In situ hybridization of nucleic acids

Hybridization of radioactive nucleic acid sequences can be carried out with DNA in cytological preparations. By autoradiography distinct DNA sequences can then be localized in eukaryotic chromosomes.     

Effects of different fixatives on detection of nucleic acids from paraffin-embedded tissues by in situ hybridization using oligonucleotide probes

Detection of nucleic acids from paraffin-embedded material by in situ hybridization with oligonucleotide probes is increasingly being used. To determine the effect of fixation on the preservation of DNA and mRNA, we studied 18 lymphoid tissues fixed in B5, formalin, OmniFix, ethanol, and Bouin's fixatives and embedded in paraffin by in situ hybridization, using biotinylated oligonucleotide poly d(T) probes and immunoglobulin light chain probes. Detection of DNA using the poly d(T) probe was most consistent and most intense in tissue fixed in formalin, followed by OmniFix and ethanol, with B5 and Bouin's fixatives yielding unsatisfactory results. Detection of mRNA, using the light chain probes, was most consistent and most intense with tissue fixed in formalin and Bouin's solution, followed by B5 fixative, with OmniFix and ethanol fixatives yielding unsatisfactory results. The results of mRNA detection using the poly d(T) probe were found not to correlate with mRNA content as determined by the light chain probes for several fixatives, possibly owing to selective degradation of portions of the mRNA molecule.     

Discrimination of bacteriophage infected cells using locked nucleic acid fluorescent in situ hybridization (LNA-FISH)

Bacteriophage–host interaction studies in biofilm structures are still challenging due to the technical limitations of traditional methods. The aim of this study was to provide a direct fluorescence in situ hybridization (FISH) method based on locked nucleic acid (LNA) probes, which targets the phage replication phase, allowing the study of population dynamics during infection. Bacteriophages specific for two biofilm-forming bacteria, Pseudomonas aeruginosa and Acinetobacter, were selected. Four LNA probes were designed and optimized for phage-specific detection and for bacterial counterstaining. To validate the method, LNA-FISH counts were compared with the traditional plaque forming unit (PFU) technique. To visualize the progression of phage infection within a biofilm, colony-biofilms were formed and infected with bacteriophages. A good correlation (r = 0.707) was observed between LNA-FISH and PFU techniques. In biofilm structures, LNA-FISH provided a good discrimination of the infected cells and also allowed the assessment of the spatial distribution of infected and non-infected populations.     

Mismatch discrimination of base-modified nucleic acids and their constituents: non-Watson-Crick base pairing induced by tautomerization

The effect of tautomerism on the mismatch discrimination was studied on 7-deazapurine and 8-aza-7-deazapurine analogues of isoguanosine. 7-Halogenated 7-deaza-2'-deoxyisoguanosines show better base pair discrimination than 2'-deoxyisoguanosine due to the more favored keto tautomer formation. 8-Aza-7-deazaisoguanosine and its 7-halogeno derivatives also show higher keto tautomer population than that of isoguanosine, but the 7-halogens do not bias the tautomeric equilibrium significantly as it is observed for the 7-deaza-2'-deoxyisoguanosine derivatives.     

Binding of RecA protein to single-stranded nucleic acids: spectroscopic studies using fluorescent polynucleotides

Binding of the recA gene product from Escherichia coli to single-stranded polynucleotides has been investigated using poly(dA) that have been modified by chloroacetaldehyde to yield fluorescent 1,N6-ethenoadenine (epsilon A) bases. A strong enhancement of the fluorescent quantum yield of poly(d epsilon A) is induced upon RecA protein binding. A 4-fold increase is observed in the absence of ATP or ATP gamma S and a 7-fold increase in the presence of either nucleoside triphosphate. RecA protein can bind to poly(d epsilon A) in the absence of both Mg2+ ions and ATP (or ATP gamma S) but Mg2+ ions are required to observe RecA protein binding in the presence of ATP (or ATP gamma S) at pH 7.5. ATP binding to the RecA-poly(d epsilon A) complex induces a dissociation of RecA from the polynucleotide followed by re-binding of [RecA-ATP-Mg2+] ternary complex. Whereas ATP-induced dissociation of RecA-poly(d epsilon A) complexes is a fast process, the subsequent binding reaction of [RecA-ATP-Mg2+] is slow. A model is proposed whereby [RecA-ATP-Mg2+] binding to poly(d epsilon A) involves slow nucleation and elongation processes along the polynucleotide backbone. The nucleation reaction is shown to involve at least a trimer or a tetramer. Polymerization of the [RecA-ATP-Mg2+] ternary complex stops when the polynucleotide is entirely covered with 6 +/- 1 nucleotides per RecA monomer. ATP hydrolysis then induces a release of RecA-ADP complexes from the polynucleotide template.     

