Increased resolution of in situ hybridization signal by electron microscopy: A comparison with fluorescence microscopy

Genome ◽  
1999 ◽  
Vol 42 (5) ◽  
pp. 1001-1007
Author(s):  
Raouf Fetni ◽  
Patrick Scott ◽  
Frédérique Tihy ◽  
Claude-Lise Richer ◽  
Nicole Lemieux
Keyword(s):  
Electron Microscopy ◽  
In Situ Hybridization ◽  
Fluorescence Microscopy ◽  
Dna Sequences ◽  
Cdna Probe ◽  
Single Copy ◽  
Resolving Power ◽  
Chromosomal Structure ◽  
Definition Of

Cytogenetic studies by in situ hybridization (ISH) have proven to be valuable for gene mapping on banded chromosomes when combined with fluorescence microscopy (FISH). However, even under the best conditions, FISH technology has a resolving power inherent to light of just 0.2 µm. Its utilization is further limited by the diffusion of light coming from the fluorescent signal which covers an area considerably larger than the target DNA sequence. The development of new ISH protocols applied to electron microscopy (EMISH) should increase the resolution for cytogenetic mapping and fine chromosomal structure studies. Despite these advances, few attempts have been made which exploit this increased resolution. Here we present a detailed analysis of ISH signals obtained by fluorescence and electron microscopy methodologies to demonstrate and define the higher sensitivity obtainable by electron microscopy. This comparative study was conducted with probes of different origins: telomeric, classical satellite, alpha satellite, and single-copy DNA sequences, which provide a good reference point for later studies. We were also able to map a 200-bp cDNA probe by EMISH. This study assesses the nature of the resolution and the better definition of the EMISH signal, which confirms the greater resolution of electron microscopy as compared with that achieved with light microscopy. It also indicates that better delineation of two closely linked sequences is achieved at the electron microscopy level.Key words: In situ hybridization, electron microscopy, fluorescence microscopy, localization, repetitive and small single-copy probes.

Fluorescence in situ hybridization on plant extended chromatin DNA fibers for single-copy and repetitive DNA sequences

2011 ◽  
Vol 30 (9) ◽  
pp. 1779-1786 ◽  
Author(s):  
Kun Yang ◽  
Hecui Zhang ◽  
Richard Converse ◽  
Yong Wang ◽  
Xiaoying Rong ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Repetitive Dna ◽  
Dna Sequences ◽  
Single Copy ◽  
Repetitive Dna Sequences

Use of fluorescence in situ hybridization to study the arrangement of DNA sequences in normal and disease-related chromosomes

1995 ◽  
Vol 53 ◽  
pp. 748-749
Author(s):  
Barbara J. F. Trask ◽  
Hillary Massa ◽  
Cynthia Friedman ◽  
Richard Esposito ◽  
Ger van den Engh ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Dna Sequences ◽  
Single Copy ◽  
Two Dimensions ◽  
Genetic Maps ◽  
Epifluorescence Microscopy ◽  
Metaphase Chromosomes ◽  
Interphase Nuclei

The sites of specific DNA sequences can be fluorescently tagged by fluorescence in situ hybridization (FISH). Different sequences can be labeled with different fluorochromes so that their arrangement can be studied using epifluorescence microscopy. The distances between points on the same or different chromosomes can be determined easily in a large number of interphase nuclei or metaphase chromosomes. A variety of probe types, ranging from single-copy sequences to highly repeated sequences can be employed. Our work has focussed on the analysis of hybridization patterns in two dimensions using conventional fluorescence microscopy.We have used FISH to study various aspects of genome organization that are difficult to study using other techniques. Examples of these applications will be presented.FISH is now the method of choice for determining the chromosomal location of DNA sequences. DNA sequences can be positioned in the genome with <1:1000 accuracy (to a 3-Mbp region within a 3000-Mbp genome). Through FISH, the cytogenetic, physical and genetic maps of chromosomes can be linked.

Histochemical aspects of nucleic acid detection

1995 ◽  
Vol 53 ◽  
pp. 746-747
Author(s):  
B. A. Hamkalo ◽  
Elizabeth R. Unger
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
High Resolution ◽  
Dna Sequences ◽  
Single Copy ◽  
Nucleic Acid Detection ◽  
Metaphase Chromosomes ◽  
Potential Applications ◽  
Nucleic Acid Sequences

This symposium brings together several approaches for the detection of specific nucleic acid sequences that have potential applications at the histochemical level.Trask et al. report on the use of fluorescence in situ hybridization (FISH) techniques to study the arrangement of DNA sequences in normal and diseaserelated chromosomes. The sites of specific DNA sequences can be fluorescently tagged. Different sequences can be labeled with different fluorochromes so that their arrangement can be studied using fluorescence microscopy. The distances between points on the same or different chromosomes can be determined in a large number of interphase nuclei or metaphase chromosomes. A variety of probe types, ranging from single-copy sequences to highly repeated sequences can be employed.Hamkalo and co-workers have used non-radioactive methods at the EM level for the detection of nucleic acid sequences by in situ hybridization. Analysis of metaphase chromosomes by electron microscopy allows for high resolution mapping of chromosomes. A variety of labelling procedures have been employed to illustrate the utility of high resolution nucleic acid sequence mapping in these preparations.

