Optimization of the conditions for the in situ hybridization of cloned DNA sequences and for the differential staining of human chromosomes

The influence of different experimental conditions on in situ hybridization of DNA and subsequent differential staining of chromosomes was studied. The most optimal conditions for chromosomal localization of cloned repetitive DNA sequences were the lack of chromosome pretreatment with acid and RNase, reduction of the denaturation time to 30 s, carrying out of hybridization at a relatively low temperature (under 37 degrees C) at the expense of the use of formamide, addition to the hybridization mixture of 10% of dextran sulfate-500. The conditions indicated permit obtaining on radioautographs the G- and C-segmentation of human chromosomes.     

Detection of legumin gene DNA sequences in pea by in situ hybridization

In order to detect low copy number sequences in pea using biotin-labelled probes we optimised some aspects of the in situ hybridization technique. We found protoplast preparations to be superior to standard squashes in terms of their signal: noise ratio. Heat and alkali denaturation of chromosomal DNA were both more effective than acid denaturation. A comparison of antibody-fluorochrome and streptavidin-enzyme conjugates showed the streptavidin-alkaline phosphatase conjugate to be the most sensitive detection system. Using the optimised method, we were able to detect a single site for a 13.5 kb legumin gene clone.     

USE OF REPEATED DNA SEQUENCES AS CYTOLOGICAL MARKERS

The majority of DNA that is found in most of the flowering plants appears to be non-coding DNA. Much of this excess DNA consists of nucleotide sequences which exist as multiple copies throughout the genome and are designated as repetitive sequences. Those sequences which are found in moderately high to high numbers of copies are observed to be of the greatest value as cytological markers. Moderately high copies may exist as sequences which are dispersed throughout the chromosomes of some species and not dispersed in other more distantly related species. By taking advantage of this characteristic and the technique of in situ hybridization with biotinylated probes, breakpoints of chromosomal translocations may be observed between species such as wheat and rye. Many of the high copy number repetitive sequences are organized in a tandem fashion in specific loci in the chromosome. Chromosomal identification may be accomplished by using the in situ hybridization technique. Upon in situ hybridization with a repetitive sequence isolated from Aegilops squarrosa, the patterns of the sites of hybridization allowed the D-genome chromosomes to be identified. The sequence was also observed only on the D-genome chromosomes of several polyploid species indicating its usefulness as a genome specific marker. Using this genome specificity, assessment of the orientation of the D-genome chromosomal segments of hexaploid wheat carrying the sequence during interphase and prophase of mitotic root tip cells was possible. Repetitive DNA sequences, therefore, provide cytological markers necessary for studies of chromosomal identification, genome allocation, and genome orientation. The use of biotin-labeled DNA probes allows the technique of in situ hybridization to be performed much more rapidly and with a greater degree of safety and reliability.     

Human bone marrow colony formation in diffusion chambers. Normal values.

In situ hybridization techniques have previously employed a series of manipulations to effect denaturation of chromosomal DNA and reannealing of DNA-RNA hybrids. This report presents a new protocol which combines the denaturation and reannealing processes. DNA is heated in a solution of 50% formamide, 50% 4 × SSC containing the RNA to be hybridized. After l h at 70 °C the preparation is slowly cooled to 37 °C over a period of 6 h and incubated at 37 °C for an additional 10 h. This technique eliminates the possibility of premature reannealing of the DNA while employing hybridization conditions which, in vitro, lead to accurate base pairing.     

Fast and Non-Toxic In Situ Hybridization without Blocking of Repetitive Sequences

Formamide is the preferred solvent to lower the melting point and annealing temperature of nucleic acid strands in in situ hybridization (ISH). A key benefit of formamide is better preservation of morphology due to a lower incubation temperature. However, in fluorescence in situ hybridization (FISH), against unique DNA targets in tissue sections, an overnight hybridization is required to obtain sufficient signal intensity. Here, we identified alternative solvents and developed a new hybridization buffer that reduces the required hybridization time to one hour (IQFISH method). Remarkably, denaturation and blocking against repetitive DNA sequences to prevent non-specific binding is not required. Furthermore, the new hybridization buffer is less hazardous than formamide containing buffers. The results demonstrate a significant increased hybridization rate at a lowered denaturation and hybridization temperature for both DNA and PNA (peptide nucleic acid) probes. We anticipate that these formamide substituting solvents will become the foundation for changes in the understanding and performance of denaturation and hybridization of nucleic acids. For example, the process time for tissue-based ISH for gene aberration tests in cancer diagnostics can be reduced from days to a few hours. Furthermore, the understanding of the interactions and duplex formation of nucleic acid strands may benefit from the properties of these solvents.     

