In situ nick translation of human metaphase chromosomes with the restriction enzymes Mspl and Hpall reveals an R-band pattern

1990 ◽  
Vol 54 (3-4) ◽  
pp. 132-136 ◽  
Author(s):  
S. Adolph ◽  
H. Hameister
Keyword(s):  
Restriction Enzymes ◽  
Band Pattern ◽  
Nick Translation ◽  
Metaphase Chromosomes ◽  
In Situ Nick Translation

Ultrastructural banding induced by DraI or HaeIII progressive digestion and in situ nick translation on human chromosomes

Genome ◽  
1994 ◽  
Vol 37 (6) ◽  
pp. 950-956
Author(s):  
Rosalba Del Coco ◽  
Liborio Stuppia ◽  
Caterina Cinti ◽  
Rita Peila ◽  
Nadir Mario Maraldi
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Restriction Enzymes ◽  
Human Chromosomes ◽  
Nick Translation ◽  
Metaphase Chromosomes ◽  
In Situ Nick Translation ◽  
Transmission Electron ◽  
Dna Loss

Fixed human metaphase chromosomes were progressively digested with DraI or HaeIII restriction enzymes, submitted to in situ nick translation, and observed by transmission electron microscopy to obtain further information on the localization of the endonuclease target sequences and on the conformational changes in chromosomal bands. This approach allows us to detect specific nick translation patterns, namely, G-banding or R-like banding after short DraI and HaeIII endonuclease digestion, respectively. Intermediate banding recognizable as C-negative banding and G + C banding are induced by longer HaeIII digestion, before the C-positive banding. These patterns appear to depend both on different target sites of the employed endonucleases and on the DNA loss at different digestion times.Key words: human chromosomes, in situ nick translation, DraI banding, HaeIII banding, electron microscopy.

Comparison of methods for the detection of in situ restriction enzyme – nick translation using fluorochromes and confocal microscopy

Genome ◽  
1995 ◽  
Vol 38 (5) ◽  
pp. 1032-1036 ◽  
Author(s):  
L. Stuppia ◽  
C. Cinti ◽  
S. Santi ◽  
R. Peila ◽  
N. M. Maraldi ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Restriction Enzyme ◽  
Restriction Enzymes ◽  
Chromosome Morphology ◽  
Nick Translation ◽  
Metaphase Chromosomes ◽  
Direct Labelling ◽  
Series Of Experiments ◽  
The Cost

A series of experiments was carried out to determine the most efficient methods for detecting incorporated nucleotides in the "in situ" restriction enzyme – nick translation technique. Different methods were tested on fixed human metaphase chromosomes using confocal microscopy for the demonstration of the patterns produced. Of the various techniques tested, that using DIG-dUTP in conjunction with FITC-labelled anti-DIG appears to show the greatest sensitivity and specificity. The use of biotinylated nucleotides with FITC-avidin gives rather less sensitivity, while direct labelling with fluorescein-dUTP produces results more rapidly with better chromosome morphology but at the cost of reduced sensitivity. Resorufin-labelled dUTP was unusable, because of the low level of fluorescence and its very rapid fading. The successful fluorescence methods are more sensitive and faster than using horseradish peroxidase or alkaline phosphatase for detection.Key words: restriction enzymes, nick translation, chromosomes, fluorochromes, confocal microscopy.

DNA sequence mapping in interphase and metaphase chromosomes by fluorescence in situ hybridization

1992 ◽  
Vol 50 (1) ◽  
pp. 496-497
Author(s):  
Barbara Trask ◽  
Susan Allen ◽  
Anne Bergmann ◽  
Mari Christensen ◽  
Anne Fertitta ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Dual Band ◽  
Nick Translation ◽  
Metaphase Chromosomes ◽  
Band Pass ◽  
Texas Red ◽  
Fluorescent Spot

Using fluorescence in situ hybridization (FISH), the positions of DNA sequences can be discretely marked with a fluorescent spot. The efficiency of marking DNA sequences of the size cloned in cosmids is 90-95%, and the fluorescent spots produced after FISH are ≈0.3 μm in diameter. Sites of two sequences can be distinguished using two-color FISH. Different reporter molecules, such as biotin or digoxigenin, are incorporated into DNA sequence probes by nick translation. These reporter molecules are labeled after hybridization with different fluorochromes, e.g., FITC and Texas Red. The development of dual band pass filters (Chromatechnology) allows these fluorochromes to be photographed simultaneously without registration shift.

scholarly journals Detection of DNA fragmentation in apoptosis: application of in situ nick translation to cell culture systems and tissue sections.

