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At least 73 records · Page 4Linked to original sources
PubMed2004

Effect of storage conditions on the extraction of PCR-quality genomic DNA from saliva.

BACKGROUND: Saliva is a potentially useful source of genomic DNA for genetic studies since it can be collected in a painless and non-invasive manner. We sought to determine whether different storage conditions of saliva samples impact our ability to extract genomic DNA that is of sufficient quality for use in the polymerase chain reaction (PCR). METHODS: Saliva was collected from healthy volunteers and 2-ml aliquots subjected to different storage conditions: S1--washing of saliva using phosphate-buffered saline (PBS) and extraction of DNA on the same day of collection; S2--washing and centrifugation to yield a pellet, which was stored at-70 degrees C for 1 week prior to DNA extraction; S3--storage of whole saliva at 4 degrees C for 7 days, followed by washing and extraction of DNA; S4--storage at 4 degrees C for 7 days, followed by washing and pellet formation. The pellet was stored at -70 degrees C for 1 month before extraction of the DNA; S5--storage at-70 degrees C for 1 month, followed by washing and extraction of DNA. DNA yield and purity was determined by spectrophotometry at 260 and 280 nm. Twenty nanograms of genomic DNA was used for the polymerase chain reaction, and the resulting PCR band was captured by digital photography and quantified. RESULTS: The amounts of DNA extracted from 2 ml of saliva varied widely under the different storage conditions, while purity of the DNA extraction, based on OD(260/280) ratios, was good and comparable. PCR resulted in the presence of a single specific product of the correct size from all samples regardless of saliva storage conditions. Quantification of PCR bands showed significant differences between the various storage conditions (P<0.05). Compared to S1 samples, PCR bands from conditions S2 and S3 were not as strong, while those amplified from S4 and S5 samples were the weakest. Post-hoc analyses showed that the means for conditions S4 and S5 were significantly different from S1-S3. Qualitatively similar results were obtained when the PCR experiment was repeated. CONCLUSIONS: Saliva can act as a useful source of genomic DNA, even when stored under less than optimal conditions.

Cold Temperature↗
PubMed2001

Prostate epithelium-derived Ets transcription factor mRNA is overexpressed in human breast tumors and is a candidate breast tumor marker and a breast tumor antigen.

PURPOSE: There is a need to identify novel breast tumor-associated molecules with a potential as diagnostic/prognostic markers of breast cancer as well as targets of vaccine and drug discovery against this cancer. EXPERIMENTAL DESIGN: We used a combination of digital differential display and reverse transcription-PCR (RT-PCR) methods to identify breast tumor-associated cDNAs. RESULTS: It was found that prostate epithelium-derived Ets transcription factor (PDEF) and five other cDNAs occur at high frequency in the cDNA libraries from normal human breast tissue and human breast tumors. In contrast, these cDNAs are either undetectable or present at low frequencies in the cDNA libraries from other normal human tissues. RT-PCR expression analysis of PDEF showed it to be overexpressed in 14 of 20 primary human breast tumors and in one metastases tested. Also, consistent with the digital differential display data, RT-PCR analysis of PDEF expression showed highly restricted expression in normal human tissues. Furthermore, we show that PDEF transcript levels are 192-fold higher in the peripheral blood of a breast cancer patient in comparison with two normal individuals and another breast cancer patient. In contrast to PDEF, RT-PCR analysis of the expression of the other three cDNAs, including MYL5, Hs.44017, and Hs.215937, showed that these cDNAs are expressed in several normal human tissues. CONCLUSIONS: These results suggest that PDEF is a breast tumor-associated cDNA and should be further evaluated for its potential as a breast tumor marker and a breast tumor antigen.

Antigens, Neoplasm↗
PubMed1997

Partial identification of spirochaetes from two dairy cows with digital dermatitis by polymerase chain reaction analysis of the 16S ribosomal RNA gene.

Specimens taken postmortem from typical lesions of digital dermatitis in two dairy cows were tested by the polymerase chain reaction (PCR) for the presence of a spirochaetal 16S rRNA gene. Seven different assays detected the gene in the samples from both cows. Two of the PCR products were sequenced and a comparison of the nucleotide sequences revealed that the spirochaete belonged to the genus Treponema and was closely related to Treponema denticola. A PCR specific for the detection of the digital dermatitis-associated treponeme was developed.

Animals↗
PubMed2006

A one-step real-time PCR assay for detection of DQA1*05, DQB1*02 and DQB1*0302 to aid diagnosis of celiac disease.

