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At least 19 recordsLinked to original sources
PubMed1989

A comparison of slot blot, southern blot, and in situ hybridization analyses for human papillomavirus DNA in genital tract lesions.

Genital tract lesions were analyzed for human papillomavirus (HPV) DNA by in situ hybridization using probes of HPVs 6/11, 16/18, and 31/33/35. All of the HPVs detected in vulvar and perianal condylomata by in situ hybridization were HPV 6/11-related, whereas the majority of HPVs detected in cervical intraepithelial grade I lesions were types 16/18- and 31/33/35-related. None of the lesions with histologic features equivocal for HPV infection had detectable HPV DNA by in situ hybridization, though some did contain HPV DNA sequences as ascertained by filter hybridization analysis. The sensitivity of in situ hybridization was compared with that of filter hybridization (slot blot and/or Southern blot). The correlation was high (28 of 30) for cases that contained HPVs 6/11 or 16, as deduced by filter hybridization, and was much less (ten of 29) for cases that contained HPVs distinct from types in the filter hybridization probe cocktail (HPVs 6/11, 16, 18, 31, 35 and 51). There was a high concordance between the results of Southern blot hybridization and slot blot hybridization analyses, especially with cases that contained HPVs 6/11 and 16. In situ hybridization, slot blot, and Southern blot hybridization analyses are all very effective in detecting the common HPV types (HPVs 6/11 or 16). In situ hybridization is useful in differentiating cervical lesions that contain HPV 6/11 from those that contain HPV 16 or other types with oncogenic potential. However, filter hybridization is superior to in situ hybridization when analyzing cases with histologic findings equivocal for HPV infection or cases that contain HPV types related to, but distinct from, the types included in the probe.

Blotting, Southern↗
PubMed1994

Molecular diagnosis of the Philadelphia chromosome translocation. A comparison of isotopic and chemiluminescent Southern blotting.

Southern blotting using radiolabeled probes is a well established technique for the detection of the Philadelphia translocation in the diagnosis of chronic myelogenous leukemia (CML). However, the use of radioisotopes in the clinical setting is often problematic. Because of this we investigated the use of a digoxigenin-labeled probe and chemiluminography in the detection of the Philadelphia translocation. In this study DNA was extracted from 19 bone marrow or blood samples from patients with CML or other malignancies and subjected to Southern blotting with a probe specific for the Philadelphia translocation, Phl/bcr3. The probe was labeled with either 32P or digoxigenin to determine the relative sensitivity and specificity of autoradiography and chemiluminography in the molecular diagnosis of the BCR/abl fusion gene. All 19 samples were tested by both methods. All blots were performed and interpreted by individuals blind to the initial patient diagnosis. In addition, 12 samples (6 positive for CML, 6 negative for CML as determined by Southern blotting with 32P-labeled probe) were subjected to triplicate Southern-blot analyses, with three separate lots of digoxigenin-labeled probe to assay batch to batch variability in the efficacy of the probe. Radiolabeled and digoxigenin-labeled probes resulted in identical diagnoses in all cases. All results obtained by molecular analysis correlated perfectly with the clinical diagnoses of the patients from whom the samples had been obtained. Reanalysis of patient samples with different batches of digoxigenin-labeled probe gave highly reproducible results. With digoxigenin-labeled probe, diagnostic results were obtained after exposure times of less than 1 h at room temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiography↗
PubMed1992

Prevalence of genital papilloma virus infections in asymptomatic and symptomatic women, studied with a combined dot-blot and Southern blot procedure.