Nonisotopic in situ hybridization and plant genome mapping: the first 10 years

Genome ◽  
1994 ◽  
Vol 37 (5) ◽  
pp. 717-725 ◽  
Author(s):  
Jiming Jiang ◽  
Bikram S. Gill
Keyword(s):  
In Situ Hybridization ◽  
Physical Mapping ◽  
Dna Sequences ◽  
Genome Mapping ◽  
Molecular Cytogenetics ◽  
Gene Families ◽  
Single Copy ◽  
Plant Genome ◽  
Metaphase Chromosomes

Nonisotopic in situ hybridization (ISH) was introduced in plants in 1985. Since then the technique has been widely used in various areas of plant genome mapping. ISH has become a routine method for physical mapping of repetitive DNA sequences and multicopy gene families. ISH patterns on somatic metaphase chromosomes using tandemly repeated sequences provide excellent physical markers for chromosome identification. Detection of low or single copy sequences were also reported. Genomic in situ hybridization (GISH) was successfully used to analyze the chromosome structure and evolution of allopolyploid species. GISH also provides a powerful technique for monitoring chromatin introgession during interspecific hybridization. A sequential chromosome banding and ISH technique was developed. The sequential technique is very useful for more precise and efficient mapping as well as cytogenetic determination of genomic affinities of individual chromosomes in allopolyploid species. A critical review is made on the present resolution of the ISH technique and the future outlook of ISH research is discussed.Key words: in situ hybridization, physical mapping, genome mapping, molecular cytogenetics.

scholarly journals Flow cytometry assay for the detection of single-copy DNA in human lymphocytes

2020 ◽  
Vol 48 (15) ◽  
pp. e86-e86
Author(s):  
Naoki Uno ◽  
Norihito Kaku ◽  
Yoshitomo Morinaga ◽  
Hiroo Hasegawa ◽  
Katsunori Yanagihara
Keyword(s):  
Flow Cytometry ◽  
In Situ Hybridization ◽  
Dna Sequences ◽  
Human Lymphocytes ◽  
Single Copy ◽  
Target Sequence ◽  
Flow Cytometry Assay ◽  
Flow Cytometric ◽  
Copy Dna

Abstract Specific nucleic acid sequences can be detected in individual cells by in situ hybridization. However, when very few copies of a target sequence are present per cell, its signal is undetectable by flow cytometry. Although various approaches have been developed to increase fluorescence signals for in situ hybridization, flow cytometric detection of specific genomic DNA sequences has not been established. Here, we present a flow cytometry assay for detection of single-copy genomic sequences in human lymphocytes using in situ PCR with universal energy transfer-labelled primers.

scholarly journals Localization of Single- and Low-Copy Sequences on Tomato Synaptonemal Complex Spreads Using Fluorescence in Situ Hybridization (FISH)

Genetics ◽  
1999 ◽  
Vol 152 (1) ◽  
pp. 427-439 ◽  
Author(s):  
Daniel G Peterson ◽  
Nora L V Lapitan ◽  
Stephen M Stack
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Synaptonemal Complex ◽  
Dna Sequences ◽  
Single Copy ◽  
Differential Staining ◽  
Rflp Markers ◽  
Metaphase Chromosomes ◽  
Fish Mapping

Abstract 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.

Identifying a single-copy DNA sequence associated with the expression of a heterochromatic gene, the light locus of Drosophila melanogaster

Genome ◽  
1990 ◽  
Vol 33 (3) ◽  
pp. 405-415 ◽  
Author(s):  
Robert H. Devlin ◽  
David G. Holm ◽  
Karen R. Morin ◽  
Barry M. Honda
Keyword(s):  
Drosophila Melanogaster ◽  
In Situ Hybridization ◽  
Dna Sequences ◽  
Polytene Chromosomes ◽  
Single Copy ◽  
Molecular Organization ◽  
Copy Dna ◽  
Single Copy Dna ◽  
P Transposon

Although little is known about the molecular organization of most genes within heterochromatin, the unusual properties of these chromosomal regions suggest that genes therein may be organized and expressed very differently from those in euchromatin. We report here the cloning, by P transposon tagging, of sequences associated with the expression of the light locus, an essential gene found in the heterochromatin of chromosome 2 of Drosophila melanogaster. We conclude that this DNA is either a segment of the light locus, or a closely linked, heterochromatic sequence affecting its expression. While other functional DNA sequences previously described in heterochromatin have been repetitive, light gene function may be associated, at least in part, with single-copy DNA. This conclusion is based upon analysis of DNA from mutations and reversions induced by P transposable elements. The cloned region is unusual in that this single-copy DNA is embedded within middle-repetitive sequences. The in situ hybridization experiments also show that, unlike most other sequences in heterochromatin, this light-associated DNA evidently replicates in polytene chromosomes, but its diffuse hybridization signal may suggest an unusual chromosomal organization.Key words: polytene chromosomes, P transposon, in situ hybridization, middle-repetitive DNA.

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