Polymerase Chain reaction generated probes for fluorescence in situ hybridization

The extended use of Fish with centromeric probes in many cytogenetic laboratories is often impaired by the cost of this technique. Polymerase Chain Reaction (PCR) constitutes a simple way to generate and label such centromeric probes at low cost. Two types of human DNA source can be used: 1--Somatic hybrid cell lines containing a unique human chromosome. The specific amplification of the human subset of alphoid DNA is realised with a primer pair specific for the consensus region of human alpha satellite sequence. 2--Total Human DNA. This time, a primer pair specific for the alpha satellite DNA of the chromosome of interest must be designed. These probes, labelled during the PCR reaction by direct incorporation of modified dUTP, are actually widely used in our laboratory, alone or mixed with other probes (chromosome painting or locus specific probes).     

Chromosomal localization of the human myeloperoxidase gene by in situ hybridization using oligonucleotide probes

Oligonucleotide probes have been used to map the myeloperoxidase (MPO) gene locus to chromosome bands 17q21-22. This is in agreement with results reported using conventional cDNA probes. No evidence for the existence of a second MPO gene locus was obtained. Six synthetic 72-base oligonucleotides, corresponding to different exon regions of the MPO gene, were tritium-labeled and used as in situ hybridization probes. Synthetic oligonucleotide probes offer a useful alternative to conventional DNA probes for gene mapping.     

Detection of avocado sunblotch viroid by hybridization with synthetic oligonucleotide probes

Two short (20 and 17 nucleotides) DNA hybridization probes, complementary to avocado sunblotch viroid (ASBV) RNA nucleotides 68-87 and 88-104 respectively (Symons, R.H., Nucleic Acid Res. 9, 6527, 1981) were synthesized. The sensitivity and specificity of these radioactively labelled probes for hybridization with RNA of several ASBV isolates are demonstrated.     

Prenatal diagnosis with repetitive in situ hybridization probes

We have used chromosome-specific repetitive sequences to detect the most common human aneuploidies prenatally. Together chromosome 21, 13, 18, X, and Y aneuploidy comprises 95% of the chromosome abnormalities that result in a high risk of abnormal phenotypes at birth. The X, Y, and 18 repetitive probes work reliably in multiple tissue types including directly examined and cultured amniocytes, chorionic villus cells, lymphocytes, and cultured fibroblasts. The probe that detects both chromosomes 13 and 21 routinely gives results in each cell type tested except directly studied amniocytes which can be interpreted in seven-ninths of the cases with protocol 1 and all tested samples with protocol 2. Our protocols diagnosed trisomy 21 in a 23-week fetus with low maternal serum AFP and a trisomy 18 in a direct chorionic villus sample 2 working days after the samples were obtained. Trisomy 21 also has been ruled out in a CVS karyotype first thought to be 47,XY,+21. These studies reflect the potential value of in situ hybridization to provide a more rapid, less expensive means to screen most at-risk fetal populations with less effort in first world cytogenetic laboratories, and to provide economical cytogenetic services in less developed countries. © 1992 Wiley-Liss, Inc.     

Detection ofLeishmania parasites by DNA in situ hybridization with non-radioactive probes

In situ hybridization techniques develop rapidly into diagnostic tools of considerable value for detection of viruses and bacteria. Here we report the application of this technique for the detection ofLeishmania parasites. Biotin-labelled total promastigote DNA was hybridized to culturedLeishmania parasites and to blood and impression smears of infected mice. In promastigotes kinetoplasts were strongly stained, nuclei somewhat more diffuse. In amastigotes both nuclear and kinetoplast DNA hybridized strongly. Amastigotes were easily detected in tissue of infected mice by their stable configuration of kinetoplast and nuclei. Cross-hybridization was observed betweenLeishmania donovani andL. tropica, but not between these two andL. braziliensis orTrypanosoma cruzi. A minor aspecific staining of host cell nuclei in the smears did not interfere with the detectability of the parasites.     

Prenatal diagnosis with repetitive in situ hybridization probes.

