scholarly journals mathFISH, a Web Tool That Uses Thermodynamics-Based Mathematical Models forIn SilicoEvaluation of Oligonucleotide Probes for FluorescenceIn SituHybridization

2010 ◽  
Vol 77 (3) ◽  
pp. 1118-1122 ◽  
Author(s):  
L. Safak Yilmaz ◽  
Shreyas Parnerkar ◽  
Daniel R. Noguera
Keyword(s):  
Mathematical Models ◽  
Oligonucleotide Probe ◽  
Theoretical Framework ◽  
Probe Design ◽  
Oligonucleotide Probes ◽  
Web Tool ◽  
Web Based ◽  
Probe Target ◽  
Probability Of Success

ABSTRACTMathematical models of RNA-targeted fluorescencein situhybridization (FISH) for perfectly matched and mismatched probe/target pairs are organized and automated in web-based mathFISH (http://mathfish.cee.wisc.edu). Offering the users up-to-date knowledge of hybridization thermodynamics within a theoretical framework, mathFISH is expected to maximize the probability of success during oligonucleotide probe design.

scholarly journals Empirical Establishment of Oligonucleotide Probe Design Criteria†

2005 ◽  
Vol 71 (7) ◽  
pp. 3753-3760 ◽  
Author(s):  
Zhili He ◽  
Liyou Wu ◽  
Xingyuan Li ◽  
Matthew W. Fields ◽  
Jizhong Zhou
Keyword(s):  
Free Energy ◽  
Oligonucleotide Probe ◽  
Binding Free Energy ◽  
Perfect Match ◽  
Oligonucleotide Array ◽  
Probe Design ◽  
Oligonucleotide Probes ◽  
Cross Hybridization ◽  
Probe Target ◽  
Sequence Identity

ABSTRACT Criteria for the design of gene-specific and group-specific oligonucleotide probes were established experimentally via an oligonucleotide array that contained perfect match (PM) and mismatch probes (50-mers and 70-mers) based upon four genes. The effects of probe-target identity, continuous stretch, mismatch position, and hybridization free energy on specificity were tested. Little hybridization was observed at a probe-target identity of ≤85% for both 50-mer and 70-mer probes. PM signal intensities (33 to 48%) were detected at a probe-target identity of 94% for 50-mer oligonucleotides and 43 to 55% for 70-mer probes at a probe-target identity of 96%. When the effects of sequence identity and continuous stretch were considered independently, a stretch probe (>15 bases) contributed an additional 9% of the PM signal intensity compared to a nonstretch probe (≤15 bases) at the same identity level. Cross-hybridization increased as the length of continuous stretch increased. A 35-base stretch for 50-mer probes or a 50-base stretch for 70-mer probes had approximately 55% of the PM signal. Little cross-hybridization was observed for probes with a minimal binding free energy greater than −30 kcal/mol for 50-mer probes or −40 kcal/mol for 70-mer probes. Based on the experimental results, a set of criteria are suggested for the design of gene-specific and group-specific oligonucleotide probes, and the experimentally established criteria should provide valuable information for new software and algorithms for microarray-based studies.

Differential sensitivity of 16S rRNA targeted oligonucleotide probes used for fluorescence in situ hybridization is a result of ribosomal higher order structure

1996 ◽  
Vol 42 (10) ◽  
pp. 1061-1071 ◽  
Author(s):  
Marc E. Frischer ◽  
Peter J. Floriani ◽  
Sandra A. Nierzwicki-Bauer
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
Fluorescence In Situ Hybridization ◽  
Differential Sensitivity ◽  
Probe Design ◽  
Oligonucleotide Probes ◽  
Whole Cells ◽  
Gene Probes ◽  
Wide Range

The use of 16S rRNA targeted gene probes for the direct analysis of microbial communities has revolutionized the field of microbial ecology, yet a comprehensive approach for the design of such probes does not exist. The development of 16S rRNA targeted oligonucleotide probes for use with fluorescence in situ hybridization (FISH) procedures has been especially difficult as a result of the complex nature of the rRNA target molecule. In this study a systematic comparison of 16S rRNA targeted oligonucleotide gene probes was conducted to determine if target location influences the hybridization efficiency of oligonucleotide probes when used with in situ hybridization protocols for the detection of whole microbial cells. Five unique universal 12-mer oligonucleotide sequences, located at different regions of the 16S rRNA molecule, were identified by a computer-aided sequence analysis of over 1000 partial and complete 16S rRNA sequences. The complements of these oligomeric sequences were chemically synthesized for use as probes and end labeled with either [γ-32P] ATP or the fluorescent molecule tetramethylrhodamine-5/-6. Hybridization sensitivity for each of the probes was determined by hybridization to heat-denatured RNA immobilized on blots or to formaldehyde fixed whole cells. All of the probes hybridized with equal efficiency to denatured RNA. However, the probes exhibited a wide range of sensitivity (from none to very strong) when hybridized with whole cells using a previously developed FISH procedure. Differential hybridization efficiencies against whole cells could not be attributed to cell wall type, since the relative probe efficiency was preserved when either Gram-negative or -positive cells were used. These studies represent one of the first attempts to systematically define criteria for 16S rRNA targeted probe design for use against whole cells and establish target site location as a critical parameter in probe design.Key words: 16S rRNA, oligonucleotide probes, in situ hybridization.

