence microscope equipped with motorized z-axis drive, shutters, and filter wheels with a scanning stage, a digital camera, and image analysis software. The relative abundances of specific microbial taxa are quantified in samples of marine picoplankton, as detected by fluorescence in situ hybridization (FISH) and catalyzed reporter deposition. Pairs of microscopic images are automatically acquired from numerous positions at two wavelengths, and microbial cells with both general DNA and FISH staining are counted after object edge detection and signal-to-background ratio thresholding. Microscopic fields that are inappropriate for cell counting are automatically excluded prior to measurements. Two nested walk paths guide the device across a series of triangular preparations until a user-defined number of total cells has been analyzed per sample. A backup autofocusing routine at incident light allows automated refocusing between individual samples and can reestablish the focal plane after fatal focusing errors at epifluorescence illumination. The system was calibrated to produce relative abundances of FISH-stained cells in North Sea samples that were comparable to results obtained by manual evaluation. Up to 28 preparations could be analyzed within 4 h without operator interference. The device was subsequently applied for the counting of different microbial populations in incubation series of North Sea waters. Automated digital microscopy greatly facilitates the processing of numerous FISH-stained samples and might thus open new perspectives for bacterioplankton population ecology. PMID:12732531

Pernthaler, Jakob; Pernthaler, Annelie; Amann, Rudolf

2003-05-01

182

Adjunct prenatal testing: patient decisions regarding ethnic carrier screening and fluorescence in situ hybridization.  

PubMed

Little has been reported regarding how women make decisions about genetic carrier screening for Ashkenazi Jewish genetic disease and cystic fibrosis (CF), and for fluorescent in situ hybridization (FISH) during pregnancy. Thirty-seven women who underwent genetic counseling and prenatal diagnosis were interviewed about their prenatal decision making. Respondents were largely Caucasian (95%), and undergoing prenatal diagnosis because of maternal age (78%). Sixty-three percent of those who reported having genetic carrier screening correctly defined it; 83% felt positively about it. Primary reasons reported for electing screening were: to get information, to be prepared, perception of risk, wanting peace of mind and percieved inability to care for an affected child. Women who declined screening felt they had very little or no risk, and some were deterred by cost. Ninety-five percent of respondents elected to have FISH; most were motivated by its speed in providing information and peace of mind or by timing of when the procedure was performed. Those who declined FISH reported being less concerned about having an affected child, receiving bad news, or waiting 2 weeks for results and slightly less affected by their "feelings toward medical testing" or physician's suggestion. These findings suggest decision-making factors differ between those electing and declining adjunct prenatal testing and increased knowledge about these factors may impact the way in which these services are offered by health care professionals. Prospective research with a larger population will be useful in further delineating the factors that influence prenatal decisions about adjunct testing measures. PMID:19731471

Sturm, Erica L; Ormond, Kelly E

2004-02-01

183

Quantitative imaging and statistical analysis of fluorescence in situ hybridization (FISH) of Aureobasidium pullulans.  

PubMed

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 or an A. pullulans 18S rRNA oligonucleotide probe in direct or indirect FISH reactions. In general, type of fixation (paraformaldehyde or methanol-acetic acid) had no apparent effect on cell integrity and minimal impact on fluorescence. Permeabilization by enzyme treatment for various times, though needed to admit high Mw detection reagents (avidin-FITC) in indirect FISH, tended to nonspecifically degrade cells and lower the signal. Digestion was unnecessary and undesirable for the directly labelled probes. Multilabelled (five fluorescein molecules) probes enhanced fluorescence about fourfold over unilabelled probes. Overall, direct FISH was preferable to indirect FISH and is recommended especially for studies of microbes on natural substrata. PMID:10192042

Spéar, R N; Li, S; Nordheim, E V; Andrews, J H

1999-03-01

184

mRNA fluorescence in situ hybridization to determine overlapping gene expression in whole-mount mouse embryos.  

PubMed

Background: Whole-mount in situ hybridization (ISH) is a prevalent tool to examine the spatial distribution of gene transcripts in intact embryos. Chromogenic-based methods of signal development are commonly used in mouse embryos because of their high sensitivity. Fluorescence techniques, however, offer several advantages over chromogenic methods including the ability to visualize multiple signals in a specimen at once. Results: We describe a procedure for fluorescence in situ hybridization (FISH) for whole mouse embryos up to embryonic day 13.5. We show that this approach successfully produces a bright expression signal for several genes, validating the procedure in multiple tissues. Further, we show that double FISH can be used to visualize the expression of two genes in a single embryo by determining that Hoxd13 and Shh are co-expressed in both the limb bud and the hindgut. Finally, we demonstrate that FISH can be paired with confocal microscopy to take optical sections of interior regions of the embryo. Conclusions: FISH is a valid alternative to chromogenic-based ISH for visualizing gene expression in whole mouse embryos. This work provides a framework to add additional fluorescence signals in the mouse such as visualizing both mRNA and protein by pairing the procedure with immunofluorescence. Developmental Dynamics 242:1094-1100, 2013. © 2013 Wiley Periodicals, Inc. PMID:23749471

Neufeld, Stanley J; Zhou, Xiaolan; Vize, Peter D; Cobb, John

2013-06-27

185

Differentiation of Candida albicans and Candida dubliniensis by Fluorescent In Situ Hybridization with Peptide Nucleic Acid Probes  

PubMed Central

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 and identification of microorganisms. Peptide nucleic acid (PNA) is a DNA mimic with improved hybridization characteristics, and the neutral backbone of PNA probes offers significant advantages in whole-cell in situ hybridization assays. In this study, we developed PNA probes targeting the rRNAs of C. albicans and C. dubliniensis and applied them to a fluorescence in situ hybridization method (PNA FISH) for differentiation between C. albicans and C. dubliniensis. Liquid cultures were smeared onto microscope slides, heat fixed, and then hybridized for 30 min. Unhybridized PNA probe was removed by washing, and smears were examined by fluorescence microscopy. Evaluation of the PNA FISH method using smears of 79 C. dubliniensis and 70 C. albicans strains showed 100% sensitivity and 100% specificity for both PNA probes. We concluded that PNA FISH is a powerful tool for the differentiation of C. albicans and C. dubliniensis.

Oliveira, Kenneth; Haase, Gerhard; Kurtzman, Cletus; Hyldig-Nielsen, Jens Jo/rgen; Stender, Henrik

2001-01-01

186

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

Microsoft Academic Search

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,

Yuri B. Yurov; Ilia V. Soloviev; Svetlana G. Vorsanova; Bertrand Marcais; Gerard Roize; Robert Lewis

1996-01-01

187

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...

188

Use of fluorescence in situ hybridization to clarify a complex chromosomal rearrangement in a child with multiple congenital anomalies  

SciTech Connect

A child with multiple congenital anomalies was referred for cytogenetic evaluation. G-banded analysis showed a complex chromosome rearrangement involving 6 different chromosomes and 10 breakpoints. Fluorescence in situ hybridization (FISH) using whole chromosome painting probes and repetitive sequence probes was performed. In most cases the painting probes alone helped to clarify the G-banded results. However, in one instance, where the terminal band of the long arm of chromosome 1 was involved, the use of a telomeric probe was essential in defining the rearrangement. 13 refs., 3 figs.

Spikes, A.S.; Hegmann, K.; Smith, J.L. [Baylor College of Medicine, Houston, TX (United States)] [and others

1995-05-22

189

Cytogenetic characterization of diffuse large cell lymphoma using multi-color fluorescence in situ hybridization.  

PubMed

We have employed multi-color fluorescence in situ hybridization (M-FISH) to characterize the cytogenetic changes in 20 diffuse large B-cell lymphomas (DLBCL), that contained complex and partially characterized karyotypes. The M-FISH analysis helped to delineate 94% of the unidentified abnormalities and assisted in redefining some unidentified/misidentified karyotypic changes. Recurrent breakpoints observed in approximately 20% cases included 14q32, 3p21, 3q27, 22q12, 1q25, and 18q21 (in decreasing order), and 1p22, 1q21, 4q31, 6q21, and 8q24 (in four cases each). Numerical gain of chromosomes 7, 9, 12, and X and loss of chromosomes 1, 4, 6, 17, and Y, were noted in approximately 20% of cases. The minimum deleted regions encompassed 6q21-q25, 1p22-p36, 1q32-q44, 2p23-p25, 4q31-q35, 13p13-q14, and 17p11-p13. Two cases presented with a sole structural abnormality, and one contained a der(17)t(9;17)(p21;p13), which has not been reported earlier as a sole abnormality in DLBCL. Upon completely characterizing the karyotypes, we observed with interesting that in 55% of the cases, more than one BCL gene bearing regions was involved in translocations. In the remaining 45%, where only one or none of the BCL gene regions was involved in a rearrangement, we observed the loss of chromosomes 6 and/or 17 or partial deletions of 6q and/or 17p or gain of 7 and/or 12. Our findings suggest that, although BCL2 and BCL6 are most often implicated in DLBCL, the possibility of the disruptions of BCL3, BCL8, BCL9, and BCL10 as a "primary event" in DLBCL cannot be ruled out. Most often, a combination of events may be necessary for the genesis of DLBCL or progression of follicular lymphoma to DLBCL. Overall, M-FISH has enhanced our ability to provide a comprehensive karyotypic analysis, and has helped in defining the importance of BCL3, BCL8, BCL9, and BCL10 carrying breakpoints in DLBCL. PMID:11850073

Dave, Bhavana J; Nelson, Marilu; Pickering, Diane L; Chan, Wing C; Greiner, Timothy C; Weisenburger, Dennis D; Armitage, James O; Sanger, Warren G

2002-01-15

190

Specific detection of green sulfur bacteria by in situ hybridization with a fluorescently labeled oligonucleotide probe.  

PubMed

An oligodeoxynucleotide probe (GSB-532) specific for green sulfur bacteria was developed. Highly stringent hybridization conditions were established using whole cells of Chlorobium limicola DSM249 immobilized on glass slides. At a formamide concentration of 10%, the optimum specificity was reached at 47 degrees C. When a conventional fixation procedure was used, a conspicuous autofluorescence developed within the cells. This autofluorescence was due to the liberation of bacteriochlorophyll by the detergent Triton X-100 and a subsequent conversion to bacteriophenophytin and related compounds. The signal-to-noise ratio could be increased by a final dehydration of the samples with methanol. Finally, the method was adapted to the hybridization of natural samples collected on polycarbonate membrane filters. In situ hybridization of pure cultures, various enrichments, and natural samples from the chemocline of a freshwater lake confirmed that probe GSB-532 hybridized exclusively to cells of green sulfur bacteria. Our protocol allows the highly specific detection of green sulfur bacteria in water samples and a rapid screening of natural bacterial communities. Employing probe GSB-532, the phylogenetic affiliation of the epibionts in "Chlorochromatium aggregatum" and "Pelochromatium roseum" could be demonstrated for the first time. PMID:10339808

Tuschak, C; Glaeser, J; Overmann, J

1999-03-01

191

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

PubMed

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

Rickerts, V

2013-11-01

192

Prediction of human cell radiosensitivity: Comparison of clonogenic assay with chromosome aberrations scored using premature chromosome condensation with fluorescence in situ hybridization  

Microsoft Academic Search

The purpose of the present investigation was to determine whether chromosome aberrations scored by premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH) can predict the radiosensitivity of human cell lines, thereby providing a possible means of assessing the in situ radiosensitivity of normal tissues and the radiocurability of individual human cancers. We used four cells lines of different

K. Sasai; J. W. Evans; M. S. Kovacs

1994-01-01

193

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

Microsoft Academic Search

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

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

2004-01-01

194

Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain  

PubMed Central

Background In recent years, mapping of overlapping and abutting regulatory gene expression domains by chromogenic two-color in situ hybridization has helped define molecular subdivisions of the developing vertebrate brain and shed light on its basic organization. Despite the benefits of this technique, visualization of overlapping transcript distributions by differently colored precipitates remains difficult because of masking of lighter signals by darker color precipitates and lack of three-dimensional visualization properties. Fluorescent detection of transcript distributions may be able to solve these issues. However, despite the use of signal amplification systems for increasing sensitivity, fluorescent detection in whole-mounts suffers from rapid quenching of peroxidase (POD) activity compared to alkaline phosphatase chromogenic reactions. Thus, less strongly expressed genes cannot be efficiently detected. Results We developed an optimized procedure for fluorescent detection of transcript distribution in whole-mount zebrafish embryos using tyramide signal amplification (TSA). Conditions for hybridization and POD-TSA reaction were optimized by the application of the viscosity-increasing polymer dextran sulfate and the use of the substituted phenol compounds 4-iodophenol and vanillin as enhancers of POD activity. In combination with highly effective bench-made tyramide substrates, these improvements resulted in dramatically increased signal-to-noise ratios. The strongly enhanced signal intensities permitted fluorescent visualization of less abundant transcripts of tissue-specific regulatory genes. When performing multicolor fluorescent in situ hybridization (FISH) experiments, the highly sensitive POD reaction conditions required effective POD inactivation after each detection cycle by glycine-hydrochloric acid treatment. This optimized FISH procedure permitted the simultaneous fluorescent visualization of up to three unique transcripts in different colors in whole-mount zebrafish embryos. Conclusions Development of a multicolor FISH procedure allowed the comparison of transcript gene expression domains in the embryonic zebrafish brain to a cellular level. Likewise, this method should be applicable for mRNA colocalization studies in any other tissues or organs. The key optimization steps of this method for use in zebrafish can easily be implemented in whole-mount FISH protocols of other organisms. Moreover, our improved reaction conditions may be beneficial in any application that relies on a TSA/POD-mediated detection system, such as immunocytochemical or immunohistochemical methods.

2011-01-01

195

Development of a fluorescence in situ hybridization protocol for the identification of micro-organisms associated with wastewater particles and flocs  

Microsoft Academic Search

Fluorescence in situ hybridization (FISH) provides a unique tool to study micro-organisms associated with particles and flocs. FISH enables visual examination of micro-organisms while they are structurally intact and associated with particles. However, application of FISH to wastewater and sludge samples presents a specific set of problems. Wastewater samples generate high background fluorescence due to their organic and inorganic content

Banu Örmeci; Karl G. Linden

2008-01-01

196

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

SciTech Connect

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.

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

197

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

SciTech Connect

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.

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

198

Quantification of target molecules needed to detect microorganisms by fluorescence in situ hybridization (FISH) and catalyzed reporter deposition-FISH.  

PubMed

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a method that is widely used to detect and quantify microorganisms in environmental samples and medical specimens by fluorescence microscopy. Difficulties with FISH arise if the rRNA content of the probe target organisms is low, causing dim fluorescence signals that are not detectable against the background fluorescence. This limitation is ameliorated by technical modifications such as catalyzed reporter deposition (CARD)-FISH, but the minimal numbers of rRNA copies needed to obtain a visible signal of a microbial cell after FISH or CARD-FISH have not been determined previously. In this study, a novel competitive FISH approach was developed and used to determine, based on a thermodynamic model of probe competition, the numbers of 16S rRNA copies per cell required to detect bacteria by FISH and CARD-FISH with oligonucleotide probes in mixed pure cultures and in activated sludge. The detection limits of conventional FISH with Cy3-labeled probe EUB338-I were found to be 370 +/- 45 16S rRNA molecules per cell for Escherichia coli hybridized on glass microscope slides and 1,400 +/- 170 16S rRNA copies per E. coli cell in activated sludge. For CARD-FISH the values ranged from 8.9 +/- 1.5 to 14 +/- 2 and from 36 +/- 6 to 54 +/- 7 16S rRNA molecules per cell, respectively, indicating that the sensitivity of CARD-FISH was 26- to 41-fold higher than that of conventional FISH. These results suggest that optimized FISH protocols using oligonucleotide probes could be suitable for more recent applications of FISH (for example, to detect mRNA in situ in microbial cells). PMID:18552182

Hoshino, Tatsuhiko; Yilmaz, L Safak; Noguera, Daniel R; Daims, Holger; Wagner, Michael

2008-06-13

199

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

PubMed

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 microg/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. PMID:18644401

Park, June-Woo; Tompsett, Amber; Zhang, Xiaowei; Newsted, John L; Jones, Paul D; Au, Doris; Kong, Richard; Wu, Rudolf S S; Giesy, John P; Hecker, Markus

2008-07-02

200

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

PubMed

A molecular biology method, fluorescent in situ hybridization (FISH), in which the pre-treatment was improved in allusion to the media of the constructed wetlands (CW), e.g. the soil and the grit, was used to investigate the vertical distribution characteristics of ammonia-oxidizing bacteria (AOB) quantity and the relation with oxidation-reduction potential (ORP) in the Typha latifolia constructed wetlands under three different loadings in summer from May to September. Results showed that the quantity of the AOB decreased in the Typha latifolia CW with the increase of vertical depth. However, the AOB quantity was 2-4 times the quantity of the control in the root area. Additionally, ORP in the rhizosphere was found to be higher than other areas, which showed that Typha latifolia CW was in an aerobic state in summer when using simulated non-point sewage at the rural area of Taihu Lake in China and small town combined sewage. PMID:16465894

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

2005-01-01

201

[Registration of stable aberrations in peripheral blood lymphocytes using G-differential chromosome staining and fluorescence in situ hybridization methods].  

PubMed

G-banding analysis and fluorescence in situ hybridization (FISH) for whole chromosomes 1, 2 and 4 were applied in comparative assay for frequency of stable chromosome aberrations in 37 individuals with previous exposure to radiation (15 clean-up workers/liquidators of Chernobyl nuclear power plant accident and 22 residents of radiocontaminated areas) and in 18 individuals of a reference group. In G-banding analysis of stable aberrations, we used classification of Ohtaki K. et al., 1992, in compliance with ISCN, 1985. FISH-assay for translocations is performed in accordance with classification of Tucker J.D. et al., 1995. Comparison of the results reveals statistical trustworthy correlation between the two assays. The results point out that FISH for translocations in as few as three chromosomes, when combined with screening of numerous metaphases, provides sensitivity comparable with that provided by G-banding which covers the whole genome. PMID:9889772

Obukhova, T N; Domracheva, E V

202

Frequency of aneuploidy in pig oocytes matured in vitro and of the corresponding first polar bodies detected by fluorescent in situ hybridization  

Microsoft Academic Search

The objectives of this study were to develop a two-color fluorescent in situ hybridization (FISH) method for evaluating aneuploidy in gilt oocytes using chromosome-specific DNA probes, and to establish baseline frequencies of aneuploidy in pig oocytes matured in vitro. The ovaries were collected from gilts at the local slaughterhouse. Immature oocytes were isolated by slicing the cortex of the ovaries.

M. Vozdová; M. Machatková; S. Kubí?ková; D. Zudová; E. Jokešová; J. Rubes

2001-01-01

203

Hybridization for human epidermal growth factor receptor 2 testing in gastric carcinoma: a comparison of fluorescence in-situ hybridization with a novel fully automated dual-colour silver in-situ hybridization method  

PubMed Central

Aims: Amplification of the human epidermal growth factor receptor 2 (HER2) gene has been reported in gastric carcinoma (GC). Accordingly, trastuzumab plus chemotherapy has recently become the new standard treatment for HER2-positive advanced GCs. The aim was to compare the alleged gold standard for hybridization [fluorescence in-situ hybridization (FISH)] with a novel, fully automated brightfield dual-colour silver-enhanced in-situ hybridization (SISH) method. Methods and results: The studies series was comprised of 166 GC samples. Additionally, tumours with discordant results obtained by FISH and SISH were analysed by real-time quantitative polymerase chain reaction (PCR) with the LightMix kit HER-2/neu. Of the samples, 17.5% and 21% were amplified by FISH and SISH, respectively. Heterogeneity was identified in up to 52% of cases. In 96.4% of cases, FISH showed the same results as SISH. All six discordant cases were positive by SISH and negative by FISH. On review of the FISH slides, all contradictory cases were polysomic and were confirmed to be negative for amplification by real-time PCR. Interestingly, all ratios in this latter group were between 2.06 and 2.50, so setting the cut-off for amplification at ?3 resulted in perfect concordance. Conclusions: Dual-colour SISH represents a novel method for the determination of HER2 status in GC.

Garcia-Garcia, Elena; Gomez-Martin, Carlos; Angulo, Barbara; Conde, Esther; Suarez-Gauthier, Ana; Adrados, Magdalena; Perna, Cristian; Rodriguez-Peralto, Jose Luis; Hidalgo, Manuel; Lopez-Rios, Fernando

2011-01-01

204

Fluorescence in situ hybridization on DNA halo preparations and extended chromatin fibres.  

PubMed

Although many fluorescence in situ hybridisation (FISH) protocols involve the use of intact, fixed nuclei, the resolution achieved is not always sufficient, especially for physical mapping. In light of this, several techniques are commonly used to create extended chromatin fibres or extruded loops of DNA. As a result, it is possible to visualise and distinguish regions of the genome at a resolution higher than that attained with conventional preparations for FISH. Such methodologies include fibre-FISH and the DNA halo preparation. While fibre-FISH involves the stretching of chromatin fibres across a glass slide, the DNA halo preparation is somewhat more complex; whereby DNA loops instead of chromatin fibres are generated from interphase nuclei. Furthermore, the DNA halo preparation coupled with FISH is a useful tool for examining interactions between the inextractable nuclear matrix and the cell's genome.In this chapter, we describe how to successfully generate extended chromatin fibres and extruded DNA loops. We will also provide detailed methodologies for coupling either procedure with two distinct FISH procedures; 2D-FISH, which allows for the visualisation of specific chromosomal regions, while telomere peptide nucleic acid (PNA) FISH, enables the detection of all telomeres present within human nuclei. PMID:20809301

Elcock, Lauren S; Bridger, Joanna M

2010-01-01

205

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

SciTech Connect

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

Giraldez, R.A. [Oncor, Inc., Gaithersburg, MD (United States); Harris, C. [Univ. of Wisconsin, WI (United States)

1994-09-01

206

Determination of HER2 amplification in primary breast cancer using dual-colour chromogenic in situ hybridization is comparable to fluorescence in situ hybridization: a European multicentre study involving 168 specimens  

PubMed Central

García-Caballero T, Grabau D, Green A R, Gregory J, Schad A, Kohlwes E, Ellis I O, Watts S & Mollerup J (2010) Histopathology56, 472–480 Determination of HER2 amplification in primary breast cancer using dual-colour chromogenic in situ hybridization is comparable to fluorescence in situ hybridization: a European multicentre study involving 168 specimens Aims: Fluorescence in situ hybridization (FISH) can be used to reveal several genomic imbalances relevant to proper cancer diagnosis and to the correct treatment regime. However, FISH requires expensive and advanced fluorescence microscopes in addition to expertise in fluorescence microscopy. To determine whether a newly developed dual-colour chromogenic in situ hybridization (CISH) method is a suitable alternative to FISH, we analysed the human epidermal growth factor receptor 2 gene (HER2) amplification level of 168 breast cancer specimens using dual-colour CISH and FISH and compared the results. Methods and results: We found 100% agreement between HER2 status determined by FISH and dual-colour CISH. Furthermore, we observed that the time used to score slides was significantly reduced by 28% in dual-colour CISH compared with the FISH protocol. Concordance between HER2 protein status and dual-colour CISH or FISH was equally good with an overall agreement of 96.8%. Correlation between the HER2/centromere 17 gene ratios obtained with dual-colour CISH and FISH was highly significant with an overall correlation coefficient (?) of 0.96. Conclusions: We conclude that dual-colour CISH and bright field microscopy are excellent alternatives to FISH when analysing the HER2 status of primary breast cancer.

Garcia-Caballero, Tomas; Grabau, Dorthe; Green, Andrew R; Gregory, John; Schad, Arno; Kohlwes, Elke; Ellis, Ian O; Watts, Sarah; Mollerup, Jens

2010-01-01

207

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

PubMed Central

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.

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

1999-01-01

208

Combined fluorescent in situ hybridization for detection of microRNAs and immunofluorescent labeling for cell-type markers.  

