Biotinylated DNA Probes Research Articles

Nonisotopic human papillomavirus DNA typing of cervical smears obtained at the initial colposcopic examination

To determine the prevalence of human papillomavirus (HPV) infection in 401 patients attending colposcopy for the first time, scraped cervical cells were investigated using dot blot hybridization and biotinylated DNA probes to HPV 6 and 11 (low-risk types) and 16, 18, and 33 (high-risk types). The HPV DNA was isolated from 52% of patients (low-risk types = 4%, high-risk types = 48%). Seventy-five percent had a cervical intraepithelial neoplasia (CIN)-condyloma. Low-risk types were infrequent (7%) and high-risk types (41%) predominant in condyloma/CIN I lesions when converse rates were expected. As CIN I lesions harboring high-risk types are at some risk of progressing to a higher grade dysplasia, colposcopic examination and treatment of this subgroup would seem justified. As expected, high-risk types were statistically associated with increasing grades of dysplasia. This hybridization method identified typeable HPV DNA in 60% of patients with a CIN-condyloma, and highlighted a unique HPV profile for this patient cohort.

Human papillomavirus type 16/18 in uterine cervical adenocarcinomain situ and adenocarcinoma a study byin situ hybridization with biotinylated DNA probes

The distribution of human papillomavirus (HPV) DNA type 16/18 in the paraffin sections of 11 adenocarcinomas (AC), 4 adenocarcinomas in situ (AIS), and 2 adenosquamous carcinomas (AC/SCC) of the cervix was examined by in situ hybridization (ISH) with biotinylated DNA probes (B-probes). Four AC, four AIS, and one AC/SCC were positive. These results show that B-probes are an acceptable alternative to both ISH with radiolabeled probes and Southern blot hybridization (SBH), which are difficult to apply in routine pathologic laboratories. Cervical tissues infiltrated with endometrial AC were negative for HPV DNA 16/18, and it is suggested that the presence of HPV DNA 16/18 in uterine AC may be helpful in discriminating cervical AC from endometrial AC.

In situ hybridisation in herpetic lesions using a biotinylated DNA probe.

In situ hybridisation was performed with a biotinylated DNA probe for herpes simplex virus (HSV) using high temperature denaturation on formalin fixed, paraffin wax sections of lung, brain, ganglion and keratinising and non-keratinising squamous epithelia. Eosinophilic viral nuclear inclusions or characteristically moulded multiple nuclei with altered chromatin, which were present in two cases of HSV encephalitis and one case of viral pneumonitis, all showed complete hybridisation visualised by an alkaline phosphatase/nitroblue tetrazolium detector system. HSV encephalitis and trigeminal ganglionitis, which were confirmed serologically or clinicopathologically but lacked nuclear changes, also gave positive dense nuclear signal in neurons, glias and satellite cells. No staining was present in the ganglion cells in trigeminal zoster, the glia in progressive multifocal leucoencephalopathy, or in a variety of cells in a lung coinfected with cytomegalovirus. In 10 herpetic blisters of squamous epithelia, infected cells hybridised strongly, while morphologically similar herpes zoster lesions remained negative. In neural tissues non-hybridisation staining was most obtrusive in corpora amylacea and seemed to reflect nonspecific probe adherence. In squamous epithelium, major non-hybridisation staining was caused by probe and antibody possibly adhering to intracellular keratin. The HSV probe permits specific detection of virus in the absence of characteristic nuclear changes and allows varicella zoster virus to be differentiated from HSV, provided that the aforementioned problems with non-hybridisation staining are borne in mind.

Synthesis in vitro and application of biotinylated DNA probes for human papilloma virus type 16 by utilizing the polymerase chain reaction

The polymerase chain reaction (PCR) has been used to incorporate biotinylated deoxynucleotide triphosphate analogues into a 120 bp sequence from the E6 region of human papilloma virus type 16 (HPV 16). No loss of amplification efficiency is observed utilizing concentrations of up to 200 microM-biotin-11-dUTP, or 180 microM-biotin-7-dATP and -biotin-16-dUTP (where the numbers refer to the number of carbon atoms in the spacer arms). Internally biotinylated PCR products can be detected following slot-blot or vacuum transfer to mitrocellulose or nylon filters without prior electrophoretic separation of the reactants, since unincorporated biotinylated analogues pass through the filter. Internally biotinylated PCR products can also be applied as hybridization probes in Southern blot analysis or in situ hybridization. This system enables detection of PCR products or target sequences at levels below that for 5'-biotinylated probes and can be applied in an 'open sandwich assay' without the need for a separate labelled probe currently required in conventional sandwich assays. However, as hybridization probes, sensitivity may be limited by the steric hindrance of strand hybridization possibly due to the spacer arms linking the nucleotides to the biotin molecule.

