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.

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.

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.

Banding of rye (Secale cereale) chromosomes using the restriction enzymes AluI, DraI, HpaII, and MspI

Genome ◽  
1993 ◽  
Vol 36 (5) ◽  
pp. 998-1002 ◽  
Author(s):  
T. Stößer ◽  
T. Günther ◽  
C. U. Hesemann
Keyword(s):  
Secale Cereale ◽  
Restriction Enzymes ◽  
Chromosome Band ◽  
Banding Technique ◽  
Recognition Sequence ◽  
Metaphase Chromosomes ◽  
Banding Patterns ◽  
Secale Cereale L ◽  
Characteristic Banding

Mitotic metaphase chromosomes of the rye inbred line L 301, which belongs to the Sortiment of the University of Hohenheim, were treated in situ with the restriction enzymes AluI (recognition sequence: 5′-AC/GT-3′), DraI (recognition sequence: 5′-TTT/AAA-3′), and the isoschizomeres HpaII and MspI (recognition sequence: 5′-C/CGG-3′) and stained with Giemsa. The chromosomes indicated similar banding patterns in comparison with the conventional Giemsa-C-banding. However, we have found in rye chromosomes after restrictase treatment that the telomeric bands were reduced in extension. In a lower degree the centromeric bands of individual chromosomes could be absent in dependence of the used restriction enzymes. The number of the intercalary bands were also reduced. Nevertheless, the tested restriction enzymes produced characteristic banding patterns of the rye genome. This uncomplicated banding technique is suited for a very quick banding method of karyotype analysis especially to obtain a first survey of the band patterns on the rye chromosomes.Key words: Secale cereale L., chromosome band pattern, in situ digestion, restriction endonuclease, restriction banding.

A Simplified Combination of DNA Probe Preparation and Fluorescence in situ Hybridization

1992 ◽  
Vol 47 (9-10) ◽  
pp. 739-747 ◽  
Author(s):  
Dino Celeda ◽  
Ulrich Bettag ◽  
Christoph Cremer
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Repetitive Dna ◽  
Human Lymphocytes ◽  
Dna Probes ◽  
Dna Probe ◽  
Nick Translation ◽  
Metaphase Chromosomes ◽  
Human Dna

Fluorescence in situ hybridization (FISH) has found widespread applications in cytogenetics. So far the standard protocols for probe amplification (and simultaneous labeling) by PCR, nick translation and in situ hybridization involve different buffer systems leading to a number of time consuming washing steps even before hybridization. In this manuscript we show a fast technique of a close combination of DNA probe preparation and in situ hybridization (ISH). This method was applied to metaphase chromosomes from human lymphocytes fixed on slides. Two specific repetitive DNA probes, the pUC 1.77 DNA probe and the DYZ 1 repetitive DNA fraction were used, amplified and labeled in different ways. Additional experiments with total genomic male human DNA as the DNA probe suggest that this method may be extended to a large variety of other probes. Moreover the ISH technique described does not require toxic denaturing agents, such as formamide.

The effect of restriction enzyme digestion of human metaphase chromosomes on C-band variants of chromosomes 1 and 9

Genome ◽  
1988 ◽  
Vol 30 (5) ◽  
pp. 652-655 ◽  
Author(s):  
Ute Hedemann ◽  
M. Schürmann ◽  
E. Schwinger
Keyword(s):  
Restriction Enzyme ◽  
Restriction Enzymes ◽  
Enzyme Digestion ◽  
Restriction Endonucleases ◽  
Restriction Enzyme Digestion ◽  
Human Chromosomes ◽  
Metaphase Chromosomes ◽  
Homologous Chromosomes

Human metaphase chromosomes, fixed on slides, have been treated with 8 different restriction endonucleases and 29 combinations of 2 restriction enzymes prior to staining with Giemsa. The endonucleases AluI and DdeI and the combinations AluI + DdeI, AluI + HaeIII, AluI + HinfI, and AluI + MboI have then been used to digest metaphase chromosomes of nine individuals with C-band variants of chromosomes 1 or 9, obtained by the CBG technique. The restriction enzyme resistant chromatin of the paracentromeric regions of chromosomes 1 and 9 has been measured and compared with the corresponding CBG-bands. The size of the enzyme resistant chromatin regions depend upon the type of enzyme(s) used. Treatment with AluI + MboI was the only digestion that acted differently on different chromosome pairs. However, within one pair of homologous chromosomes, all digestions revealed the same variations as conventional C-banding.Key words: C-band variants, heterochromatin, human chromosomes, restriction endonucleases.