PubMed

Identification of the cell type of origin for normal or aberrant gene expression is critical for many studies, and poses a significant problem for some regulatory RNAs such as microRNAs. MicroRNAs are small non-coding RNAs that regulate cellular function by targeting specific mRNAs and reducing the level of their protein product. Aberrant expression of miRNAs in cell-types where they are not normally expressed occurs in several disease conditions. Therefore, it is important to determine not only the expression level of microRNAs, but also where they are expressed. Here we describe a detailed method for fluorescent in situ hybridization (FISH) combined with immunofluorescent labeling for cell-type markers in formalin fixed paraffin embedded (FFPE) sections along with modifications required to adapt the protocol for primary neurons grown in culture. We have combined the specificity and stability of locked nucleic acid (LNA) probes with tyramide signal amplification. To prevent loss of small RNA species, we performed post-fixation with ethylcarbodiimide (EDC). Additionally by omitting protease digestion and using only high temperature with sodium citrate buffer for FFPE sections, we were able to perform immunolabeling for proteins concurrently with in situ hybridization without compromising efficacy of either procedure. PMID:24065888

Chaudhuri, Amrita D; Yelamanchili, Sowmya V; Fox, Howard S

2013-09-23

209

Original Articles Double-Target Fluorescence In Situ Hybridization Distinguishes Multiple Genetically Aberrant Clones in Head and Neck Squamous Cell Carcinoma  

Microsoft Academic Search

Genomic heterogeneity has been observed in several solid tumor types. To investigate this phenomenon in head and neck squamous cell carcinoma (HNSCC), we analyzed macroscopically distinct tissue samples of 12 resected tumors by a combination of fluorescence in situ hybridization (FISH) and DNA flow cytometry. Using a panel of centromeric DNA probes, numerical chromosomal aberrations were detected in 10 tumors,

Joris A. Veltman; Anton H. N. Hopman; Saskia A. van der Vlies; Fredrik J. Bot; Frans C. S. Ramaekers; Johannes J. Manni

210

Small cell osteosarcoma with Ewing sarcoma breakpoint region 1 gene rearrangement detected by interphase fluorescence in situ hybridization.  

PubMed

Because of its characteristic morphologic appearance, small cell osteosarcoma (SCO) can be confused with other small round cell malignancies of the bone, most importantly with Ewing sarcoma, making this distinction difficult. A specific tool used in separating SCO from Ewing sarcoma has been the detection of Ewing sarcoma breakpoint region 1 (EWSR1) gene rearrangements in Ewing sarcoma and their absence in SCO. However, there are rare case reports that have documented the existence of EWSR1 gene rearrangement in SCO. In this report, we describe another case of SCO with an EWSR1 gene rearrangement detected by interphase fluorescence in situ hybridization. Our finding adds support to the existing evidence that SCO is a tumor that can be characterized by EWSR1 gene arrangements. Therefore, we caution the pathology community not to rely solely on molecular studies in distinguishing SCO from Ewing sarcoma. PMID:22971270

Dragoescu, Ema; Jackson-Cook, Colleen; Domson, Gregory; Massey, Davis; Foster, William C

2012-09-10

211

Detection of aneuploidy in interphase nuclei from non-small cell lung carcinomas by fluorescence in situ hybridization using chromosome-specific repetitive DNA probes  

Microsoft Academic Search

Interphase fluorescence in situ hybridization (FISH) is particularly useful for detecting chromosome changes in tumors exhibiting a low mitotic index, as is the case in many human non-small cell lung carcinomas (NSCLCs). A panel of centromeric DNA probes specific for the autosomes 6, 7, 8, 9, 12, 17, and 18 was used to analyze 17 primary NSCLCs. Evidence for aneuploidy

Takahiro Taguchi; Jian-yuan Zhou; Madelyn Feder; Samuel Litwin; Andres J. P. Klein-Szanto; Joseph R. Testa

1996-01-01

212

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

PubMed Central

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.

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

2013-01-01

213

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

PubMed

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

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

2013-03-27

214

Prenatal detection of structural abnormalities of chromosome 18: associations with interphase fluorescence in situ hybridization (FISH) and maternal serum screening.  

PubMed

We describe two cases of prenatally ascertained isochromosome 18. Case 1 included both an isochromosome 18p and an isochromosome 18q, while Case 2 involved only an isochromosome 18q. Both of these cases were associated with a positive maternal serum triple screen trisomy 18 risk (greater than 1 in 100 risk). In addition, fluorescence in situ hybridization (FISH) was performed on uncultured amniotic fluid interphase cells in both cases looking for aneuploidy for chromosomes 13, 18, 21, X and Y. The results of the interphase analyses support the common knowledge that careful interpretation of interphase FISH analysis is necessary and that results should be followed by full cytogenetic analysis. To our knowledge these are the first reported cases of structurally abnormal chromosomes 18 being associated with a positive maternal serum triple screen for trisomy 18. PMID:12210569

Graf, Michael D; Gill, Prabhcharan; Krew, Michael; Schwartz, Stuart

2002-08-01

215

Fluorescence in situ hybridization as adjunct to cytology improves the diagnosis and directs estimation of prognosis of malignant pleural effusions  

PubMed Central

Background The identification of malignant cells in effusions by conventional cytology is hampered by its limited sensitivity and specificity. The aim of this study was to investigate the value of fluorescence in situ hybridization (FISH) as adjuncts to conventional cytologic examination in patients with malignant pleural effusions. Methods We conducted a retrospective cohort study of 93 inpatients with pleural effusions (72 malignant pleural effusions metastatic from 11 different organs and 21 benign) over 23 months. All the patients came from Chinese northeast areas. Aspirated pleural fluid underwent cytologic examination and fluorescence in situ hybridization (FISH) for aneuploidy. We used FISH in single-colour or if appropriate in dual-colour evaluation to detect chromosomal aberrations (chromosomes 7, 11, and 17) in effusion cells as markers of malignancy, to raise the diagnostic yield and identified the efficiency by diagnostic biopsy. Predominant cytogenetic anomalies and patterns of intratumor cytogenetic heterogeneity were brought in relation to overall survival rate. Results Cytology alone confirmed malignant pleural effusions in 45 of 72 patients (sensitivity 63%), whereas FISH alone positively identified 48 of 72 patients (sensitivity 67%). Both tests had high specificity in predicting benign effusions. If cytology and FISH were considered together, they exhibited 88% sensitivity and 94.5% specificity in discriminating benign and malignant effusions. Combined, the two assays were more sensitive than either test alone. Although the positive predictive value of each test was 94.5%, the negative predictive value of cytology and FISH combined was 78%, better than 47% and 44% for FISH and cytology alone, respectively. There was a significantly prolonged survival rate for patients with aneuploidy for chromosome 17. Conclusions FISH in combination with conventional cytology is a highly sensitive and specific diagnostic tool for detecting malignant cells in pleural effusions . The high sensitivity and specificity may be associated with geographic area and race. Simple numeric FISH anomalies may be prognostic.

2012-01-01

216

Peptide nucleic acid fluorescence in situ hybridization for rapid detection of Klebsiella pneumoniae from positive blood cultures.  

PubMed

This study evaluated a novel peptide nucleic acid (PNA) probe targeting a region of the 23S rRNA gene of Klebsiella pneumoniae by fluorescence in situ hybridization (FISH). Analytical performance was determined using 39 reference strains and other well-characterized strains of Klebsiella spp. and Enterobacter aerogenes. The probe was found to be specific for the K. pneumoniae complex (K. pneumoniae including Klebsiella ozaenae and Klebsiella variicola). The diagnostic accuracy was evaluated with 264 blood cultures containing Gram-negative rods. Using conventional identification as the reference, performance specifications were as follows: sensitivity 98.8 %, specificity 99.5 %, positive predictive value 98.8 % and negative predictive value 99.5 %. Discrepancies were resolved by PNA FISH retest and phenotypic tests. In conclusion, the K. pneumoniae probe provided an accurate diagnosis within 3 h and may supplement other methods for direct identification of Gram-negative bacteria. PMID:17577055

Søgaard, Mette; Hansen, Dennis S; Fiandaca, Mark J; Stender, Henrik; Schønheyder, Henrik C

2007-07-01

217

Fluorescence in situ hybridization for identification of Tritrichomonas foetus in formalin-fixed and paraffin-embedded histological specimens of intestinal trichomonosis  

Microsoft Academic Search

In the present study a highly species-specific oligonucleotide sequence of Tritrichomonas foetus 18S rRNA was used to design an antisense probe for identification of T. foetus in formalin-fixed and paraffin-embedded histological specimens by means of fluorescence in situ hybridization (FISH). Using archival histological specimens from several species with light microscopic evidence of intestinal trichomonosis, and under optimized hybridization conditions, the

J. L. Gookin; M. R. Stone; M. J. Yaeger; D. K. Meyerholz; Peter Moisan

2010-01-01

218

Chromosomal assignment of human nuclear envelope protein genes LMNA, LMNB1, and LBR by fluorescence in situ hybridization  

SciTech Connect

We have used fluorescence in situ hybridization to establish precise chromosomal localizations for three human genes encoding four different nuclear envelope proteins. Lamin A/C (LMN1, HGMW-approved symbol LMNA) mapped to 1q21.2-q21.3, with a most probable gene assignment to 1q21.3; lamin B receptor (LBR) was localized to 1q42.1; and lamin B1 (LMNB1) was mapped to the interface of bands 5q23.3-q31.1. Assignments were determined by direct placement of signals relative to high-resolution DAPI or G-bands. Comparison of these results of band positions predicted from fractional length measurements to signal placement indicated that more accurate predictions are made using Francke idiograms and that measurement strategy avoids variance due to polymorphic chromosome segments. 30 refs., 2 figs., 1 tab.

Wydner, K.L.; McNeil, J.A. [Univ. of Masssachusetts Medical Center, Worcester, MA (United States); Lin, Feng [Columbia Univ., New York, NY (United States)] [and others

1996-03-05

219

Improved technique for analysis of formalin-fixed, paraffin-embedded tumors by fluorescence in situ hybridization  

SciTech Connect

Fluorescence in situ hybridization (FISH) and specific DNA probes for pericentromeric repeat regions and unique sequence loci have made it possible to study chromosomal aberrations from interphase tumor nuclei. Large-scale retrospective studies on the prognostic value of interphase cytogenetics would become feasible if these techniques were readily applicable to nuclei from archival formalin-fixed tumor tissues. The authors describe here an improved technique for interphase FISH analysis of tumors that have been extensively fixed in formalin. The protocol aims at improving probe penetration and hybridization efficiency by inducing chromatin decondensation and swelling of the nuclei with a heat treatment in a 90{degrees}C glycerol solution prior to hybridization. Using this cell pretreatment, FISH results on the detection of chromosome copy number aberrations and amplification of the c-erbB-2 oncogene from formalin-fixed, paraffin-embedded tissues were highly concordant with those from fresh tissues. In contrast to previously described methods, separate adjustments of denaturation of proteinase K digestion are not required for each sample. This method facilitates retrospective analyses of large species of tumors and is also useful for applying FISH to routine diagnostic purposes using formalin-fixed material. 32 refs., 1 fig., 2 tabs.

Hyytinen, Eija; Visakorpi, T.; Kallioniemi, O.P. [Tampere Univ. Hospital (Finland)] [and others

1994-06-01

220

Fluorescent in situ hybridization of DNA probes in the interphase and metaphase stages of the cell cycle.  

PubMed

In the past decade, fluorescent in situ hybridization (FISH) has been used routinely in detecting molecular abnormalities in the interphase and metaphase stages of the cell cycle. Many of the molecular anomalies which are detected in this manner are diagnostic of a prenatal, postnatal, or neoplastic genetic disorder. With the continuous isolation of commercially available DNA probes specific to a particular chromosome region, FISH analysis has become standardized in its ability to detect characteristic chromosomal anomalies in association with genetic and neoplastic diseases. In recent years, FISH has also become automated to accommodate the increased volume of slide preparations necessary for the number of DNA probes needed to detect characteristic molecular anomalies in cancer tissues and bone marrow samples. FISH technology provides essential information to the physician regarding the diagnosis, response to treatment, and ultimately the prognosis of their patients' disorder. It has become an important source of information routinely used in conjunction with chromosome analyses, and presently to confirm molecular alterations detected by array comparative genomic hybridization (aCGH) analyses. In this chapter we describe the methods for performing FISH analyses in order to determine the presence or the absence of genetic abnormalities which define whether the patient has either a genetic syndrome or malignant disease. PMID:23179826

Cannizzaro, Linda A

2013-01-01

221

Localization of T-DNA Insertions in Petunia by Fluorescence in Situ Hybridization: Physical Evidence for Suppression of Recombination.  

PubMed Central

Using fluorescence in situ hybridization (FISH) with metaphase preparations, we localized a 4-kb single-copy T-DNA sequence in a group of petunia transformants. The selected T-DNAs previously had been shown to be linked to the phenotypic marker FI on chromosome II. Linkage analysis had revealed that recombination around the FI locus is suppressed in a wide cross relative to an inbred recombination assay. The localization of six FI-linked T-DNAs and the FI locus itself, using FISH, revealed a number of aspects of recombination in petunia: (1) the central region of chromosome II showed at least a 10-fold suppression of recombination in wide crosses relative to the distal region; (2) recombination in wide hybrids over two-thirds of the chromosome was extremely low; and (3) recombination between completely homologous chromosomes in an inbred cross also was suppressed in the central region. In addition, the T-DNAs were not evenly distributed along the chromosome, suggesting a possible preference for a distal position for T-DNA integration. Implications for such a preference are discussed.

Ten Hoopen, R; Robbins, TP; Fransz, PF; Montijn, BM; Oud, O; Gerats, A; Nanninga, N

1996-01-01

222

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

Microsoft Academic Search

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

Jan Jezbera; Karel Hornak; Karel Simek

223

Analysis of fluorescence in situ hybridization, mtDNA quantification, and mtDNA sequence for the detection of early bladder cancer  

Microsoft Academic Search

We designed this study to test the sensitivities of cytology, the nuclear matrix protein 22 (NMP22) assay, and fluorescence in situ hybridization (FISH) in the early detection of urothelial carcinoma, and to identify mtDNA alterations in urinary epithelial cells. We collected 41 urine samples and 26 corresponding peripheral blood samples from patients with clinically suspected urothelial carcinoma. The FISH and

Jong-Ha Yoo; Borum Suh; Tae Sung Park; Myung-Geun Shin; Yeung Deuk Choi; Chang Hoon Lee; Jong Rak Choi

2010-01-01

224

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

National Technical Information Service (NTIS)

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 ena...

G. J. Vora K. L. Robertson

2012-01-01

225

Automated detection and analysis of fluorescent in situ hybridization spots depicted in digital microscopic images of Pap-smear specimens  

NASA Astrophysics Data System (ADS)

Fluorescence in situ hybridization (FISH) technology has been widely recognized as a promising molecular and biomedical optical imaging tool to screen and diagnose cervical cancer. However, manual FISH analysis is time-consuming and may introduce large inter-reader variability. In this study, a computerized scheme is developed and tested. It automatically detects and analyzes FISH spots depicted on microscopic fluorescence images. The scheme includes two stages: (1) a feature-based classification rule to detect useful interphase cells, and (2) a knowledge-based expert classifier to identify splitting FISH spots and improve the accuracy of counting independent FISH spots. The scheme then classifies detected analyzable cells as normal or abnormal. In this study, 150 FISH images were acquired from Pap-smear specimens and examined by both an experienced cytogeneticist and the scheme. The results showed that (1) the agreement between the cytogeneticist and the scheme was 96.9% in classifying between analyzable and unanalyzable cells (Kappa=0.917), and (2) agreements in detecting normal and abnormal cells based on FISH spots were 90.5% and 95.8% with Kappa=0.867. This study demonstrated the feasibility of automated FISH analysis, which may potentially improve detection efficiency and produce more accurate and consistent results than manual FISH analysis.

Wang, Xingwei; Zheng, Bin; Li, Shibo; Zhang, Roy; Mulvihill, John J.; Chen, Wei R.; Liu, Hong

2009-03-01

226

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

Microsoft Academic Search

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

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

1988-01-01

227

An improved fluorescence in situ hybridization protocol for the identification of bacteria and archaea in marine sediments.  

PubMed

In situ identification of prokaryotic cells in subsurface sediments is hampered by the low cellular rRNA contents of the target organisms. Fluorescence in situ hybridization with catalyzed reporter deposition (CARD-FISH) has the potential to overcome this limitation, and was therefore optimized for a 40 cm deep sediment core sampled from a tidal sandy flat of the German Wadden Sea. Treatment with methanol and H(2)O(2) inactivated endogenous peroxidases and effectively reduced the background signal. Percentage of DAPI stained cells detected with the probe combination EUB(I-III), targeting nearly all the Bacteria, were comparable for CARD-FISH with a horseradish peroxidase (HRP)-labeled probe and FISH with a fluorescently monolabeled probe in the 2-3 cm depth interval (92% and 82%, respectively), but significantly higher with the HRP-labeled probe at 35-40 cm, the deepest layer sampled (63% with HRP vs. 26% with monolabeled probe). With CARD-FISH Alphaproteobacteria and the Desulfobulbaceae group of sulfate-reducing bacteria were detected only in the upper layers. In contrast, Desulfosarcinales, the Bacteroidetes group, Planctomycetes, Betaproteobacteria, and Gammaproteobacteria were found at all depths. Archaea were detectable with ARCH915-HRP after achromopeptidase treatment. Surprisingly, aggregates of Bacteria and Archaea were found, below 12 cm depth, that strongly resemble consortia involved in anoxic oxidation of methane that have previously been found in sediments near methane hydrate deposits. With the optimized CARD-FISH protocol, microbial populations could also be detected in deeper sediment horizons. Furthermore, the intensity of the CARD-FISH signals improved detection of rare organisms such as Archaea. PMID:19712361

Ishii, Kousuke; Mussmann, Marc; MacGregor, Barbara J; Amann, Rudolf

2004-11-01

228

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

PubMed

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

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

229

Triplex in-situ hybridization  

DOEpatents

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.

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

2002-01-01

230

Multicenter Evaluation of a Candida albicans Peptide Nucleic Acid Fluorescent In Situ Hybridization Probe for Characterization of Yeast Isolates from Blood Cultures  

PubMed Central

We evaluated aliquots from 244 clinical blood culture bottles that demonstrated yeasts on Gram stain using a Candida albicans peptide nucleic acid (PNA) fluorescent in situ hybridization (FISH) probe. The sensitivity, specificity, positive predictive value, and negative predictive value of the C. albicans PNA FISH test in this study were 99%, 100%, 100%, and 99.3%, respectively.

Wilson, D. A.; Joyce, M. J.; Hall, L. S.; Reller, L. B.; Roberts, G. D.; Hall, G. S.; Alexander, B. D.; Procop, G. W.

2005-01-01

231

Non-invasive early prenatal diagnosis using fluorescent in situ hybridization on transcervical cells: comparison of two different methods for retrieval  

Microsoft Academic Search

Objective: We compared the efficiencies of uterine and endocervical lavage to retrieve fetal cells from first trimester pregnancies for further analysis with fluorescent in situ hybridization (FISH). Study Design: Transcervical cell (TCC) samples were collected at 7–10 weeks of gestations by uterine lavage (13 women) and by endocervical lavage (12 women) who were scheduled for volunteer termination of pregnancy. A

Tolga Erg?n; Volkan Baltaci; Hulusi B. Zeynelo?lu; E. Hakan Duran; Mehmet H. Ergenel?; Sertaç Batio?lu

2001-01-01

232

Mapping of alpha- and beta-globin genes on Antarctic fish chromosomes by fluorescence in-situ hybridization.  

PubMed

The pathways and mechanisms of genomic change that have led to the peculiar haemoglobinless phenotype of the white-blooded Antarctic icefishes (16 species in the family Channichthyidae) constitute an important model for understanding the rapid diversification of the Antarctic notothenioid fish flock. To provide complementary structural information on genomic change at globin-gene loci in Antarctic fish species, cytogenetic studies and in-situ chromosomal mapping have been undertaken. Using a DNA probe containing one alpha- and one beta-globin gene from the embryonic/juvenile globin gene cluster of the red-blooded species Notothenia coriiceps, we mapped the cluster on the chromosomes of Antarctic teleosts by fluorescence in-situ hybridization. As anticipated on the basis of its molecular organization, the cluster was located on a single chromosome pair in all of the red-blooded fish species probed (N. coriiceps, N. angustata, Trematomus hansoni, T. pennellii). In contrast, the alpha/beta-globin probe did not recognize complementary sequences on the chromosomes of the white-blooded species Chionodraco hamatus and Channichthys rhinoceratus. These results represent the first example of chromosomal mapping of embryonic/juvenile globin genes in teleostean fishes. Beyond its relevance to the evolutionary history of Antarctic notothenioids, this work contributes to our understanding of the evolution of the chromosomal loci of globin genes in fishes and other vertebrates. PMID:14516071

Pisano, Eva; Cocca, Ennio; Mazzei, Federico; Ghigliotti, Laura; di Prisco, Guido; Detrich, H William; Ozouf-Costaz, Catherine

2003-01-01

233

Characterization of DNA immobilization and subsequent hybridization using in situ quartz crystal microbalance, fluorescence spectroscopy, and surface plasmon resonance  

Microsoft Academic Search

We have characterized the immobilization of thiol-modified oligomers on Au surfaces and subsequent hybridization with a perfectly matched or single-base mismatched target using a quartz crystal microbalance (QCM) and fluorescence spectroscopy. The surface density of immobilized probe molecules and the hybridization efficiency depending on the type of buffer and salt concentration were investigated. We observed some ambiguities in surface coverage

Yoon-Kyoung Cho; Sunhee Kim; Young A Kim; Hee Kyun Lim; Kyusang Lee; DaeSung Yoon; Geunbae Lim; Y. Eugene Pak; Tai Hwan Ha; Kwan Kim

2004-01-01

234

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

PubMed Central

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.

Robertson, Kelly L.

2012-01-01

235

Array-based comparative genomic hybridization is more informative than conventional karyotyping and fluorescence in situ hybridization in the analysis of first-trimester spontaneous abortion  

PubMed Central

Background Array-based comparative genomic hybridization (aCGH) is a new technique for detecting submicroscopic deletions and duplications, and can overcome many of the limitations associated with classic cytogenetic analysis. However, its clinical use in spontaneous abortion needs comprehensive evaluation. We used aCGH to investigate chromosomal imbalances in 100 spontaneous abortions and compared the results with G-banding karyotyping and fluorescence in situ hybridization (FISH). Inconsistent results were verified by quantitative fluorescence PCR. Results Abnormalities were detected in 61 cases. aCGH achieved the highest detection rate (93.4%, 57/61) compared with traditional karyotyping (77%, 47/61) and FISH analysis (68.9%, 42/61). aCGH identified all chromosome abnormalities reported by traditional karyotyping and interphase FISH analysis, with the exception of four triploids. It also detected three additional aneuploidy cases in 37 specimens with ‘normal’ karyotypes, one mosaicism and 10 abnormalities in 14 specimens that failed to grow in vitro. Conclusions aCGH analysis circumvents many limitations in traditional karyotyping or FISH. The accuracy and efficiency of aCGH in spontaneous abortions highlights its clinical usefulness for the future. As aborted tissues have the potential to be contaminated with maternal cells, the threshold value of detection in aCGH should be lowered to avoid false negatives.

2012-01-01

236

Evaluation of the environmental specificity of Fluorescence In Situ Hybridization (FISH) using Fluorescence-Activated Cell Sorting (FACS) of probe (PSE1284)-positive cells extracted from rhizosphere soil.  