A technique for the detection and typing of genital infections with human papilloma virus (HPV) is described. Following a non-invasive sampling and a simplified preparation procedure, the analysis was performed as a combined dot-blot and Southern blot analysis, where the former test was used to exclude cases without demonstrable content of HPV DNA. The subgenomic probes used in the Southern blot analyses have a higher specificity than can be achieved with genomic ones, and only one band is obtained for each of HPV types 6, 11, 16, 18, 31, 33 and 35. Extra bands indicating the presence of cross-reacting HPV of undetermined type occurred in only 0.7% of the 1,268 cases tested. HPV was demonstrated in 7.8% of the 612 health control samples and in 16% of 634 consecutive samples from patients with symptoms. All but type 18 occurred more commonly in symptomatic than in asymptomatic patients. Presence of HPV also correlated to cervical intraepithelial neoplasia (CIN) in simultaneously collected smears. The HPV was demonstrated in 13% of patients with normal cytology, in 45% with CIN and in 82% of patients with invasive carcinoma. All types of HPV occurred more commonly in CIN patients, while only types 16, 18, 31 and 33 were found in the patients with cancer.

Blotting, Southern↗
PubMed1993

Detection and typing of human papillomavirus DNA in the uterine cervix of Japanese women by nonradioactive dot blot and Southern blot hybridization.

HPV infection was examined with Cytobrush in exfoliated cervical cells sampled from 347 women. HPV DNA analysis was conducted in two steps. The presence of HPV DNA was demonstrated by dot blotting and typing of HPV DNA was made by Southern blotting using biotinylated probes. Of 167 cases with cervical intra-epithelial neoplasia (CIN) and cervical carcinoma, HPV DNA was detected and typed in 25 cases (15.0%). HPV 16, 18, and 33 were found mainly, and no HPV 6 or 11 was detected. The frequency of HPV DNA was 7.1% of CIN I, 28.6% of CIN II, 54.5% of CIN III, and 50.0% of invasive carcinoma. HPV occurred in patients newly diagnosed as CIN at 25.8% and in those of diagnosed as CIN in the past and followed up, 7.6%. Of 180 healthy women, the screening test of FO8830 was positive for HPV DNA in four women (2.2%). The present system proved to be useful for facilitating large-scale clinical research.

Blotting, Southern↗
PubMed1992

Detection of human papillomavirus (HPV) infections in Japanese women with and without abnormal cervical cytology by dot blot and Southern blot hybridization.

The frequency of human papillomavirus (HPV) was investigated in 245 Japanese women (100 showing normal cytology and 145 showing abnormal cytology). A previously presented method for the detection and typing of HPV DNA was used, where a dot blot test excluded samples that did not hybridize with HPV DNA. Positive samples were further analyzed by a southern blot procedure, to give the HPV type. A mixture of subgenomic probes of the types 6, 11, 16, 18, 31, 33 and 35 was used. HPV DNA was detected in only 2% (2/100) of patients without abnormal cytology. In patients with abnormal cytology, the frequency was 30% (43/145). HPV was detected in 100% (2/2) of the patients with condyloma acuminatum, 39% (26/66) of those with mild to moderate dysplasia, 44% (7/16) of those with severe dysplasia to carcinoma in situ, and 70% (7/10) of those with invasive carcinoma. Young women (20-29 yr.) had the highest HPV frequency (71%). HPV types 16 and 18 were found in 39% of the patients with mild to moderate dysplasia, in 71% of those with severe dysplasia to carcinoma in situ and in 86% of those with invasive carcinoma. These may be considered as high risk types for development to cancer. HPV type 31 may be considered as a moderate risk, as it was detected in 37% (16/43) of the patients. HPV types 6 and 11 were found in 100% of patients with condyloma acuminatum (2/2). To clarify the natural history of the HPV infection in the uterine cervix, it is necessary to conduct further studies.

Blotting, Southern↗
PubMed1992

Detection of human papillomavirus (HPV) using dot blot and Southern blot, hybridizing with a mixture of seven probes.