We have used chromosome-specific repetitive sequences to detect the most common human aneuploidies prenatally. Together chromosome 21, 13, 18, X, and Y aneuploidy comprises 95% of the chromosome abnormalities that result in a high risk of abnormal phenotypes at birth. The X, Y, and 18 repetitive probes work reliably in multiple tissue types including directly examined and cultured amniocytes, chorionic villus cells, lymphocytes, and cultured fibroblasts. The probe that detects both chromosomes 13 and 21 routinely gives results in each cell type tested except directly studied amniocytes which can be interpreted in seven-ninths of the cases with protocol 1 and all tested samples with protocol 2. Our protocols diagnosed trisomy 21 in a 23-week fetus with low maternal serum AFP and a trisomy 18 in a direct chorionic villus sample 2 working days after the samples were obtained. Trisomy 21 also has been ruled out in a CVS karyotype first thought to be 47,XY, +21. These studies reflect the potential value of in situ hybridization to provide a more rapid, less expensive means to screen most at-risk fetal populations with less effort in first world cytogenetic laboratories, and to provide economical cytogenetic services in less developed countries.     

New localizations of VH sequences by in situ hybridization with biotinylated probes

The chromosomal localization of genes of three VH families (VH 1-3) was performed using in situ hybridization with biotinylated probes. Significantly strong signals were observed on chromosome 14, band 14q32, and on bands 16p11 and 15q11, although less frequently. Signal intensity and frequency were more important on chromosome 14 with all three probes, and on chromosome 16 with the VH2 and VH3 probes, while chromosome 15 was more marked than 16 with the VH1 probe. The localization of VH gene on chromosomes other than 14 suggests that several genes of the VH family had been simultaneously translocated in evolution and that the newly localized VH sequences may be pseudogenes.     

In situ hybridization of immunoglobulin light chain mRNA in paraffin sections using biotinylated or hapten-labelled oligonucleotide probes

An in situ hybridization technique has been developed for the detection of immunoglobulin light chain mRNA in routine pathology specimens. The method detects kappa or lambda constant region sequences using a cocktail of synthetic oligonucleotide probes labelled with biotin or fluorescein 5-isothiocyanate (FITC) reporter molecules. The probes were labelled at flanking sites chemically by primary amine directed acylation and by 'homopolymer tailing' with terminal deoxynucleotidyl transferase using non-radioactive nucleotide analogues. The mRNA was unmasked in the formalin-fixed tissue sections by digestion with varying concentrations of proteinase K, and the hybrids were demonstrated using alkaline phosphatase with either a streptavidin/biotin based four-stage system or an anti-FITC antibody based detection system. Alkaline phosphatase was visualized using a Fast Red naphthol-capture method and the sections were counterstained with haematoxylin. The results confirm that the method is specific for kappa or lambda mRNA and show that specific mRNAs can be detected in routine formalin-fixed sections using non-radioactive techniques with retention of good morphology. The method reliably detects light chain mRNA in cells expressing secretory immunoglobulin. The protocol can also be applied to tissue rich in endogenous biotin by using hapten-labelled probes.     

Fluorescence in situ hybridization in diagnostic cytology

Fluorescence in situ hybridization (FISH) is a technique that uses fluorescently labeled DNA probes to detect chromosomal alterations in cells. FISH can detect various types of cytogenetic alterations including aneusomy (ie, abnormalities of chromosome copy number), duplication, amplification, deletion, and translocation. Because tumor cells generally contain chromosomal alterations, FISH is able to detect cells that have chromosomal abnormalities consistent with neoplasia in exfoliative and aspiration cytology specimens. This review will discuss the utility of FISH for the detection of bladder, lung, pancreatobiliary, and esophageal carcinoma in cytologic specimens.     

Detection and typing of human papillomaviruses by in situ hybridization with biotinylated oligonucleotide mixtures

The value of biotinylated Oligonucleotide probes for screening and typing by in situ hybridization of the most frequent genital human papillomavirus infections (HPVs 6,11,16,18, 31, and 33) was assessed. Optimal hybridization conditions were defined on a panel of paraffin-embedded tissue sections previously characterized with HPV full genome probes. Mixtures of oligonucleotides rather than single oligonucleotides were used to improve sensitivity and specificity. All HPV-positive specimens were detected by the screening mixture with a sensitivity and specificity similar to that of full genome probes. Typing mixtures were highly specific for each HPV type. This study confirms the potential of Oligonucleotide probes for detecting and typing HPV infections. © 1995 Wiley-Liss, Inc.     

