Epi-fluorescence Microscopy of Single Molecule DNA Denaturation in situ

Mapping Intimacies ◽

10.1101/368423 ◽

2018 ◽

Author(s):

Pulkit Sharma

Keyword(s):

Single Molecule ◽

In Situ Hybridisation ◽

Fluorescence In Situ Hybridisation ◽

Experimental Conditions ◽

Target Dna ◽

Short Probe ◽

Fibre Fish ◽

First Time

AbstractDNA can be denatured by two main methods which are: a) denaturation in solution (invitro) and b) denaturation on a slide surface (in-situ). Additionally, DNA can also be denatured in gels with urea. The method to be used depends on various factors such as the application, the source of the DNA, the length, and the techniques available to confirm the extent of denaturation. Verification of the extent of denaturation is important because of the following factors: 1) increases the chances of hybridization (especially for short probes), 2) prevents the loss of expensive probes (if the target site is not denatured then, the probes will not hybridize and will only cause a high a background), 3) a higher degree of denaturation allows for more probes to be used and therefore, more information can be derived after hybridization, and 4) essential to maximize due to extremely short probe length. It is important to ensure that DNA morphology is preserved after denaturation in order for the probes to hybridise and also for ensuring proper statistical analysis for high throughput applications. In this work, various experimental conditions for in situ denaturation of single molecule DNA is presented.Significance StatementThe significance of this work is that it emphasizes on the importance of denaturation of target genomic DNA in DNA fibre FISH (fluorescence in situ hybridisation) experiments. If the quality of the target DNA is poor after denaturation or the target DNA is not properly denatured, then it will be very difficult or impossible to hybridize the probe DNA during FISH experiments. This will affect the final results for DNA FISH. Additionally, it is the first time that single DNA combed molecules have been shown to be denatured in situ. Most of the past work has been on gels only. Thus the work is both unique and significant.

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Genome size, chromosome number, and rDNA organisation in Algerian populations of Artemisia herba-alba (Asteraceae), a basic plant for animal feeding facing overgrazing erosion

Anales del Jardín Botánico de Madrid ◽

10.3989/ajbm.2415 ◽

2016 ◽

Vol 73 (2) ◽

pp. 043

Author(s):

Youcef Bougoutaia ◽

Sònia Garcia ◽

Teresa Garnatje ◽

Meriem Kaid-Harche ◽

Joan Vallès

Keyword(s):

Chromosome Number ◽

Genome Size ◽

In Situ Hybridisation ◽

Fluorescence In Situ Hybridisation ◽

Ploidy Levels ◽

Rdna Genes ◽

Basic Plant ◽

Tetraploid Cytotypes ◽

First Time

Artemisia herba-alba is a largely-distributed and often landscape-dominating taxon in arid areas of the Mediterranean and Irano-Turanian regions. In Algeria, in 2010 its communities covered 10% of the steppe territory, but its populations have been subjected to overgrazing. A karyological study based on 22 populations together with a cytogenetic characterisation of this species has been performed for the first time in Algerian materials, through genome size and chromosome number determination. Fluorescence in situ hybridisation (FISH) was also used to assess the rDNA loci number and distribution in the two ploidy levels detected. The studied accessions are diploid (2n = 2x = 18 chromosomes, 6 populations) or tetraploid (2n = 4x = 36 chromosomes, 15 populations). One population, occupying a more or less central geographic position among the studied area, presented both cytotypes. Genome size reflects well the two ploidy levels, with no evidence of downsizing with polyploidy. The karyotypes are rather symmetric (2A Stebbins’ class). FISH analyses detected four signals (2 loci) in diploid and eight signals (4 loci) in tetraploid cytotypes for both ribosomal DNA genes, which present an L-type (linked) organisation, i.e. with loci from both rDNA genes colocalised. The presence of two ploidy levels suggest a genomic dynamism and even a possible differentiation underlying the morphological uniformity and despite the dramatic decrease experienced by this plant in Algeria in terms of surface coverage.

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Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains

10.1101/128785 ◽

2017 ◽

Cited By ~ 2

Author(s):

Lu Yang ◽

Joshua S. Titlow ◽

Darragh Ennis ◽

Carlas Smith ◽

Jessica Mitchell ◽

...

Keyword(s):

Transcriptional Regulation ◽

Single Molecule ◽

Single Gene ◽

In Situ Hybridisation ◽

Brain Structures ◽

Fluorescence In Situ Hybridisation ◽

Stems Cells ◽

Post Transcriptional Regulation ◽

The Brain

AbstractRNA in situ hybridization can be a powerful method to investigate post-transcriptional regulation, but analysis of intracellular mRNA distributions in thick, complex tissues like the brain poses significant challenges. Here, we describe the application of single-molecule fluorescent in situ hybridization (smFISH) to quantitate primary transcription and post-transcriptional regulation in whole-mount Drosophila larval and adult brains. Combining immunofluorescence and smFISH probes for different regions of a single gene, i.e., exons, 3’UTR, and introns, we show examples of a gene that is regulated post-transcriptionally and one that is regulated at the level of transcription. We also show that the method can be used to co-visualise a variety of different transcripts and proteins in neuronal stems cells as well as deep brain structures such as mushroom body neuropils. Finally, we introduce the use of smFISH as asensitivealternative to conventional antibody labelling to mark specific neural stem cell populations in the brain.