Celiac disease is an autoimmune disorder that develops after dietary exposure of the small intestine to gluten peptides in cereals. Celiac disease has a strong genetic component associated with HLA-DQ2 and HLA-DQ8, and testing for absence of these genetic markers is useful when serological tests and biopsies are indeterminate, as it renders celiac disease highly unlikely. We have developed a new real-time PCR assay, using sequence-specific primers (PCR-SSP) and TaqMan probes, for detection of DQB1*05, DQB1*02 (coding for DQ2) and DQB1*0302 (coding for DQ8). PCR amplification and detection of DQ2 and DQ8 was accurately and unambiguously performed from genomic DNA isolated from cell lines and human DNA. Amplification was scored digitally, without laboratory manipulation of amplified PCR products and with a higher accuracy than PCR-SSP. This assay should increase accuracy and throughput, and reduce risks of contamination in laboratories where testing for HLA DQ2 and DQ8 is performed as part of diagnosis of celiac disease.

Celiac Disease↗
PubMed2026

Potato Black Scurf and Stem Canker: Pathogen Biology, Global Distribution, and Traditional and Modern Diagnostics.

Rhizoctonia solani is a soil- and seed-borne fungal pathogen of potatoes. It is a persistent threat to potato production worldwide. The symptoms appear as black scurf on tubers and stem canker, causing severe yield and quality losses of potatoes. The pathogen reproduces asexually via hyphae and sclerotia. Its genetic diversity is organized into anastomosis groups (AGs), with AG3-PT being the predominant group on potato. The global trade of seed potatoes is very important for agricultural development; however, it has facilitated the dissemination of the pathogen across regions. Moreover, disease development is affected by environmental and agronomic factors, causing variable symptom severity and differential economic impacts. Given the pathogen's genetic complexity, accurate diagnosis is very important, necessitating a transition from traditional culture-based and biochemical methods toward molecular, genomic, and emerging digital technologies. Methods such as PCR, isothermal amplification, sequencing, sensor-based biosensing, and artificial intelligence-driven imaging have improved the detection, quantification, and noninvasive monitoring of the pathogen. Combining these diagnostic methods into a tiered framework will be helpful for precision disease surveillance, informed disease management decision-making, and the development of sustainable potato production systems.

black scurf↗
PubMed1992

[DNA typing and analysis of the D1S8 (MS 32) allele in the Japanese population by the minisatellite variant repeat (MVR) mapping by polymerase chain reaction (PCR) assay].

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) of the D1S8 (MS32) locus from total genomic DNA and size separated alleles in the Japanese population was performed. DNA was extracted from blood in healthy non-related Japanese individuals. The digital data obtained were computer analysed, and from this single assay all unrelated individuals tested could be unambiguously distinguished. The average difference in diploid codes between unrelated individuals was 36 exclusions over the first 60 repeat unit positions. The mean proportions of a-, t-, and 0-type repeat units were 73.5%, 24.5% and 2.1%, respectively. MVR-PCR gives digital profiles which show extreme levels of individual variation and should prove very useful for personal identification. The advantages and further applications of MVR-PCR are also discussed.

Alleles↗
PubMed2005

sgk1, a member of an RNA cluster associated with cell death in a model of Parkinson's disease.

In an effort to gain deeper insight into the molecular processes underlying neurodegeneration in Parkinson's disease, we performed gene expression profiling at several early time points after MPTP-injection into old (1-year) mice. We used a PCR-based gene expression profiling method, digital expression pattern display (DEPD), a method of very high sensitivity and reproducibility, which displays almost all transcripts of a tissue. To identify cell death-associated genes, we defined clusters of differentially expressed transcripts with expression behaviour that correlated with the temporal profile of cell death progression and characterized one of these cell death clusters further. We selected one of the strongest regulated genes, the serum and glucocorticoid-regulated kinase 1 (sgk1), and validated its differential expression by Northern blot analysis, semiquantitative PCR and in situ hybridization. Up-regulation of sgk1 (i) coincides with the onset of dopaminergic cell death in both the 8-week acute and 1-year subacute MPTP models, (ii) spans the entire brain, (iii) is attenuated by the l-deprenyl-mediated inhibition of the MPTP conversion to its active metabolite MPP+ and (iv) is not induced by dehydration. This study demonstrated that the combination of the DEPD technology, clustering analysis and a detailed histopathology is a useful tool for elucidating molecular pathways in neurodegenerative diseases.

Age Factors↗
PubMed2025

Advances in diagnosis of diseases causing diarrhea in newborn calves.