A previously presented method for the detection and typing of human papillomavirus (HPV) DNA has been modified for the simultaneous analysis of HPV types 6, 11, 16, 18, 31, 33 and 35. The method has two steps, where a dot blot test is used to exclude cases that do not hybridize with HPV-DNA. The remaining cases are then analysed by a Southern blot procedure, using a mixture of subgenomic probes for the simple and accurate analysis of HPV types. When the procedure was used for the analysis of clinical samples, patient groups at varying risk were found to differ with regard to the prevalence of HPV infections. Thus, the virus was detected in only 8.8% of otherwise healthy young women - i.e. women without clinical signs of HPV-related disease - as compared to 48% of women who also had cervical intraepithelial neoplasia (CIN). A similarly high prevalence was found in patients in whom CIN persisted (42%) as compared to those in whom the morphological lesion regressed to normal (8%).

Blotting, Southern↗
PubMed1998

Telomere length distribution and Southern blot analysis.

Southern blot analysis of terminal restriction fragments (TRFs) is the standard method for quantitative examination of telomere length distributions. Since TRFs contain a subtelomeric component, central parameters of the TRF distribution n(L) such as the arithmetic mean (M) or the median (Me) cannot be derived directly from Southern blot data, i.e. from the optical density distribution OD(L). Several estimates have been applied instead; the seeming arithmetic mean A, the "center of mass" C, and the positions of maximal (P) and half-maximal optical density (P(1/2)). We show that C> A> M for any non-truncated distributions n(L), and P> M> P1/2 for any symmetrical unimodal n(L). Symmetric appearance on a Southern blot, however, suggests positive skewness of n(L). Thus, a lognormal form of n(L) may be considered. Then, C> A> M> P=Me> P(1/2). Alternatively, a Weibull distribution may be assumed. The latter is compatible with negative feedback-regulation of the telomere lengths. Using the maximum likelihood method we compare these distributions with FISH-data on telomere lengths in different cell types. The fit of the lognormal distribution is clearly superior. Lognormal genesis may relate to telomere breakage and recombination. Truncation of the upper end of the TRF distribution is possible due to Southern blot artifacts. Thereby, the order of the estimates may change to P> C> A. Having minimal sensitivity to truncation, P seems to be the optimal choice. however, the variability of P is high since peakedness of OD(L) and DNA length resolution are inversely related.

Animals↗
PubMed1990

Use of cervical Cytobrush samples for dot-blot detection and Southern blot typing of human papillomaviruses using subgenomic probes.

The use of Cytobrush samples for the detection and typing of cervical human papillomavirus (HPV) infections was analyzed. The average yield of squamous cells from Cytobrush samples was on the order of 10(6) cells when the sample was collected in a lytic collection buffer, which was approximately double the content of (1) samples collected in ethanol and (2) an average biopsy specimen. The material obtained could be used for sensitive detection and typing of HPV infections using, respectively, a nonradioactive dot-blot method and the Southern blot procedure performed with subgenomic probes, which permitted a simple interpretation even in cases of mixed infections. A sample containing at least 500,000 viral copies was required for the detection and typing. At this level of sensitivity, the frequencies of HPV obtained in different risk groups varied from 6% (in "healthy" young women) to 53% (in women with abnormal cytologic findings in simultaneous smears). The noninvasive nature of the sampling procedure and the relative simplicity of the test should allow this method to be applied in large-scale studies.

Blotting, Southern↗
PubMed1992

Validation of hybridization assays: correlation of filter in situ, dot blot and PCR with Southern blot.

A number of validation experiments have compared the most commonly used HPV hybridization methods with the accepted gold standard, Southern blot hybridization. The methods discussed are filter in situ hybridization (FISH), dot blot hybridization (ViraPap/ViraType), and polymerase chain reaction (PCR). FISH now appears to be too inaccurate to be recommended for future epidemiological studies. ViraPap/ViraType compares well to Southern blot, but is limited to the detection of seven genital HPV types. PCR-based methods may be more sensitive than Southern blot and are likewise capable of detecting most known genital HPV types. Direct comparisons of the several available PCR methods should be performed. Currently, there is no perfect method for HPV testing, because Southern blot itself is prone to some errors in performance and interpretation. Given that the scientific and clinical usefulness of HPV tests depends on the repeatability and accuracy of the assays, more intra- and inter-assay comparisons should be done to establish reference standards applicable to this area of molecular diagnostics.