PubMed

We explicitly tested for the first time the 'environmental specificity' of traditional 16S rRNA-targeted Fluorescence In Situ Hybridization (FISH) through comparison of the bacterial diversity actually targeted in the environment with the diversity that should be exactly targeted (i.e. without mismatches) according to in silico analysis. To do this, we exploited advances in modern Flow Cytometry that enabled improved detection and therefore sorting of sub-micron-sized particles and used probe PSE1284 (designed to target Pseudomonads) applied to Lolium perenne rhizosphere soil as our test system. The 6-carboxyfluorescein (6-FAM)-PSE1284-hybridized population, defined as displaying enhanced green fluorescence in Flow Cytometry, represented 3.51±1.28% of the total detected population when corrected using a nonsense (NON-EUB338) probe control. Analysis of 16S rRNA gene libraries constructed from Fluorescence Activated Cell Sorted-recovered fluorescent populations (n=3), revealed that 98.5% (Pseudomonas spp. comprised 68.7% and Burkholderia spp. 29.8%) of the total sorted population was specifically targeted as evidenced by the homology of the 16S rRNA sequences to the probe sequence. In silico evaluation of probe PSE1284 with the use of RDP-10 probeMatch justified the existence of Burkholderia spp. among the sorted cells. The lack of novelty in Pseudomonas spp. sequences uncovered was notable, probably reflecting the well-studied nature of this functionally important genus. To judge the diversity recorded within the FACS-sorted population, rarefaction and DGGE analysis were used to evaluate, respectively, the proportion of Pseudomonas diversity uncovered by the sequencing effort and the representativeness of the Nycodenz(®) method for the extraction of bacterial cells from soil. PMID:22264503

Gougoulias, Christos; Shaw, Liz J

2012-01-20

237

Microsieve lab-chip device for rapid enumeration and fluorescence in situ hybridization of circulating tumor cells.  

PubMed

Herein we present a lab-chip device for highly efficient and rapid detection of circulating tumor cells (CTCs) from whole blood samples. The device utilizes a microfabricated silicon microsieve with a densely packed pore array (10(5) pores per device) to rapidly separate tumor cells from whole blood, utilizing the size and deformability differences between the CTCs and normal blood cells. The whole process, including tumor cell capture, antibody staining, removal of unwanted contaminants and immunofluorescence imaging, was performed directly on the microsieve within an integrated microfluidic unit, interconnected to a peristaltic pump for fluid regulation and a fluorescence microscope for cell counting. The latter was equipped with a dedicated digital image processing program which was developed to automatically categorize the captured cells based on the immunofluorescence images. A high recovery rate of >80% was achieved with defined numbers of MCF-7 and HepG2 cancer cells spiked into human whole blood and filtered at a rapid flow rate of 1 mL min(-1). The device was further validated with blood drawn from various cancer patients (8 samples). The whole process, from sample input to result, was completed in 1.5 h. In addition, we have also successfully demonstrated on-microsieve fluorescence in situ hybridization for single cell molecular analysis. This simple method has great potential to supplant existing complex CTC detection schemes for cancer metastasis analysis. PMID:22930096

Lim, Li Shi; Hu, Min; Huang, Mo Chao; Cheong, Wai Chye; Gan, Alfred Tau Liang; Looi, Xing Lun; Leong, Sai Mun; Koay, Evelyn Siew-Chuan; Li, Mo-Huang

2012-11-01

238

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce  

PubMed Central

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55°C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (? 500 ?L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37°C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalapeño peppers.

Bisha, Bledar; Brehm-Stecher, Byron F.

2010-01-01

239

The human sorbitol dehydrogenase gene: cDNA cloning, sequence determination, and mapping by fluorescence in situ hybridization  

SciTech Connect

The cDNA for human sorbitol dehydrogenase (SORD) has been cloned and sequenced. It translates into a peptide of 356 amino acid residues, one more than the sequence previously reported from peptide analysis. An extra alanine was found at the acetyl-blocked N-terminal, between positions 1 and 4. This matches the rat cDNA, which also has 356 amino acids, with an extra proline at position 3. Four other mismatches were also observed, but these are all amino acid substitutions that occur outside proposed functionally important regions. Further work must be performed to determine whether these discrepancies represent polymorphic forms of the enzyme. The SORD gene was mapped by fluorescence in situ hybridization and found to occupy a single site on chromosome 15q15, indicating that it is a single-copy gene. This was confirmed by Southern blot hybridization. SORD is thought to be involved in the etiology of diabetic complications, and its deficiency has been linked to congenital cataracts. The cloned gene could be used as a probe to study the role of this enzyme in the pathogenesis of these diseases. 24 refs., 4 figs.

Lee, F.K.; Chung, S. (Univ. of Hong Kong (Hong Kong)); Cheung, M.C. (Univ. of California, San Francisco, CA (United States))

1994-05-15

240

Assignment of the human aggrecan gene (AGC1) to 15q26 using fluorescence in situ hybridization analysis  

SciTech Connect

The large aggregating proteoglycan aggrecan is a major structural component of the extracellular matrix of articular cartilage. Recent cDNA cloning of the human aggrecan gene (AGC1) reveals a core protein of at least 2316 amino acids characterized by several distinct structural domains. Two globular domains, termed G1 and G2, are present at the amino terminus of the molecule and a third, termed G3, is present at the carboxy terminus. The G1 domain is homologous in structure to the cartilage link protein and accounts for the aggregating potential of aggrecan through its ability to interact with hyaluronic acid. The aggrecan gene is known to consist of 15 exons, with each exon encoding a distinct functional region of the mature protein. However, while the link protein gene is known to reside on chromosome 5 in the human, the location of the aggrecan gene is currently undetermined in any species. The probe (pAGG2) for the aggrecan gene was mapped on chromosome band 15q26, most likely in the subregion of 15q26.1, using fluorescence in situ hybridization. Clear signals were noted on both chromatids of chromosome band 15q26 in over 80% of the 300 metaphase cells examined in three independent experiments using pAGG2. No other sites of hybridization were noted on both chromatids of any other chromosome band. The precise band location was identified by using chromsomes of about 650 bands and employing fluorescence reverse banding with chromomycin A3 and distamycin. 14 refs., 1 fig.

Korenberg, J.R.; Chen, X.N. [Univ. of California, Los Angeles, CA (United States); Doege, K. [Shriners Hospital for Crippled Children, Portland, OR (United States); Grover, J.; Roughley, P.J. [McGill Univ., Montreal, Quebec (Canada)

1993-05-01

241

Paternal-age effects on sperm aneuploidy investigated in mice and humans by three-chromosome fluorescence in situ hybridization  

SciTech Connect

We conducted a cross-species comparison of the effects of paternal age on sperm aneuploidy in mice and humans. A new murine assay was developed to detect sperm hyperhaploidy and polyploidy for chromosomes X, Y, and 8 using fluorescence in situ hybridization with chromosome-specific DNA probes, to serve as a direct corollate to the three-chromosome method developed early for human sperm. Sperm aneuploidy was evaluated in eight male B6C3F1 male mice (aged 22.5-30.5 mo) and compared to young controls (2.4 mo). The aged group showed significant ({approximately}2.0-fold) increases in hyperhaploidies involving chromosomes X, Y and 8, with the greatest effects seen in the oldest animals. Sperm aneuploidy was also evaluated in two groups of healthy men who differed in mean age [46.8{plus_minus}3.1 (n=4) vs. 28.5{plus_minus}5.0 (n=10) yrs], using the three-chromosome method. The older group showed a statistically significant increase in hyperhaploid sperm for both sex chromosomes. Additional controlled human studies are planned. Taken together, the murine and human data are consistent with a positive effect of paternal age on sperm aneuploidy. In both species, the strongest age effect was observed for hyperhaploidies of chromosome Y. Future studies are needed to investigate the shape of the age-effect curve and to evaluate chromosomal differences, especially for humans in their late reproductive years.

Wyrobek, A.J.; Lowe, X. [Lawrence Livermore National Lab., Livermore, CA (United States); Holland, N.T. [Unvi. of California, Berkeley, CA (United States)] [and others

1994-09-01

242

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

SciTech Connect

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.

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

2006-07-15

243

Multiplex Ligation-Dependent Probe Amplification Versus Multiprobe Fluorescence in Situ Hybridization To Detect Genomic Aberrations in Chronic Lymphocytic Leukemia  

PubMed Central

Cytogenetic abnormalities play a major role in the prognosis of patients with chronic lymphocytic leukemia (CLL). Several methods have emerged to try to best identify these abnormalities. We used fluorescence in situ hybridization (FISH) to determine the frequency of cytogenetic changes in our CLL patient population. We also evaluated the effectiveness of multiplex ligation-dependent probe amplification (MLPA) in detecting these abnormalities. Sixty-two B-CLL patients and 20 healthy controls were enrolled, and FISH and MLPA analyses were performed on peripheral blood samples. Using FISH, genomic aberrations were found in 73% of patients and presented as follows: single 13q14.3 deletion (60%), trisomy 12 (7%), ATM deletion (6%), 17p13.1 deletion (2%). MLPA analyses done on 61/62 patients showed sensitivity and specificity values of 90% and 100% respectively. MLPA revealed several additional copy number changes, the most common being 19p13 (LDLR and CDKN2D). Moreover, the cost for MLPA analysis, including technical time and reagents, is 86% less than FISH. In conclusion, cytogenetic abnormalities are a common finding in CLL patients, and MLPA is a reliable approach that is more cost effective and faster than FISH. Despite MLPA limitations of sensitivity, it can be used as a first-line screen and complementary test to FISH analysis.

Al Zaabi, Eiman A.; Fernandez, Louis A.; Sadek, Irene A.; Riddell, D. Christie; Greer, Wenda L.

2010-01-01

244

Temporal and spatial distribution of Bacillus and Clostridium histolyticum in swine manure composting by fluorescent in situ hybridization (FISH).  

PubMed

The temporal and spatial distribution of the genus Bacillus and Clostridium histolyticum group in swine manure composting was determined by fluorescent in situ hybridization using fluorescently labeled 16S rRNA-targeted oligonucleotide probes LGC353b and Chis150, respectively. The temporal distribution of total bacteria, Bacillus and C. histolyticum, detected in each layer of the composting pile was noticeable in that the number of them detected at the high-temperature stage was higher than that of the cooling stage. The number detected at the cooling stage was higher than that of the temperature-rising stage. The number of the total bacteria distributed in three locations achieved balance at the stage of cooling. The spatial distribution of the genus Bacillus cells was that the number and the relative abundance of Bacillus cells detected in the middle layer of composting pile were the lowest at each stage of composting. However, the minimum value of the relative abundance exceeded 8%. Compared with Bacillus spp., the C. histolyticum group displayed higher relative abundance in the same layer at different stages of composting except in the top layer at the stage of high temperature. However, the characteristic of the spatial distribution was not noticeable. The detected limits of the genus Bacillus and C. histolyticum group were both found to be the high cell density of 10(6) cells g(-1) (wet weight). These results indicated that the genus Bacillus and C. histolyticum group were the predominant bacteria in the swine manure composting process and may play important role in this complex environment. PMID:21881890

Yi, Jing; Zheng, Rong; Li, Fenge; Chao, Zhe; Deng, Chang Yan; Wu, Jian

2011-09-01

245

A break-apart fluorescence in situ hybridization assay for detecting RET translocations in papillary thyroid carcinoma.  

PubMed

At least 15 different translocations have been described activating RET in papillary thyroid carcinomas. A break-apart fluorescence in situ hybridization (FISH) assay should detect many translocations involving the RET gene without requiring knowledge of the partner gene. G-banding and spectral karyotyping was performed to further characterize the papillary carcinoma cell line TPC-1. BAC clones flanking the 5' and 3' regions of RET were labeled with SpectrumRed and biotin detected with avidin-AMCA (blue). In addition to the previously described chromosomal t(1;10;21), TPC-1 was found to have del(7)(q22q31) and der(8)t(8;8)(p21;q11.2). With the BAC probes, TPC-1 interphase nuclei showed the expected signal pattern of one paired red-blue signal as well as separated red and blue signals from the rearranged RET gene in 93% of cells. Interphase nuclei from normal human lymphocytes showed two paired red-blue signals in 97% of cells. TPC-1 cells were found to have the previously described chromosomal rearrangement that involves chromosome 10, with few other cytogenetically detectable genomic alterations. RET rearrangement can be detected by a break-apart BAC FISH probe set in the interphase nuclei of TPC-1 cells. This assay can potentially detect clinically relevant translocations involving RET. PMID:17954268

Chen, Frank; Clark, Douglas P; Hawkins, Anita L; Morsberger, Laura A; Griffin, Constance A

2007-10-15

246

Infertility in a new 46, XX male with positive SRY confirmed by fluorescence in situ hybridization: a case report.  

PubMed

The 46, XX male syndrome (de la Chapelle syndrome or 46, XX testicular disorder of sex development) is a rare form of sex reversal with complex mechanisms leading to a large spectrum of clinical manifestations ranging from ambiguous genitalia in the newborn to normal male phenotype. Therefore, diagnosis is established either pre- or early postnatal, or in adult life due to male infertility. In some cases, subtle clinical signs during childhood and puberty may be overlooked. A 28-year-old married man presented with azoospermia without erectile dysfunction. Between 9-14 years he was examined for the small testes and under-masculinized external genitalia but the diagnosis was not further clarified. At presentation, hormonal laboratory evaluation revealed hypergonadotropic hypogonadism. Chromosome analysis showed a 46, XX karyotype and translocation of SRY (testis-determining factor) from chromosome Y to chromosome X was identified by fluorescence in situ hybridization (FISH). Despite early subtle clinical signs of abnormal sexual development in this new 46, XX male syndrome, medical investigations were triggered by infertility. PMID:19205451

Pepene, C E; Coman, I; Mihu, D; Militaru, M; Duncea, I

2008-01-01

247

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

PubMed Central

Background & objectives: Microdeletion syndromes are characterized by small (<5 Mb) chromosomal deletions in which one or more genes are involved. These are frequently associated with multiple congenital anomalies. The phenotype is the result of haploinsufficiency of genes in the critical interval. Fluorescence in situ hybridization (FISH) technique is commonly used for precise genetic diagnosis of microdeletion syndromes. This study was conducted to assess the role of FISH in the diagnosis of suspected microdeletion syndrome. Methods: FISH was carried out on 301 clinically suspected microdeletion syndrome cases for the confirmation of clinical diagnosis using non-commercial probes. Of these, 177 cases were referred for 22q11.2 microdeletion, 42 cases were referred for William syndrome, 38 cases were referred for Prader Willi/Angelman and 44 cases were referred for other suspected microdeletion syndromes. Results: FISH was confirmatory in 23 cases only (7.6%). There were 17 cases of 22q11.2 microdeletion, four cases of Prader Willi syndrome and two cases of William syndrome. Interpretation & conclusion: We conclude that FISH should not be the method of choice for clinically suspected microdeletion syndromes. We propose to follow strict clinical criteria for FISH testing or preferably to follow better methods (genotype first approach). Whole genome screening may be used as first line of test and FISH may be used for confirmation of screening result, screening of family members and prenatal diagnosis.

Halder, Ashutosh; Jain, Manish; Chaudhary, Isha; Gupta, Neerja; Kabra, Madhulika

2013-01-01

248

Comprehensive Analysis of ETS Family Members in Melanoma by Fluorescence In Situ Hybridization Reveals Recurrent ETV1 Amplification.  

PubMed

E26 transformation-specific (ETS) transcription factors are known to be involved in gene aberrations in various malignancies including prostate cancer; however, their role in melanoma oncogenesis has yet to be fully explored. We have completed a comprehensive fluorescence in situ hybridization (FISH)-based screen for all 27 members of the ETS transcription factor family on two melanoma tissue microarrays, representing 223 melanomas, 10 nevi, and 5 normal skin tissues. None of the melanoma cases demonstrated ETS fusions; however, 6 of 114 (5.3%) melanomas were amplified for ETV1 using a break-apart FISH probe. For the six positive cases, locus-controlled FISH probes revealed that two of six cases were amplified for the ETV1 region, whereas four cases showed copy gains of the entire chromosome 7. The remaining 26 ETS family members showed no chromosomal aberrations by FISH. Quantitative polymerase chain reaction showed an average 3.4-fold (P value = .00218) increased expression of ETV1 in melanomas, including the FISH ETV1-amplified cases, when compared to other malignancies (prostate, breast, and bladder carcinomas). These data suggest that a subset of melanomas overexpresses ETV1 and amplification of ETV1 may be one mechanism for achieving high gene expression. PMID:23908683

Mehra, Rohit; Dhanasekaran, Saravana M; Palanisamy, Nallasivam; Vats, Pankaj; Cao, Xuhong; Kim, Jung H; Kim, David Sl; Johnson, Timothy; Fullen, Douglas R; Chinnaiyan, Arul M

2013-08-01

249

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

PubMed

Background & objectives: Microdeletion syndromes are characterized by small (<5 Mb) chromosomal deletions in which one or more genes are involved. These are frequently associated with multiple congenital anomalies. The phenotype is the result of haploinsufficiency of genes in the critical interval. Fluorescence in situ hybridization (FISH) technique is commonly used for precise genetic diagnosis of microdeletion syndromes. This study was conducted to assess the role of FISH in the diagnosis of suspected microdeletion syndrome. Methods: FISH was carried out on 301 clinically suspected microdeletion syndrome cases for the confirmation of clinical diagnosis using non-commercial probes. Of these, 177 cases were referred for 22q11.2 microdeletion, 42 cases were referred for William syndrome, 38 cases were referred for Prader Willi/Angelman and 44 cases were referred for other suspected microdeletion syndromes. Results: FISH was confirmatory in 23 cases only (7.6%). There were 17 cases of 22q11.2 microdeletion, four cases of Prader Willi syndrome and two cases of William syndrome. Interpretation & conclusion: We conclude that FISH should not be the method of choice for clinically suspected microdeletion syndromes. We propose to follow strict clinical criteria for FISH testing or preferably to follow better methods (genotype first approach). Whole genome screening may be used as first line of test and FISH may be used for confirmation of screening result, screening of family members and prenatal diagnosis. PMID:24056568

Halder, Ashutosh; Jain, Manish; Chaudhary, Isha; Gupta, Neerja; Kabra, Madhulika

2013-07-01

250

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

PubMed Central

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.

Strapoc, Dariusz; Huizinga, Brad; Lidstrom, Ulrika; Ashby, Matt; Macalady, Jennifer L.

2012-01-01

251

Quantitative fluorescence in situ hybridization analysis of microbial consortia from a biogenic gas field in Alaska's Cook Inlet basin.  

PubMed

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, CO(2), 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

Dawson, Katherine S; Str?po?, Dariusz; Huizinga, Brad; Lidstrom, Ulrika; Ashby, Matt; Macalady, Jennifer L

2012-03-16

252

Detection of chromosomal abnormalities in uterine leiomyoma using conventional cytogenetic method and interphase fluorescence in situ hybridization.  

PubMed

Seventy-nine uterine leiomyomas were examined using a conventional cytogenetic method and fluorescence in situ hybridization (FISH) for detection of chromosomal abnormalities of chromosome 12. Nine (17.6%) of 51 tumor samples examined showed chromosomal abnormalities by conventional cytogenetic analysis. Rearrangements of chromosome 12 were detected in two tumors. Other tumors showed abnormalities affecting chromosomes 1, 4, 6, 7, 10, 13, 14, and 22. For FISH, the whole-chromosome painting probe and the D12Z3 probe specific for the centromeric region were used to detect structural and numerical abnormalities of chromosome 12. Of forty-one tumor samples, six showed structural aberrations and four showed numerical aberrations of chromosome 12 by FISH analysis. Of the tumors with structural aberrations identified by FISH, two had normal karyotypes, two showed structural rearrangements of chromosome 12 cytogenetically, and two could not be analyzed because of an insufficient number of metaphases. There were no correlations between the cytogenetic data and clinical parameters. The results indicate that chromosomal abnormalities are important in the biology of at least some types of uterine leiomyomas, and that FISH is a useful complement to conventional cytogenetic analysis in the study of solid tumors. PMID:8697434

Hayashi, S; Miharu, N; Okamoto, E; Samura, O; Hara, T; Ohama, K

1996-07-15

253

Abundance and Phylogenetic Affiliation of Iron Reducers in Activated Sludge as Assessed by Fluorescence In Situ Hybridization and Microautoradiography  

PubMed Central

Microautoradiography (MAR) was used to enumerate acetate-consuming bacteria under Fe(III)-reducing conditions in activated sludge. This population is believed to consist of dissimilatory iron-reducing bacteria, because the applied incubation conditions and the use of specific inhibitors excluded consumption of radiolabeled acetate by other physiological groups such as sulfate reducers. By use of this approach, dissimilatory iron reducers were found in a concentration of 1.1 × 108 cells per ml, corresponding to approximately 3% of the total cell count as determined by DAPI (4?,6?-diamino-2-phenylindoledihydrochloride-dilactate) staining. The MAR enumeration method was compared to the traditional most-probable-number (MPN) method (FeOOH-MPN) and a modified MPN method, which contains Ferrozine directly within the MPN dilutions to determine the production of small amounts of ferrous iron (Ferrozine-MPN). The Ferrozine-MPN method yielded values 6 to 10 times higher than those obtained by the FeOOH-MPN method. Nevertheless, the MAR approach yielded counts that were 100 to 1,000 times higher than those obtained by the Ferrozine-MPN method. Specific in situ Fe(III) reduction rates per cell (enumerated by the MAR method) were calculated and found to be comparable to the respective rates for pure cultures of dissimilatory iron-reducing bacteria, suggesting that the new MAR method is most reliable. A combination of MAR and fluorescence in situ hybridization was used for phylogenetic characterization of the putative iron-reducing bacteria. All activated-sludge cells able to consume acetate under iron-reducing conditions were targeted by the bacterial oligonucleotide probe EUB338. Around 20% were identified as gamma Proteobacteria, and 10% were assigned to the delta subclass of Proteobacteria.

Nielsen, Jeppe Lund; Juretschko, Stefan; Wagner, Michael; Nielsen, Per Halkjaer

2002-01-01

254

Automated segmentation and analysis of fluorescent in situ hybridization (FISH) signals in interphase nuclei of pap-smear specimens  

NASA Astrophysics Data System (ADS)

Interphase fluorescence in situ hybridization (FISH) technology is a potential and promising molecular imaging tool, which can be applied to screen and detect cervical cancer. However, manual FISH detection method is a subjective, tedious, and time-consuming process that results in a large inter-reader variability and possible detection error (in particular for heterogeneous cases). Automatic FISH image analysis aims to potentially improve detection efficiency and also produce more accurate and consistent results. In this preliminary study, a new computerized scheme is developed to automatically segment analyzable interaphase cells and detect FISH signals using digital fluorescence microscopic images acquired from Pap-smear specimens. First, due to the large intensity variations of the acquired interphase cells and overlapping cells, an iterative (multiple) threshold method and a feature-based classifier are applied to detect and segment all potentially analyzable interphase nuclei depicted on a single image frame. Second, a region labeling algorithm followed up a knowledge-based classifier is implemented to identify splitting and diffused FISH signals. Finally, each detected analyzable cell is classified as normal or abnormal based on the automatically counted number of FISH signals. To test the performance of this scheme, an image dataset involving 250 Pap-smear FISH image frames was collected and used in this study. The overall accuracy rate for segmenting analyzable interphase nuclei is 86.6% (360/424). The sensitivity and specificity for classifying abnormal and normal cells are 88.5% and 86.6%, respectively. The overall cell classification agreement rate between our scheme and a cytogeneticist is 86.6%. The testing results demonstrate the feasibility of applying this automated scheme in FISH image analysis.

Wang, Xingwei; Zheng, Bin; Li, Shibo; Zhang, Roy R.; Li, Yuhua; Mulvihill, John J.; Chen, Wei R.; Liu, Hong

2009-02-01

255

Analysis of methanogenic activity in a thermophilic-dry anaerobic reactor: Use of fluorescent in situ hybridization  

SciTech Connect

Methanogenic activity in a thermophilic-dry anaerobic reactor was determined by comparing the amount of methane generated for each of the organic loading rates with the size of the total and specific methanogenic population, as determined by fluorescent in situ hybridization. A high correlation was evident between the total methanogenic activity and retention time [-0.6988Ln(x) + 2.667] (R{sup 2} 0.8866). The total methanogenic activity increased from 0.04 x 10{sup -8} mLCH{sub 4} cell{sup -1}day{sup -1} to 0.38 x 10{sup -8} mLCH{sub 4} cell{sup -1}day{sup -1} while the retention time decreased, augmenting the organic loading rates. The specific methanogenic activities of H{sub 2}-utilizing methanogens and acetate-utilizing methanogens increased until they stabilised at 0.64 x 10{sup -8} mLCH{sub 4} cell{sup -1}day{sup -1} and 0.33 x 10{sup -8} mLCH{sub 4} cell{sup -1}day{sup -1}, respectively. The methanogenic activity of H{sub 2}-utilizing methanogens was higher than acetate-utilizing methanogens, indicating that maintaining a low partial pressure of hydrogen does not inhibit the acetoclastic methanogenesis or the anaerobic process.