Denaturation, renaturation, and loss of DNA during in situ hybridization procedures

With the aim of optimizing in situ hybridization methods, alkaline, acid, and thermal denaturation procedures have been studied for their ability to separate the DNA strands of nuclear DNA and for the DNA losses they induce. Isolated methanol/acetic acid-fixed mouse liver nuclei have been used as a biological object. The results, obtained with acridine orange staining and microfluorometry, show that all denaturations studied lead to almost complete strand separation. Quantitative DNA staining and cytometry indicated that with heat and alkaline denaturation about 40% of the DNA is lost. Acid denaturation led to about 20% DNA loss. For the alkaline denaturation, the DNA retention could be improved to a 20% DNA loss by adding 70% ethanol to the denaturation medium. During hybridization, another 20% DNA loss occurs. When denatured nuclei are brought under annealing conditions, a rapid renaturation of a considerable fraction of the remaining DNA occurs. The extent of renaturation was dependent on the type of denaturation used. For the ethanolic alkaline denaturation, it was estimated to be 35%. Quantitative nonautoradiographic in situ hybridization experiments with acetylaminofluorene-modified mouse satellite DNA showed that alkaline denaturation procedures are superior to the heat and acid denaturation. As proven by acridine orange fluorescence measurements, hybridization conditions can be designed that permit DNA.RNA hybridization under in situ DNA.DNA denaturing conditions. These conditions should be very useful, especially for in situ hybridization with single-stranded RNA probes.     

Molecular cytotaxonomy of primates by chromosomal in situ suppression hybridization

A new strategy for analyzing chromosomal evolution in primates is presented using chromosomal in situ suppression (CISS) hybridization. Biotin-labeled DNA libraries from flow-sorted human chromosomes are hybridized to chromosome preparations of catarrhines, platyrrhines, and prosimians. By this approach rearrangements of chromosomes that occurred during hominoid evolution are visualized directly at the level of DNA sequences, even in primate species with pronounced chromosomal shuffles.     

Localization of specific repetitive DNA sequences in individual rice chromosomes

This paper describes the characterization and chromosomal distribution of three different rice (Oryza sativa) repetitive DNA sequences. The three sequences were characterized by sequence analysis, which gave 355, 498 and 756 bp for the length of the repeat unit in Os48, OsG3-498 and OsG5-756, respectively. Copy number determination by quantitative DNA slot-blot hybridization analysis showed 4000, 1080 and 920 copies, respectively, per haploid rice genome for the three sequences. In situ DNA hybridization analysis revealed that 95% of the silver grains detected with the Os48 probe were localized to euchromatic ends of seven long arms and one short arm out of the 12 rice chromosomes. For the OsG3-498 repetitive sequence, the majority of silver grains (58%) were also clustered at the same chromosomal ends as that of Os48. The minority (28%) of silver grains were located at heterochromatic short arms and centromeric regions. For the OsG5-756 repetitive sequence, 81% of the silver grains labeled the heterochromatic short arms and regions flanking all of the 12 centromeres. Thus, each of these three repetitive sequences was distributed at specific defined chromosomal locations rather than randomly at many chromosomal locations. The approximate copy number of a given repetitive DNA sequence at any specific chromosomal location was calculated by combining the information from in situ DNA hybridization analysis and the total copy number as determined by DNA slot-blot hybridization.by J. Huberman     

Histological Detection of Chicken δ-Crystallin DNA Sequences Introduced into Mouse Teratocarcinoma Cell Lines

In situ hybridization techniques to detect specific DNA sequences in histological sections were developed for the purpose of analyzing experimental chimeras produced by combination of mouse teratocarcinoma (TCC) cells stably carrying chicken δ-crystallin DNA sequences and normal mouse embryos. Various hybridization conditions for detection of exogenous DNA sequences were compared in samples of solid tumors of TCC lines. Of the conditions examined, denaturation of DNA in alkali and hybridization at 68°C in 6x SSC in the presence of dextran sulphate was the best for detecting δ-crystallin DNA sequences. With ³H-labelled probe under these conditions, virtually all nuclei containing more than 100 copies of chicken δ-crystallin sequences were labelled sufficiently to be distinguishable from nuclei without chicken sequences. This technique could be applied to other experimental chimeras in which specific DNA sequences can be used as markers of certain cell lineages.     