Blotting, Southern↗
PubMed1992

Abnormalities of chromosomes 7 and 22 in human malignant pleural mesothelioma: correlation between Southern blot and cytogenetic analyses.

Southern blot hybridization studies were performed on 23 mesothelioma primary tumor specimens to detect chromosome 1-, 7-, and 22-specific numerical changes, gene amplifications, and gene rearrangements. The molecular findings were compared with previous cytogenetic findings. No gene rearrangements or amplifications were detected. A numerical abnormality of chromosome 7 was detected by Southern blot analysis in two cases in which no numerical abnormality had been detected by the previous chromosome study. A numerical aberration of chromosome 22 was detected in five cases, which was compatible with the cytogenetic finding of monosomy 22 in these cases. The Southern blot results for the copy numbers of chromosomes 7 and 22 were concordant with the cytogenetic findings in 65%-80% of the cases.

Blotting, Southern↗
PubMed1991

Human papillomavirus infection of the male diagnosed by Southern-blot hybridization and polymerase chain reaction: comparison between urethra samples and penile biopsy samples.

A group of 108 healthy men aged 18 to 23 years underwent penoscopy after application of acetic acid. In 39 (37%) aceto-white lesions were found and a biopsy was taken. From 105 patients a urethra sample was collected with a brush. The prevalence of HPV-DNA by dot-blot and Southern-blot hybridizations (DBH/SBH) and polymerase chain reaction (PCR) as well as the correlation between results from urethra samples and penile aceto-white epithelium were examined. In urethra, HPV-DNA was detected in four of the 66 patients (6%) with normal epithelium and in three of the 39 (8%) with aceto-white epithelium by using DBH/SBH. By PCR the corresponding findings were eight (12%) and 10 (26%) HPV-DNA-positive patients, respectively. Out of the 39 biopsy samples HPV-DNA was detected in six (15%) by DBH/SBH and in 17 (44%) by PCR. Twenty-two (56%) of the patients with aceto-white lesions were by PCR positive in either the urethra sample or biopsy. All samples positive by DBH/SBH were also positive by PCR except for one biopsy with HPV 35, a type not tested for by PCR. HPV 11 was the most common finding in urethra and HPV 6 and 16 in aceto-white epithelium.

Adolescent↗
PubMed1999

Easy detection of all T cell receptor gamma (TCRG) gene rearrangements by Southern blot analysis: recommendations for optimal results.

Southern blot analysis of T cell receptor (TCR) gene rearrangements has proven to be a helpful tool to establish clonality in T cell leukemias and lymphomas. To improve the detection of clonal TCR gamma (TCRG) gene rearrangements by Southern blot analysis, we designed four new Jgamma probes and determined the most optimal restriction enzymes to be used with these probes. Based on detailed analysis of the sequences as well as on hybridization experiments with the TCRGJ21 probe, the Jgamma1.2 and Jgamma2.1 downstream areas were found to be highly homologous, suggesting that during evolution the duplication of the Jgamma region was followed by deletion of the tentative Jgamma2.2 gene segment. Southern blot analysis of 51 T cell acute lymphoblastic leukemias (T-ALL) revealed that all TCRG gene rearrangements can be detected by use of the TCRGJ13 probe in EcoRI digests and the TCRGJ21 probe in PstI digests. Additional probes and digests allow a more precise identification of the exact type of TCRG gene rearrangements in the majority of cases. Almost 90% of the TCRG gene rearrangements in T-ALL involved the Jgamma2 region (16% Jgamma2.1 and 72% Jgamma2.3), whereas Jgamma1 region rearrangements were particularly found in TCRgammadelta+ T-ALL. This information has implications for design of primer sets for PCR analysis at diagnosis and for PCR target choice in detection of minimal residual disease during follow-up of T-ALL patients.