Regularities of in situ nick translation labeling pattern of cereal metaphase chromosomes

Genome ◽  
1991 ◽  
Vol 34 (1) ◽  
pp. 131-138 ◽  
Author(s):  
M. V. Kost ◽  
N. L. Bolsheva ◽  
N. S. Badaev ◽  
A. V. Zelenin
Keyword(s):  
Active Regions ◽  
Nucleolar Organizer ◽  
Nucleolar Organizer Regions ◽  
Nick Translation ◽  
Active Chromatin ◽  
Metaphase Chromosomes ◽  
B Genome ◽  
Structural Conformation ◽  
Labeling Pattern

A method of nick translation in situ that makes it possible to detect regions hypersensitive to DNase I in metaphase chromosomes has been modified for plants. Such regions have a peculiar structural conformation typical of transcriptionally active chromatin. The distribution of transcriptionally active regions labeled with [3H]TTP along the length of individual chromosomes of rye and B genome of wheat was studied. The following regularities of this distribution were found: a weak labeling of heterochromatic regions; an increase in the intensity of labeling in regions located at the border of euchromatin and heterochromatin and in the nucleolar organizer regions. Each chromosome of the B and R genomes has its own labeling pattern, but some similarities in the labeling pattern in all chromosomes have been revealed. Such similarities are characteristic of homoeologous chromosomes.Key words: wheat, modified nick translation, rye.

scholarly journals Restriction enzyme digestion chromosome banding in Crassostrea and Ostrea species: comparative karyological analysis within Ostreidae

Genome ◽  
2004 ◽  
Vol 47 (5) ◽  
pp. 781-788 ◽  
Author(s):  
A Leitão ◽  
R Chaves ◽  
S Santos ◽  
H Guedes-Pinto ◽  
P Boudry
Keyword(s):  
Restriction Enzyme ◽  
Chromosome Banding ◽  
Genome Mapping ◽  
Genetic Research ◽  
Restriction Enzymes ◽  
Restriction Enzyme Digestion ◽  
Banding Patterns ◽  
Crassostrea Angulata ◽  
Restriction Enzyme Banding

Reliable banding techniques are a major necessity for genetic research in oysters. In this study, we carried out the cytogenetic characterization of four oyster species (family Ostreidae) using restriction endonuclease treatments. Chromosomes were treated with three different restriction enzymes, stained with Giemsa, and examined for banding patterns. The following species were studied: Crassostrea gigas (2n = 20; total number of bands with ApaI, 74; HaeIII, 61; PstI, 76), Crassostrea angulata (2n = 20; ApaI, 62; HaeIII, 61; PstI, 55) (subfamily Crassostreinae), Ostrea edulis (2n = 20; ApaI, 82; HaeIII, 59; PstI, 66), and Ostrea conchaphila (2n = 20; ApaI, 68; HaeIII, 62; PstI, 69) (subfamily Ostreinae). Treatment of samples with ApaI, HaeIII, and PstI produced specific banding patterns, which demonstrates the potential of these enzymes for chromosome banding in oysters. This is of special interest, since it has been recently shown in mammalian chromosomes that restriction enzyme banding is compatible with fluorescence in situ hybridization. This study therefore provides a fundamental step in genome mapping of oysters, since chromosome banding with restriction enzymes facilitates physical gene mapping in these important aquaculture species. The analysis of the banded karyotypes revealed a greater similarity within the genera of Crassostrea and Ostrea than between them.Key words: Ostreidae, Crassostrea, Ostrea, chromosome banding, in situ restriction enzyme banding.

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