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.

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

Distribution of TaqI sites along human chromosomes revealed by in situ enzyme-nick translation

Genome ◽  
1993 ◽  
Vol 36 (1) ◽  
pp. 202-205 ◽  
Author(s):  
I. Tagarro ◽  
J. J. Gonzalez-Aguilera ◽  
A. M. Fernandez-Peralta ◽  
G. F. de Stefano ◽  
L. Ferrucci
Keyword(s):  
Complete Information ◽  
Human Chromosomes ◽  
Nick Translation ◽  
In Situ Nick Translation ◽  
Translation Procedure

We have studied the relative richness of TaqI sites along human chromosomes by means of a nonradioactive in situ enzyme-nick translation procedure. Regions with a higher content of these sequences are shown to be the noncentromeric heterochromatin blocks, whereas within euchromatin, terminal R-bands are the domains more enriched in these sites. Results obtained suggest that the method of performing enzyme digestions using time as a variable, and then in situ nick translation, provides much more complete information about the distribution of enzyme sequences along chromosomes than standard enzyme digestions.Key words: TaqI, nick translation, biotin, T-bands, heterochromatin.

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.

The distribution of genes on human chromosomes as studied by in situ nick translation

Genome ◽  
1992 ◽  
Vol 35 (5) ◽  
pp. 890-894 ◽  
Author(s):  
J. de la Torre ◽  
A. T. Sumner ◽  
J. Gosalvez ◽  
L. Stuppia
Keyword(s):  
Cpg Islands ◽  
Dnase I ◽  
Human Chromosomes ◽  
Nick Translation ◽  
X Chromosomes ◽  
Dnase I Hypersensitivity ◽  
In Situ Nick Translation ◽  
Inactive X Chromosome ◽  
Active Genes

We have studied the distribution of potentially active genes on human chromosomes, using two methods: DNAse I hypersensitivity and restriction enzyme – nick translation with enzymes sensitive to methylation of CpG doublets. DNAse hypersensitivity is known to be associated with potentially active genes, and, when the reaction is detected by "in situ" nick translation, produces an R-banding pattern. Digestion of chromosomes with HpaII or CfoI, both of which should preferentially cut unmethylated sequences in the CpG islands associated with the majority of genes, also produces R-banding patterns. Deviations are attributable to overdigestion of the chromosomes, leading to extraction of DNA and loss of the specific sites that were to be detected. Contrary to the results of a number of previous workers, we have failed to demonstrate any differences between the DNAse I hypersensitivity or the degree of methylation of the active and inactive X chromosomes in metaphases from females.Key words: human chromosomes, gene distribution, DNAse I hypersensitivity, in situ nick translation, R-banding, CpG islands, DNA methylation, inactive X chromosome.

In situ observations of electromigration induced void behavior

1995 ◽  
Vol 53 ◽  
pp. 244-245
Author(s):  
T. Marieb ◽  
J. C. Bravman ◽  
P. Flinn ◽  
D. Gardner ◽  
M. Madden
Keyword(s):  
Electron Microscopy ◽  
Void Nucleation ◽  
Plan View ◽  
Transmission Electron ◽  
Nucleation Sites ◽  
Microelectronics Industry ◽  
In Situ Observations ◽  
Backscatter Electron Detector ◽  
Voltage Scanning

Electromigration and stress voiding have been active areas of research in the microelectronics industry for many years. While accelerated testing of these phenomena has been performed for the last 25 years[1-2], only recently has the introduction of high voltage scanning electron microscopy (HVSEM) made possible in situ testing of realistic, passivated, full thickness samples at high resolution.With a combination of in situ HVSEM and post-testing transmission electron microscopy (TEM) , electromigration void nucleation sites in both normal polycrystalline and near-bamboo pure Al were investigated. The effect of the microstructure of the lines on the void motion was also studied.The HVSEM used was a slightly modified JEOL 1200 EX II scanning TEM with a backscatter electron detector placed above the sample[3]. To observe electromigration in situ the sample was heated and the line had current supplied to it to accelerate the voiding process. After testing lines were prepared for TEM by employing the plan-view wedge technique [6].

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.

Epitaxy and texture analysis of Bi-Sr-Ca-Cu-O thin films on (100) MgO using Transmission Electron Microscopy

1990 ◽  
Vol 48 (4) ◽  
pp. 68-69
Author(s):  
J. T. Sizemore ◽  
D. G. Schlom ◽  
Z. J. Chen ◽  
J. N. Eckstein ◽  
I. Bozovic ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Thin Films ◽  
Transmission Electron Microscopy ◽  
Critical Currents ◽  
Unit Cell ◽  
X Ray Diffraction ◽  
Cell Axis ◽  
Transmission Electron ◽  
Synthesis Procedure

Investigators observe large critical currents for superconducting thin films deposited epitaxially on single crystal substrates. The orientation of these films is often characterized by specifying the unit cell axis that is perpendicular to the substrate. This omits specifying the orientation of the other unit cell axes and grain boundary angles between grains of the thin film. Misorientation between grains of YBa2Cu3O7−δ decreases the critical current, even in those films that are c axis oriented. We presume that these results are similar for bismuth based superconductors and report the epitaxial orientations and textures observed in such films.Thin films of nominally Bi2Sr2CaCu2Ox were deposited on MgO using molecular beam epitaxy (MBE). These films were in situ grown (during growth oxygen was incorporated and the films were not oxygen post-annealed) and shuttering was used to encourage c axis growth. Other papers report the details of the synthesis procedure. The films were characterized using x-ray diffraction (XRD) and transmission electron microscopy (TEM).

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