Thus, faster kinetics and specific binding were observed with different FFPE tissue specimens and with cytological metaphase spreads. To investigate if the increased hybridization rate was caused by the change in the relative buffer composition, i.
However, the signal intensities using the EC buffer were still considerably stronger. The increased signal intensities observed at lower formamide concentration supports the notion that the traditional formamide concentration hinders fast hybridization kinetics [26] and that high salt concentrations facilitates fast hybridization kinetics.
Next, we examined if classic heat-denaturation was required to obtain efficient hybridization with the EC buffer. After denaturation, the complementary strands are brought together under conditions that favor hybridization.
This can be achieved by creating an environment with high probe concentration at a lowered temperature. In classic formamide buffers, the dextran sulfate is believed to raise the effective probe concentration and thereby increase the kinetics [4] , [27] , [28]. This suggests that the solvents have a lower affinity for attachment to the bases through hydrogen bonds. This lower affinity towards base pairing could be a part of the mechanism for faster hybridization of the strands.
The strands can gain easier access and bind to their complementary strands when there are no hydrogen bonds from the solvent disturbing the base pairing [5] , [26]. An analogy is a zipper that can be opened and closed fast, unless objects, in this case formamide, are stuck in the zipper and thereby hindering the assembly until they are removed.
The stability of the DNA helix is primarily caused by hydrophobic stacking and not by base pairing [29] , [30]. The base pairing ensures that the bases are closely packed such that stacking can occur. The observed effect of re-annealing without prior heat-induced denaturation or with a low heat denaturation temperature supports the notion that polar aprotic solvent buffers decrease the stability of the helix. We propose that the mechanism of the solvents is not by attacking hydrogen bonds, but instead by diminishing the hydrophobic stacking of bases and thereby decreasing the stability of the helix.
Repetitive sequences re-anneal much faster than unique gene sequences [31] — [33]. A decreased stability of the DNA helix might result in an even faster re-annealing of the repetitive sequences in both the probe and the genome after denaturation.
Therefore, we suggest that they re-anneal to themselves, and as a consequence, DNA blocking with Cot-1 can be omitted. The new hybridization solvents are strong candidates to replace the use of classic formamide as the preferred solvent in molecular biology due to their properties to lower the melting temperature, increase the hybridization rate and decrease health risks.
In addition to the results shown in this paper, they also work well for e. The shortened hybridization time of the IQISH technology will have a major impact on ISH based cancer diagnostic as the turnaround time from sample to diagnosis makes a difference for the patient.
Manual scoring of signal intensities in time-chase experiments using formamide and ethylene carbonate buffers. HSP considerations in relation to the suggested base stacking mechanism.
Please also see Table S1 and S2. We thank A. Al-Abdullah and M. Pham for technical assistance; A. Petersen, J. Lohse and J. Mollerup for discussions and review; S. Rudbeck for comments and suggestions regarding the manuscript.
CMH has worked as consultant for Dako. CMH holds financial interest in the application. SHM holds no financial interest in the patent applications. All applications are owned by Dako. Funding: These authors have no support or funding to report.
National Center for Biotechnology Information , U. PLoS One. Published online Jul Steen H. Hansen 2. Charles M. Hansen 2 Charles M. Konradin Metze, Editor. Author information Article notes Copyright and License information Disclaimer.
Received Mar 6; Accepted Jun Copyright Matthiesen, Hansen. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. This article has been cited by other articles in PMC. Figure S2: Time-chase comparison between formamide buffer and EC buffer.
Figure S3: Manual scoring of signal intensities in time-chase experiments using formamide and ethylene carbonate buffers. Discussion S1: HSP considerations in relation to the suggested base stacking mechanism. Abstract Formamide is the preferred solvent to lower the melting point and annealing temperature of nucleic acid strands in in situ hybridization ISH.
Introduction For the past 30 years, formamide has been the solvent of choice in in situ hybridization ISH for lowering the melting point by destabilizing the double-stranded structure of the nucleic acid helix [1] — [6]. Materials and Methods Specimens Breast carcinoma, tonsil and colon tissue were obtained from Department of Pathology, Odense University Hospital, Region South, Denmark, fixed in formaldehyde prior to embedding for 24 hours, 24 hours, and 72 hours, respectively.