Diarrhea in newborn calves is a serious global health problem. It poses challenges for animal industry, veterinarians and researchers due to the rapid onset of dehydration. Mixed infections make treatment complicated, and many young calves suffer high rates of illness and death from this condition. Numerous enteropathogens are associated with diarrhea in newborn calves, encompassing viruses, bacteria, parasites, and protozoa. Their occurrence differs by region, yet the most prevalent infections include E. coli, Salmonella species, Clostridium perfringens, Clostridium difficile, Rotavirus, Coronavirus, Cryptosporidium, Toxocara, Giardia and Eimeria. This review outlines the diagnostic techniques for diseases that lead to diarrhea in newborn calves. Diagnosis is based on clinical manifestations; however, the laboratory identification of etiological items is the only valid way for detecting the illness's aetiology and initiating treatment protocols. Classic methods such as bacterial culturing, fecal flotation, direct microscopy, and virus isolation help us understand pathogens better. Immunological assays like ELISA and immunochromatography are fast, accurate, affordable, and useful for on-farm detection. They help identify specific antigens or antibodies efficiently. Molecular methods including PCR (standard, multiplex, real time and digital), LAMP assays, DNA microarrays and whole-genome sequencing allow highly accurate and sensitive detection. They can identify pathogens effectively, even at very low levels. Nanotechnology-based assays introduce a novel level of sensitivity and specificity, often yielding quick results with minimal sample volumes. In conclusion, accurate and rapid diagnosis using advanced techniques is critical for managing and preventing diseases that lead to diarrhea in newborn calves.

Animals↗
PubMed1997

Can prostate-specific antigen reverse transcriptase-polymerase chain reaction be used as a prospective test to diagnose prostate cancer?

The present study addressed the question as to whether prostate-specific antigen reverse transcriptase-polymerase chain reaction (PSA RT-PCR) could be used to identify prospectively men who have prostate cancer and to help determine which patients with an initially negative biopsy would benefit from rebiopsy. PSA RT-PCR was performed prospectively on 90 patients who were to have a prostate biopsy because of an elevated PSA level, an abnormal digital rectal examination, or both. PSA RT-PCR was performed, and the sensitivity of the test was enhanced by hybridization of the PCR with a 32P-labeled PSA cDNA probe (exons 3-5). Of the 90 men, 36 (40%) had prostate cancer on biopsy. Of these 36 men, 5 (13.9%) had a positive PSA RT-PCR finding, whereas 31 (84.1%) tested negative. Of 54 men with negative biopsies, 8 (14.8%) had a positive PSA RT-PCR result. The sensitivity of PSA RT-PCR for the detection of biopsy-proven prostate cancer was 13.9% and the specificity was 85.2%. Only 3 of 12 (25%) patients with advanced disease had a positive test result. The sensitivity of PSA RT-PCR for the detection of biopsy-proven prostate adenocarcinoma in men suspected of having prostate cancer is poor. Indeed, men without biopsy-proven prostate cancer are just as likely to have a positive result in the PSA RT-PCR as are men with cancer. Whether these men with negative prostate biopsies and positive PSA RT-PCR findings may eventually develop prostate cancer remains to be determined. At this time, PSA RT-PCR for the prospective detection of prostate cancer should be considered investigational.

Adenocarcinoma↗
PubMed1994

Molecular staging of prostate cancer with the use of an enhanced reverse transcriptase-PCR assay.

OBJECTIVE: Because up to 40 percent of surgically treated patients with prostate cancer are subsequently found to be clinically understaged, a more sensitive staging modality to identify extraprostatic disease prior to surgery is required. METHODS: We describe an enhanced reverse transcriptase [RT] polymerase chain reaction (PCR) assay utilizing oligonucleotide primers specific for the human prostate-specific antigen (PSA). This assay identifies PSA-synthesizing cells from reverse transcribed mRNA. This assay was applied to RNAs extracted from the peripheral blood lymphocytes of 65 patients with clinically localized prostate cancer. In addition, blood from 20 women, 20 young men, 25 age-matched control men under treatment for benign prostatic hyperplasia (BPH), and 18 men with established, untreated metastatic prostate cancer was tested. RESULTS: An RT-PCR assay for PSA can recognize one PSA-expressing cell diluted into one hundred thousand lymphocytes. The sensitivity of this assay can be enhanced by the addition of digoxigenin-modified nucleotides to the PCR reaction and this assay was applied to RNAs extracted from the peripheral lymphocyte fraction of 148 prostate cancer patients and controls at this institution. Although no specimen from women or men without cancer was positive in this assay, 14 of 18 metastatic prostate cancer patients were positive (77.8%). Additionally, 25 of 65 (38.5%) patients with clinically localized disease (T1-2b) were positive from blood specimens obtained prior to surgery. Final pathologic results from this group of patients identified a correlation between positivity on this assay and the presence of capsular tumor penetration (sensitivity, 68%; specificity, 84%) as well as strong correlation with the finding of carcinoma at the surgical margin (sensitivity, 87%; specificity, 76%). Logarithmic regression analysis of the results of the RT-PCR assay indicates its remarkable superiority to digital rectal examination, computed tomography scan, endorectal coil magnetic resonance imaging, PSA, prostate-specific antigen density, or Gleason score for predicting the true pathologic stage of prostate cancer in these surgically treated patients. CONCLUSIONS: An RT-PCR assay using PSA primers to detect prostate cells in the peripheral circulation of surgical-candidate patients is significantly correlated with capsular penetration and tumor-positive surgical margins. This molecular assay provides a sensitive and specific means to stage correctly apparent localized prostate cancer prior to radical prostatectomy.