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Single molecule fluorescence in situ hybridisation for quantitating post-transcriptional regulation in Drosophila brains

Methods ◽

10.1016/j.ymeth.2017.06.025 ◽

2017 ◽

Vol 126 ◽

pp. 166-176 ◽

Cited By ~ 17

Author(s):

Lu Yang ◽

Josh Titlow ◽

Darragh Ennis ◽

Carlas Smith ◽

Jessica Mitchell ◽

...

Keyword(s):

Transcriptional Regulation ◽

Single Molecule ◽

In Situ Hybridisation ◽

Fluorescence In Situ Hybridisation ◽

Single Molecule Fluorescence ◽

Post Transcriptional Regulation

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Tissue microarray (TMA) applications: implications for molecular medicine

Expert Reviews in Molecular Medicine ◽

10.1017/s1462399403006781 ◽

2003 ◽

Vol 5 (26) ◽

pp. 1-12 ◽

Cited By ~ 20

Author(s):

Ronald Simon ◽

Guido Sauter

Keyword(s):

Tissue Microarray ◽

Statistical Power ◽

In Situ Hybridisation ◽

Basic Research ◽

Molecular Medicine ◽

Fluorescence In Situ Hybridisation ◽

Lead Discovery ◽

Experimental Conditions ◽

Expression Screening

Modern expression-screening platforms such as complementary DNA (cDNA) arrays allow for high-throughput lead discovery in cancer and other diseases. For evaluation of promising candidate genes, however, in situ analysis of high numbers of clinical tissues samples – for example, by immunohistochemistry or fluorescence in situ hybridisation – is mandatory. Tissue microarray (TMA) technology greatly facilitates such analysis. Minute tissue cores (diameter 0.6 mm) are removed from up to a thousand different conventional paraffin blocks and re-assembled in a single empty paraffin block at predefined positions. Sections of the resulting TMA can be utilised for the range of research applicable to conventional tissue sections. Important advantages of the TMA technology are speed (parallel analysis of up to a thousand tissues), cost efficiency (the same amount of reagents required for a single large-section analysis is sufficient for a thousand samples), and standardisation (the same experimental conditions are applied to all samples). Because of the high numbers of samples usually included in TMAs, they are optimally suited to detect genotype–phenotype associations with high statistical power. Thus, TMA technology will markedly accelerate the transition from basic research to clinical applications.

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Rapid Multi-Hybridisation FISH Screening for Balanced Porcine Reciprocal Translocations Suggests a Much Higher Abnormality Rate Than Previously Appreciated

Cells ◽

10.3390/cells10020250 ◽

2021 ◽

Vol 10 (2) ◽

pp. 250

Author(s):

Rebecca E O’Connor ◽

Lucas G Kiazim ◽

Claudia C Rathje ◽

Rebecca L Jennings ◽

Darren K Griffin

Keyword(s):

Semen Analysis ◽

In Situ Hybridisation ◽

Economic Loss ◽

Environmental Damage ◽

Fluorescence In Situ Hybridisation ◽

Reciprocal Translocations ◽

Chromosome Translocations ◽

Farrowing Rate ◽

Significant Economic Loss

With demand rising, pigs are the world’s leading source of meat protein; however significant economic loss and environmental damage can be incurred if boars used for artificial insemination (AI) are hypoprolific (sub-fertile). Growing evidence suggests that semen analysis is an unreliable tool for diagnosing hypoprolificacy, with litter size and farrowing rate being more applicable. Once such data are available, however, any affected boar will have been in service for some time, with significant financial and environmental losses incurred. Reciprocal translocations (RTs) are the leading cause of porcine hypoprolificacy, reportedly present in 0.47% of AI boars. Traditional standard karyotyping, however, relies on animal specific expertise and does not detect more subtle (cryptic) translocations. Previously, we reported development of a multiple hybridisation fluorescence in situ hybridisation (FISH) strategy; here, we report on its use in 1641 AI boars. A total of 15 different RTs were identified in 69 boars, with four further animals XX/XY chimeric. Therefore, 4.5% had a chromosome abnormality (4.2% with an RT), a 0.88% incidence. Revisiting cases with both karyotype and FISH information, we reanalysed captured images, asking whether the translocation was detectable by karyotyping alone. The results suggest that chromosome translocations in boars may be significantly under-reported, thereby highlighting the need for pre-emptive screening by this method before a boar enters a breeding programme.

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