Blotting, Southern↗
PubMed1996

One-day detection of PCR amplified Chlamydia trachomatis DNA in clinical samples: ELISA versus Southern blot hybridisation.

AIMS: To compare ELISA and Southern blot hybridisation for the detection of PCR amplified Chlamydia trachomatis DNA extracted from clinical samples; to assess the value of the ELISA method in a clinical setting. METHODS: DNA was extracted from urogenital samples of 508 patients, purified and amplified using C trachomatis specific primers, one of which was endlabelled with biotin. Amplification products were detected by Streptavidin biotin based ELISA and non-radioactive Southern blotting. RESULTS: Of the 508 samples, 29 were positive and 479 negative by both methods. No discrepant results were observed. CONCLUSION: Streptavidin biotin based ELISA and Southern blotting were equally sensitive for detecting PCR amplified C trachomatis DNA. Using ELISA, test results could be generated within a single day.

Base Sequence↗
PubMed1989

Primary malignant lymphoma of the brain: demonstration of immunoglobulin gene rearrangements in four cases by the Southern blot hybridization technique.

Using the Southern blot hybridization technique, four cases of the primary malignant lymphomas of the brain, histologically diffuse large cell lymphoma, were examined for the immunoglobulin gene rearrangements. In three lymphomas, the rearrangements were observed in both heavy and light chain genes, providing strong evidence for a B cell lineage of these tumors. On the other hand, in the remaining lymphoma, the rearrangement was observed only in the heavy chain gene. Despite this, immunohistochemical examination revealed positive stainings for heavy and light chain immunoglobulins in tumor cells, suggesting the occurrence of light chain gene rearrangement at the undetectable level. Thus, B lymphocytic differentiation at the gene level was demonstrated in three, or possibly all, of the primary intracerebral malignant lymphomas examined. Since no more than two rearrangements were detected in each immunoglobulin gene, these lymphomas were considered to be monoclonal in nature.

Adult↗
PubMed1990

Neoplastic angioendotheliosis: demonstration of immunoglobulin gene rearrangements by the Southern blot hybridization technique.

Using the Southern blot hybridization technique, two cases of 'neoplastic angioendotheliosis' (NAE) were examined for immunoglobulin gene rearrangements. For DNA analysis, frozen kidney (Case 1) and lung (Case 2) tissue, in which intravascular neoplastic cells were abundant, were used. In each case rearrangements were observed in both heavy and light chain genes. On examination of the heavy chain gene, the JH DNA probe detected one rearranged band in Case 1 and two in Case 2 after EcoRI digestion. On examination of the light chain genes, the C kappa DNA probe revealed one rearranged band in Case 1 and two in Case 2 after BamHI digestion, while the C lambda 2 DNA probe revealed only germline configurations after EcoRI digestion in each case. Thus, B-lymphocytic differentiation at the gene level was demonstrated in neoplastic cells in both cases. Since no more than two rearrangements were detected in each immunoglobulin gene, the intravascular neoplastic cells were considered to be monoclonal in nature.

Aged↗
PubMed1993

Detection of immunoglobulin heavy-chain gene rearrangements by Southern blot analysis: recommendations for optimal results.

Southern blot analysis of immunoglobulin (Ig) and T-cell receptor genes has proven to be important for detection of clonal rearrangements in patients with lymphoproliferative diseases. To improve the detection of clonal Ig heavy-chain (IgH) gene rearrangements, we carefully determined the precise restriction map of the joining (J)H and constant (C)mu region of the IgH locus, and evaluated relevant combinations of restriction enzymes with JH and C mu probes. Our extensive Southern blot analyses revealed that rearrangements in the JH region are optimally detectable by use of a JH probe in combination with at least two restriction enzyme digests which are not affected by polymorphisms, and which produce small germline bands (e.g. BglII and BamHI/HindIII), thereby reducing the chance of comigration of germline and/or rearranged bands. Application of a JH or a C mu probe in combination with BamHI or EcoRI digests should be avoided, because of the large size of the restriction fragments and the occurrence of polymorphisms. Comparison of different types of JH probes demonstrated that optimally reproducible signals, independent of the rearranged JH gene segment, are only obtained if the JH probe is complementary to the 3' flanking sequences of the JH gene region, such as our IGHJ6 probe.