Montero, B. [Department of Chemical Engineering, Food and Environmental Technologies, Faculty of Marine and Environmental Sciences, University of Cadiz, Campus Rio San Pedro s/n, 11510-Puerto Real, Cadiz (Spain)], E-mail: blanca.montero@uca.es; Garcia-Morales, J.L.; Sales, D.; Solera, R. [Department of Chemical Engineering, Food and Environmental Technologies, Faculty of Marine and Environmental Sciences, University of Cadiz, Campus Rio San Pedro s/n, 11510-Puerto Real, Cadiz (Spain)

2009-03-15

256

Potential errors with rapid analysis techniques: partial duplication 21q resulting from a paternal paracentric insertion uncovered in chorionic villus sampling by fluorescence in situ hybridization.  

PubMed

We report on partial duplication 21q resulting from a paternal insertion identified during prenatal diagnosis. While performing interphase fluorescence in situ hybridization (I-FISH), we were able to identify 3 signals of the LSI 21 Spectrum Orange probe with chorionic villus sampling. Using standard cytogenetic analysis, I-FISH and GTG banding, structural aberrations in 21q in the parents and in the fetus could not be reliably determined. Applying metaphase fluorescence in situ hybridization (M-FISH), we identified a recombinant chromosome 21 carrying an interstitial duplication of the Down syndrome critical region inherited from the father. Both data from our analysis and published literature recommend the use of rapid testing methods such as I-FISH and standard cytogenetic analysis in prenatal diagnosis. It became obvious that I-FISH would not detect such a particular aberration. Thus, karyotyping, I-FISH and M-FISH should be performed in all Down syndrome cases. PMID:20029221

Ehrhardt, N; Kujat, A; Faber, R; Horn, L-C; Froster, U G

2009-12-17

257

Bacterial activity and community composition in stream water and biofilm from an urban river determined by fluorescent in situ hybridization and DGGE analysis  

Microsoft Academic Search

Physiologic activity and community structure of planktonic and biofilm microbial communities in an urban river were analyzed using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) staining, fluorescent in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction (PCR)-amplified 16S rDNA fragments. Respiring bacteria estimated by CTC reduction were higher in biofilms (20%) than in stream water samples (12%).

Ruben Araya; Katsuji Tani; Tatsuya Takagi; Nobuyasu Yamaguchi; Masao Nasu

2003-01-01

258

Quantification of uncultured Ruminococcus obeum-like bacteria in human fecal samples with fluorescent in situ hybridization and flow cytometry using 16S ribosomal RNA targeted probes  

Microsoft Academic Search

A 16S rRNA-targeted probe was designed and validated in order to quantify the number of uncultured Ruminococcus obeum-like bacteria by fluorescent in situ hybridization (FISH). These bacteria have frequently been found in 16S ribosomal DNA clone libraries prepared from bacterial communities in the human intestine. Thirty-two reference strains from the human intestine, including a phylogenetically related strain and strains of

E. G. Zoetendal; K. Ben-Amor; H. J. M. Harmsen; F. Schut; A. D. L. Akkermans; Vos de W. M

2002-01-01

259

Quantification of Uncultured Ruminococcus obeum-Like Bacteria in Human Fecal Samples by Fluorescent In Situ Hybridization and Flow Cytometry Using 16S rRNA-Targeted Probes  

Microsoft Academic Search

A 16S rRNA-targeted probe was designed and validated in order to quantify the number of uncultured Ruminococcus obeum-like bacteria by fluorescent in situ hybridization (FISH). These bacteria have frequently been found in 16S ribosomal DNA clone libraries prepared from bacterial communities in the human intestine. Thirty-two reference strains from the human intestine, including a phylogenetically related strain and strains of

Erwin G. Zoetendal; Kaouther Ben-Amor; Hermie J. M. Harmsen; Frits Schut; Antoon D. L. Akkermans; Willem M. de Vos

2002-01-01

260

Potential Errors with Rapid Analysis Techniques: Partial Duplication 21q Resulting from a Paternal Paracentric Insertion Uncovered in Chorionic Villus Sampling by Fluorescence in situ Hybridization  

Microsoft Academic Search

We report on partial duplication 21q resulting from a paternal insertion identified during prenatal diagnosis. While performing interphase fluorescence in situ hybridization (I-FISH), we were able to identify 3 signals of the LSI 21 Spectrum Orange probe with chorionic villus sampling. Using standard cytogenetic analysis, I-FISH and GTG banding, structural aberrations in 21q in the parents and in the fetus

N. Ehrhardt; A. Kujat; R. Faber; L.-C. Horn; U. G. Froster

2009-01-01

261

Detection and Identification of Candida Species in Experimentally Infected Tissue and Human Blood by rRNA-Specific Fluorescent In Situ Hybridization  

Microsoft Academic Search

Two 18S rRNA-targeted oligonucleotide probes specific for Candida albicans and Candida parapsilosis were used to detect and identify by fluorescent in situ hybridization these medically important Candida species in deep organs of mice after experimental systemic infection. The C. albicans-specific probe detected fungal cells in kidney, spleen, and brain sections of a mouse infected with C. albicans but not in

AXEL LISCHEWSKI; MARIANNE KRETSCHMAR; HERBERT HOF; RUDOLF AMANN; JORG HACKER; JOACHIM MORSCHHAUSER

1997-01-01

262

Quantification of C-ERB-B 2 gene amplification in breast cancer cells using fluorescence in situ hybridization and digital image analysis  

Microsoft Academic Search

Fluorescence in situ hybridization (FISH) allows detection of the intercellular heterogeneity of C-ERB-B2 gene amplification in uncultured breast cancer cells. Nevertheless, because high levels of amplification result in coalescence of signals, direct microscopy quantification is restricted to cells with low levels of amplification or with dispersed signals. A methodology of digital image analysis, using surface and grey-level FISH signals as

JoséLuis Fernández; Vicente Goyanes; Carmen López-Fernández; Ismael Buñot; Jaime Gosálvez

1996-01-01

263

Concurrent use of flow cytometry and fluorescence in-situ hybridization techniques for detecting faulty meiosis in a human sperm sample  

Microsoft Academic Search

5To whom correspondence should be addressed 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

R. Weissenberg; A. Aviram; R. Golan; L. M. Lewin; J. Levron; I. Madgar; J. Dor; G. Barkai; B. Goldman

1998-01-01

264

Archaeal Community Revealed by 16s rRNA and Fluorescence in situ Hybridization in a Sulphuric Hydrothermal Hot Spring, Northern Taiwan  

Microsoft Academic Search

Summary  The archaeal community composition of Yangmingshan National Park in northern Taiwan was investigated by 16S rRNA and fluorescence\\u000a in situ hybridization (FISH). Optimization of tetrameric restriction enzyme (TRE) was performed to achieve efficient digestion and\\u000a differentiation in the restriction fragment length polymorphism (RFLP) fragments, and AciI, BstUI and RsaI were shown to be the optimal TREs for TRE-RFLP. Nine clones

Chang-Chai Ng; Chen-Chin Chang; Yuan-Tay Shyu

2005-01-01

265

Investigation of bone marrow involvement in malignant lymphoma using fluorescence in situ hybridization: possible utility in the detection of micrometastasis.  

PubMed

We evaluated the usefulness of interphase fluorescence in situ hybridization (FISH) for the detection of bone marrow involvement of lymphoma, comparing the results with those of microscopic examination. Bone marrow aspirates obtained for staging work-up from 150 patients with non-Hodgkin lymphoma were used in this study. Interphase FISH study using four probes and conventional G-banding were performed on bone marrow aspirates. The four probes included locus specific identifier (LSI) immunoglobulin heavy chain (IGH) dual-color break-apart rearrangement probe, an LSI p16 SpectrumOrange/CEP 9 SpectrumGreen probe, an LSI BCL6 dual-color break-apart rearrangement probe. Among 150 cases, 29 cases (19.3%) showed infiltration of neoplastic lymphoid cells by microscopic examination. Chromosomal aberrations were detected by FISH in eight patients and by conventional cytogenetic study in three patients. FISH study showed 14q32 rearrangement in four patients (4/126, 3.2%), 9q21 rearrangement in no patients (0/144, 0%), 3q27 rearrangement in four patients (4/131. 3.1%), and a gain of 1q21q32 in two patients (2/115, 1.7%). Among eight patients with abnormal FISH patterns, six had normal karyotypes or no analyzable metaphase according to the conventional cytogenetic study. Seven patients with FISH abnormality showed bone marrow involvement of lymphoma by microscopic examination. One patient, who was defined as having no evidence of bone marrow involvement by microscopic examination, showed a 3q27 aberration in the FISH study. Although the number of patients with BM involvement that was detected by FISH was low, abnormal FISH patterns were detected in six patients who did not have abnormal karyotypes. Therefore, FISH analysis would be beneficial in cytogenetic diagnosis and follow-up study of minimal residual diseases, once the cytogenetic changes are detected at initial diagnosis. PMID:18786435

Huh, Hee Jin; Min, Hyun Chung; Cho, Han Ik; Chae, Seok Lae; Lee, Dong Soon

2008-10-01

266

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

SciTech Connect

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.

Korenberg, J.R.

1993-12-31

267

A New Method to Extract Nuclei from Paraffin-Embedded Tissue to Study Lymphomas Using Interphase Fluorescence in Situ Hybridization  

PubMed Central

Fluorescence in situ hybridization (FISH) is difficult to accomplish using thin-sections of paraffin-embedded lymphoid tissue because of the high cellularity and truncated cells that interfere with accurate scoring of individual nuclei. We modified and tested a new technique to isolate individual nuclei from tissue cores of paraffin-embedded tissue processed with xylene, proteinase K, citric acid, and pepsin. The efficacy of this method to study paraffin-embedded tissue was investigated in six normal lymph nodes or tonsils and 32 malignant lymphomas including five mantle cell, five follicular, five Burkitt, five extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue, five anaplastic large-cell, and seven diffuse large B-cell. Fusion of CCND1 and IgH, BCL2 and IgH, c-myc and IgH, and MALT1 and API2 were detected using probes with a dual-fusion FISH strategy. Anomalies involving ALK and BCL6 were detected using break-apart FISH probes. FISH studies were successful for each of the 38 specimens. Chromosome anomalies were detected in each malignant specimen, but not in the normal lymphoid tissue. The correct chromosome anomaly was detected in 22 of 22 specimens with genetic abnormalities that were established by other genetic techniques. This FISH technique is useful to detect chromosome anomalies with high sensitivity and specificity in paraffin-embedded tissue and may provide important diagnostic and prognostic genetic information.

Paternoster, Sarah F.; Brockman, Stephanie R.; McClure, Rebecca F.; Remstein, Ellen D.; Kurtin, Paul J.; Dewald, Gordon W.

2002-01-01

268

Aneuploidy study of human oocytes first polar body comparative genomic hybridization and metaphase II fluorescence in situ hybridization analysis  

Microsoft Academic Search

BACKGROUND: The object of this study was to determine the mechanisms that produce aneuploidy in oocytes and establish which chromosomes are more prone to aneuploidy. METHODS: A total of 54 oocytes from 36 women were analysed. The whole chromosome complement of the first polar body (1PB) was analysed by com- parative genomic hybridization (CGH), while the corresponding metaphase II (MII)

C. Gutierrez-Mateo; J. Benet; D. Wells; P. Colls; M. G. Bermudez; J. F. Sanchez-Garcia; J. Egozcue; J. Navarro; S. Munne ´

2004-01-01

269

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

Microsoft Academic Search

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 fluoro- chrome Cy3 or horseradish peroxidase-modified oligonucleotide probes. For both basins, significant differ- ences in total prokaryotic abundance and phylogenetic composition were observed among oxic, anoxic, and transitional (redoxcline) waters. Epsilon-proteobacteria,

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

2006-01-01

270

Fluorescence in situ hybridization assessment of the telomeric regions of jumping translocations in a case of aggressive B-cell non-Hodgkin lymphoma  

Microsoft Academic Search

We report a jumping translocation involving a donor chromosome 1 long arm in a case of aggressive B-cell non-Hodgkin lymphoma (NHL). Conventional cytogenetic banding studies demonstrated a breakpoint distal to the heterochromatic region of the donor 1q chromosome. Characterization by fluorescence in situ hybridization (FISH) of the jumping translocation demonstrated an apparent telomeric sequence loss of the recipient chromosomes. Additional

Brian A. Gray; Angela Bent-Williams; Julie Wadsworth; Russell L. Maiese; Andres Bhatia; Robert T. Zori

1997-01-01

271

Detection of EWS-FLI-1 fusion in Ewing's sarcoma\\/peripheral primitive neuroectodermal tumor by fluorescence in situ hybridization using formalin-fixed paraffin-embedded tissue  

Microsoft Academic Search

The balanced translocation t(11;22)(q24;q12) is specific for the Ewing's sarcoma\\/peripheral primitive neuroectodermal tumors (ESPNETs) and results in the EWSFLI-1 fusion transcript, which can be detected by reverse transcription polymerase chain reaction (RT-PCR). Recent studies also have used fluorescence in situ hybridization (FISH) to show the translocation; however, most of these have been performed on cell lines or touch preparations and

Shimareet Kumar; Svetlana Pack; Dhruv Kumar; Robert Walker; Martha Quezado; Zhengping Zhuang; Paul Meltzer; Maria Tsokos

1999-01-01

272

Detection of recombinant Pseudomonas putida in the wheat rhizosphere by fluorescence in situ hybridization targeting mRNA and rRNA  

Microsoft Academic Search

A method was developed to detect a specific strain of bacteria in wheat root rhizoplane using fluorescence in situ hybridization\\u000a and confocal microscopy. Probes targeting both 23S rRNA and messenger RNA were used simultaneously to achieve detection of\\u000a recombinant Pseudomonas putida (TOM20) expressing toluene o-monooxygenase (tom) genes and synthetic phytochelatin (EC20). The probe specific to P. putida 23S rRNA sequences

Cindy H. Wu; Yu-Chen Hwang; Wonkyu Lee; Ashok Mulchandani; Thomas K. Wood; Marylynn V. Yates; Wilfred Chen

2008-01-01

273

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

Microsoft Academic Search

The ability to determine spatial and temporal microRNA (miRNA) accumulation at the tissue, cell and subcellular levels is essential for understanding the biological roles of miRNAs and miRNA-associated gene regulatory networks. This protocol describes a method for fast and effective detection of miRNAs in frozen tissue sections using fluorescence in situ hybridization (FISH). The method combines the unique miRNA recognition

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

2007-01-01

274

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

Microsoft Academic Search

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

Tomonori Kindaichi; Tsukasa Ito; Satoshi Okabe

2004-01-01

275

Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques  

Microsoft Academic Search

The ribosomal-RNA (rRNA) approach to microbial evolution and ecology has become an integral part of environmental microbiology. Based on the patchy conservation of rRNA, oligonucleotide probes can be designed with specificities that range from the species level to the level of phyla or even domains. When these probes are labelled with fluorescent dyes or the enzyme horseradish peroxidase, they can

Rudolf Amann; Bernhard M. Fuchs

2008-01-01

276

FastFISH: technique for ultrarapid fluorescence in situ hybridization on uncultured amniocytes yielding results within 2 h of amniocentesis.  

PubMed

Rapid aneuploidy detection methods allow prenatal diagnosis results to be released within 48 h, but not on the same day as the invasive test. We aimed to develop a rapid fluorescence in situ hybridization (FISH) method (FastFISH) that releases accurate results on the same day as amniocentesis. FastFISH was optimized to be completed within 2 h of sample collection using CEP and LSI probes for chromosomes 13, 18, 21, X, Y and DiGeorge syndrome (DGS). The technique was tested on 100 consecutive amniotic fluid samples in a blinded study. It was also validated as a 1-day molecular genetic test on three representative fetal tissue samples: chorionic villus, amniotic fluid and fetal blood. In the blinded study, FastFISH results were ready within 2 h of sample collection. Of the 100 amniotic fluid samples, 49 male and 50 female fetuses were identified. One fetus was 47, XXY (Klinefelter syndrome). Three fetuses had trisomy 21. One fetus suspected of DGS by ultrasound was identified as normal. Results of FastFISH analyses in all 100 cases were concordant with their karyotypes (100% accuracy; lower 95% CI, 97.05%). In the 1-day test validation, all results were released on the same day and were concordant with their respective karyotypes. FastFISH allows results to be released on the same day as amniocentesis. It represents the necessary development for a 1-day prenatal diagnosis service. PMID:17430982

Choolani, M; Ho, S S Y; Razvi, K; Ponnusamy, S; Baig, S; Fisk, N M; Biswas, A

2007-04-12

277

Fluorescence in situ hybridization on paraffin-embedded abortion material as a means of retrospective chromosome analysis.  

PubMed

A fluorescence in situ hybridization (FISH) procedure was used to detect chromosome abnormalities in archival abortion material. Nuclei were isolated from 50-microns-thick tissue blocks from 18 selected and karyotyped abortions. Five probes for repetitive centromeric sequences of chromosomes 1, 16, 18, X and Y were used. For each chromosome, at least 200 nuclei were scored blindly, i.e. without knowledge of the karyotype. The FISH results obtained were compatible with the cytogenetic data in 14 cases. There were four discrepancies. Two of these were observed for cases karyotyped as trisomy 16. Furthermore, FISH results showed trisomy 18 in two cases having normal chromosomes 18 and 18q+, respectively. The latter case was not discrepant if the structural rearrangement involved chromosome 18 material. The remaining discrepancies could be explained by chromosomal mosaicism. Admixture of normal maternal cells was also noted. It is concluded that FISH can be used to study retrospectively the presence of chromosome abnormalities in abortion material. However, the quality obtained after the use of fresh material is superior. PMID:7959687

van Lijnschoten, G; Albrechts, J; Vallinga, M; Hopman, A H; Arends, J W; Geraedts, J

1994-11-01

278

Replication Timing of Human Telomeric DNA and Other Repetitive Sequences Analyzed by Fluorescence in Situ Hybridization and Flow Cytometry  

Microsoft Academic Search

The replication timing of telomeres seems to differ between species. Yeast telomeres are late replicating, whereas limited data from very few human cell lines have indicated telomere replication throughout S phase. In the present study a series of permanent cell lines and patient samples was investigated using a flow cytometric approach for telomere length determination based on in situ hybridization

M. Hultdin; E. Grönlund; K.-F. Norrback; T. Just; K. Taneja; G. Roos

2001-01-01

279

Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization.  

PubMed

Chromogenic (CISH) and fluorescent (FISH) in situ hybridization have emerged as reliable techniques to identify amplifications and chromosomal translocations. CISH provides a spatial distribution of gene copy number changes in tumour tissue and allows a direct correlation between copy number changes and the morphological features of neoplastic cells. However, the limited number of commercially available gene probes has hindered the use of this technique. We have devised a protocol to generate probes for CISH that can be applied to formalin-fixed, paraffin-embedded tissue sections (FFPETS). Bacterial artificial chromosomes (BACs) containing fragments of human DNA which map to specific genomic regions of interest are amplified with phi29 polymerase and random primer labelled with biotin. The genomic location of these can be readily confirmed by BAC end pair sequencing and FISH mapping on normal lymphocyte metaphase spreads. To demonstrate the reliability of the probes generated with this protocol, four strategies were employed: (i) probes mapping to cyclin D1 (CCND1) were generated and their performance was compared with that of a commercially available probe for the same gene in a series of 10 FFPETS of breast cancer samples of which five harboured CCND1 amplification; (ii) probes targeting cyclin-dependent kinase 4 were used to validate an amplification identified by microarray-based comparative genomic hybridization (aCGH) in a pleomorphic adenoma; (iii) probes targeting fibroblast growth factor receptor 1 and CCND1 were used to validate amplifications mapping to these regions, as defined by aCGH, in an invasive lobular breast carcinoma with FISH and CISH; and (iv) gene-specific probes for ETV6 and NTRK3 were used to demonstrate the presence of t(12;15)(p12;q25) translocation in a case of breast secretory carcinoma with dual colour FISH. In summary, this protocol enables the generation of probes mapping to any gene of interest that can be applied to FFPETS, allowing correlation of morphological features with gene copy number. PMID:16446704

Lambros, Maryou B K; Simpson, Pete T; Jones, Chris; Natrajan, Rachael; Westbury, Charlotte; Steele, Dawn; Savage, Kay; Mackay, Alan; Schmitt, Fernando C; Ashworth, Alan; Reis-Filho, Jorge S

2006-04-01

280

Use of fluorescence in situ hybridization (fish) to study chromosomal damage induced by radiation and bromodeoxyuridine in human colon cancer cells  

Microsoft Academic Search

Although the thymidine analog radiation sensitizer bromodeoxyuridine (BrdUrd) increases radiation-induced chromosomal aberrations, it is not known whether these aberrations are uniformly distributed among chromosomes. Using fluorescence in situ hybridization, we carried out a study to test the hypothesis that BrdUrd-induced radiosensitization may be mediated by nonuniform chromsomal damage. Log phase HT29 human colon cancer cells were exposed to 10 μM

S. R. Wilt; A. C. Burgess; T. S. Lawrence

1994-01-01

281

Chromosomal localization of sixty autosomal loci in sheep (Ovis aries, 2n = 54) by fluorescence in situ hybridization and R-banding  

Microsoft Academic Search

Sixty autosomal loci (5 type I and 55 type II) from 24 bovine syntenic groups, and previously FISH-mapped to goat and river buffalo chromosomes, were localized by fluorescence in situ on sheep (Ovis aries, 2n = 54) chromosomes, thereby notably extending the cytogenetic map of this economically important species. Caprine BAC clones were hybridized to R-banded chromosome preparations. FITC-signals and

L. Iannuzzi; A. Perucatti; G. P. Di Meo; L. Schibler; D. Incarnato; E. P. Cribiu

2003-01-01

282

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

Microsoft Academic Search

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

Daming Zhang; Tao Sang

1999-01-01

283

Characterization of double minute chromosomes' DNA content in a human high grade astrocytoma cell line by using comparative genomic hybridization and fluorescence in situ hybridization.  

PubMed

The presence of double minute chromosomes (dmin) in cancer cells is known to be correlated with gene amplifications. In human high grade astrocytomas or glioblastomas, about 50% of cytogenetically characterized cases display dmin. G5 is a cell line which has been established from a human glioblastoma containing multiple dmin. In order to identify the DNA content of these dmin, three techniques were successively used: conventional cytogenetic analysis, comparative genomic hybridization (CGH). and fluorescent in situ hybridization (FISH). The karyotype of G5 cells showed numerical chromosome changes (hypertriploidy), several marker chromosomes, and multiple dmin. CGH experiments detected two strong DNA amplification areas located in 9p21-22 and 9p24, as well as an underrepresentation of chromosomes 6, 10, 11, 13, 14, and 18q. By using FISH with a chromosome 9-specific painting probe to metaphase chromosomes of the G5 cell line, dmin were shown to contain DNA sequences originating from chromosome 9. This study demonstrates the usefulness of a combination of classical karyotyping, CGH, and FISH to identify the chromosomal origin of amplified DNA sequences in dmin. PMID:8707292

Giollant, M; Bertrand, S; Verrelle, P; Tchirkov, A; du Manoir, S; Ried, T; Mornex, F; Doré, J F; Cremer, T; Malet, P

1996-09-01

284

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

PubMed

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. PMID:8533833

Juyal, R C; Greenberg, F; Mengden, G A; Lupski, J R; Trask, B J; van den Engh, G; Lindsay, E A; Christy, H; Chen, K S; Baldini, A

1995-09-11

285

Variation of wheatgrass chromosomes in wheat-wheatgrass alien addition line “TAI27” revealed by fluorescence in situ hybridization (FISH)  

Microsoft Academic Search

The Karyotyp of the primary wheat-whastgrass alien addition line TAI-27 was 2n = 44 in which all d the chromosomes were metacentric\\u000a and subrmetacentric. However, in the progeny of TAI-27 a pair of chromosomes had become small chromosomea in the two morphologically\\u000a different plants. Fluorescence in situ hybridizstionm (FISH) technique was used to analyze the two different plants. The observations

Fangpu Han; Xiangqi Zhang; Xiuling Bu; Mengyuan He; Shui Hao; Youzhi Ma; Zhiyong Xin

1998-01-01

286

Tubular and endothelial chimerism in renal allografts using fluorescence and chromogenic in situ hybridization (FISH, CISH) technology.  