Detection of calcitonin-encoding mRNA by radioactive and non-radioactive in situ hybridization: improved colorimetric detection and cellular localization of mRNA in thyroid sections.

The localization of mRNA encoding calcitonin was studied by in situ hybridization using 35S-labeled RNA probes and biotin-labeled DNA probes. Radiolabeled probes were detected by autoradiography and biotin-labeled probes by streptavidin-biotin-peroxidase. To intensify the colorimetric signal, the indirect avidin-biotin complex (ABC) method was performed. However, the results were often variable. To improve the sensitivity, the peroxidase reaction signal was enhanced with a gold-silver deposit intensification reaction. To shorten the incubation times and to enhance the colorimetric reaction, several reaction steps were performed in a microwave oven. The localization of calcitonin mRNA in thyroid tissue, as detected with in situ hybridization, was confirmed by immunohistochemical localization of the calcitonin polypeptide. The results of in situ hybridization using biotinylated probes were compared to in situ hybridization using radioactive probes. Our data show that the results of in situ hybridization applied on frozen and paraffin-embedded sections using biotinylated DNA probes, detected with an indirect streptavidin-biotin-peroxidase reaction and intensified by silver-gold enhancement, were comparable to those obtained with radioactive probes. The localization of calcitonin encoding mRNA was in agreement with the localization of the calcitonin polypeptide.

Combined beta-galactosidase and immunogold/silver staining for immunohistochemistry and DNA in situ hybridization.

A combination of beta-galactosidase enzyme and the immunogold/silver staining method was studied for evaluation of double-staining experiments. Applications are shown for immunohistochemical double staining using two monoclonal antibodies and for combined immunohistochemistry and DNA in situ hybridization in one tissue section. The following advantages for the present double-staining method were evaluated: superior sensitivity of the immunogold/silver staining method for at least one epitope, which also allows detection of biotinylated DNA probes. The structure of the indolyl precipitate after revelation of beta-galactosidase activity did not show a concealing effect during a sequential double-staining method, as compared with the visualization of peroxidase with diaminobenzidine. These factors, and the sharply contrasting colored reaction products of beta-galactosidase (blue-green) and the immunogold/silver staining method including silver enhancement (brown-black), allow clear distinction of mixed-stained cell constituents.

Aneuploidy assay on diethylstilbestrol by means of in situ hybridization of radioactive and biotinylated DNA probes on interphase nuclei

Aneuploidy tests by means of in situ hybridization with chromosome-specific DNA probes on interphase nuclei have been carried out on human lymphocytes treated with diethylstilbestrol (DES). A DNA probe specific for chromosome Y (Y97), either radioactive or biotinylated, was used for the assays. Autoradiography or FITC-conjugated antibiotin antibodies were employed to visualize the hybridization sites. A significant increase of hyperdiploid nuclei was obtained with both procedures and a dose-related effect was revealed using the biotinylated probe. The results obtained, while giving further support to the evidence that DES is able to induce aneuploidy in cultured human cells, also indicate that the sensitivity of the assay can be improved by using biotinylated probes coupled with fluorescent antibodies.