1993 ◽  
Vol 41 (7) ◽  
pp. 1023-1030 ◽  
Author(s):  
R Gold ◽  
M Schmied ◽  
G Rothe ◽  
H Zischler ◽  
H Breitschopf ◽  
...  
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Specific Cell ◽  
Nick Translation ◽  
Proliferating Cells ◽  
Cell Surface Antigens ◽  
Simultaneous Application ◽  
Tissue Sections ◽  
In Situ Nick Translation

Since DNA fragmentation is a key feature of programmed cell death (PCD) and also occurs in certain stages of necrosis, we have adapted the methodology of in situ nick-translation (ISNT) to detect DNA fragmentation on a single-cell level. We first established the technique for cell preparations. Apoptosis was induced by gamma-irradiation on freshly isolated rat thymocytes. After fixation procedures, ISNT was performed by overnight incubation either with fluorescein-12-dUTP or with digoxigenin-labeled 11-dUTP and DNA polymerase I. The enzymatic incorporation of labeled nucleotides at sites of DNA fragmentation was detected by flow cytometry either directly or indirectly with fluorescein-conjugated anti-digoxigenin. The quantitative results demonstrated close correlation with morphological essays for apoptosis, DNA gel electrophoresis, and ISNT. Proliferating cells determined by bromodeoxyuridine immunofluorescence were not labeled by ISNT. Immunocytochemistry for cell surface antigens in combination with ISNT allowed the identification of specific cell types undergoing PCD. Furthermore, the simultaneous application of photolabeling techniques with ethidium monoazide and ISNT led to the identification of DNA fragmentation in cells with still intact membranes. Extending ISNT to tissue sections of paraformaldehyde-fixed, paraffin-embedded material reliably revealed labeling of cells with typical morphological features of apoptosis. However, this technique was not useful in detecting early stages of necrotic cell death.

Microprocedure for in situ nick translation of chromosomes

1992 ◽  
Vol 62 (2) ◽  
pp. 150-153
Author(s):  
Li Zhengang ◽  
Howard L. Hosick ◽  
Kang Fan
Keyword(s):  
Nick Translation ◽  
In Situ Nick Translation

DNA single-strand breaks in postischemic gerbil brain detected by in situ nick translation procedure

1995 ◽  
Vol 200 (2) ◽  
pp. 129-132 ◽  
Author(s):  
Muneshige Tobita ◽  
Isao Nagano ◽  
Shozo Nakamura ◽  
Yasuto Itoyama ◽  
Kyuya Kogure
Keyword(s):  
Single Strand ◽  
Nick Translation ◽  
Strand Breaks ◽  
In Situ Nick Translation ◽  
Gerbil Brain ◽  
Single Strand Breaks ◽  
Translation Procedure

In situ nick translation of meiotic chromosomes to demonstrate homologous heterochromatin heterogeneity

Genome ◽  
1993 ◽  
Vol 36 (2) ◽  
pp. 268-270 ◽  
Author(s):  
J. de la Torre ◽  
C. López-Fernández ◽  
P. Herrero ◽  
J. Gosálvez
Keyword(s):  
Tandem Duplication ◽  
Dna Recombination ◽  
Nick Translation ◽  
Meiotic Chromosomes ◽  
In Situ Nick Translation ◽  
Living Species ◽  
Longitudinal Differentiation ◽  
Translation Procedure ◽  
Restriction Endonuclease Cleavage

The in situ nick translation procedure performed on fixed meiotic chromosomes partially cleaved with restriction endonucleases shows a different staining of homologous heterochromatic regions, which could be explained through a differential restriction endonuclease cleavage. Mutations occurring before massive tandem duplication and involving those DNA motifs that produce these heterochromatic blocks, together with the absence of DNA recombination that characterizes these particular regions, could explain the observed results. This method for chromosome labelling is most useful to demonstrate a certain level of heterochromatin heterogeneity that is present in the genome of living species but remained cryptic to other techniques that are also able to induce longitudinal differentiation of the chromosomes.Key words: cytogenetics, nick translation, meiosis, heterochromatin.

A new banding pattern of human chromosomes by in situ nick translation using ECO RI and biotin-dUTP

2008 ◽  
Vol 28 (2) ◽  
pp. 173-176 ◽  
Author(s):  
Jörn Bullerdiek ◽  
Jürgen Dittmer ◽  
Angelika Faehre ◽  
Sabine Bartnitzke ◽  
Volker Kasche ◽  
...  
Keyword(s):  
Banding Pattern ◽  
Human Chromosomes ◽  
Nick Translation ◽  
In Situ Nick Translation ◽  
Eco Ri
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