Rapid detection of a plant virus by solution hybridization using oligonucleotide probes

A novel solution hybridization method for the diagnosis of a plant virus was evaluated. Synthetic oligonucleotide probes were used for the detection of potato virus X (PVX) in crude leaf sap extracts by hybridization in solution. Three 40-nucleotide-long oligonucleotide probes complementary to RNA sequences of potato virus X near the 3' end were synthesized. Two probes were 32P-labelled and one biotinylated. The three probes were allowed to form hybrids with the target viral nucleic acid in solution, and the formed hybrids were isolated with the aid of the biotinylated capture probe using avidin polystyrene beads after the reaction. Alternatively, hybrids were captured from the poly(A) tail of the viral RNA on oligo(dT) cellulose. The maximum signal was obtained after 4 h hybridization. About 70% of the maximum signal was obtained after 2 h hybridization. Sensitivity with the two 32P-labelled oligonucleotide probes was 1-5 x 10(7) molecules of PVX RNA. This corresponds to 0.6-3 ng of the virus. Crude leaf sap did not interfere with the detection of the virus. These results suggest that this solution hybridization method permits rapid detection of a plant virus in crude plant sap without sample pretreatment and may thus open new avenues for the development of a nucleic-acid-based ELISA-like diagnostic test for the detection of plant viruses.     

A novel diagnostic method of Pneumocystis carinii. In situ hybridization of ribosomal ribonucleic acid with biotinylated oligonucleotide probes

The reactivity and specificity of the in situ hybridization of ribosomal RNA in the diagnosis of Pneumocystis carinii were investigated. Three complementary oligonucleotide probes, 22 and 25 nucleotides long, corresponding to the species specific regions of 5S and 18S ribosomal RNA of Pneumocystis carinii were synthesized and labeled with biotinylated dUPT at the 3' termini. In situ hybridization was performed on formalin-fixed paraffin-embedded human lung tissues using the mixture of these probes and detected with the avidin-biotin peroxidase complex method. The reactions were positive in all 12 cases of Pneumocystis carinii pneumonia, but in none of the infections with other pathogenic agents, including virus (6 cases), mycobacteria (4 cases), protozoa (4 cases) and fungi (8 cases). The reactivity and specificity of this method was comparable with that of immunohistochemistry using a monoclonal anti-human Pneumocystis carinii antibody. Because the specificity of in situ hybridization is based on nucleotide sequences of ribosomal RNAs, that are constant among species, contrary to morphology of protista or expression of antigens, it should complement conventional staining and immunohistochemical methods, and provide a useful tool for the diagnosis of Pneumocystis carinii.     

Aneuploidy assays on interphase nuclei by means of in situ hybridization with DNA probes

A method has been developed to detect chemically induced aneuploidyin interphase nuclei by means of in situ hybridization withchromosome-specific DNA probes. Lymphocyte cultures were treatedwith two known aneuploidy inducers, Benomyl and Griseofulvin.Two DNA fragments, QP23 and Y97, homologous to repetitive sequences,localized in the pericentromeric region of chromosome 9 andin the centromeric region of Y chromosome respectively, wereused as probes. Following autoradiography, grain clusters, revealingthe presence of the target chromosomes, were scored in restingnuclei. A marked increase in the frequency of cells with supernumeraryautoradiographic signals was observed with both probes at allconcentrations of the test compounds. The assay procedure appearedto be reproducible, sensitive and efficient in scoring largecell samples. It may therefore provide a useful tool for preliminaryscreening of potential aneuploidy inducers.     

Fluorescence in situ hybridization with peptide nucleic acid probes for rapid identification of Candida albicans directly from blood culture bottles

A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C. albicans 26S rRNA. The PNA probe is added to smears made directly from the contents of the blood culture bottle and hybridized for 90 min at 55 degrees C. Unhybridized PNA probe is removed by washing of the mixture (30 min), and the smears are examined by fluorescence microscopy. The specificity of the method was confirmed with 23 reference strains representing phylogenetically related yeast species and 148 clinical isolates covering the clinically most significant yeast species, including C. albicans (n = 72), C. dubliniensis (n = 58), C. glabrata (n = 5), C. krusei (n = 2), C. parapsilosis (n = 4), and C. tropicalis (n = 3). The performance of the C. albicans PNA FISH method as a diagnostic test was evaluated with 33 routine and 25 simulated yeast-positive blood culture bottles and showed 100% sensitivity and 100% specificity. It is concluded that this 2.5-h method for the definitive identification of C. albicans directly from yeast-positive blood culture bottles provides important information for optimal antifungal therapy and patient management.     