Detection of human cytomegalovirus early and late antigen and DNA production in cell culture and the effects of dimethyl sulfoxide, dexamethasone, and DNA inhibitors on early antigen induction

Recently a conventional method for the laboratory diagnosis of human cytomegalovirus (HCMV) infection was improved by using centrifugation culture to enhance viral adsorption and by detecting HCMV early antigen and DNA. Comparison of the sensitivity of three rapid methods using commercial diagnostic reagents for the detection of HCMV early antigen (EA), late antigen (LA), and DNA was quantitatively evaluated in centrifugation cultures of human fibroblast cells (MRC-5) infected with HCMV. HCMV-EA was first detected 4 hours after infection, and the number of antigen-positive cells increased rapidly thereafter. Using biotinylated DNA probe, viral DNA was first detected 12 hours postinfection; the number of DNA-positive cells increased slowly. HCMV-LA was first seen 48 hours postinfection, and the number of LA-positive cells also increased thereafter. Thus detection of HCMV-EA was the most rapid and sensitive method for HCMV diagnosis. Several chemical compounds have been used to enhance HCMV replication. The effect of dimethyl sulfoxide (DMSO), dexamethasone (DEX), 5-bromo-2-deoxyuridine (BrdU), 5-fluoro-deoxyuridine (FdU), and cytosine arabinoside (Ara-C) on HCMV-EA induction was evaluated in centrifugation cultures of MRC-5 cells infected with HCMV. Infected cells treated with 1% DMSO alone or with DMSO plus DEX (10(-5) M) have been shown to increase the number of HCMV-EA-positive cells three- to fivefold over the untreated control cultures. The enhancing effects of Ara-C, BrdU, and BrdU plus FdU were demonstrated only occasionally.

DNA-hybridization electron microscopy: Localization of five regions of 16 S rRNA on the surface of 30 S ribosomal subunits

DNA-hybridization electron microscopy has been used to locate five regions of 16 S rRNA on the surface of 30 S ribosomal subunits. Biotinylated DNA probes that are complementary to selected regions of 16 S rRNA were hybridized to activated 30 S ribosomal subunits. These hybridized probes were reacted with avidin and localized by electron microscopy. The specificity of DNA binding was monitored with RNase H, which recognizes RNA-DNA hybrids and cleaves the RNA. Three of the five sequences examined were mapped on the platform. These sequences are 686–703, 714–733 and 787–803. Region 1492–1505 is mapped in the cleft and region 518–533 is at the neck on the side opposite the platform, respectively.

Detection of human papillomavirus in skin and genital lesions of renal allograft recipients by in situ hybridization

Renal allograft recipients have an increased incidence of malignancy including squamous carcinoma of cervix and skin. There is growing evidence that human papillomavirus (HPV) has a part to play in malignant transformation at these sites. We have previously identified HPV DNA in the skin and genital lesions of such patients by dot and Southern blotting. In situ hybridization studies, using biotinylated DNA probes for HPV 4, 5 and 8 in skin lesions and 6, 11, 16 and 18 in genital lesions, were performed on tissues derived from the same group of patients. In the cutaneous lesions, only 25% of the specimens probed were found to contain virus by in situ hybridization; 60% of these specimens were found to harbour virus by dot and Southern blotting. In situ hybridization revealed HPV 16 and/or 18 in 86% of the genital lesions probed.

The demonstration of human papillomavirus 16 genomes in the nuclei of genital cancers using two different methods ofin situ hybridization

Biopsy specimens of 16 invasive genital cancers (two vulval carcinomas, two carcinomas of the vagina, and 12 cervix carcinomas) were examined for the presence and distribution of human papillomavirus (HPV) DNA by in situ hybridization with 3H-labeled and biotinylated DNA probes of HPV 6, HPV 11, and HPV 16. None of the tumors reacted with HPV 6 or HPV 11. Using in situ hybridization with 3H-labeled DNA probes nine of the 16 cancers gave positive results with HPV 16. Only three of the nine were positive for HPV 16 by in situ hybridization with biotinylated probes. Currently, the method of in situ hybridization with commercial biotinylated probes is less sensitive than in situ hybridization with 3H-labeled HPV DNA.

Detection of human papillomavirus 16 and 18 DNA in cervical dysplasia and cervical carcinoma by in situ hybridization.