PubMed

The role of endothelial and tubular chimerism in renal allograft adaptation and rejection varies in different studies. We addressed the correlation between different clinico-pathological settings and sex-chromosomal endothelial and/or tubular chimerism in renal allografts. We examined the presence or absence of the X and Y chromosomes by fluorescence and chromogenic in situ hybridization (FISH, CISH) methodology on paraffin embedded kidney biopsies in 16 gender mismatched renal transplants (1 to 12 years post-transplantation). Twelve patients were male, four female. Four groups were selected: (i) Vascular calcineurin inhibitor toxicity without rejection; (ii) T-cell mediated vascular rejection; (iii) antibody mediated rejection; and (iv) C4d-positivity in AB0-incompatible transplants with or without rejection. Twelve non-transplant kidney biopsies (8 female, 4 male) were used as controls. Tubular chimerism was detected more frequently (69%) than endothelial chimerism (12%) in renal transplants. One of 12 control patients had tubular and endothelial chimeric cells (8%). The Y chromosome occurred in 8/12 male recipients (67%) in tubular epithelial cells and in 5/12 male recipients (42%) in endothelial cells. Double X chromosomes were detected in 3/4 female recipients in tubular epithelium. Tubular chimerism occurred more often with endothelial chimerism and capillaritis without correlation with other parameters, such as rejection. Combined Y chromosomal tubular and lymphatic endothelial chimerism correlated with T-cell mediated vascular rejection in two out of three patients (66%). Combined Y chromosomal tubular and peritubular capillary chimerism correlated with antibody mediated C4d+ rejection in one out of two patients (50%). Tubular and/or endothelial chimerism occur frequently in gender mismatched renal allografts and, when combined, this is associated with T-cell mediated rejection. PMID:22449229

Varga, Zsuzsanna; Gaspert, Ariana; Behnke, Silvia; von Teichman, Adriana; Fritzsche, Florian; Fehr, Thomas

2012-03-04

287

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

Microsoft Academic Search

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

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

1999-01-01

288

Sequential multiple probe fluorescence in-situ hybridization analysis of human oocytes and polar bodies by combining centromeric labelling and whole chromosome painting  

Microsoft Academic Search

The incidence of chromosomal aneuploidy was analysed in 104 unfertilized human oocytes and 56 first polar bodies using a double-label fluorescence in-situ hybridization (FISH) procedure. Combinations of centromeric (or locus-specific) DNA probes and whole chromosome painting probes for chromosomes 9, 13, 16, 18, 21 and X were applied on oocyte preparations, in a sequential FISH protocol. This combined approach allowed

T. Anahory; B. Andreo; G. Regnier-Vigouroux; J. P. Soulie; M. Baudouin; J. Demaille; F. Pellestor

2003-01-01

289

Use of a modified fluorescent in situ hybridization procedure to improve the identification of Streptococcus pneumoniae in blood cultures.  

PubMed

Streptococcus pneumoniae is an important causative agent for bacteremia. Fluorescent in situ hybridization (FISH) is a helpful molecular technique for the rapid identification of S. pneumoniae in positive blood cultures. There are many reports concerning the application of an enzymatic treatment with lysozyme in the FISH procedure for partial cell wall digestion of S. pneumoniae. However, this study was aimed to test the FISH procedure without enzymatic treatment for the identification of S. pneumoniae in blood culture specimens. Seventy-seven positive blood culture specimens containing Gram-positive cocci were examined by both the conventional laboratory methods and FISH. Detection of S. pneumoniae was performed by two FISH procedures: one procedure was performed with an enzymatic treatment step and the other one was done without enzymatic treatment. In addition, the specimens were tested by the FISH procedure with enzymatic treatment to detect Streptococcus pyogenes and Enterococcus. The specificity of FISH in comparison with conventional culture methods was 100%. The sensitivity of the FISH procedure with enzymatic treatment for the detection of S. pneumoniae was 90%, whereas, the sensitivity of the FISH procedure without enzymatic treatment was 100%. In fact, by omission of enzymatic treatment, detection of S. pneumoniae was improved in 6 specimens. The results of the FISH and culture methods for the detection of S. pyogenes and Enterococcus were compatible. Altogether, FISH procedure without enzymatic treatment step seems to improve the detection of S. pneumoniae in some cases. Thus, for successful detection of S. pneumoniae, we suggest the application of both FISH procedures (the procedure with enzymatic treatment and the procedure without enzymatic treatment) for each blood culture specimen. PMID:24060554

Tajbakhsh, Saeed; Gharibi, Somayyeh; Zandi, Keivan; Yaghobi, Ramin

2013-09-01

290

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

SciTech Connect

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

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

1994-09-01

291

Multicolor fluorescence in situ hybridization analysis of the spermatozoa of a male heterozygous for a reciprocal translocation t(11;22)(q23;q11)  

Microsoft Academic Search

A reciprocal translocation between chromosomes 11 and 22 is a site-specific translocation that has been seen in many families\\u000a with no common ancestry. This translocation is of particular interest because balanced carriers have a 0.7–3.7% risk of having\\u000a children with the supernumerary der(22), resulting from a 3:1 segregation. We have used a three color fluorescence in situ\\u000a hybridization (FISH) with

Anna M. Estop; Kathy M. Cieply; Santiago Munne; Eleanor Feingold

1999-01-01

292

Segmentation and Classification of Dot and Non-Dot-Like Fluorescence in situ Hybridization Signals for Automated Detection of Cytogenetic Abnormalities  

Microsoft Academic Search

Signal segmentation and classification of fluorescence in situ hybridization (FISH) images are essential for the detection of cytogenetic abnormalities. Since current methods are limited to dot-like signal analysis, we propose a methodology for segmenta- tion and classification of dot and non-dot-like signals. First, nuclei are segmented from their background and from each other in order to associate signals with specific

Boaz Lerner; Lev Koushnir; Josepha Yeshaya

2007-01-01

293

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

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

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

2007-01-01

294

Assignment of the human prosaposin gene (PSAP) to 10q22.1 by fluorescence in situ hybridization. Giraffidae, okapi (Okapiajohnstoni), and giraffe (Giraffa camelopardalis): evidence for ancestral telomeres at the okapi polymorphic rob (4;26) fusion site.  

PubMed

The human prosaposin gene (PSAP) was previously localized to 10q21-->q22 by isotopic in situ hybridization using a human prosaposin cDNA as a probe. The present study, using fluorescence in situ hybridization with a mouse genomic prosaposin fragment as probe, confirms the localization of PSAP and precisely maps it to band 10q22.1. PMID:8641138

Bar-Am, I; Avivi, L; Horowitz, M

1996-01-01

295

Identification of neurofibromatosis 1 (NF1) homologous loci by direct sequencing, fluorescence in situ hybridization, and PCR amplification of somatic cell hybrids  

SciTech Connect

Using fluorescence in situ hybridization (FISH), we have identified seven NF1-related loci, two separate loci on chromosome 2, at bands 2q21 and 2q33-q34, and one locus each on five other chromosomes at bands 14q11.2, 15q11.2, 18p11.2, 21q11.2-q21, and 22q11.2. Application of PCR using NF1 primer pairs and genomic DNA from somatic cell hybrids confirmed the above loci, identified additional loci on chromosomes 12 and 15, and showed that the various loci do not share homology beyond NF1 exon 27b. Sequenced PCR products representing segments corresponding to NF1 exons from these loci demonstrated greater than 95% sequence identity with the NF1 locus. We used sequence differences between bona fide NF1 and NF1-homologous loci to strategically design primer sets to specifically amplify 30 of 36 exons within the 5{prime} end of the NF1 gene. These developments have facilitated mutation analysis at the NF1 locus using genomic DNA as template. 41 refs., 3 figs., 3 tabs.

Purandare, S.M.; Neil, S.M.; Brothman, A. [Univ. of Utah, Salt Lake City (United States)]|[Jikei Univ., Tokyo (Japan)] [and others

1995-12-10

296

Preparations of meiotic pachytene chromosomes and extended DNA fibers from cotton suitable for fluorescence in situ hybridization.  

PubMed

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

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

2012-03-19

297

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

PubMed Central

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.

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

2012-01-01

298

Fluorescence in situ Hybridization method using Peptide Nucleic Acid probes for rapid detection of Lactobacillus and Gardnerella spp.  

PubMed Central

Background Bacterial vaginosis (BV) is a common vaginal infection occurring in women of reproductive age. It is widely accepted that the microbial switch from normal microflora to BV is characterized by a decrease in vaginal colonization by Lactobacillus species together with an increase of Gardnerella vaginalis and other anaerobes. Our goal was to develop and optimize a novel Peptide Nucleic Acid (PNA) Fluorescence in situ Hybridization assay (PNA FISH) for the detection of Lactobacillus spp. and G. vaginalis in mixed samples. Results Therefore, we evaluated and validated two specific PNA probes by using 36 representative Lactobacillus strains, 22 representative G. vaginalis strains and 27 other taxonomically related or pathogenic bacterial strains commonly found in vaginal samples. The probes were also tested at different concentrations of G. vaginalis and Lactobacillus species in vitro, in the presence of a HeLa cell line. Specificity and sensitivity of the PNA probes were found to be 98.0% (95% confidence interval (CI), from 87.8 to 99.9%) and 100% (95% CI, from 88.0 to 100.0%), for Lactobacillus spp.; and 100% (95% CI, from 92.8 to 100%) and 100% (95% CI, from 81.5 to 100.0%) for G. vaginalis. Moreover, the probes were evaluated in mixed samples mimicking women with BV or normal vaginal microflora, demonstrating efficiency and applicability of our PNA FISH. Conclusions This quick method accurately detects Lactobacillus spp. and G. vaginalis species in mixed samples, thus enabling efficient evaluation of the two bacterial groups, most frequently encountered in the vagina.

2013-01-01

299

Duplication of intrachromosomal insertion segments 4q32->q35 confirmed by comparative genomic hybridization and fluorescent in situ hybridization  

PubMed Central

A 35-year-old man with infertility was referred for chromosomal analysis. In routine cytogenetic analysis, the patient was seen to have additional material of unknown origin on the terminal region of the short arm of chromosome 4. To determine the origin of the unknown material, we carried out high-resolution banding, comparative genomic hybridization (CGH), and FISH. CGH showed a gain of signal on the region of 4q32?q35. FISH using whole chromosome painting and subtelomeric region probes for chromosome 4 confirmed the aberrant chromosome as an intrachromosomal insertion duplication of 4q32?q35. Duplication often leads to some phenotypic abnormalities; however, our patient showed an almost normal phenotype except for congenital dysfunction in spermatogenesis.

Kim, Jin Woo; Park, Ju Yeon; Oh, Ah Rum; Choi, Eun Young; Ryu, Hyun Mee; Kang, Inn Soo; Koong, Mi Kyoung

2011-01-01

300

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

PubMed Central

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.

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

1999-01-01

301

IDENTIFICATION OF SEX CHROMOSOME MOLECULAR MARKERS USING RAPDS AND FLUORESCENT IN SITU HYBRIDIZATION IN RAINBOW TROUT  

EPA Science Inventory

The goal of this work is to identify molecular markers associated with the sex chromosomes in rainbow trout to study the mode of sex determination mechanisms in this species. Using the RAPD assay and bulked segregant analysis, two markers were identified that generated polymorphi...

302

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

SciTech Connect

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.

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

1994-09-01

303

Sequential numerical changes of chromosomes 7 and 18 in diffuse-type stomach cancer cell lines: combined comparative genomic hybridization, fluorescence in situ hybridization, and ploidy analyses.  

PubMed

Sequential changes of chromosomal copy number were analyzed retrospectively in five diffuse-type gastric cancer cell lines by comparative genomic hybridization (CGH), DNA cytometry, and fluorescence in situ hybridization (FISH) with centromeric and painting probes. By CGH, we found loss of 18q21 in all of the cell lines and gains of 7p11-q31, 20q, and 22 in four of the five cell lines. Actual copy numbers of chromosomes 7 and 18 were determined by FISH: disomy 18 with (partial) loss of 18q in the two DNA-diploid cell lines (AGS and MKN-45), trisomy 7 in MKN-45, disomy 18 and tetrasomy 7 with one-copy loss of 7p and one-copy gain of 7q tip in DNA-triploid HSC-39/40A, and trisomy 18 and hexasomy 7 with one-copy loss of 7q in DNA-tetraploid KATO-III. Because the DNA aneuploidy is thought to result through tetraploidization, and the duplicated chromosomal changes in DNA aneuploid tumors seem to precede tetraploidization, the duplicated gain of chromosome 7 and one-copy loss of 7q in KATO-III were inferred to have occurred before and after tetraploidization, respectively. Similarly, HSC-39/40A were inferred to be preceded by the DNA-diploid stage with disomy 7 and monosomy 18. As the loss of 18q21 and the gain of 7p11-q31 were inferred to have occurred already in the DNA diploid stage in at least four and two of the cell lines, respectively, the 18q21 loss may be more important than the 7q gain as an earlier event in the genesis of diffuse-type stomach cancer. The combined CGH, FISH, and ploidy analyses thus give us a clue to extract important earlier events from the chromosomal changes that were screened by CGH alone. PMID:10748289

Okada, K; Sugihara, H; Bamba, M; Bamba, T; Hattori, T

2000-04-15

304

Constitutional von Hippel-Lindau (VHL) gene deletions detected in VHL families by fluorescence in situ hybridization.  

PubMed

von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited cancer syndrome predisposing to a variety of tumor types that include retinal hemangioblastomas, hemangioblastomas of the central nervous system, renal cell carcinomas, pancreatic cysts and tumors, pheochromocytomas, endolymphatic sac tumors, and epididymal cystadenomas [W. M. Linehan et al., J. Am. Med. Assoc., 273: 564-570, 1995; E. A. Maher and W. G. Kaelin, Jr., Medicine (Baltimore), 76: 381-391, 1997; W. M. Linehan and R. D. Klausner, In: B. Vogelstein and K. Kinzler (eds.), The Genetic Basis of Human Cancer, pp. 455-473, McGraw-Hill, 1998]. The VHL gene was localized to chromosome 3p25-26 and cloned [F. Latif et al., Science (Washington DC), 260: 1317-1320, 1993]. Germline mutations in the VHL gene have been detected in the majority of VHL kindreds. The reported frequency of detection of VHL germline mutations has varied from 39 to 80% (J. M. Whaley et al., Am. J. Hum. Genet., 55: 1092-1102, 1994; Clinical Research Group for Japan, Hum. Mol. Genet., 4: 2233-2237, 1995; F. Chen et al., Hum. Mutat., 5: 66-75, 1995; E. R. Maher et al., J. Med. Genet., 33: 328-332, 1996; B. Zbar, Cancer Surv., 25: 219-232, 1995). Recently a quantitative Southern blotting procedure was found to improve this frequency (C. Stolle et al., Hum. Mutat., 12: 417-423, 1998). In the present study, we report the use of fluorescence in situ hybridization (FISH) as a method to detect and characterize VHL germline deletions. We reexamined a group of VHL patients shown previously by single-strand conformation and sequencing analysis not to harbor point mutations in the VHL locus. We found constitutional deletions in 29 of 30 VHL patients in this group using cosmid and P1 probes that cover the VHL locus. We then tested six phenotypically normal offspring from four of these VHL families: two were found to carry the deletion and the other four were deletion-free. In addition, germline mosaicism of the VHL gene was identified in one family. In sum, FISH was found to be a simple and reliable method to detect VHL germline deletions and practically useful in cases where other methods of screening have failed to detect a VHL gene abnormality. PMID:10554035

Pack, S D; Zbar, B; Pak, E; Ault, D O; Humphrey, J S; Pham, T; Hurley, K; Weil, R J; Park, W S; Kuzmin, I; Stolle, C; Glenn, G; Liotta, L A; Lerman, M I; Klausner, R D; Linehan, W M; Zhuang, Z

1999-11-01

305

'Genetic heterogeneity' in HER2/neu testing by fluorescence in situ hybridization: a study of 2,522 cases.  

PubMed

Amplification for the ERBB2 oncogene encoding the HER2/neu protein (HER2) is of predictive and prognostic importance in breast carcinoma. Fluorescence in situ hybridization (FISH) is a widely accepted method for determining HER2 amplification status. A HER2-amplified tumor is defined as having a ratio of HER2 signals to chromosome 17 centromeric probe signals (HER2/CEP17 ratio) exceeding 2.2. However, the presence of scattered cells demonstrating HER2 amplification is of unclear significance. A 2009 panel guideline defined a tumor with 'genetic heterogeneity' as having at least 5% but fewer than 50% of (non-clustered) tumor nuclei with a ratio >2.2. The study objective was to examine the statistical distribution of breast tumors tested by FISH for HER2 amplification, after implementation of this 2009 guideline. We identified 2522 consecutive breast carcinoma cases (2009-2011) tested for HER2 amplification. All cases were tested by FISH using a standard clinical protocol, adhering to established guidelines. For each case, data on cell counts were retrieved electronically. Each tumor was compared with a theoretical normal distribution by quantile-quantile analysis. Of 2522 FISH tests for HER2, 1900 (75%) were non-amplified, 394 (16%) were amplified, and 228 (9%) were HER2-equivocal. A total of 666 (26%) had 'genetic heterogeneity.' Among these 'genetically heterogeneous' cases, the ratio was non-amplified in 430 (64.5%), amplified in 24 (4%), and equivocal in 212 (31.5%). The amplified subpopulation in 'genetically heterogeneous' tumors was larger if the overall ratio was close to 2.2. However, the percentage of nuclei >2.2 in a 'genetically heterogeneous' tumor was not informative of the underlying tumor-cell distribution. We conclude that the proportion of HER2-amplified nuclei within a tumor does not contribute information independent of the actual HER2/CEP17 ratio. Reassessment of the definition of 'genetic heterogeneity' in HER2 testing is warranted. PMID:22282306

Chang, Martin C; Malowany, Janet I; Mazurkiewicz, Julita; Wood, Martha

2012-01-27

306

Localization of a candidate colon tumor-suppressor gene (DRA) to 7q22-q31. 1 by fluorescence in situ hybridization  

SciTech Connect

The authors have previously reported that the DRA gene is located on chromosome 7. This assignment was based on Southern blot hybridization of a DRA cDNA to genomic DNA from rodent-human somatic cell hybrids. In this report, they localize the DRA gene to chromosome band 7q22-q31.1 by fluorescence in situ hybridization (FISH) with a full-length (2.9 kb) cDNA as probe. Metaphase spreads from normal human lymphocytes were prepared according to the method of Fan et al. The cDNA clone 611C was labeled with biotin-11-dUTP using a nick-translation kit (Oncor) followed by purification on a Sephadex G50-fine column. FISH and detection of immunofluorescence were performed according to the technique of Pinkel et al. with minor modifications. The chromosome preparations were stained with both diamidino-2-phenylindole and propidium iodide (Oncor) and observed with a Zeiss Axiophot fluorescence microscope. Hybridization was detected on chromosome 7 in 22 of 47 spreads examined. Of 89 fluorescent signals on all chromosomes, 44 (49%) were located on 7q. All signals on chromosome 7 appeared to be located at 7q22-q31.1. Hybridization is in the vicinity of the met protooncogene locus at 7q31.

Taguchi, T.; Testa, J.R. (Fox Chase Cancer Center, Philadelphia, PA (United States)); Papas, T.S.; Schweinfest, C. (Medical Univ. of South Carolina, Charleston, SC (United States))

1994-03-01

307

The detection of contiguous gene deletions at the neurofibromatosis 1 locus with fluorescence in situ hybridization  

Microsoft Academic Search

Neurofibromatosis type 1 (NFl) is a common genetic disorder characterized primarily by the development of multiple neurofibromas and pigmentary changes. The recent identification of contiguous gene deletions in NF1 a previously unrecognized molecular basis for this disorder, raises important questions regarding deletion frequency in the patient population and the role that contiguous genes may play in the physical manifestations of

K. A. Leppig; D. Viskochil; S. Neil; A. Rubenstein; V. P. Johnson; X. L. Zhu; A. R. Brothman; K. Stephens

1996-01-01

308

Toward an automated system for the analysis of cytogenetic abnormalities using fluorescence in situ hybridization technique  

Microsoft Academic Search

In order to build an automatic system that searches, acquires and analyses chromosomal aberrations, we have coupled a commercial system, Metafer4 (MetaSystems, Germany), with an original software (VRAIC) that is able to separate normal from aberrant metaphase images. Images of metaphases are first stained with a FISH technique and then acquired in three different channels: a BLUE channel, where all

Ezio Catanzariti; Raffaele D. Esposito; Roberta Santilli; Matteo Santoro

2003-01-01

309

Fluorescence in situ Hybridization (FISH) as a Method to Detect Aneuploid Cells  

Microsoft Academic Search

Objectives: To test the sensitivity and specificity of various FISH probes for detecting male and aneuploid cells and to determine the percentage of fetal cells that must be present in a sample in order to use the probes for prenatal diagnosis. Methods: Adult human lymphocytes were cultured and harvested. Twelve different proportions of male to female cells and 5 different

Hsiao-Jui Wei; Tsung-Hsien Su; Chung-Liang Chien; Chii-Ruey Tzeng

1997-01-01

310

Combined Fluorescent-Chromogenic In Situ Hybridization for Identification and Laser Microdissection of Interphase Chromosomes  

PubMed Central

Chromosome territories constitute the most conspicuous feature of nuclear architecture, and they exhibit non-random distribution patterns in the interphase nucleus. We observed that in cell nuclei from humans with Down Syndrome two chromosomes 21 frequently localize proximal to one another and distant from the third chromosome. To systematically investigate whether the proximally positioned chromosomes were always the same in all cells, we developed an approach consisting of sequential FISH and CISH combined with laser-microdissection of chromosomes from the interphase nucleus and followed by subsequent chromosome identification by microsatellite allele genotyping. This approach identified proximally positioned chromosomes from cultured cells, and the analysis showed that the identity of the chromosomes proximally positioned varies. However, the data suggest that there may be a tendency of the same chromosomes to be positioned close to each other in the interphase nucleus of trisomic cells. The protocol described here represents a powerful new method for genome analysis.

Paz, Nerea; Zabala, Amaia; Royo, Felix; Garcia-Orad, Africa; Zugaza, Jose L.; Parada, Luis A.

2013-01-01

311

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

Microsoft Academic Search

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

M. McCorquodale; O. Bereziouk; D. J. McCorquodale

1994-01-01

312

Meiotic segregation analysis of a 14;21 Robertsonian translocation carrier by fluorescence in situ hybridization.  

PubMed

Meiotic segregation of chromosomes 14 and 21 in sperm from a 14;21 Robertsonian translocation carrier was analyzed with dual-color FISH using two locus-specific DNA probes (Tel 14q and LSI 21). The frequency of normal or chromosomally balanced sperm, resulting from alternate segregation, was 88.42%. The frequency of unbalanced sperm, resulting from adjacent segregation, was 11.25%. These observed frequencies deviated significantly from the theoretical frequencies (33.33% and 66.67%, respectively) based on random chromosome segregation, with sperm resulting from alternate segregation being preferentially produced in the translocation carrier. With respect to the chromosomally unbalanced sperm, the frequency of 21q disomic sperm was 2.45%, which is in agreement with the frequencies of unbalanced fetuses or offspring at the time of amniocentesis or at term (0-4.3%) reported by others. Although the frequency of 14 or 21 nullisomic sperm should be theoretically equal to that of 14q or 21q disomic sperm in both the carrier and controls, the frequency of nullisomic sperm was significantly higher than that of disomic sperm in the carrier (P=0.0009 for chromosome 14, P<0.0001 for chromosome 21) but not in the controls (P=0.091 for chromosome 14, P=0.74 for chromosome 21). This evidence suggests the occurrence of maturation arrest during spermatogenesis of the carrier. PMID:10746560

Honda, H; Miharu, N; Samura, O; He, H; Ohama, K

2000-02-01

313

Rapid method for measuring clastogenic fingerprints using fluorescence in situ hybridization  

DOEpatents

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.