Chromosomal insertion of human papillomavirus 18 sequences in HeLa cells detected by nonisotopic in situ hybridization and reflection contrast microscopy

Summary Genomic insertion of human papillomavirus (HPV) sequences is associated with the genesis of cervical carcinoma, and HPV-induced incipient cellular alterations may also present a requisite for the establishment of cell lines such as HeLa. Considering the theoretical importance of specific viral integration sites, we attempted to detect in HeLa cells the chromosomal location of DNA sequences homologous to HPV-16 and HPV-18 sequences by a nonisotopic high resolution in situ hybridization technique. Chromosome identification following in situ hybridization was possible by counterstaining of the same preparation with Chromomycin A₃, Distamycin A, and DAPI. Using this approach, we have assigned HPV-18 integration in HeLa cells to band 8q24 (a site including the locus of the myc-protooncogene), to an abnormal chromosome 22, and to a not yet identified marker chromosome possibly neighboring other oncogenic or activating sites. The sensitive detection technique described in this study presents a new approach involving in situ chromosome hybridization with biotinylated DNA probes in combination with reflection contrast microscopy and subsequent fluorescent R-and C-banding. The method allowed the assignment of a 7-kb HPV-18 DNA probe to human chromosomal sites important in growth regulation and cancerogenesis. It should prove useful in a number of similar studies using other viral and oncogenic DNA probes.     

Rapid Denaturation Improves Chromosome Morphology and Permits Multiple Hybridizations During Fluorescence in Situ Hybridization

Denaturation of chromosomal DNA for fluorescence in situ hybridization (FISH) is an essential step in a procedure associated with a number of variables. In our experience, shorter denaturation time in 70% formamide/2 × SSC at 72 C provides sufficient denaturation, where the hydrogen bonds are broken between the purines and pyrimidines of the double helix. This shortened exposure improves retention of morphology of human chromosomes from lymphocytes, aminocytes, fibroblasts and bone marrow, and allows the same metaphases to be denatured repeatedly and rehybridized with different probes. This approach is useful in investigations where sample volume is limited.     

In situ hybridization and chromosome banding in mammalian species

Chromosome banding is often required in conjunction with fluorescent in situ hybridization of labelled probes for chromosome painting, satellite DNA and low-copy sequences to allow identification of chromosomes and simultaneous probe localization. Here, we present a method that reveals both patterns with only one observation step. The band pattern is produced by restriction-enzyme digestion of chromosomes, followed by fixation with paraformaldehyde in PBS, a short chromosome denaturation step in hybridization solution, and then standard in situ hybridization, washing and detection protocols. Using a range of different mammalian species, chromosome-banding patterns were immediately recognizable, although synchronisation procedures normally required for high- resolution G-banding were not needed. Unlike other methods available, only one round of observation is required using a conventional fluorescence microscope, the method works without modification in many species, and in situ hybridization is not used for chromosome identification (allowing multiple targets and minimizing background). The banding pattern is probably generated by a combination of DNA dissolution and heterochromatin reorganisation after enzyme digestion, followed by paraformaldehyde fixation of the new chromatin structure and incomplete denaturation. The method is of widespread utility in comparative genomics and genome organization programmes.     

Evolution of Y chromosomal lampbrush loop DNA sequences of Drosophila

The evolutionary conservation of Y chromosomal DNA sequences of Drosophila hydei in different species of the genus Drosophila was studied by in situ hybridization and on genomic DNA blots of restriction enzyme digested DNA. We demonstrated that Y specific DNA sequences, which form major parts of lampbrush loops related to the male fertility genes, are only retained in a few closely related species during evolution. Other Y chromosomal DNA sequences, also present in lampbrush loops but with homology to autosomal and X chromosomal locations, were found in distant species. We propose a model for the evolution of the Y chromosomal lampbrush loops.     