We improved the in situ hybridization method using biotinylatyed probes for the detection of human papillomavirus (HPV) 16 and 18 DNA in formalin-fixed and paraffin-embedded tissue sections. Our method included HCl treatment, digestion with 30μg/ml proteinase K, post-fixation with 70% ethanol and incubation with a streptavidin-biotinylated alkaline phosphatase conjugate for visualization of biotin molecules on DNA probes. The sensitivity of this method using biotinylated DNA probes was comparable to using 35S labeled RNA probes. Using this method, 72 cases of cervical dysplasia and 51 cases of squamous cell carcinoma of cervix were examined for the presence of HPV 16 and 18 DNA. HPV 16 DNA was detected by in situ hybridization in 19 (26%) cases: 8 of 17 (47%) with mild dysplasia, 4 of 24 (16%) with moderate dysplasia and 7 of 30 (23%) with severe dysplasia. In 25 cases of carcinoma in situ and 26 cases of invasive carcinoma, HPV 16 or 18 DNA was detected in 8 cases of which 4 cases had an associated nonmalignant lesion. All four cases showed that the intensity in the malignant lesions was less than the dysplasia or benign lesions in the same patients. These findings suggest that replication of HPV DNA might be suppressed in the transformed cells.

Cytomegalovirus and Human Herpesvirus-6 in Sudden Infant Death Syndrome: An in situ Hybridization Study

Thirty lung, spleen, and thymus sections and four lymph node sections from 40 sudden infant death syndrome (SIDS) cases were probed with biotinylated DNA probes specific for human herpesvirus-6 (HHV-6) and cytomegalovirus (CMV). Control material gave a strong, distinct signal with little or no background staining and no cross-reactivity. No staining was found with the SIDS material. We find no evidence to implicate an overwhelming infection by either virus in SIDS.

Human type II collagen gene (COL2A1) assigned to chromosome 12q13.1-q13.2 by in situ hybridization with biotinylated DNA probe.

We have made a regional assignment of the type II collagen gene (COL2A1) on human chromosome 12 by means of an in situ hybridization technique with a biotinylated DNA probe. The precise localization of the signal was mapped to the band 12q13.1-q13.2. This result was in agreement with the previous mapping by isotopic in situ hybridization technique (12q13.1-q13.2), but not with the result of Southern hybridization analysis using somatic cell hybrids (12q14.3).

Evaluation of a Non-Radioactive DNA Probe for Confirmatory Identification of Neisseria gonorrhoeae

A biotinylated DNA probe combined with a streptavidin-peroxidase complex for the identification of culture isolates of N. gonorrhoeae (Ortho diagnostic systems, Neckargemünd, FRG) was compared with the conventional carbohydrate utilisation test as reference. All 118 strains identified by the reference method as N. gonorrhoeae also gave positive reactions with the DNA hybridisation assay. However, with this test 2 of 23 non-gonococcal Neisseria or Branhamella species were identified as N. gonorrhoeae as well. The study shows that the DNA hybridisation technique can principally be used for the confirmatory identification of N. gonorrhoeae, but since specificity is particularly essential for confirmatory identification, the DNA hybridisation assay evaluated cannot be recommended for routine diagnosis.

Detection and Typing of Human Papillomavirus in Histologic Specimens by In Situ Hybridization with Biotinylated DNA Probes

Eighty tissue biopsies from 73 women suspected of having papillomavirus (HPV) infection of the lower genital tract were examined by in situ hybridization with biotinylated DNA probes derived from the complete genomes of four HPV types (6, 11, 16, and 18) and restriction analysis of the extracted DNA on Southern blots. In a subset of 52 samples, the in situ test had a 90.4% sensitivity (47 of 52) in detecting the presence or absence of virus, whereas Southern blot analysis detected HPV with a sensitivity of 98.1% (51 of 52). For 51 samples, in which the viral type was determined by restriction analysis, comparison of the signals separately generated by the four probes after in situ hybridization allowed a correct identification of the infecting HPV type in 86.2% (44 of 51) of cases.

A comparison of methods for the detection of human papillomavirus DNA by in situ hybridization with biotinylated probes on human carcinoma cell lines: Application to wart sections

