However, successful results in these systems may not be consistent with experiments on plant material. The presence of a cell wall significantly impedes in situ procedures. It is very difficult to remove the cytoplasm and cell wall debris from plant chromosome spreads.
Half of the success in FISH results depends on a good quality preparation, and obtaining it is a considerable challenge. Here, we compared the influence of different concentrations of ethylene carbonate or formamide in a hybridization mixture with the results of FISH on rye Secale vavilovii chromosomes.
Two different times 90 min or 16 h of hybridization were tested. Probes complementary to the rye repetitive sequences JNK and Bilby were used in this analysis. The results obtained after in situ hybridization in a mixture containing EC were very similar to results of hybridization performed under analogous conditions; however, using a mixture containing FA. Hybridization time did not affect the result. Regardless of whether the hybridization was carried out for 16 h or 90 min, distinct hybridization signals were visible in the chromosomes and cell nuclei.
Each of the method modifications obtained specific signals in the centromeric region of all rye chromosomes Bilby probe or on 2RL chromosomes JNK probe , according to the Bilby Francki, Francki, M. Figure 1 — Fluorescent in situ hybridization in S. Golczyk Golczyk, H. A simple non-toxic ethylene carbonate fluorescence in situ hybridization EC-FISH for simultaneous detection of repetitive DNA sequences and fluorescent bands in plants.
Protoplasma — The use of JNK and Bilby probes allowed to obtain positive hybridization results after 90 min of its duration.
Thus, it seems that EC-FISH does not only enable avoidance of toxic compounds, but it may also be a time-saving procedure. However, it should be noted that only repetitive DNA probes were tested and positive results were obtained on the high-quality preparations. All modifications showed clear hybridization signals. The hybridization and post-hybridization conditions were different from those in our protocol, indicating that the EC-method is flexible and can be adapted to various research profiles.
Both hybridization and post-hybridization parameters should be determined experimentally. Matthiesen and Hansen Matthiesen, S. Tafe et al. Journal of Clinical Pathology The application of ethylene carbonate shortened the hybridization time and did not require the use of blocking DNA.
Attempts were also made to eliminate formamide from the FISH procedure without using a substitute. Celeda et al. A simplified combination of DNA probe preparation and fluorescence in situ hybridization. Section C Optimization of fast-fluorescence in situ hybridization with repetitive alpha-satellite probes.
Rapid fluorescence in situ hybridization with repetitive DNA probes: quantification by digital image analysis. Cytometry A rapid FISH technique for quantitative microscopy. BioTechniques Chromosome painting using repetitive DNA sequence as probes for somatic chromosome identification in maize.
V; Leitch, A. Extensive chromosomal variation in a recently formed natural allopolyploid species, Tragopogon miscellus Asteraceae. Formamide-free genomic in situ hybridization ff-GISH allows unambiguous discrimination of highly similar parental genomes in diploid hybrids and allopolyploids. Cytogenetic Genome Research — It should be emphasized that most probes used were complementary to repetitive DNA sequences in these studies. The lack of denaturing agent in the hybridization mixture allows shortening the hybridization step.
However, to ensure stringency, this hybridization is usually performed at higher temperatures. Coronavirus Resources. Authors: Emanuela V. Volpi 1. Emanuela V. The denaturation map also shows the repetitive domain dissociating in two -state fashion. The small difference between observed and calculated temperature scales can probably be attributed to small errors in the ratios of upon for one or more of the 10 nearest neighbors This curve corresponds to an expanded view of the subtransition labeled no.
The plasmid was linearized for melting at the Eco RV locus bp away from the insert, so that the insert melts as a closed loop in two-state fashion. The curve was obtained as described in the text and in the legend to Figure 1. The curve punctuated by diamond-shaped symbols every 0. The T m was Residuals for the difference between experimental and van't Hoff curves are plotted below the melting curve, and indicate a totally random standard error of 3.
The curve punctuated by cross-shaped symbols every 0. The calculated transition for the insert domain was isolated from the transition background emanating from other parts of the plasmid DNA, and superimposed on the experimental curve in this figure.
The variation of with C F for the repetitive sequence domain is shown in Figure 5. Thereafter the enthalpy drops only 0. Since T m 1 drops monotonically with formamide concentration, the initial drop at low C F is almost perfectly compensated by a similar drop in , as shown in Figure 6 , where it is assumed that.
The thermodynamic effects associated with DNA melting can be apportioned to chemical and solvation effects, where the origin of the linear compensation is attributable to perturbations of the latter by formamide. The initial drop therefore suggests a small enthalpic loss associated with the exchange of formamide for bound water and possibly counterion to the helical state. Although decreases over the wide range of C F examined in this study, we did not find a corresponding decrease in the cooperativity of melting.
The inherent cooperativity of melting, the strong dependence of the conformational state of base pairs on those of their neighbors from hydrogen bonding and stacking forces, is unaffected by the denaturant. Other results indicate that the denaturant both destabilizes the helical state and stabilizes the coil state.
Between 0 and 1. We interpret these local differences as arising from sequence-dependent variations in the energy of hydration at selected sites. Because results were obtained at high resolution, T m of a large number of domains of different base compositions were obtained simultaneously from the same plasmid DNA specimen in a single melting experiment. Since the coil states are affected equally, the helical states must be destabilized to different extents, reflecting differences in hydration patterns and hydration energies for different helical conformations of synthetic duplexes.
We suggest that this tightly bound layer of hydration is not displaced by formamide, leading to a reduced sensitivity. This indicates the unusual hydration pattern and stability associated with these tracts at melting temperatures does not take place in the duplex until some critical length between three and six AT base pairs. The model includes the potential enhancement of hydrogen bonding through resonance-assistance between base pairs as well as between bases and formamide.
Results of this study are consistent with formamide destabilizing the helical state through displacement of loosely and uniformly bound hydrate. Formamide is a strongly associating liquid 23 , capable of four hydrogen bonds, the same as water. It is a strong donor and a stronger acceptor than water Displacement of hydrate leads to destabilization of the helix, presumably because of the propensity of formamide to form hydrogen bonded networks and oligomeric chains in aqueous solution Displacement of hydrate is the key process.
The geometric relationships of donor and acceptor groups of these two molecules are also very different 24— DNA hydrate can be categorized as either tightly or loosely bound. One-third bind in a sequence-independent fashion with sufficient strength they become immobilized, exhibiting low levels of exchange with the surrounding solvent 32 , Waters binding to condensed cations and anionic phosphate oxygens bind most firmly, while the hydration of sugars and backbone ester oxygens is of more moderate strength 35— The remaining two-thirds are found primarily at groove sites, and are more weakly bound, binding tightly only under special circumstances and only in the narrow groove Presumably the weakly bound waters include a readily exchangeable class that is displaced by formamide.
If this class of hydrate is found in the grooves, and in the narrow groove in particular, displacement by formamide would alter the ionic potential, leading to changes in the numbers of sodium ions condensed to helical and coil states. Such changes are included in the first of the two schemes above, 8 , because the amount bound to the helix with its high charge density, may be affected by formamide. Rather, the effect depends on displacement of a uniform class of bound water.
Displacement may alter hydrate that contributes to the stability of the duplex, or it may alter the local dielectric, thereby altering counterion screening or the numbers of condensed counterion According to this scheme, the phenomenological effects of formamide are equivalent to lowering the bulk concentration of counterion.
In , chemist S. They produced among many other compounds guanine, adenine, cytosine, and uracil—the four nitrogen bases that make up DNA. Formamide is found in great quantities throughout the observable universe, giving credibility to the idea that life on Earth could have originated outside the planet.
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