Fluorescence in situ hybridization: A brief review

Fluorescence in situ hybridization (FISH) is used for many purposes, including analysis of chromosomal damage, gene mapping, clinical diagnostics, molecular toxicology and cross-species chromosome homology. FISH allows an investigator to identify the presence and location of a region of cellular DNA or RNA within morphologically preserved chromosome preparations, fixed cells or tissue sections. This report describes in situ hybridization, and discusses the past, present and future applications of this method for genetic analysis and molecular toxicology. © 1996 Wiley-Liss, Inc.     

Detection of immunoglobulin light chain mRNA in nodular sclerosing Hodgkin''s disease by in situ hybridization with biotinylated oligonucleotide probes compared with immunohistochemical staining with poly- and monoclonal antibodies

In order to elucidate the origin of the Hodgkin's and Reed-Sternberg cells, the expression of immunoglobulin kappa- and lambda light chain mRNA in 23 cases of nodular sclerosing and two cases of mixed cellularity Hodgkin's disease was examined by in situ hybridization using biotinylated oligonucleotide probes and compared with immunohistochemical staining with mono- and polyclonal antibodies against immunoglobulin kappa- and lambda light chains. No hybridization signals were seen in Hodgkin's or Reed-Sternberg cells in any of the cases. Polyclonal staining with polyclonal anti-immunoglobulin light chain antibodies was seen in Hodgkin's and Reed-Sternberg cells in 12 cases of nodular sclerosis and in two cases of mixed cellularity and with monoclonal antibodies in three cases of nodular sclerosis, but in no cases of mixed cellularity. In all cases, there was polyclonal labelling of plasma cells with both the oligonucleotide probes and the antibodies. In five cases, the Hodgkin's and Reed-Sternberg cells were also stained with one of the B-cell antibodies L26, MB2 or LN1. Lack of mRNA signals in Hodgkin's and Reed-Sternberg cells might indicate that these cells in Hodgkin's disease of the nodular sclerosis subtype are either not B-cell derived or they are early B-cells (precursor B-cells) not yet able to produce immunoglobulin light chain mRNA, at least not at a level detectable by in situ hybridization. Immunohistochemical staining of Hodgkin's and Reed-Sternberg cells, however, with antibodies against immunoglobulin kappa and lambda light chains may be explained by cellular uptake of the light chains, but the difference in reactivity between poly- and monoclonal antibodies cannot be explained at present.     

The distribution of histidine decarboxylase mRNA in the rat brain: an in situ hybridization study using synthetic oligonucleotide probes

L-Histidine decarboxylase catalyzes the formation of histamine from the amino acid L-histidine. We have studied the distribution of neurons expressing mRNA for histidine decarboxylase in adult rat brain using in situ hybridization with synthetic oligonucleotide probes. The expression of mRNA for histidine decarboxylase was detected in the hypothalamic tuberomammillary nucleus that has been shown to contain histidine decarboxylase-like and histamine-like immunoreactivity, but not in any other brain area. This method may prove useful in studying the physiological role of central histaminergic neurons.     

Synthetic oligonucleotide probes for the detection of human papilloma viruses by in situ hybridisation

Using the published nucleotide sequence data for human papilloma virus (HPV) types 1, 6 and 16, sequences of 30 bases in length from the beginning of the E6 open reading frame (ORF) were selected. Oligonucleotides were synthesised on an Oswel Gene Synthesizer and labelled at the 3' end with biotin using the enzyme terminal transferase. In situ hybridisation was carried out on paraffin sections of wart and cervical tissues mounted on silanated slides. A 2 h hybridisation step allowed the whole process to be completed within one working day. The technique successfully demonstrated the presence of HPV-1 in skin warts, and of HPV-6 and HPV-16 in genital warts and cervical lesions. This simple approach has diagnostic potential for the detection and typing of papilloma viruses in biopsy material.     

In situ hybridization with nonisotopic probes using different detection systems.

In order to evaluate a sensitive nonisotopic in situ hybridization method for routine work in pathology laboratories, we compared seven different detection systems, using digoxigenin- and biotin-labeled probes. The sensitivity of these methods was tested on four cases of cervical condyloma all known to be positive for HPV 6. Four of these methods gave satisfactory results without any background staining. The single biotin method and the single digoxigenin method were equally sensitive, while the two triple biotin methods, using mouse anti-biotin/anti-mouse IgG/alkaline phosphatase mouse anti-alkaline phosphatase or mouse anti-biotin/alkaline phosphatase anti-mouse IgG/alkaline phosphatase mouse anti-alkaline phosphatase as the detection systems, tremendously improved the sensitivity. The enhanced sensitivity of the nonisotopic in situ hybridization method make it useful in investigation of pathologic tissues.