Lucas, Joe N. (San Ramon, CA)

2000-01-01

314

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

Microsoft Academic Search

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

Tariq Moatter; Zahida Khilji; Farzana Murad; Shama Munim

315

Identification of Prader-Willi Syndrome mosaicism by fluorescent in situ hybridization  

SciTech Connect

Prader-Willi Syndrome (PWS) is a microdeletion syndrome involving an interstitial deletion of region q11-q13 on the paternal chromosome 15. We report 2 cases of PWS that were analyzed using FISH and were found to be mosaic for a normal cell line and a deleted cell line. Case 1 was diagnosed as an atypical PWS who was cytogenetically normal. She is a 38 y.o. white female displaying some but not all of the features of PWS. Case 2 is a 23 y.o. white male with a classical deletion of chromosome 15q11-q13. He displays very typical features of PWS. He was also noted to be albino. FISH analysis was performed on PHA stimulated lymphocytes. We examined four loci: D15S11, SNRPN, D15S10, and GABRB3. The number of cells examined for each locus ranged from 46 to 75. Case 1 was deleted at 3 of the 4 loci (D15S11, SNRPN and GABRB3) in 30% of her cells. The D15S10 locus was not deleted. This may account for the atypical features displayed by this patient. It also suggests that this chromosome is rearranged resulting in the retention of the interstitial locus. The exact nature of the rearrangement needs to be determined. Case 2 was deleted at all four loci in 60% of the cells analyzed. This result was unexpected because his deletion was identified cytogenetically, but mosaicism was not detected. These are the first reported cases of mosaic PWS diagnosed using FISH. The use of cytogenetics alone requires high resolution banding to accurately identify the deletion. This makes the detection of small deletions in every cell difficult and the determination of mosaicism almost impossible. Our results suggest that mosaicism may be occurring more frequently than previously thought and may account for some of the atypical cases. Studies are in progress to determine the effect of mosaicism on methylation at genes located within this region which are imprinted and are thought to be involved in the etiology of Prader-Willi Syndroms.

Mowery-Rushton, P.A.; Surti, U. [Magee Womens Hospital, Pittsburgh, PA (United States); Hanchett, J.M. [Rehabilitation Institute, Pittsburgh, PA (United States)

1994-09-01

316

Fluorescence in situ hybridization and spectral imaging analysisof human oocytes and first polar bodies  

Microsoft Academic Search

We investigated the frequencies of abnormalities involving either chromosome 1, 16, 18 or 21 in failed-fertilized human oocytes.While abnormalities involving chromosome 16 showed an age-dependant increase, results for the other chromosomes did not show statistically significant differences between the three age groups <35 yrs, 35-39 yrs, and >39 yrs. The scoring of four chromosomes is likely to underestimate the true

Heinz-Ulli G. Weier; Jingly F. Weier; Maria Oter Renom; Xuezhong Zheng; Pere Colls; Aida Nureddin; Chau D. Pham; Lisa W. Chu; Catherine Racowsky; Santiago Munne

2004-01-01

317

Fluorescence In Situ Hybridization and Spectral Imaging Analysis of Human Oocytes and First Polar Bodies  

Microsoft Academic Search

We investigated the frequencies of abnormalities involving either chromosome 1, 16, 18, or 21 in failed-fertilized human oocytes. Although abnormalities involving chromosome 16 showed an age-dependent increase, results for the other chromosomes did not show statistically significant differences among the three age groups, <35 years, 35–39 years, and >39 years. The scoring of four chromosomes is likely to underestimate the

Heinz-Ulli G. Weier; Jingly F. Weier; Maria Oter Renom; Xuezhong Zheng; Pere Colls; Aida Nureddin; Chau D. Pham; Lisa W. Chu; Catherine Racowsky; Santiago Munné

2005-01-01

318

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

SciTech Connect

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

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

1994-09-01

319

Fluorescence in situ hybridization and spectral imaging analysisof human oocytes and first polar bodies  

SciTech Connect

We investigated the frequencies of abnormalities involving either chromosome 1, 16, 18 or 21 in failed-fertilized human oocytes.While abnormalities involving chromosome 16 showed an age-dependant increase, results for the other chromosomes did not show statistically significant differences between the three age groups <35 yrs, 35-39 yrs, and >39 yrs. The scoring of four chromosomes is likely to underestimate the true rate of aneuploid cells. Thus, for a pilot study investigating a more comprehensive analysis of oocytes and their corresponding first polar bodies (1PBs), we developed a novel 8-probe chromosome enumeration scheme using FISH and SIm.

Weier, Heinz-Ulli G.; Weier, Jingly F.; Oter Renom, Maria; Zheng,Xuezhong; Colls, Pere; Nureddin, Aida; Pham, Chau D.; Chu, Lisa W.; Racowsky, Catherine; Munne, Santiago

2004-10-06

320

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

SciTech Connect

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.

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

1994-09-01

321

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

PubMed

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

Bisht, M S; Mukai, Y

2000-12-01

322

Electron Paramagnetic Resonance and Fluorescence In Situ Hybridization-Based Investigations of Individual Doses for Persons Living at Metlino in the Upper Reaches of the Techa River  

SciTech Connect

Waterborne releases to the Techa River from the Mayak Production Association in Russia during 1949-1956 resulted in significant doses to persons living downstream; the most contaminated village was Metlino, about 7 km from the site of release. Internal and external doses have been estimated for these residents using the Techa River Dosimetry System-2000 (TRDS-2000); the primary purpose is to support epidemiological studies of the members of the Extended Techa River Cohort. Efforts to validate the calculations of external and internal dose are considered essential. One validation study of the TRDS-2000 system has been performed by the comparison of calculated doses to quartz from bricks in old buildings at Metlino with those measured by luminescence dosimetry. Two additional methods of validation considered here are electron paramagnetic resonance (EPR) measurements of teeth and fluorescence in situ hybridization (FISH) measurements of chromosome translocations in circulating lymphocytes. For electron paramagnetic resonance, 36 measurements on 26 teeth from 16 donors from Metlino were made at the GSF-National Research Center for Environment and Health (16 measurements) and the Institute of Metal Physics (20 measurements); the correlation among measurements made at the two laboratories has been found to be 0.99. Background measurements were also made on 218 teeth (63 molars, 128 premolars, and 27 incisors). Fluorescence in situ hybridization measurements were made for 31 residents of Metlino. These measurements were handicapped by the analysis of a limited number of cells; for several individuals no stable translocations were observed. Fluorescence in situ hybridization measurements were also made for 39 individuals believed to be unexposed. The EPR- and FISH-based estimates agreed well for permanent residents of Metlino: 0.67 +/- 0.21 Gy and 0.48 +/- 0.18 Gy (mean +/- standard error of the mean), respectively. Results of the two experimental methods also agreed well with the estimates derived from the use of the TRDS-2000. For all persons investigated according to each technique, the EPR-measured dose to enamel was 0.55 +/- 0.17 Gy, and the TRDS-2000 prediction for the dose to enamel for these individuals is 0.55 +/- 0.07 Gy. The fluorescence in situ hybridization-based dose, 0.38 +/- 0.10 Gy, compared well to the TRDS-2000 prediction of external dose, 0.31 +/- 0.03 Gy, to red bone marrow for these persons. Validation of external doses at the remaining villages is an active area of investigation.

Degteva, M. O.; Anspaugh, L. R.; Akleyev, A V.; Jacob, Peter; Ivanov, Denis V.; Wieser, Albrecht; Vorobiova, M I.; Shishkina, Elena A.; Shved, Valentina A.; Vozilova, Alexandra; Bayankin, Sergey N.; Napier, Bruce A.

2005-02-01

323

3p22.1 and 10q22.3 Deletions Detected by Fluorescence In Situ Hybridization (FISH)  

PubMed Central

Background Our objective was to study the feasibility of detecting chromosomal deletions at 3p22.1 and 10q22.3 by fluorescent in situ hybridization (FISH) and to examine their distribution in different areas of the airway in patients with non-small cell lung cancer. Methods Brush biopsies from the mainstem bronchus on the normal side contralateral to the tumor (NBB) and mainstem bronchus on the tumor side (TBB) were obtained from 122 patients who underwent surgical resection. Touch preparations from the tumor (TTP), normal lung parenchyma, and bronchi adjacent to the tumor were also obtained. Two FISH assays using probes complementary to 3p22.1 and 10q22.3 were used to detect deletions. Results NBB showed a relatively low deletion rate of 3p22.1 and 10q22.3 compared with TTP (p < 0.0001). TBB showed a significantly higher rate of deletions compared with NBB but lower than TTP from the tumor (p < 0.05) for both 3p22.1 and 10q22.3. A significantly higher deletion rate was seen at TTP compared with normal lung parenchyma at both the 3p22.1 and 10 q22.3 (p < 0.0001). Correlations were seen between the deletion rates of TTP and TBB at 3p22.1 (? = 0.61, p < 0.0001) and between TTP and bronchi adjacent to the tumor at 10q22.3 (? = 0.64, p < 0.0001). Conclusion Deletions of the 3p22.1 and 10q22.3 regions can be reliably detected by FISH. As one progresses from the contralateral normal bronchus to the bronchus on the side of tumor and the tumor itself, the percentage of chromosomal deletions increases in a statistically significant fashion. This suggests that, FISH analysis of bronchoscopic brushes may be useful for identifying patients at high risk for developing non-small cell lung cancer.

Yendamuri, Sai; Vaporciyan, Ara A.; Zaidi, Tanweer; Feng, Lei; Fernandez, Ricardo; Bekele, Nebiyou B.; Hofstetter, Wayne L.; Jiang, Feng; Mehran, Reza J.; Rice, David C.; Spitz, Margaret R.; Swisher, Stephen G.; Walsh, Garrett L.; Roth, Jack A.; Katz, Ruth L.

2012-01-01

324

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

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.

Robson, L.; Jackson, J.; Cowell, C.; Sillence, D.; Smith, A. [Children`s Hospital, Camperdown (Australia)

1994-04-15

325

[Application of Flow Cytometry-Fluorescence in situ Hybridization in the Measurement of Relative Telomere Length of Bone Marrow CD34(+) Cells in Patients with Myelodysplastic Syndrom].  

PubMed

This study was aimed to investigate the feasibility of flow cytometry-fluorescence in situ hybridization (Flow-FISH) in measuring the telomere length of bone marrow cell subgroups in patients with myelodysplastic syndrome (MDS). Seven newly diagnosed patients with low-risk MDS and seven nutritional anemia patients who were matched with age and sex, were enrolled in this study. Heparinized bone marrow were sampled. Taking Molt-4 cell line as internal control cells, leukocytes isolated from whole bone marrow were labeled with CD34-Alexa Fluork ® 647, then denatured by high temperature and hybridized with FITC-conjugated telomere probe. The DNA was conterstained and the relative tolemere length (RTL) of nucleated cells and CD34(+) cells in bone marrow were measured by four-color flow cytometry. The results showed that CD34(+) cells could be gated for the measurement of RTL in both groups, undergoing the denaturation and hybridization. Primary analysis indicated that the RTL of bone marrow CD34(+) cells in MDS patients was significantly shorter than that of bone marrow nucleated cells (P = 0.001), and the RTL of both CD34(+) cells and nucleated cells in bone marrow of MDS patients were significantly shorter than that of control group (P = 0.020, 0.002). It is concluded that the application of Flow-FISH in the measurement of RTL of certain cell subgroup is feasible by labeling the cell with thermostable fluorescence-conjugated antibody, and this technique is worthy to be investigated further. PMID:24156433

Guo, Tian-Jiao; Sun, Wan-Ling; Zhang, Wei; Ma, Xiao-Cai; Liu, Cong-Yan; He, Jing-Juan; Xu, Juan

2013-09-01

326

Development of automated brightfield double In Situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) for breast carcinomas and an assay performance comparison to manual dual color HER2 fluorescence In Situ hybridization (FISH)  

PubMed Central

Background Human epidermal growth factor receptor 2 (HER2) fluorescence in situ hybridization (FISH) is a quantitative assay for selecting breast cancer patients for trastuzumab therapy. However, current HER2 FISH procedures are labor intensive, manual methods that require skilled technologists and specialized fluorescence microscopy. Furthermore, FISH slides cannot be archived for long term storage and review. Our objective was to develop an automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) and test the assay performance with dual color HER2 FISH evaluated breast carcinomas. Methods The BDISH assay was developed with the nick translated dinitrophenyl (DNP)-labeled HER2 DNA probe and DNP-labeled CEN 17 oligoprobe on the Ventana BenchMark® XT slide processing system. Detection of HER2 and CEN 17 signals was accomplished with the silver acetate, hydroquinone, and H2O2 reaction with horseradish peroxidase (HRP) and the fast red and naphthol phosphate reaction with alkaline phosphatise (AP), respectively. The BDISH specificity was optimized with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified HER2 gene) and BT-474 (amplified HER2 gene). Then, the BDISH performance was evaluated with 94 routinely processed breast cancer tissues. Interpretation of HER2 and CEN 17 BDISH slides was conducted by 4 observers using a conventional brightfield microscope without oil immersion objectives. Results Sequential hybridization and signal detection for HER2 and CEN 17 ISH demonstrated both DNA targets in the same cells. HER2 signals were visualized as discrete black metallic silver dots while CEN 17 signals were detected as slightly larger red dots. Our study demonstrated a high consensus concordance between HER2 FISH and BDISH results of clinical breast carcinoma cases based on the historical scoring method (98.9%, Simple Kappa = 0.9736, 95% CI = 0.9222 – 1.0000) and the ASCO/CAP scoring method with the FISH equivocal cases (95.7%, Simple Kappa = 0.8993%, 95% CI = 0.8068 – 0.9919) and without the FISH equivocal cases (100%, Simple Kappa = 1.0000%, 95% CI = 1.0000 – 1.0000). Conclusion Automated BDISH applications for HER2 and CEN 17 targets were successfully developed and it might be able to replace manual two-color HER2 FISH methods. The application also has the potential to be used for other gene targets. The use of BDISH technology allows the simultaneous analyses of two DNA targets within the context of tissue morphological observation.

Nitta, Hiroaki; Hauss-Wegrzyniak, Beatrice; Lehrkamp, Megan; Murillo, Adrian E; Gaire, Fabien; Farrell, Michael; Walk, Eric; Penault-Llorca, Frederique; Kurosumi, Masafumi; Dietel, Manfred; Wang, Lin; Loftus, Margaret; Pettay, James; Tubbs, Raymond R; Grogan, Thomas M

2008-01-01

327

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

SciTech Connect

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.

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

328

Non-fluorescent RNA In Situ Hybridization Combined with Antibody Staining to Visualize Multiple Gene Expression Patterns in the Embryonic Brain of Drosophila.  

PubMed

In Drosophila, the brain arises from about 100 neural stem cells (called neuroblasts) per hemisphere which originate from the neuroectoderm. Products of developmental control genes are expressed in spatially restricted domains in the neuroectoderm and provide positional cues that determine the formation and identity of neuroblasts. Here, we present a protocol for non-fluorescent double in situ hybridization combined with antibody staining which allows the simultaneous representation of gene expression patterns in Drosophila embryos in up to three different colors. Such visible multiple stainings are especially useful to analyze the expression and regulatory interactions of developmental control genes during early embryonic brain development. We also provide protocols for whole mount and flat preparations of Drosophila embryos, which allow a more detailed analysis of gene expression patterns in relation to the cellular context of the early brain (and facilitate the identification of individual brain neuroblasts) using conventional light microscopy. PMID:24048924

Jussen, David; Urbach, Rolf

2014-01-01

329

Comparing two diagnostic laboratory tests for Williams syndrome: fluorescent in situ hybridization versus multiplex ligation-dependent probe amplification.  

PubMed

Most people with Williams syndrome (WS) have a heterozygous 1.55 Mb deletion on chromosome 7q11.23. For diagnostic purposes, fluorescence in situ hybridisation (FISH) with commercial FISH probes is commonly used to detect this deletion. We investigated whether multiplex ligation-dependent probe amplification (MLPA) is a reliable alternative for FISH. The MLPA kit (SALSA P029) contains probes for eight genes in the WS critical region: FKBP6, FZD9, TBL2, STX1A, ELN, LIMK1, RFC2, and CYLN2. The experimental FISH assay that was used consists of four probes covering the WS critical region. A total number of 63 patients was tested; in 53 patients, a deletion was detected both with FISH and MLPA(P029), in 10 patients both techniques failed to demonstrate a deletion. In only one patient, a deletion was detected which was not previously detected by two commercial FISH probes. This patient appeared to carry a small, atypical deletion. We conclude that MLPA is a reliable technique to detect WS. Compared with FISH, MLPA is less time consuming and has the possibility to detect also smaller, atypical deletions and duplications in the WS critical region. PMID:17949295

van Hagen, Johanna M; Eussen, Hubertus J F M M; van Schooten, Ron; van Der Geest, Josef N; Lagers-van Haselen, Gerardina C; Wouters, Cokkie H; De Zeeuw, Chris I; Gille, Johan J P

2007-01-01

330

Clinical application and limitations of the fluorescence in situ hybridization (FISH) assay in the diagnosis and management of melanocytic lesions: a report of 3 cases.  

PubMed

Histopathologic examination is the gold standard for the diagnosis of melanocytic lesions, including melanoma, and guides management options and disease prognosis based on the depth of invasion. Although most melanomas can be readily distinguished from benign nevi, some pigmented lesions are more ambiguous and can be challenging to interpret as truly benign or truly malignant. Unfortunately, misclassification can render severe consequences for the patient, making it imperative to explore further analysis to determine the true nature of an ambiguous lesion. A relatively new technique known as fluorescence in situ hybridization (FISH) has become prevalent in dermatopathology for distinguishing between benign and malignant pigmented lesions; however, there are few reports on the application of FISH results in the clinical setting. We present 3 cases in which a FISH assay was utilized to assist in the diagnosis and management of ambiguous pigmented lesions. We also provide a review of the most recent literature regarding this diagnostic modality. PMID:23259206

Nijhawan, Rajiv I; Votava, Henry J; Mariwalla, Kavita

2012-10-01

331

Specific Detection of Dehalococcoides Species by Fluorescence In Situ Hybridization with 16S rRNA-Targeted Oligonucleotide Probes  

PubMed Central

Dehalococcoides ethenogenes is the only known cultivated organism capable of complete dehalogenation of tetrachloroethene (PCE) to ethene. The prevalence of Dehalococcoides species in the environment and their association with complete dehalogenation of chloroethenes suggest that they play an important role in natural attenuation of chloroethenes and are promising candidates for engineered bioremediation of these contaminants. Both natural attenuation and bioremediation require reliable and sensitive methods to monitor the presence, distribution, and fate of the organisms of interest. Here we report the development of 16S rRNA-targeted oligonucleotide probes for Dehalococcoides species. The two designed probes together encompass 28 sequences of 16S rRNA genes retrieved from the public database. Except D. ethenogenes and CBDB1, all the others are environmental clones obtained from sites contaminated with chlorinated ethenes. They are all closely related and form a unique cluster of Dehalococcoides species. In situ hybridization of probe Dhe1259t with D. ethenogenes strain 195 and two enrichment cultures demonstrated the applicability of the probe to monitoring the abundance of active Dehalococcoides species in these enrichment samples.

Yang, Yanru; Zeyer, Josef

2003-01-01

332

Use of fluorescence in situ hybridization (fish) to study chromosomal damage induced by radiation and bromodeoxyuridine in human colon cancer cells  

SciTech Connect

Although the thymidine analog radiation sensitizer bromodeoxyuridine (BrdUrd) increases radiation-induced chromosomal aberrations, it is not known whether these aberrations are uniformly distributed among chromosomes. Using fluorescence in situ hybridization, we carried out a study to test the hypothesis that BrdUrd-induced radiosensitization may be mediated by nonuniform chromsomal damage. Log phase HT29 human colon cancer cells were exposed to 10 {mu}M BrdUrd (or media alone) for one cell cycle, and the G1 cells were separated by centrifugal elutriation. Half of the control and BrdUrd samples were irradiated with 8 Gy. Cells were then incubated for 24-28 h, and metaphase spreads were prepared. Fluorescence in situ hybridization was performed using paint probes for chromosomes 1 and 4. We found that radiation induced 0.20 aberrations per chromosome in chromosome 4. Based on the ratio of the relative lengths of chromosome 1-4(1.34), it was predicted that chromosome 1 would have {approx}0.26 aberrations per chromosome. However, we observed 0.39 aberrations per chromosome 1, which was significantly greater than the predicted (p<0.001 by chi-square). Incubation with BrdUrd prior to irradiation significantly increased the aberrations found in chromosome 1 (by a factor of 1.4) and chromosome 4 (by a factor of 1.9) compared to radiation alone (p<0.001 for both chromosome 1 and 4). This study demonstrates that individual chromosomes in human colon cancer cells show significantly different rates of aberration after irradiation. Furthermore, the BrdUrd-mediated increase in radiation-induced chromosomal aberrations may not be uniform among chromosomes. 20 refs., 4 figs., 1 tab.

Wilt, S.R.; Burgess, A.C.; Lawrence, T.S. [ Univ. of Michigan Medical Center, Ann Arbor, MI (United States)] [and others

1994-11-15

333

Identification of t(17;22)(q22;q13) (COL1A1/PDGFB) in dermatofibrosarcoma protuberans by fluorescence in situ hybridization in paraffin-embedded tissue microarrays.  

PubMed

Dermatofibrosarcoma protuberans is genetically characterized by the translocation t(17;22)(q22;q13) resulting in the PDGFB/COL1A1 fusion gene. Fluorescence in situ hybridization with specific probes enables a rapid detection of this gene. In this study, the presence of the translocation t(17;22)(q22;q13) by fluorescence in situ hybridization in paraffin-embedded tissue microarrays was analyzed. Two tissue microarrays including 40 cases of dermatofibrosarcoma protuberans and 20 dermatofibromas were evaluated. Fluorescence in situ hybridization analyses were performed using a dual-color dual-fusion noncommercial probe. Clinical and histopathologic features were examined, and the association with fluorescence in situ hybridization results was assessed. A total of 29 samples of dermatofibrosarcoma protuberans and 16 of dermatofibromas were successfully evaluated. Twenty-five (86%) dermatofibrosarcoma protuberans samples were positive for the translocation, which was absent in all samples of dermatofibromas. Two of the negative dermatofibrosarcoma protuberans showed unusual, hypercellular areas with marked cytologic atypia, whereas 1 case exhibited overlap features with dermatofibroma. Tumors with fibrosarcomatous areas seemed to have a higher percentage of positive cells and the number of copies of the COL1A1/PDFGB gene. In conclusion, the COL1A1/PDGFB fusion gene was present in most of the dermatofibrosarcoma protuberans tissue samples. The detection of the translocation may be an additional diagnostic tool in cases of dermatofibrosarcoma protuberans showing nonconclusive histologic features. PMID:21111450

Segura, Sonia; Salgado, Rocío; Toll, Agustí; Martín-Ezquerra, Gemma; Yébenes, Mireia; Sáez, Amparo; Solé, Francesc; Barranco, Carlos; Umbert, Pablo; Espinet, Blanca; Pujol, Ramón M

2010-12-15

334

Differentiation of Methanosaeta concilii and Methanosarcina barkeri in Anaerobic Mesophilic Granular Sludge by Fluorescent In Situ Hybridization and Confocal Scanning Laser Microscopy†  

PubMed Central

Oligonucleotide probes, designed from genes coding for 16S rRNA, were developed to differentiate Methanosaeta concilii, Methanosarcina barkeri, and mesophilic methanogens. All M. concilii oligonucleotide probes (designated MS1, MS2, and MS5) hybridized specifically with the target DNA, but MS5 was the most specific M. concilii oligonucleotide probe. Methanosarcina barkeri oligonucleotide probes (designated MB1, MB3, and MB4) hybridized with different Methanosarcina species. The MB4 probe specifically detected Methanosarcina barkeri, and the MB3 probe detected the presence of all mesophilic Methanosarcina species. These new oligonucleotide probes facilitated the identification, localization, and quantification of the specific relative abundance of M. concilii and Methanosarcina barkeri, which play important roles in methanogenesis. The combined use of fluorescent in situ hybridization with confocal scanning laser microscopy demonstrated that anaerobic granule topography depends on granule origin and feeding. Protein-fed granules showed no layered structure with a random distribution of M. concilii. In contrast, a layered structure developed in methanol-enriched granules, where M. barkeri growth was induced in an outer layer. This outer layer was followed by a layer composed of M. concilii, with an inner core of M. concilii and other bacteria.