Results New FISH Hybridization Buffer In the search for formamide substitutes that are less toxic and can reduce the hybridization time, the Hansen solubility parameters for DNA [18] were used as a guidance to identify potential replacement candidates.
Open in a separate window. Figure 1. Examples of alternative solvents for FISH hybridization buffer. Time-course Testing A time-course experiment was performed on FFPE breast carcinoma tissue to examine the signal intensities obtained using EC and formamide buffers at different hybridization time points Figure 2 and Figure S2.
Figure 2. Effect of EC on hybridization rate. Multi-Specimens To examine if the fast hybridization was a specimen phenomenon, different types of specimens were tested Figure S4. Reproductive Toxicology — Evaluation of the developmental toxicity of formamide in Sprague-Dawley CD rats. Toxicological Sciences Evaluation of the developmental toxicity of formamide in New Zealand white rabbits. Toxicological Sciences — A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species.
Developmental Biology The negative effects of formamide lead to search for less toxic equivalents, while ensuring its advantages in the hybridization process Durm et al. Fast-FISH technique for rapid, simultaneous labeling of all human centromeres. Cytometry 53— Fast and non-toxic in situ hybridization without blocking of repetitive sequences. PLoS One 7: e Application of locked nucleic acid-based probes in fluorescence in situ hybridization. Applied Microbiology and Biotechnology — Of all reagents tested, ethylene carbonate proved to be the most optimal for the ISH process and its application reduced hybridization time, denaturation temperature, and background noise.
Rye, Secale vavilovii , was the experimental material. Two molecular probes were used to locate sequences characteristic of the genus of Secale : JNK and Bilby. This study compared the results of FISH analyses using a hybridization mixture containing either formamide or ethylene carbonate at 90 min and 16 h hybridization times. The study also intended to demonstrate whether the replacement of toxic formamide for non-toxic ethylene carbonate affected the intensity and quality of hybridization signals.
We performed studies with two inbred lines of Secale vavilovii Grossh. Journal of Applied Genetics Polymorphism of heterochromatin bands on chromosomes of rye Secale vavilovii Grossh. Acta Biologica Cracoviensia series Botanica Biologia Plantarum DNA was isolated from 1 g of freshly collected rye coleoptiles. Primer3 on the WWW for general users and for biologist programmers.
Methods in Molecular Biology Tracking of intercalary DNA sequences integrated into tandem repeat arrays in Secale vavilovii. Acta Societatis Botanicorum Poloniae It was located on the long arm of chromosome 2R in the Japanese S. A novel repetitive sequence, termed the JNK repeat family, located on an extra heterochromatic region of chromosome 2R of Japanese rye. Chromosome Research 6: Identification of Bilby, a diverged centromeric Ty1-copia retrotransposon family from cereal rye Secale cereale L.
Genome — Planta Signal amplification was repeated once. The preparations were analyzed with an epifluorescence microscope Axio Imager Z2. Over the years, numerous in situ hybridization protocols have been developed and adapted to specific needs, such as the detection of repetitive DNA sequences, short DNA sequences, whole genomes, non-coding RNA or mRNA Sinigaglia et al.
Most of them recommend using formamide FA as a solvent. Trends in modern science strive for the elimination of toxic substances used during experiments, shortening the test procedure, and development of easy-to-use, yet repeatable protocols. The scientific field is constantly looking for a substance to replace formamide during in situ hybridization and that can provide a comparable result.
Ethylene carbonate EC appears to be a promising alternative to formamide. Rapid diagnosis of acute promyelocytic leukemia with the PML-RARA fusion gene using a combination of droplet-reverse transcription-polymerase chain reaction and instantquality fluorescence in situ hybridization.
Clinica Chimica Acta 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. Formamide is used as a feedstock in the manufacture of formate esters, as an ionizing solvent, as an RNA stabilizer in gel electrophoresis, and in tissue preservation.
More intriguingly, it may be a key compound in the origin of life on Earth. 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. Learn more about this molecule from CAS , the most authoritative and comprehensive source for chemical information. If your favorite molecule is not in our archive , please send an email to motw acs. The molecule can be notable for its current or historical importance or for any quirky reason.
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