Application of primed in situ DNA synthesis (PRINS) with telomere human commercial kit in molecular cytogenetics of Equus caballus and Sus scrofa scrofa

Recently, molecular techniques have become an indispensable tools for cytogenetic research. Especially, development of in situ techniques made possible detection at the chromosomal level, genes as well as repetitive sequences like telomeres or the DNA component of telomeres. One of these methods is primed in situ DNA synthesis (PRINS) using an oligonucleotide primer complementary to the specific DNA sequence. In this report we described application of PRINS technique with telomere human commercial kit to telomere sequences identification. This commercial kit may be use to visualization of interstitial telomeric signal in pig genome. PRINS is attractive complement to FISH for detection of DNA repetitive sequences and displays lower level of non-specific hybridization than conventional FISH.     

Genomic distribution of heterochromatic sequences in equids: implications to rapid chromosomal evolution.

We describe a molecular model for rapid chromosomal evolution that proposes tandemly repeated DNA sequences as a driving force. A prediction of this model is that when extensive rearrangements of euchromatin have been facilitated by heterochromatin, genomes will be characterized by tandemly repeated sequences that have actively changed chromosomal fields by intragenomic movement. Alternatively, it is proposed that in conservative chromosomal lineage each class of tandemly repeated sequences will be restricted to a specific chromosomal field. To provide baseline data to test this model we examined four classes of tandemly repeated elements in six species of equids (Equus). Distribution of these sequences among species, as determined from slot blot analysis, and restriction site variation, shown by Southern blot hybridization, document that these sequences are in an evolutionarily dynamic state, and in situ hybridization documents extensive intragenomic movement among nonhomologous chromosomes and chromosomal fields. These data are interpreted as being compatible with the predictions of this model. Although this is clearly not the sole molecular factor driving chromosomal evolution, the model appears to be viable as an explanation of certain patterns of chromosomal evolution such as karyotypic megaevolution and some types of karyotypic orthoselection.     

Identifying a single-copy DNA sequence associated with the expression of a heterochromatic gene, the light locus of Drosophila melanogaster

Although little is known about the molecular organization of most genes within heterochromatin, the unusual properties of these chromosomal regions suggest that genes therein may be organized and expressed very differently from those in euchromatin. We report here the cloning, by P transposon tagging, of sequences associated with the expression of the light locus, an essential gene found in the heterochromatin of chromosome 2 of Drosophila melanogaster. We conclude that this DNA is either a segment of the light locus, or a closely linked, heterochromatic sequence affecting its expression. While other functional DNA sequences previously described in heterochromatin have been repetitive, light gene function may be associated, at least in part, with single-copy DNA. This conclusion is based upon analysis of DNA from mutations and reversions induced by P transposable elements. The cloned region is unusual in that this single-copy DNA is embedded within middle-repetitive sequences. The in situ hybridization experiments also show that, unlike most other sequences in heterochromatin, this light-associated DNA evidently replicates in polytene chromosomes, but its diffuse hybridization signal may suggest an unusual chromosomal organization.     

Detection of single-copy genes by nonisotopic in situ hybridization on human chromosomes

Summary A technique of in situ hybridization on metaphase chromosomes with biotinylated DNA probes is described. This technique was used to localize unique DNA sequences on chromosomes and allowed a localization of two probes 1.8 and 1.3 kb long. The hybridization signal appears like two, twin, spots on the two sister chromatids, allowing a clear distinction from the background. Moreover a chromosomal localization is possible by counting a relatively small number of mitoses compared with the technique using ³H-labeled DNA probes.     

PCR amplification of chromosome-specific DNA isolated from flow cytometry-sorted chromosomes.

We have established a method for amplifying and obtaining large quantities of chromosome-specific DNA by linker/adaptor ligation and polymerase chain reaction (PCR). Small quantities of DNA isolated from flow cytometry-sorted chromosomes 17 and 21 were digested with MboI, ligated to a linker/adaptor, and then subjected to 35 cycles of PCR. Using this procedure, 20 micrograms of chromosome-specific DNA can be obtained. Southern blot analysis using several DNA probes previously localized to chromosomes 17 and 21 indicated that these gene sequences were present in the amplified chromosome-specific DNA. A small quantity of the chromosome-specific DNA obtained from the first round of PCR amplification was used to amplify DNA for a second, third, and fourth round of PCR (30 cycles), and specific DNA sequences were still detectable. Fluorescence in situ hybridization using these chromosome-specific DNA probes clearly indicated the hybridization signals to the designated chromosomes. We showed that PCR-amplified chromosome 17-specific DNA can be used to detect nonrandom chromosomal translocation of t(15;17) in acute promyelocytic leukemia by fluorescence in situ hybridization.