Base Sequence↗
PubMed2000

Immunoglobulin heavy-chain gene rearrangement studies by Southern blot using DNA extracted from formalin-fixed, paraffin-embedded tissue.

BACKGROUND: In most clinical molecular diagnostics laboratories, Southern blots for gene rearrangement studies are not routinely performed on formalin-fixed, paraffin-embedded (FFPE) tissue samples. In this study, immunoglobulin heavy-chain gene rearrangements by Southern blot using DNA extracted from FFPE tissue samples were studied. METHODS AND RESULTS: Eleven paired freshly frozen and FFPE tissue samples were evaluated for immunoglobulin gene rearrangements by PCR and Southern blot analyses. An additional 14 selected samples sent to our laboratory to rule out lymphoma, for which only FFPE tissue (no frozen tissue) was available and for which PCR was interpreted as negative, were evaluated by the same techniques. Southern blots generated from DNA extracted from FFPE tissues were qualitatively identical to those generated from DNA extracted from fresh or freshly frozen tissue and correlated well with the final diagnoses. Ten interpretable Southern blots were generated in the 14 cases in which no frozen tissue was available. Four of these ten blots were interpreted as positive for an immunoglobulin gene rearrangement. Although the number of samples analyzed is small, success with Southern blotting correlated with increased sample size and sample width (1.17 vs 0.49 cm(2); P <.024; 0.71 vs 0.43 cm; P <. 049, respectively). CONCLUSION: DNA extracted from FFPE tissue samples using the simple, efficient, and nontoxic techniques described in this report can be used in many cases for Southern blotting for the detection of clonality by gene rearrangement studies.

Blotting, Southern↗
PubMed2001

Usefulness of genus-specific PCR and Southern blot species-specific hybridization for the detection of imported malaria cases in Italy.

A PCR method involving a genus-specific oligonucleotides set and Southern blot hybridization with four species-specific probes to P. falciparum, P. vivax, P. malariae and P. ovale was evaluated for the detection of malaria parasites in blood samples from 101 patients with clinically suspect malaria infection imported to Italy. Plasmodium falciparum was the main species detected. As determined by microscopy, 53 (52.4%) patients had malaria and of these: 40 (75.5%) were infected with P. falciparum; 7 (13.2%) with P. vivax; 1 (1.9%) with P. ovale; 3 (5.7%) with P. malariae; 1 (1.9%) with P. vivax or P. ovale; and 1 (1.9%) with P. falciparum or P. vivax. Ninety-seven out 101 blood samples were submitted to ParaSight-F test which showed a sensitivity of 94.73%, and a specificity of 93.22%, as compared to microscopy. The PCR assay using the genus-specific oligonucleotide primer set (pg-PCR) was able to detect 53 (52.4%) infections and showed a sensitivity of 100% and a specificity of 100%, when compared to microscopy. The parasite species were identified by Southern blot hybridization using species-specific probes and 40 (75.5%) samples were P. falciparum positive, 5 (9.4%) P. vivax positive, 4 (7.5%) P. ovale positive, and 2 (3.8%) P. malariae positive. When the Southern blot results were compared to those of blood-film diagnosis, we observed some disagreement. In particular, compared to Southern blot, microscopy underestimated P. ovale infection; blood film analysis recognised only 1 P. ovale sample, whereas Southern blot recognised 4 P. ovale positive samples (by microscopy, 2 of these were detected as P. vivax, 1 as P. ovale or P. vivax, and the other as P. falciparum or P. vivax). Southern blot hybridization was unable to identify one P. falciparum and one P. vivax positive case detected by microscopy. We also plan to use a reference nested-PCR assay to clarify the disagreement observed between microscopy and Southern blot hybridization.

Animals↗