Study on Specificity and Sensitivity of Oligonucleotide Probes for Fluorescence In Situ Hybridization

2012 ◽  
Vol 518-523 ◽  
pp. 628-631
Author(s):  
Xiao Dan Wang ◽  
Jiang Zhu ◽  
Takashi Someya
Keyword(s):  
Detection Rate ◽  
Oligonucleotide Probe ◽  
The Other ◽  
Oligonucleotide Probes ◽  
Bacterial Strains ◽  
Detection Rates ◽  
Average Value ◽  
Other Hand ◽  
Specificity And Sensitivity

Sixteen bacterial strains were used to analyze the specificity and sensitivity of the commonly used oligonucleotide probe for FISH. The results showed that GAM42a probe was not only hybridized with γ-subclass proteobacteria strains, but also hybridized with other subclass proteobacteria strains. The specificity of GAM42a probe therefore would be improved. On the other hand, the average value of detection rates of ALF1b, HGCGP and LGCGP354B probe was less than 50%. After 10min lysozyme treatment, only the detection rate of LGCGP354B probe was improved.

scholarly journals Comparison of the sensitivities of ribonucleic acid and oligonucleotide probes for in situ detection of Theiler's virus mRNA.

1993 ◽  
Vol 41 (7) ◽  
pp. 1099-1103 ◽  
Author(s):  
C Aubert ◽  
S Ozden
Keyword(s):  
Ribonucleic Acid ◽  
Oligonucleotide Probe ◽  
Virus Genome ◽  
Oligonucleotide Probes ◽  
Theiler's Virus ◽  
Infected Cells ◽  
Rna Probe ◽  
In Situ Detection

To evaluate the sensitivity of in situ detection of the Theiler's virus genome, we hybridized BHK-21-infected cells with antisense ribo- and oligonucleotide 35S-labeled probes. The sensitivity achieved with the anti-sense 280-nucleotide riboprobe was similar to that obtained with a 93-mer oligonucleotide probe. However, more reproducible and accurate results were obtained with the riboprobe. With long exposure times, the background was higher with the oligonucleotide probe than with the RNA probe. The background was improved by using freshly labeled oligonucleotide probe.

scholarly journals Double Labeling of Oligonucleotide Probes for Fluorescence In Situ Hybridization (DOPE-FISH) Improves Signal Intensity and Increases rRNA Accessibility

2009 ◽  
Vol 76 (3) ◽  
pp. 922-926 ◽  
Author(s):  
Kilian Stoecker ◽  
Christiane Dorninger ◽  
Holger Daims ◽  
Michael Wagner
Keyword(s):  
In Situ Hybridization ◽  
Signal Intensity ◽  
Probe Design ◽  
Oligonucleotide Probes ◽  
Direct Identification ◽  
Double Labeling ◽  
Clinical Specimens ◽  
Target Sites ◽  
Fish Signal

ABSTRACT Fluorescence in situ hybridization (FISH) with singly labeled rRNA-targeted oligonucleotide probes is widely applied for direct identification of microbes in the environment or in clinical specimens. Here we show that a replacement of singly labeled oligonucleotide probes with 5′-, 3′-doubly labeled probes at least doubles FISH signal intensity without causing specificity problems. Furthermore, Cy3-doubly labeled probes strongly increase in situ accessibility of rRNA target sites and thus provide more flexibility for probe design.

scholarly journals Flow Cytometric Analysis of the In Situ Accessibility of Escherichia coli 16S rRNA for Fluorescently Labeled Oligonucleotide Probes