Rocheleau, Sylvie; Greer, Charles W.; Lawrence, John R.; Cantin, Christiane; Laramee, Louise; Guiot, Serge R.

1999-01-01

335

Differentiation of Methanosaeta concilii and Methanocarcina barkeri in anaerobic mesophilic granular sludge by fluorescent in situ hybridization and confocal scanning laser microscopy  

SciTech Connect

Oligonucleotide probes, designed from genes coding for 16S rRNA, were developed to differentiate Methanosaeta concilii, Methanosarcina barkeri, and mesophilic methanogens. All M. concilii oligonucleotide probes (designated MS1, MS2, and MS5) hybridized specifically with the target DNA, but MS5 was the most specific M. concilii oligonucleotide probe. Methanosarcina barkeri oligonucleotide probes (designated MB1, MB3, and MB4) hybridized with different Methanosarcina species. The MB4 probe specifically detected Methanosarcina barkeri, and the MB3 probe detected the presence of al mesophilic Methanosarcina species. These new oligonucleotide probes facilitated the identification, localization, and quantification of the specific relative abundance of M. concilii and Methanosarcina barkeri, which play important roles in methanogenesis. The combined use of fluorescent in situ hybridization with confocal scanning laser microscopy demonstrated that anaerobic granule topography depends on granule origin and feeding. Protein-fed granules showed no layered structure with a random distribution of M. concilii. In contrast, a layered structure developed in methanol-enriched granules, where M. barkeri growth was induced in an outer layer. This outer layer was followed by a layer composed of M. concilii, with an inner core of M. concilii and other bacteria.

Rocheleau, S.; Greer, C.W.; Cantin, C.; Laramee, L.; Guiot, S.R. [National Research Council Canada, Montreal, Quebec (Canada). Biotechnology Research Inst.; Lawrence, J.R. [National Water Research Inst., Saskatoon, Saskatchewan (Canada)

1999-05-01

336

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

PubMed Central

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.

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

1998-01-01

337

Quantification of Uncultured Ruminococcus obeum-Like Bacteria in Human Fecal Samples by Fluorescent In Situ Hybridization and Flow Cytometry Using 16S rRNA-Targeted Probes  

PubMed Central

A 16S rRNA-targeted probe was designed and validated in order to quantify the number of uncultured Ruminococcus obeum-like bacteria by fluorescent in situ hybridization (FISH). These bacteria have frequently been found in 16S ribosomal DNA clone libraries prepared from bacterial communities in the human intestine. Thirty-two reference strains from the human intestine, including a phylogenetically related strain and strains of some other Ruminococcus species, were used as negative controls and did not hybridize with the new probe. Microscopic and flow cytometric analyses revealed that a group of morphologically similar bacteria in feces did hybridize with this probe. Moreover, it was found that all hybridizing cells also hybridized with a probe specific for the Clostridium coccoides-Eubacterium rectale group, a group that includes the uncultured R. obeum-like bacteria. Quantification of the uncultured R. obeum-like bacteria and the C. coccoides-E. rectale group by flow cytometry and microscopy revealed that these groups comprised approximately 2.5 and 16% of the total community in fecal samples, respectively. The uncultured R. obeum-like bacteria comprise about 16% of the C. coccoides-E. rectale group. These results indicate that the uncultured R. obeum-like bacteria are numerically important in human feces. Statistical analysis revealed no significant difference between the microscopic and flow cytometric counts and the different feces sampling times, while a significant host-specific effect on the counts was observed. Our data demonstrate that the combination of FISH and flow cytometry is a useful approach for studying the ecology of uncultured bacteria in the human gastrointestinal tract.

Zoetendal, Erwin G.; Ben-Amor, Kaouther; Harmsen, Hermie J. M.; Schut, Frits; Akkermans, Antoon D. L.; de Vos, Willem M.

2002-01-01

338

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

Microsoft Academic Search

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

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

339

Optimization of the fluorescence in situ hybridization (FISH) technique for high detection efficiency of very small proportions of target interphase nuclei.  

PubMed

Using commercially available fluorochrome-labeled probes specific for chromosomes X, Y, 13, 18, and 21, we optimized the technical protocols for fluorescence in situ hybridization (FISH) so that the highest sensitivity and specificity were achieved. Also, we compared the optical properties of different types of fluorescent labels in an effort to develop the most efficient FISH protocol, including the determination of which types of labels are the easiest to count accurately. The lymphocytes were purified from blood of normal male and female newborns, normal male and female adults, and a trisomy 21 male adult. Male and female lymphocytes were mixed in five different combinations. For each combination, the male lymphocytes either from newborns or from adults were diluted with female lymphocytes in seven different proportions. For each of these 35 different cell mixtures, 100,000 nuclei were analyzed and scored in a blind fashion. Among the different fluorochrome-labeled probes, the highest sensitivity and specificity were achieved when SpectrumAqua CEP-Y/SpectrumOrange CEP X probe mixture, SpectrumAqua CEP-18, SpectrumOrange LSI-13, and SpectrumOrange LSI-21 were hybridized. The hybridization sensitivity and specificity were higher than 99% for the identification of chromosomes X, Y, 13, and 18, and higher than 98% for the detection of trisomy 21. The proportion of false-positive signals was under 0.005% for XY detection and lower than 0.14% for autosome detection. With these high hybridization sensitivities and specificities, the optimized FISH protocol developed in our laboratory has the potential to detect very rare events, e.g., when the proportion of cells being sought is lower than 0.01%. In other words, our protocol allows the specific detection of one male cell sunken among 10,000 female cells. PMID:11076056

Yan, J; Guilbault, E; Massé, J; Bronsard, M; DeGrandpré, P; Forest, J C; Drouin, R

2000-10-01

340

Mapping of the human thromboxane synthase gene (TBXAS1) to chromosome 7q34-q35 by two-color fluorescence in situ hybridization  

SciTech Connect

Thromboxane synthase (TS) catalyzes the conversion of the prostaglandin endoperoxide into thromboxane A[sub 2] (TxA[sub 2]), a potent vasoconstrictor and inducer of platelet aggregation. In concert with prostacyclin, TxA[sub 2] plays a pivotal role in the maintenance of hemostasis. Deficiency of platelet TS activity has been shown to result in bleeding disorders. The potent effect of TxA[sub 2] on platelet function and vascular activity suggests a possible involvement of TS in normal and pathophysiological conditions such as cardiovascular disease. To aid in establishing the correlation of TS to disease states, the authors localized the human TS gene (TBXAS1) to chromosome 7q34-q35 using dual-color fluorescence in situ hybridization. 21 refs., 2 figs.

Chase, M.B.; Baek, Seung Joon; Purtell, D.C.; Schwartz, S. (Univ. of Maryland Medical School, Baltimore, MD (United States)); Shen, Rong Fong (Univ. of Maryland Medical School, Baltimore, MD (United States) Maryland Biotechnology Institute, Baltimore, MD (United States))

1993-06-01

341

Determination of sex chromosomal constitution and chromosomal origin of drumsticks, drumstick-like structures, and other nuclear bodies in human blood cells at interphase by fluorescence in situ hybridization  

Microsoft Academic Search

The sex chromosomal constitution has been determined in various types of human leukocytes at interphase by use of fluorescence in situ hybridization with X- and\\/or Y-specific DNA probes. It is found that during aging and differentiation of myelocytes into polymorphs there is no significant change in the relative frequency of various types of male and female cells with a specific

Asit B. Mukherjee; Nasser Z. Parsa

1990-01-01

342

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

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

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

2000-01-01

343

Ploidy assessment of porcine haploid and diploid parthenogenetic embryos by fluorescent in situ hybridization detecting a chromosome 1-specific sequence, Sus scrofa Mc1 satellite DNA.  

PubMed

The aim of the present study was to examine the feasibility of fluorescent in situ hybridization (FISH) for detecting a chromosome 1-specific sequence as a means of assessing the ploidy of porcine parthenotes. In vitro-matured oocytes with the first polar body (PB) were electrically activated; some were treated with cytochalasin B to prevent second PB extrusion (1PB embryos), and the others extruded the second PB (2PB embryos). At the 2-cell stage, one and two FISH signals were detected in each nucleus of 2PB and 1PB embryos, respectively. Almost all cells of blastocysts derived from 1PB embryos retained two signals. In contrast, cells of blastocysts derived from 2PB embryos had two signals. These data demonstrate that FISH analysis allows precise ploidy assessment of porcine parthenogenetic embryos, hence providing a practical means of detecting ploidy transition during parthenogenetic embryogenesis. PMID:21157121

Sembon, Shoichiro; Fuchimoto, Dai-ichiro; Iwamoto, Masaki; Suzuki, Shun-ichi; Onishi, Akira

2010-12-09

344

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

SciTech Connect

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.

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

345

Visual demonstration of the organization of the human complement C4 and 21-hydroxylase genes by high-resolution fluorescence in situ hybridization  

SciTech Connect

We analyzed the gene organization in the complement component C4 and 21-hydroxylase (21OH) gene region of the human major histocompatibility complex using visual mapping of stretched DNA by multicolor fluorescence in situ hybridization (FISH). Normally, this region contains a duplicated 21OH-C4 gene unit are known to occur frequently. Biotin-labeled cDNA of the C4 gene and digoxigenin-labeled cDNA of the 21OH gene were hybridized to decondensed nuclei of peripheral blood lymphocytes obtained from individuals with various 21OH-C4 haplotypes. Hybridization signals of the C4 and 21OH probes were detected with fluorescein isothiocyanate (green) and rhodamine (red), respectively. Two linear green and red signal clusters were observed in each nucleus showing the normal haplotype. Gene duplication and deletion were visualized as addition and deletion of the signal cluster, respectively. The DNA types of the 21OH-C4 region determined by FISH were concordant with the results previously obtained by conventional molecular studies. Our high-resolution FISH technique is found to be useful for screening gene duplications and deletions. 14 refs., 1 fig.

Suto, Yumiko; Tokunaga, Katsushi; Watanabe, Yoshihisa; Hirai, Momoki [Univ. of Tokyo (Japan)

1996-04-15

346

Detection and Enumeration of Methanotrophs in Acidic Sphagnum Peat by 16S rRNA Fluorescence In Situ Hybridization, Including the Use of Newly Developed Oligonucleotide Probes for Methylocella palustris  

Microsoft Academic Search

Two 16S rRNA-targeted oligonucleotide probes, Mcell-1026 and Mcell-181, were developed for specific detection of the acidophilic methanotroph Methylocella palustris using fluorescence in situ hybridization (FISH). The fluorescence signal of probe Mcell-181 was enhanced by its combined application with the oligonucleotide helper probe H158. Mcell-1026 and Mcell-181, as well as 16S rRNA oligonucleotide probes with reported group specificity for either type

SVETLANA N. DEDYSH; MANIGEE DERAKSHANI; WERNER LIESACK

2001-01-01

347

Characterization of microbial community structures and their activities in single anaerobic granules by beta imaging, microsensors and fluorescence in situ hybridization.  

PubMed

The spatial distribution of microorganisms and their in situ activities in anaerobic granules were investigated by fluorescence in situ hybridization (FISH), beta imaging and microsensors. FISH results revealed a layered structure of microorganisms in the granule, where Chloroflexi was present in the outermost layer, Smithella spp. and Syntrophobacter spp. were found in a depth of ca. 100 ?m, and Archaea was restricted to the inner layer (below ca. 300 ?m from the surface). Substrate uptake patterns elucidated by beta imaging demonstrated that glucose uptake was highest at 50 ?m depth, whereas propionate uptake had a peak at 200 ?m depth. In addition, microsensor measurements revealed that acid was produced mainly at 100 ?m depth and H(2) production was detected at a depth from 100 to 200 ?m. H(2) consumption and corresponding CH(4) production were found below 200 ?m from the surface. Direct comparison of these results implied sequential degradation of complex organic compounds in anaerobic granules; Chloroflexi contributed to fermentation of organic compounds and acid production in the outermost layer, volatile fatty acids were oxidized and H(2) was produced mainly by Smithella spp. and Syntrophobacter spp. at a depth from 100 to 200 ?m, and Archaea produced CH(4) below ca. 300 ?m from the surface. PMID:22643406

Satoh, H; Tsushima, I; Miura, Y; Ito, T; Okabe, S

2012-01-01

348

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

PubMed

Aquaculture, especially shrimp farming, has played a major role in the growth of Thailand's economy in recent years, as well as in many South East Asian countries. However, the nutrient discharges from these activities have caused adverse impacts on the quality of the receiving waterways. In particular nitrogenous compounds, which may accumulate in aquaculture ponds, can be toxic to aquatic animals and cause environmental problems such as eutrophication. The mineralization process is well known, but certain aspects of the microbial ecology of nitrifiers, the microorganisms that convert ammonia to nitrate, are poorly understood. A previously reported enrichment of nitrifying bacteria (ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) from a shrimp farm inoculated in a sequencing batch reactor (SBR) was studied by molecular methods. The initial identification and partial quantification of the nitrifying bacteria (AOB and NOB) were carried out by fluorescence in situ hybridization (FISH) using previously published 16S rRNA-targeting oligonucleotide probes. The two dominant bacterial groups detected by FISH were from the Cytophaga-Flavobacterium-Bacteroides and Proteobacteria (beta subdivision) phyla. Published FISH probes for Nitrobacter and Nitrospira did not hybridize to any of the bacterial cells. Therefore it is likely that new communities of NOBs, differing from previously reported ones, exist in the enrichments. Molecular genetic techniques (cloning, sequencing, and phylogenetic analysis) targeting the 16S rRNA genes from the nitrifying enrichments were performed to identify putative AOBs and NOBs. PMID:17115448

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

2007-07-01

349

Chromosomes of Brants' whistling rat and genome conservation in the Otomyinae revealed by G-banding and fluorescence in situ hybridization.  

PubMed

Conventional G- and C-banding were used to describe the chromosomes of the whistling rat, Parotomys brantsii. This species has a diploid number of 42 chromosomes. C-banding showed that large blocks of pericentromeric heterochromatin or entirely heterochromatic short arms characterize most chromosomes. G-banding and fluorescence in situ hybridization (FISH) incorporating two laboratory mouse chromosome paints were used to compare the genomes of P. brantsii, the bush karoo rat (Otomys unisulcatus; 2n = 28), and the vlei rat (O. irroratus; 2n = 28-31). The FISH results showed that sequences corresponding to mouse chromosome 2 are conserved as a single chromosome in O. irroratus but are found on two separate chromosomes in P. brantsii and O. unisulcatus. In contrast, a mouse chromosome 6 paint showed hybridization to single chromosomes in all three species examined. When taken together, the FISH and cytogenetic results produce two important findings: (1) previously undetected homoeologies between the two Otomys species can be identified and (2) a high proportion of conserved chromosomes exists between P. brantsii and O. unisulcatus. This, in conjunction with previously reported allozyme and immunoblot data, raises serious questions about the validity of the current generic taxonomy of the Otomyinae. PMID:9465891

Rambau, R V; Harrison, W R; Elder, F F; Robinson, T J

1997-01-01

350

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

PubMed Central

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

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

2012-01-01

351

Malignant pheochromocytoma in a young adult forming the structure simulating Homer Wright rosette: differentiation from neuroblastoma on repeating fluorescence in situ hybridization.  

PubMed

A peculiar adrenal tumor was analyzed using immunohistochemistry, electron microscopy, and fluorescence in situ hybridization (FISH) with multiple bacterial artificial chromosome (BAC) probes. The patient was a 34-year-old woman with a mass above the left kidney and multiple metastases. Her serum and urine dopamine level were elevated, and a diagnosis of malignant pheochromocytoma was made. The patient died approximately 3 years after her first visit. On post-mortem an adrenal tumor composed of small round cells forming Homer Wright rosette-like structures, a feature rarely observed in pheochromocytoma, was found. Immunohistochemistry was positive for chromogranin A and synaptophysin, and negative for cytokeratin, vimentin and neurofilaments. Because these results did not rule out a diagnosis of neuroblastoma, the tumor was further characterized on FISH with multiple BAC probes for loci known to be altered in neuroblastoma or pheochromocytoma, according to information in the literature that was for the most part obtained using comparative genomic hybridization. FISH demonstrated loss of heterozygosity at 11p, and gains at 16p, 19p, and 19q, a profile that favored a diagnosis of malignant pheochromocytoma over neuroblastoma. This case demonstrates that repeating FISH is useful for differential diagnosis. PMID:18705773

Mori, Hiroki; Nagata, Masao; Nishijima, Nariaki; Nagura, Kiyoko; Igarashi, Hisaki; Hamazaki, Minoru; Ozono, Seiichiro; Sugimura, Haruhiko

2008-08-01

352

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

SciTech Connect

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.

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

2007-12-01

353

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

Microsoft Academic Search

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

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

354

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

SciTech Connect

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.

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

1993-07-01

355

Alignment of the genomes of Brachypodium distachyon and temperate cereals and grasses using bacterial artificial chromosome landing with fluorescence in situ hybridization.  

PubMed

As part of an initiative to develop Brachypodium distachyon as a genomic "bridge" species between rice and the temperate cereals and grasses, a BAC library has been constructed for the two diploid (2n = 2x = 10) genotypes, ABR1 and ABR5. The library consists of 9100 clones, with an approximate average insert size of 88 kb, representing 2.22 genome equivalents. To validate the usefulness of this species for comparative genomics and gene discovery in its larger genome relatives, the library was screened by PCR using primers designed on previously mapped rice and Poaceae sequences. Screening indicated a degree of synteny between these species and B. distachyon, which was confirmed by fluorescent in situ hybridization of the marker-selected BACs (BAC landing) to the 10 chromosome arms of the karyotype, with most of the BACs hybridizing as single loci on known chromosomes. Contiguous BACs colocalized on individual chromosomes, thereby confirming the conservation of genome synteny and proving that B. distachyon has utility as a temperate grass model species alternative to rice. PMID:16489232

Hasterok, Robert; Marasek, Agnieszka; Donnison, Iain S; Armstead, Ian; Thomas, Ann; King, Ian P; Wolny, Elzbieta; Idziak, Dominika; Draper, John; Jenkins, Glyn

2006-02-19

356

Mapping chromosomal homology between humans and the black-handed spider monkey by fluorescence in situ hybridization  

Microsoft Academic Search

We hybridized human chromosome-specific DNA probes to metaphases of the New World monkey Ateles geoffroyito map the chromosomal homology between these two species. In the haploid Ateles geoffroyi karyotype the total number of signals was 51 for the 22 human autosomal probes used. Compared with Old World monkeys, the number of translocations found in the black-handed spider monkey karyotype was

M. A. Morescalchi; W. Schempp; S. Consigliere; F. Bigoni; J. Wienberg; R. Stanyon

1997-01-01

357

Analysis and direct quantification of Saccharomyces cerevisiae and Hanseniaspora guilliermondii populations during alcoholic fermentation by fluorescence in situ hybridization, flow cytometry and quantitative PCR.  

PubMed

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 methods, such as fluorescence in situ hybridization (FISH) and quantitative PCR (QPCR). Species-specific FISH probes labeled with fluorescein (FITC) were used to directly hybridize Sc and Hg cells from single and mixed cultures that were enumerated by epifluorescence microscopy and flow cytometry. Static and agitated fermentations were performed in synthetic grape juice and cell density as well as sugar consumption and ethanol production were determined throughout fermentations. Cell density values obtained by FISH and QPCR revealed the presence of high populations (10?-10? cells/ml) of Sc and Hg throughout fermentations. Plate counts of both species did not show significant differences with culture-independent results in pure cultures. However, during mixed fermentations Hg lost its culturability after 4-6 days, while Sc remained culturable (about 10? cells/ml) throughout the entire fermentation (up to 10 days). The rRNA content of cells during mixed fermentations was also analyzed by flow cytometry in combination with FISH probes. The fluorescence intensity conferred by the species-specific FISH probes was considerably lower for Hg than for Sc. Moreover, the rRNA content of Hg cells, conversely to Sc cells, remained almost unchanged after boiling, which showed that rRNA stability is species-dependent. PMID:21925033

Andorra, Imma; Monteiro, Margarida; Esteve-Zarzoso, Braulio; Albergaria, Helena; Mas, Albert

2011-08-12

358

Frequency of aneuploidy in in vitro–matured MII oocytes and corresponding first polar bodies in two dairy cattle ( Bos taurus) breeds as determined by dual-color fluorescent in situ hybridization  

Microsoft Academic Search

The current study was undertaken to investigate the aneuploidy rates in in vitro–matured meiosis II (MII) oocytes and corresponding first polar bodies in two dairy cattle (Bos taurus) breeds by using dual-color fluorescent in situ hybridization (FISH). A total of 159 and 144 in vitro–matured MII oocytes of the Italian Friesian and Italian Brown breeds, respectively, were obtained according to

D. Nicodemo; A. Pauciullo; G. Cosenza; V. Peretti; A. Perucatti; G. P. Di Meo; L. Ramunno; L. Iannuzzi; J. Rubes; D. Di Berardino

2010-01-01

359

The genes for the {alpha}-type HC3 (PMSA2) and {beta}-type HC5 (PMSB1) subunits of human proteasomes map to chromosomes 6q27 and 7p12-p13 by fluorescence in situ hybridization  

SciTech Connect

The authors have determined the locations of the genes for the two subunits, HC3 and HC5, by fluorescence in situ hybridization (FISH). Chromosome spreads were obtained from phytohemagglutinin-stimulated blood lymphocytes of a healthy donor after thymidine synchronization and bromodeoxyuridine incorporation by the method of Takahashi et al. Genomic DNA fragments of HC3 (4.3 kb, including exons 3, 4, and 5) and HC5 (7.5 kb including exons 1 and 2) (11) were labeled with biotin-16-dUTP by nick-translation. In situ hybridization was performed according to Lichter et al. in the presence of COT-1 DNA as a competitor. Hybridized probe was detected with FITC-conjugated avidin without further signal amplification. Comparison of the fluorescence signals and the banding patterns of the chromosomes indicated that the HC3 and HC5 genes were located on chromosome band 6q27 and 7p12-p13, respectively.

Okumura, Katsuzumi; Nogami, Masahiro; Taguchi, Hiroshi [Mie Univ., Tsu (Japan)] [and others

1995-05-20

360

Double-color fluorescence in situ hybridization (FISH) for the detection of Bacillus anthracis spores in environmental samples with a novel permeabilization protocol.  

PubMed

For anti-bioterrorism measures against the use of Bacillus anthracis, a double-color fluorescence in situ hybridization (FISH) is proposed, for the rapid and specific detection of B. anthracis. The probes were designed based on the differences in 16S and 23S rRNA genes of B. cereus group. A new permeabilization protocol was developed to enhance the permeability of FISH probes into B. anthracis spores. The highest detection rate (90.8 ± 0.69) of B. anthracis spores by FISH was obtained with successive incubation steps with 50% ethanol at 80 °C, a mixture of SDS/DTT solution (10mg/ml SDS, 50mM DTT) at 65 °C and finally in a lysozyme solution (20mg/ml) at 37 °C for 30 min each. This protocol was evaluated for the detection of B. anthracis spores in soil and air samples after adding formalin-fixed spores at different densities. The results have proven the success of double-color FISH in detecting B. anthracis spores in air samples in the range of 10(3) spores/m(3) and above. Conversely, for detecting B. anthracis spores in a soil sample, the lowest detection limit was found to be 10(7) spores/g dry soils. These results confirm the applicability of the developed permeabilization protocol, combined with the double-color FISH technique in specific detection of B. anthracis in soil and air samples. PMID:23523967

Weerasekara, M L M A W; Ryuda, Noriko; Miyamoto, Hiroshi; Okumura, Toru; Ueno, Daisuke; Inoue, Koichi; Someya, Takashi

2013-03-20

361

DNA\\/DNA in situ hybridization with enzyme linked probes  

Microsoft Academic Search

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

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

1987-01-01

362

Numerical chromosome alterations in colorectal carcinomas detected by fluorescence in situ hybridization. Relationship to 17p and 18q allelic losses.  