Mechanistic approach to the problem of hybridization efficiency in fluorescent in situ hybridization

In fluorescent in situ hybridization (FISH), the efficiency of hybridization between the DNA probe and the rRNA has been related to the accessibility of the rRNA when ribosome content and cell permeability are not limiting. Published rRNA accessibility maps show that probe brightness is sensitive to the organism being hybridized and the exact location of the target site and, hence, it is highly unpredictable based on accessibility only. In this study, a model of FISH based on the thermodynamics of nucleic acid hybridization was developed. The model provides a mechanistic approach to calculate the affinity of the probe to the target site, which is defined as the overall Gibbs free energy change (DeltaG degrees overall) for a reaction scheme involving the DNA-rRNA and intramolecular DNA and rRNA interactions that take place during FISH. Probe data sets for the published accessibility maps and experiments targeting localized regions in the 16S rRNA of Escherichia coli were used to demonstrate that DeltaG degrees overall is a strong predictor of hybridization efficiency and superior to conventional estimates based on the dissociation temperature of the DNA/rRNA duplex. The use of the proposed model also allowed the development of mechanistic approaches to increase probe brightness, even in seemingly inaccessible regions of the 16S rRNA. Finally, a threshold DeltaG degrees overall of -13.0 kcal/mol was proposed as a goal in the design of FISH probes to maximize hybridization efficiency without compromising specificity.     

Chromosome analysis using different staining techniques and fluorescent in situ hybridization in Cerithium vulgatum (Gastropoda: Cerithiidae)

In the present paper one population of the large" subtidal mollusc Cerithium vulgatum Bruguière, 1792 (Gastropoda: Cerithiidae) from the Northwestern coast of Sicily was investigated from a karyological point of view. The chromosome complement was Giemsa stained, conventionally karyotyped in 18 homomorphic chromosome pairs (10 bi-armed and 8 mono-armed), and subsequently analysed using silver, CMA3 and DAPI staining, and fluorescent in situ hybridization (FISH) with three repetitive DNA probes [ribosomal DNA (rDNA), (TTAGGG)n and (GATA)n]. FISH with the rDNA probe consistently mapped major ribosomal sites (18S-28S rDNA) in the terminal region of the short arms of one small sized mono-armed chromosome pair. Ribosomal DNA was transciptionally active as indicated by its preferential impregnation with silver nitrate (Ag-NOR) and did not contain a high amount of GC base pairs as suggested by the lack of a bright CMA3 fluorescence. The (TTAGGG)n telomeric probe was hybridized to the termini of nearly all chromosomes, thus demonstrating that, in C. tulgatum, this sequence has been conserved during the genomic evolution. The finding of the telomeric hexanucleotide in six species belonging to the three high taxa of Gastropoda supports the notion that this sequence is widespread within this class. The (GATA)n probe did not label any chromosome regions except for a minute terminal area of a single bivalent at pachytene stage.     

New approaches to in situ detection of nucleic acids

The present paper reviews recent results obtained by different molecular biology-based, immunocytological approaches to the localization and identification of nucleic acids in sections of biological material. Examples of sensitive, high-resolution detection methods for RNA, DNA or specialized DNA regions are presented. Special emphasis is placed on the potential values and limitations of these new methods.Presented at the XXXVII Symposium of the Society for Histochemistry, 23 September 1995, Rigi Kaltbad, Switzerland     

Gene mapping by fluorescent in situ hybridization

We describe a new method for the mapping of mammalian genes, utilizing in situ hybridization of mRNA to DNA of chromosomes. It involves the hydrogen bonding of the polyadenylic acid at the 3' end of hybridized mRNA to the polyuridylic acid tail of a highly fluorescent latex microsphere. The resultant double hybrid can be visualized by fluorescence microscopy. The chromosomal localization of human alpha + beta globin genes has been explored by this method. Our data point ot the long arms of chromosomes 4 and 5 as the loci for the human globin genes.     

In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)

Endopolyploidy, i.e. amplification of the genome in the absence of mitosis, occurs in many plant species and happens along with organ and cell differentiation. Deciphering the functional roles of endopolyploidy is hampered by the fact that polyploid tissues generally comprise cells with various ploidy levels. In some fleshy fruits (amongst them tomato fruit) the ploidy levels present at the end of development range from 2C to 256C in the same tissue. To investigate the temporal and spatial distribution of endopolyploidy it is necessary to address the DNA content of individual nuclei in situ. Conventional methods such as fluorometry or densitometry can be used for some tissues displaying favorable characteristics, e.g. small cells, small nuclei, organization in a monolayer, but high levels of varying polyploidy are usually associated with large sizes of nuclei and cells, in a complex three dimensional (3-D) organization of the tissues. The conventional methods are inadequate for such tissue, becoming semi-quantitative and imprecise. We report here the development of a new method based on fluorescent in situ bacterial artificial chromosome hybridizations that allows the in situ determination of the DNA ploidy level of individual nuclei. This method relies on the counting of hybridization signals and not on intensity measurements and is expected to provide an alternative method for mapping endopolyploidy patterns in mature, 3-D organized plant tissues as illustrated by the analysis of ploidy level and cell size in pericarp from mature green tomato fruit.     

A novel approach to nonradioactive hybridization assay of nucleic acids using stained latex particles.