1998 ◽  
Vol 64 (12) ◽  
pp. 4973-4982 ◽  
Author(s):  
Bernhard Maximilian Fuchs ◽  
Günter Wallner ◽  
Wolfgang Beisker ◽  
Ines Schwippl ◽  
Wolfgang Ludwig ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Flow Cytometric Analysis ◽  
Dimensional Structure ◽  
Cell Periphery ◽  
Oligonucleotide Probes ◽  
Probe Target ◽  
Target Sites ◽  
Signal Intensities

ABSTRACT In situ identification of whole fixed bacterial cells by hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes is often limited by low signal intensities. In addition to an impermeability of the cell periphery and a low cellular rRNA content, the three-dimensional structure of the ribosome may hinder the access of oligonucleotides to their target sites. Until now, a systematic study on the accessibility of 16S rRNA target sites had not been done. Here, we report fluorescence intensities obtained with more than 200 oligonucleotide probes (mostly 18-mers) used with whole fixed cells ofEscherichia coli DSM 30083T. Two overlapping sets of adjacent oligonucleotides, 171 in total, were designed to cover the full length of the 16S rRNA. The two sets are shifted by 5 to 13 nucleotides. The probes were labeled with carboxyfluorescein, and signal intensities of hybridized cells were quantified by flow cytometry. Care was taken that the signal intensity of cells was dependent solely on the in situ accessibility of probe target sites. The brightest signal resulted from probe Eco1482, complementary to positions 1482 to 1499. With this probe, the fluorescence was 1.7 times brighter than that of the standard bacterial probe EUB338 and 44 times brighter than that of the worst probe, Eco468. The distribution of probe-conferred cell fluorescence in six arbitrarily set brightness classes (classes I to VI; 100 to 81%, 80 to 61%, 60 to 41%, 40 to 21%, 20 to 6%, and 5 to 0% of the brightness with Eco1482, respectively) was as follows: I, 4%; II, 14%; III, 21%; IV, 29%, V, 19%; and VI, 13%. A more detailed analysis of helices 6, 18, and 23 with additional probes demonstrated that a shift of the target region by only a few bases could result in a decline of cell fluorescence from >80 to <10%. Considering the high evolutionary conservation of 16S rRNA, the in situ accessibility map of E. coli should facilitate a more rational selection of probe target sites for other species as well.

scholarly journals RNA In Situ Hybridization for Detecting Gene Expression Patterns in the Abdomens and Wings of Drosophila Species

2021 ◽  
Vol 4 (1) ◽  
pp. 20
Author(s):  
Mujeeb Shittu ◽  
Tessa Steenwinkel ◽  
William Dion ◽  
Nathan Ostlund ◽  
Komal Raja ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Expression Patterns ◽  
Drosophila Species ◽  
Gene Expression Patterns ◽  
Probe Design ◽  
Tissue Preparation ◽  
Design And Synthesis ◽  
Rna In Situ Hybridization

RNA in situ hybridization (ISH) is used to visualize spatio-temporal gene expression patterns with broad applications in biology and biomedicine. Here we provide a protocol for mRNA ISH in developing pupal wings and abdomens for model and non-model Drosophila species. We describe best practices in pupal staging, tissue preparation, probe design and synthesis, imaging of gene expression patterns, and image-editing techniques. This protocol has been successfully used to investigate the roles of genes underlying the evolution of novel color patterns in non-model Drosophila species.

scholarly journals Selected In Situ Hybridization Methods: Principles and Application

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3874
Author(s):  
Dominika Veselinyová ◽  
Jana Mašlanková ◽  
Katarina Kalinová ◽  
Helena Mičková ◽  
Mária Mareková ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Imaging Techniques ◽  
Cell Communication ◽  
Probe Design ◽  
Rapid Progress ◽  
In Situ Techniques ◽  
Different Types ◽  
Laboratory Procedures ◽  
The Individual

We are experiencing rapid progress in all types of imaging techniques used in the detection of various numbers and types of mutation. In situ hybridization (ISH) is the primary technique for the discovery of mutation agents, which are presented in a variety of cells. The ability of DNA to complementary bind is one of the main principles in every method used in ISH. From the first use of in situ techniques, scientists paid attention to the improvement of the probe design and detection, to enhance the fluorescent signal intensity and inhibition of cross-hybrid presence. This article discusses the individual types and modifications, and is focused on explaining the principles and limitations of ISH division on different types of probes. The article describes a design of probes for individual types of in situ hybridization (ISH), as well as the gradual combination of several laboratory procedures to achieve the highest possible sensitivity and to prevent undesirable events accompanying hybridization. The article also informs about applications of the methodology, in practice and in research, to detect cell to cell communication and principles of gene silencing, process of oncogenesis, and many other unknown processes taking place in organisms at the DNA/RNA level.

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