PubMed

This study concerns DNA ploidy, numerical changes of chromosomes 7, 8, 10, 17 and 18, and allelic losses at chromosomes 17p13.3 (flanking the p53 gene) and 18q21 (location of the DCC gene) in 31 freshly resected colorectal tumours. Cytological smears were used to determine DNA ploidy by image analysis, and chromosome numbers by fluorescence in situ hybridization (FISH) using chromosome-specific pericentromeric alpha-satellite DNA probes. Allelic losses were assessed by Southern blotting and by the polymerase chain reaction loss of heterozygosity method. Approximately 50% of the tumours were aneuploid. There was heterogeneity with respect to chromosome numbers, but gains and losses of chromosomes, or both, were detected in all carcinomas examined, including 10 that were nonaneuploid by image analysis. Trisomy 7 was found in 74% of the tumours, and monosomy of chromosome 18 in 32%. Allelic loss at chromosome 17p13.3 was evident in 13 of 26 informative cases, and only one case exhibited monosomy 17. In comparison monosomy 18 was found in 10 cases; 7 of them corresponded to approximately half of the cases with allelic loss within the DCC gene, and the other three were noninformative. These findings indicate that the loss of one chromosome 18 is an important mechanism producing allelic deletion of the DCC gene in colorectal carcinomas. Our data also suggest that monosomy 18 is a useful indicator for studying colorectal cancer progression on a cell by cell basis. PMID:8764933

Ooi, A; Huang, C D; Mai, M; Nakanishi, I

1996-07-01

363

Deletions of the region 17p11-13 in advanced melanoma revealed by cytogenetic analysis and fluorescence in situ hybridization  

PubMed Central

The significance of the p53 tumour-suppressor gene in the oncogenesis of a variety of malignant tumours has been demonstrated over recent years. However, the role of p53 in human malignant melanoma is still unclear. Therefore, we investigated melanoma metastases from 11 patients cytogenetically and with fluorescence in situ hybridization (FISH) after short-term culture, employing a p53 region-specific probe for 17p13.1 and a probe detecting the centromere of chromosome 17. Furthermore, paraffin-embedded tissue samples from nine of these patients were investigated immunohistochemically for expression of the p53 protein. Deletions of the short arm of chromosome 17 were seen in six melanomas in cytogenetic analysis. With FISH, three malignant melanomas had clones with only one p53-allele and an additional four malignant melanomas showed a reduced number of signals at the p53 tumour-suppressor gene locus compared with signals for the centromeric region of chromosome 17. This was confirmed by immunohistochemistry. Our results suggest that the 17p11–13 region is frequently deleted in malignant melanomas and that p53 or other genes located on this band might contribute to the malignant potential of advanced melanoma. © 1999 Cancer Research Campaign

Okamoto, I; Pirc-Danoewinata, H; Ackermann, J; Drach, J; Wadl, H Schlagbauer; Jansen, B; Wolff, K; Pehamberger, H; Marosi, C

1999-01-01

364

Low Level of Her-2 Locus Amplification by Fluorescent In Situ Hybridization Does Not Correlate with Her-2 Protein Overexpression by Immunohistochemistry in Barrett's Esophagus  

PubMed Central

An accurate evaluation of the Her-2 status has important prognostic and therapeutic implications in many carcinomas. The aim of the study was to correlate Her-2 locus (17q11.2) amplification and chromosome 17 gains as assessed by fluorescent in situ hybridization (FISH) with Her-2 protein overexpression by immunohistochemistry (IHC) in patients with Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). We analyzed 34 patients with Her-2 amplification and/or chromosome 17gains using FISH on brush cytology specimens. Seven patients (21%) showed high Her-2 locus amplification (Her-2: Cep17 ? 5?:?1), 5 (15%) showed low Her-2 locus amplification (Her-2: Cep17 ? 2 < 5?:?1), and 22 (65%) displayed gains of chromosome 17 only. Further, we confirmed Her-2 amplification on corresponding biopsies that were taken at the same occasion as the cytologybrushings. Then, we compared the FISH results with IHC data obtained from the corresponding biopsies and showed that low level of Her-2 amplification does not correlate with Her-2 protein overexpression (score +3/+2; P = 1), in contrast to the high amplification level (P = .001). Thus, in our population of BE and EAC patients, low level of Her-2 amplification does not result in detectable level of Her-2 protein as assessed by IHC.

Rygiel, Agnieszka M.; Milano, Francesca; ten Kate, Fiebo J.; Bergman, Jacques J. G. H. M.; Krishnadath, Kausillia K.

2010-01-01

365

Patterns of BCR/ABL Gene Rearrangements in Chronic Myeloid Leukemia with Complex t(9;22) Using Fluorescence In Situ Hybridization (FISH).  

PubMed

Chronic myeloid leukemia (CML) is characterized by the reciprocal translocation t(9;22)(q34;q11.2) which fuses the ABL1 oncogene on chromosome 9 with the BCR gene on chromosome 22. It is the BCR/ABL protein that drives the neoplasm and the ABL/BCR is not necessary for the disease. In the majority of CML cases, the BCR/ABL fusion gene is cytogenetically recognizable as a small derivative chromosome 22(der 22), which is known as the Philadelphia (Ph) chromosome. However, approximately 2-10% of patients with CML involve cryptic or complex variant translocations with deletions on the der(9) and/or der(22) occuring in roughly 10-15% of CML cases. Fluorescence in situ hybridization (FISH) analysis can help identify deletions and complex or cryptic rearrangements. Various BCR/ABL FISH probes are available, which include dual color single fusion, dual color extra signal (ES), dual color dual fusion and tri color dual fusion probes. To test the utility of these probes, six patients diagnosed with CML carrying different complex variant Ph translocations were studied by G-banding and FISH analysis using the BCR/ABL ES, BCR/ABL dual color dual fusion, and BCR/ABL tricolor probes. There are differences among the probes in their ability to detect variant rearrangements, with or without accompanying chromoso me 9 and/or 22 deletions, and low level disease. PMID:22421568

Esan, Olukemi A; Senft, Jamie R; Wenger, Sharon L

2012-01-01

366

Fluorescence in situ hybridization gene amplification analysis of EGFR and HER2 in patients with malignant salivary gland tumors treated with lapatinib  

PubMed Central

Aim Gene amplification status of the epidermal growth factor receptor (EGFR) and the human epidermal growth factor receptor 2 (HER2) were analyzed and correlated with clinical outcome in patients with progressive malignant salivary glands tumors (MSGT) treated with the dual EGFR/Her2 tyrosine kinase inhibitor lapatinib Methods Fluorescence in situ hybridization (FISH) analysis for both EGFR and HER2 gene amplification was performed successfully in the archival tumor specimens of 20 patients with adenoid cystic carcinomas (ACC) and 17 patients with non-ACC, all treated with lapatinib. Results For ACC, no EGFR or HER2 amplifications were detected. For non-ACC, no EGFR gene amplifications were detected but 3 patients (18%) were HER2 amplified and all had stained 3+ for both EGFR and HER2 by immunohistochemistry (IHC) in their archival specimens. Two of these patients had time-to-progression (TTP) durations of 8.3 months and 18.4 months respectively. Interestingly, patients with low and high HER2/chromosome-specific centromeric enumeration probe (CEP) 17 ratio had a prolonged TTP than those with moderate ratios for both ACC and non-ACC subtypes. Conclusions HER2 to CEP17 FISH ratio may predict which patients with MSGT have an increased likelihood to benefit from lapatinib. The finding of HER2:CEP17 ratio as a predictive marker of efficacy to lapatinib warrants further investigation.

Vidal, Laura; Tsao, Ming S; Pond, Gregory R; Cohen, Ezra EW; Cohen, Roger B; Chen, Eric X; Agulnik, Mark; Hotte, Sebastien; Winquist, Eric; Laurie, Scott; Neil Hayes, D; Ho, James; Dancey, Janet; Siu, Lillian L.

2009-01-01

367

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

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.

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

1994-09-01

368

Variations of Bacterial Populations in Human Feces Measured by Fluorescent In Situ Hybridization with Group-Specific 16S rRNA-Targeted Oligonucleotide Probes  

PubMed Central

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 for the genera Streptococcus and Lactococcus, and the final probe was designed for the species of the Clostridium coccoides-Eubacterium rectale group. The temperature of dissociation of each of the probes was determined. The specificities of the probes for a collection of target and reference organisms were tested by dot blot hybridization and fluorescent in situ hybridization (FISH). The new probes were used in initial FISH experiments to enumerate human fecal bacteria. The combination of the two Bacteroides-specific probes detected a mean of 5.4 × 1010 cells per g (dry weight) of feces; the Clostridium coccoides-Eubacterium rectale group-specific probe detected a mean of 7.2 × 1010 cells per g (dry weight) of feces. The Clostridium histolyticum, Clostridium lituseburense, and Streptococcus-Lactococcus group-specific probes detected only numbers of cells ranging from 1 × 107 to 7 × 108 per g (dry weight) of feces. Three of the newly designed probes and three additional probes were used in further FISH experiments to study the fecal flora composition of nine volunteers over a period of 8 months. The combination of probes was able to detect at least two-thirds of the fecal flora. The normal biological variations within the fecal populations of the volunteers were determined and indicated that these variations should be considered when evaluating the effects of agents modulating the flora.

Franks, Alison H.; Harmsen, Hermie J. M.; Raangs, Gerwin C.; Jansen, Gijsbert J.; Schut, Frits; Welling, Gjalt W.

1998-01-01

369

Vertical distribution of Archaea and Bacteria in a meromictic lake as determined by fluorescent in situ hybridization.  

PubMed

The prokaryotic cells distribution in the water column of the coastal saline meromictic Lake Faro (Messina, Italy) was investigated by microscopic counting techniques. Water samples were collected at a central station from the surface to the bottom, when waters were characterized by a marked stratification. A "red-water" layer, caused by a dense growth of photosynthetic sulfur bacteria, was present at a depth of 15 m, defining a transition area between oxic (mixolimnion) and anoxic (monimolimnion) layers. Fluorescently labeled 16S rRNA oligonucleotide, group-specific probes were used to determine the abundance of Bacteria and Archaea, and their subgroups, Green Sulfur Bacteria (GSB), Sulfate Reducing Bacteria (SRB), Cyanobacteria and Chromatium okenii, and Crenarchaeota and Euryarchaeota, as key elements of the microbial community. Bacteria decreased from surface to bottom, while Archaea increased with depth and reached the maximum value at 30 m, where they outnumbered the Bacteria. Bacteria and picophytoplankton prevailed in the mixolimnion. At the chemocline high numbers of prokaryotic cells were present, mainly represented by Cyanobacteria, Chromatium okenii and Euryarchaeota. GSB, SRB, and Crenarchaeota prevailed below the chemocline. Although Archaea constitute a minor fraction of microbial community, they could represent active contributors to the meromictic Lake Faro ecosystem. PMID:22006072

Lentini, Valeria; Gugliandolo, Concetta; Maugeri, Teresa L

2011-10-18

370

Characterization of Brazilian accessions of wild Arachis species of section Arachis (Fabaceae) using heterochromatin detection and fluorescence in situ hybridization (FISH).  

PubMed

The cytogenetic characterization of Arachis species is useful for assessing the genomes present in this genus, for establishing the relationship among their representatives and for understanding the variability in the available germplasm. In this study, we used fluorescence in situ hybridization (FISH) to examine the distribution patterns of heterochromatin and rDNA genes in 12 Brazilian accessions of five species of the taxonomic section Arachis. The heterochromatic pattern varied considerably among the species: complements with centromeric bands in all of the chromosomes (A. hoehnei) and complements completely devoid of heterochromatin (A. gregoryi, A. magna) were observed. The number of 45S rDNA loci ranged from two (A. gregoryi) to eight (A. glandulifera), while the number of 5S rDNA loci was more conserved and varied from two (in most species) to four (A. hoehnei). In some species one pair of 5S rDNA loci was observed adjacent to 45S rDNA loci. The chromosomal markers revealed polymorphism in the three species with more than one accession (A. gregoryi, A. magna and A. valida) that were tested. The previous genome assignment for each of the species studied was confirmed, except for A. hoehnei. The intraspecific variability observed here suggests that an exhaustive cytogenetic and taxonomic analysis is still needed for some Arachis species. PMID:24130444

Custódio, Adriana Regina; Seijo, Guillermo; Valls, José Francisco Montenegro

2013-08-30

371

Characterization of Brazilian accessions of wild Arachis species of section Arachis (Fabaceae) using heterochromatin detection and fluorescence in situ hybridization (FISH)  

PubMed Central

The cytogenetic characterization of Arachis species is useful for assessing the genomes present in this genus, for establishing the relationship among their representatives and for understanding the variability in the available germplasm. In this study, we used fluorescence in situ hybridization (FISH) to examine the distribution patterns of heterochromatin and rDNA genes in 12 Brazilian accessions of five species of the taxonomic section Arachis. The heterochromatic pattern varied considerably among the species: complements with centromeric bands in all of the chromosomes (A. hoehnei) and complements completely devoid of heterochromatin (A. gregoryi, A. magna) were observed. The number of 45S rDNA loci ranged from two (A. gregoryi) to eight (A. glandulifera), while the number of 5S rDNA loci was more conserved and varied from two (in most species) to four (A. hoehnei). In some species one pair of 5S rDNA loci was observed adjacent to 45S rDNA loci. The chromosomal markers revealed polymorphism in the three species with more than one accession (A. gregoryi, A. magna and A. valida) that were tested. The previous genome assignment for each of the species studied was confirmed, except for A. hoehnei. The intraspecific variability observed here suggests that an exhaustive cytogenetic and taxonomic analysis is still needed for some Arachis species.

Custodio, Adriana Regina; Seijo, Guillermo; Valls, Jose Francisco Montenegro

2013-01-01

372

Improved fluorescent in situ hybridization method for detection of bacteria from activated sludge and river water by using DNA molecular beacons and flow cytometry.  

PubMed

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 data presented here demonstrate that DNA MBs are suitable for at least some FISH applications in complex samples, providing superior discriminatory power compared to that of corresponding linear DNA-FISH probes. The use of DNA MBs for flow cytometric detection of Pseudomonas putida resulted in approximately double the signal-to-noise ratio of standard linear DNA probes when using laboratory-grown cultures and yielded improved discrimination of target cells in spiked environmental samples, without a need for separate washing steps. DNA MBs were also effective for the detection and cell sorting of both spiked and indigenous P. putida from activated sludge and river water samples. The use of DNA MB-FISH presents another increase in sensitivity, allowing the detection of bacteria in environmental samples without the expense of PNA MBs or multilaser flow cytometry. PMID:17277208

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

2007-02-02

373

Non-target sites with single nucleotide insertions or deletions are frequently found in 16S rRNA sequences and can lead to false positives in fluorescence in situ hybridization (FISH).  

PubMed

Fluorescence in situ hybridization (FISH) has impacted profoundly on our knowledge of the in situ ecophysiology and biodiversity of bacteria in natural communities. However, it has many technical challenges including the possibility of false positives from the binding of probes to non-target rRNA sequences. We show here that probe target sites containing single-base insertions or deletions can lead to false FISH positives, the result of hybridization with a bulge around the missing base. Experimental and in silico data suggest this situation occurs at a surprisingly high frequency. The existence of such sites is not currently considered during most FISH probe design processes. We describe software to identify potential non-target sites resulting from single-base insertions or deletions in rRNA sequences. This software also provides an estimate of the FISH probe hybridization efficiency to these sites. PMID:20649647

McIlroy, Simon J; Tillett, Daniel; Petrovski, Steve; Seviour, Robert J

2011-01-01

374

Prenatal diagnosis using interphase fluorescence in situ hybridization (FISH): 2-year multi-center retrospective study and review of the literature.  

PubMed

Since 1993, the position of the American College of Medical Genetics (ACMG) has been that prenatal interphase fluorescence in situ hybridization (FISH) is investigational. In 1997, the FDA cleared the AneuVysion assay (Vysis, Inc.) to enumerate chromosomes 13, 18, 21, X and Y for prenatal diagnosis. Data is presented from the clinical trial that led to regulatory clearance (1379 pregnancies) and from retrospective case review on 5197 new pregnancies. These studies demonstrated an extremely high concordance rate between FISH and standard cytogenetics (99.8%) for specific abnormalities that the AneuVysion assay is designed to detect. In 29 039 informative testing events (6576 new and 22 463 cases in the literature) only one false positive (false positive rate = 0.003%) and seven false negative results (false negative rate = 0.024%) occurred. A historical review of all known accounts of specimens tested is presented (29 039 using AneuVysion and 18 275 specimens tested with other probes). These performance characteristics support a prenatal management strategy that includes utilization of FISH for prenatal testing when a diagnosis of aneuploidy of chromosome 13, 18, 21, X or Y is highly suspected by virtue of maternal age, positive maternal serum biochemical screening or abnormal ultrasound findings. PMID:11288120

Tepperberg, J; Pettenati, M J; Rao, P N; Lese, C M; Rita, D; Wyandt, H; Gersen, S; White, B; Schoonmaker, M M

2001-04-01

375

A Straightforward DOPE (Double Labeling of Oligonucleotide Probes)-FISH (Fluorescence In Situ Hybridization) Method for Simultaneous Multicolor Detection of Six Microbial Populations  

PubMed Central

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is an essential tool for the cultivation-independent identification of microbes within environmental and clinical samples. However, one of the major constraints of conventional FISH is the very limited number of different target organisms that can be detected simultaneously with standard epifluorescence or confocal laser scanning microscopy. Recently, this limitation has been overcome via an elegant approach termed combinatorial labeling and spectral imaging FISH (CLASI-FISH) (23). This technique, however, suffers compared to conventional FISH from an inherent loss in sensitivity and potential probe binding biases caused by the competition of two differentially labeled oligonucleotide probes for the same target site. Here we demonstrate that the application of multicolored, double-labeled oligonucleotide probes enables the simultaneous detection of up to six microbial target populations in a straightforward and robust manner with higher sensitivity and less bias. Thus, this newly developed technique should be an attractive option for all researchers interested in applying conventional FISH methods for the study of microbial communities.

Behnam, Faris; Vilcinskas, Andreas; Stoecker, Kilian

2012-01-01

376

Peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) assay for specific detection of Mycobacterium immunogenum and DNA-FISH assay for analysis of pseudomonads in metalworking fluids and sputum  

Microsoft Academic Search

Specific and rapid detection and quantification of mycobacteria in contaminated metalworking fluid (MWF) are problematic due to complexity of the matrix and heavy background co-occurring microflora. Furthermore, cross-reactivity among neighboring species of Mycobacterium makes species differentiation difficult for this genus. Here, we report for the first time a species-specific peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) method for Mycobacterium immunogenum,

Suresh B. Selvaraju; Renuka Kapoor; Jagjit S. Yadav

2008-01-01

377

Cancer cells with p53 deletion detected by fluorescent in situ hybridization in peritoneal drainage fluid is correlated with early peritoneal seeding in resectable pancreatic cancer  

PubMed Central

Purpose Free tumor cells in peritoneal fluid in patients with pancreatic cancer may have prognostic significance but there are few reports on methods for the effective detection of free tumor cells. The aims of this study were to identify free cancer cells in peritoneal fluid with fluorescent in situ hybridization (FISH) technique and to investigate its prognostic significance. Methods Twenty-eight patients with resectable pancreatic cancer who underwent surgical resection were included. Peritoneal washing and peritoneal drainage fluid were examined by FISH for p53 deletion. Results Among the study subjects, the R0 resection rate was 75%. None of the patients had positive cytology with Papanicolaou's method. p53 deletion was detected in 9 peritoneal washings (32.1%) and in 5 peritoneal drainage fluids (17.9%). After a median of 18 months of follow-up, 25 patients (89.3%) experienced recurrence and 14 patients (50.0%) had peritoneal seeding. Patients with p53 deletion detected in the peritoneal drainage fluid had positive radial margin (60.0% vs. 17.4%, P = 0.046) more frequently and a lower peritoneal metastasis free survival (median, 11.1 months vs. 30.3 months; P = 0.030). Curative resection (P < 0.001) and p53 deletion in peritoneal drainage fluid (P = 0.030) were independent risk factors of peritoneal metastasis free survival after multivariate analysis. Conclusion FISH technique detects free cancer cells with higher sensitivity compared to Papanicolaou's method. p53 deletion detected in peritoneal drainage fluid is correlated with positive radial resection margin and results in early peritoneal seeding. Patients with p53 deletion in peritoneal drainage fluid need more aggressive adjuvant treatment.

Kang, Mee Joo; Han, Sung-Sik; Park, Jae Woo; Kwon, Wooil; Chang, Ye Rim

2013-01-01

378

Possibilities and limitations of fluorescence in situ hybridization technique in retrospective detection of low dose radiation exposure in post-chernobyl human cohorts.  

PubMed

Cytogenetic analysis using the fluorescence in situ hybridisation (FISH) technique was performed late time after the Chernobyl accident in groups of liquidators, evacuees from 30 km exclusive zone, residents of radioactively contaminated areas and control donors age-matched to exposed persons. Stable and unstable chromosome type exchanges were recorded using a hybrid conventional-PAINT nomenclature. The mean yield of stable chromosome exchanges in liquidators did not correlate with registered radiation doses but had a clear negative dependence on the duration of liquidators' staying in Chernobyl zone, that was in a good agreement with early data based on conventional dicentrics plus rings analysis. The overspontaneous excess for stable chromosome exchange level appeared to be higher in evacuees 16-40 years old than that of senior persons, whereas no age-dependent difference occurred for initially induced dicentrics plus rings yields in this cohort. The stable chromosome exchange yield, as well as combined yield of dicentrics plus rings and potentially unstable incomplete translocations in residents of radioactively contaminated areas showed a reasonable positive correlation with levels of 137Cs contamination. The observed yields of stable chromosome exchanges in all three exposed groups appeared to be somewhat lower than those of expected from unstable exchange-based doses which were referred to an in vitro dose response of stable exchanges outcome in human lymphocytes. Thus, FISH analysis can be successfully applied for qualitative cytogenetic indication of past and chronic radiation exposure to low doses but further refinement of FISH-based system for quantitative dose assessment is still required. Some practical approaches of solving this task are discussed. PMID:16396328

Maznyk, N A; Vinnikov, V A

379

Assessment of Fluorescent In Situ Hybridization and PCR-Based Methods for Rapid Identification of Burkholderia cepacia Complex Organisms Directly from Sputum Samples? †  

PubMed Central

Several species within the Burkholderia cepacia complex (BCC) have emerged as significant opportunistic pathogens of patients with cystic fibrosis (CF). BCC infection is typically associated with a poor clinical prognosis and decreased survival. These factors, combined with the existence of highly transmissible epidemic strains, have resulted in strict segregation of BCC- and non-BCC-infected patients to minimize cross infection. Accurate and rapid diagnosis of infections is essential to enable appropriate patient management. However, the rapidly evolving taxonomy of BCC poses a considerable challenge to diagnostics. In the present study, we assessed a commercially available fluorescent in situ hybridization (FISH) assay (seaFAST Cystic Fibrosis I kit) and a novel rRNA gene-based PCR assay for the rapid identification of BCC-positive sputa, irrespective of the BCC species. We report that, while the FISH assay fails to identify all BCC species, it does identify the majority of species, including the two most clinically relevant species, B. multivorans and B. cenocepacia. The sensitivity of the assay applied to sputum was limited by nonspecific background fluorescence. While sputum processing was optimized to minimize background, the resulting sensitivity for BCC detection was 8 × 105 CFU/ml. In contrast, the novel PCR assay reported herein exhibits 100% sensitivity and specificity for all BCC species and can detect 104 CFU/ml when applied to sputum. This novel rRNA gene-based assay is currently the most sensitive BCC-specific PCR assay for the detection of BCC direct from clinical samples and as such is a valuable addition to the field of BCC diagnostics.

Brown, A. R.; Govan, J. R. W.

2007-01-01

380

Nucleic acid in-situ hybridization detection of infectious agents  

NASA Astrophysics Data System (ADS)

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