We compared nine different techniques for the detection of biotinylated DNA— DNA HPV hybrids on HeLa cells with 10–50 copies of HPV 18 DNA per cell, CaSki cells with 600 copies of HPV 16 DNA per cell and tissue sections from frozen or paraffin-embedded biopsy specimens. The cell samples were either cell deposits or cytocentrifuged or cultured slides. In most cases, the samples (cell deposits and tissue sections) were denatured with hybridization mixture prepared under stringent conditions ( T m = −17°C) containing biotinylated DNA probes (cloned HPV types 1,2,6,11,16 and 18), at 90°C for 10 min. In other cases (cytocentrifuged or cultured cells), the denaturation was performed by HCl hydrolysis and mild heating at 50°C; the probes were denatured separately by heating. All the samples were further incubated overnight at 37°C. For HPV DNA detection, three amplification levels were used on cell deposits. Only the techniques involving a three-step reaction (a rabbit anti-biotin antibody — a biotinynlated goat anti-rabbit antibody — a complex of streptavidin-alkaline phosphatase or streptavidin-gold or streptavidin-fluorescein) gave satisfactory results, on both cell lines. With the one step reaction (an avidin-horseradish peroxidase, or streptavidin-alkaline phosphatase or streptavidin-fluorescein complex), no labeling of HeLa cells was observed with any of the HPV probes, including HPV 18. The techniques involving four steps (avidin or streptavidin — anti-avidin goat antibody or anti-streptavidin rabbit antibody — a biotinylated anti-goat (or anti-rabbit) antibody — a complex of avidin-biotin-peroxidase or streptavidin-biotin-alkaline phosphatase or streptavidin-biotin-horseradish peroxidase) resulted in high background on both cell lines. For the reproducible detection of low copy number of HPV DNA (less than 50 copies) such as occur in HeLa cells our data suggested that the three-step technique with the streptavidin-alkaline phosphatase complex was the method of choice. The most intense labeling was always obtained with cell deposits and the technique was successfully applied to frozen and paraffin-embedded tissue sections from typical warts.

Highly sensitive and specific non-radioactive biotinylated probes for dot-blot, Southern and colony hybridizations

We describe a simple method for the incorporation of biotin into nucleic acid probes. This method has been improved and optimized to produce biotinylated DNA probes for the detection of DNA by dot-blot. Southern and colony hybridization techniques. The sensitivity of this method has been particularly improved to allow detection of DNA quantities under one femtogram. Probes prepared by this method are highly specific for target DNA even in crude bacterial lysates.

In situ localization of specific RNAs in developing fruit bodies of the basidiomycete Schizophyllum commune

cDNA clones representing mRNAs abundantly expressed during fruiting of Schizophyllum commune were used to detect the cellular localization of these mRNAs in freeze-microtome sections of developing fruit bodies. An 18 S rRNA clone was isolated and used as a probe for total RNA. Both RNA and DNA probes with different labels were found suitable but the procedure finally adopted involved in situ hybridization with nick-translated biotinylated DNA probes. To permit the probes to permeate the cell walls it was necessary to treat the sections with RNasedepleted Trichoderma harzianum wall-lytic enzymes before hybridization. Hybridization at different developmental stages showed that the specific mRNAs were abundantly expressed in specific areas of the fruit bodies.

Enhanced endothelial cytopathogenicity induced by a cytomegalovirus strain propagated in endothelial cells

Endothelial involvement has been implicated in cytomegalovirus (CMV) infection, a source of major complications in immunosuppressed individuals (e.g., those with acquired immune deficiency syndrome [AIDS] and organ transplants). Traditionally, CMV has been grown in fibroblasts; however, propagation in these cells may alter characteristics of the virus. In developing an in vitro model system of CMV/endothelial cell interaction, we have addressed this issue by propagating a clinical isolate, CMV VHL 1, in human umbilical vein endothelial (HUVE) cells by serial cocultivation of heavily infected cultures with fresh HUVE monolayers and have compared its infectious properties with those of the fibroblast-raised strain, CMV AD169. In situ hybridization using a biotinylated DNA probe, as well as immunofluorescent staining for CMV-specific antigen, has confirmed infection of HUVE cells inoculated with either strain of the virus. Infection of HUVE by VHL was accompanied by dramatic cytopathology not observed in AD169-infected cells. Plaque assay of culture supernatants revealed greater virus production in VHL-infected HUVE as compared with equivalently inoculated fibroblasts. In contrast, AD169 production in inoculated fibroblasts exceeded that in HUVE. These studies demonstrate the suitability of cultured endothelial cells as a substrate for CMV propagation and suggest that a strain of virus thus propagated may offer an accurate model of CMV/endothelial cell interaction in human disease.