The paper describes a sensitive latex hybridization assay (LHA) method applied for indirect detection of biotinylated nucleic acid hybrids immobilized on a synthetic membrane. The biotinylated hybrids were visualized by means of latex particles containing the fluorescent dye pyronine G and coated with streptavidin; 1.6 and 0.3 pg of lambda-phage DNA was detected by dot blot hybridizations on nylon membrane and polyethyleneimine-cellophane, respectively. The assay sensitivity was increased by three orders of magnitude over that with fluorescently labeled probes due to encapsulation of the fluorescent dye in polymer particles. LHA is simple (single-stage detection procedure), fast, and more sensitive than any of the other nonradioactive hybridization methods.     

Localization of nucleic acid sequences by EM in situ hybridization using colloidal gold labels

It is possible to combine hybridization to specimens on electron-microscope grids of nucleic-acid probes labelled nonisotopically with immunogold detection of hybrid sites to map the position of target sequences rapidly and precisely. The basic technique is described, and examples are provided to illustrate the types of questions which can be approached in the general area of higher-order chromosome organization and function. A combination of two differentially labelled probes and two different-sized gold particles permits the simultaneous detection of closely linked or interspersed sequences.     

AcroM fluorescent in situ hybridization analyses of marker chromosomes

The presence of a de novo supernumerary marker chromosome (SMC) poses problems in genetic counseling. The consequences of the additional chromosomal material may range from harmless to detrimental. As the composition of a SMC cannot be deciphered by traditional banding analysis, sophisticated methods are needed for their rapid and detailed analyses. A new strategy is presented, which allows the elucidation of the composition of SMCs in one or two hybridizations. One hybridization, termed AcroM-FISH, involves a newly generated probe mix, which consists of painting probes for all acrocentric chromosomes, centromere probes for chromosomes 13/21, 14/22, 15, and a probe specific for rDNA, each labeled with a specific combination of fluorochromes. This probe mix is sufficient to characterize approximately 80% of all SMCs. For the other 20% of SMCs, chromosomes can be analyzed in a second hybridization by multicolor karyotyping, for example, multiplex FISH (M-FISH), to check for the presence of euchromatin of other chromosomes. The potential of AcroM-FISH was tested in various applications.     

Assessing chromosomal abnormalities in two-cell bovine in vitro-fertilized embryos by using fluorescent in situ hybridization with three different cloned probes

The aim of this study was to assess the efficiency of fluorescent in situ hybridization (FISH) for detecting chromosomal abnormalities in in vitro-fertilized (IVF) bovine embryos as early as the 2-cell stage. Three different cloned probes were used, two derived from a unique sequence specific to the subtelomeric (D1S48) or subcentromeric regions (19C10) of chromosome 1 and the third (H1A clone) derived from a repetitive sequence that hybridizes to the subcentromeric regions of three other chromosomes (14, 20, 25). Our results show that the incidence of chromosomal abnormalities in 2-cell bovine IVF embryos varied from 28% to 44% according to the probes used for the analysis. Whereas the efficiency of FISH was high with somatic nuclei, it appeared to be highly variable with the 2-cell embryos. FISH efficiency depended firstly on the probe sequence (repetitive or unique sequence), secondly on the chromosomal target region (centromeric or telomeric regions), and thirdly on the embryo cell cycle phase. With a unique sequence probe (19C10) specific to the subcentromeric regions, FISH efficiency was better on nuclei in the S-phase cycle than on those in the G-phase. In S-phase 2-cell embryos, the overall incidence of chromosomal abnormalities was more accurately assessed. It reached 13% and was represented by 1n/2n mixoploidies.     

Chromosomal rearrangements in children with idiopathic mental retardation using subtelomeric fluorescent in situ hybridization

To screen a selected group of children with idiopathic mental retardation for subtelomeric abnormalities using the fluorescent in situ hybridization (FISH), which has been reported to be cost-effective in routine applications. We also aimed to assess the availability of the scoring system which is used for selection of those children for FISH analysis. A total of 30 children aged 3-16 years with idiopathic mental retardation (moderate to severe) and normal karyotypes were included in this study. The children whose parents had consanguineous marriages were excluded from the study. All cases were evaluated using the scoring system published by de Vries et al. (5) Forty-one subtelomeric regions for each case were analyzed by fluorescent in situ hybridization. One case with a score value 5 presented terminal deletion of chromosome 9p by FISH (3.3 %). Analyzing chromosomes of the same case with higher resolution G-banding showed the same abnormality. The frequency of subtelomeric abnormalities in our study group was much lower than the frequencies reported in other studies and the scoring criterions suggested by de Vries et al. have not effectively increased our subtelomeric deletion detection rates. Autosomal recessive disorders may be a more common reason compared to subtelomeric abnormalities in this group of patients in the countries where consanguinity rate is high. Laboratories may be encouraged to analyze high-resolution G-banded karyotypes in those cases. Moreover more effective selection criteria for FISH are suggested by establishing thorough genotype-phenotype correlations besides case reports with different subtelomeric abnormalities.