Adult↗
PubMed2001

Rapid identification of laboratory contamination with Mycobacterium tuberculosis using variable number tandem repeat analysis.

Compared with solid media, broth-based mycobacterial culture systems have increased sensitivity but also have higher false-positive rates due to cross-contamination. Systematic strain typing is rarely undertaken because the techniques are technically demanding and the data are difficult to organize. Variable number tandem repeat (VNTR) analysis by PCR is rapid and reproducible. The digital profile is easily manipulated in a database. We undertook a retrospective study of Mycobacterium tuberculosis isolates collected over an 18-month period following the introduction of the BACTEC MGIT 960 system. VNTR allele profiles were determined with early positive broth cultures and entered into a database with the specimen processing date and other specimen data. We found 36 distinct VNTR profiles in cultures from 144 patients. Three common VNTR profiles accounted for 45% of true-positive cases. By combining VNTR results with specimen data, we identified nine cross-contamination incidents, six of which were previously unsuspected. These nine incidents resulted in 34 false-positive cultures for 29 patients. False-positive cultures were identified for three patients who had previously been culture positive for tuberculosis and were receiving treatment. Identification of cross-contamination incidents requires careful documentation of specimen data and good communication between clinical and laboratory staff. Automated broth culture systems should be supplemented with molecular analysis to identify cross-contamination events. VNTR analysis is reproducible and provides timely results when applied to early positive broth cultures. This method should ensure that patients are not placed on unnecessary tuberculosis therapy or that cases are not falsely identified as treatment failures. In addition, areas where existing procedures may be improved can be identified.

Culture Media↗
PubMed2006

Notch3 gene amplification in ovarian cancer.

Gene amplification is one of the common mechanisms that activate oncogenes. In this study, we used single nucleotide polymorphism array to analyze genome-wide DNA copy number alterations in 31 high-grade ovarian serous carcinomas, the most lethal gynecologic neoplastic disease in women. We identified an amplicon at 19p13.12 in 6 of 31 (19.5%) ovarian high-grade serous carcinomas. This amplification was validated by digital karyotyping, quantitative real-time PCR, and dual-color fluorescence in situ hybridization (FISH) analysis. Comprehensive mRNA expression analysis of all 34 genes within the minimal amplicon identified Notch3 as the gene that showed most significant overexpression in amplified tumors compared with nonamplified tumors. Furthermore, Notch3 DNA copy number is positively correlated with Notch3 protein expression based on parallel immunohistochemistry and FISH studies in 111 high-grade tumors. Inactivation of Notch3 by both gamma-secretase inhibitor and Notch3-specific small interfering RNA suppressed cell proliferation and induced apoptosis in the cell lines that overexpressed Notch3 but not in those with minimal amount of Notch3 expression. These results indicate that Notch3 is required for proliferation and survival of Notch3-amplified tumors and inactivation of Notch3 can be a potential therapeutic approach for ovarian carcinomas.

Chromosomes, Human, Pair 19↗
PubMed2003

Selectively increased expression and functions of chemokine receptor CCR9 on CD4+ T cells from patients with T-cell lineage acute lymphocytic leukemia.

In a total of 38 typical T-cell lineage acute lymphocytic leukemia (T-ALL) and T-cell lineage chronic lymphocytic leukemia (T-CLL) cases investigated, we found that CC chemokine receptor CCR9 was selectively and frequently expressed on T-ALL CD4+ T cells, was moderately expressed on T-CLL CD4+ T cells, and was rarely expressed on normal CD4+ T cells. These findings were demonstrated at protein and mRNA levels using flow cytometry and real-time quantitative reverse transcription-PCR technique and were verified by digital confocal microscopy and Northern blotting. Thymus-expressed chemokine, a ligand for CCR9, selectively induced T-ALL CD4+ T-cell chemotaxis and adhesion. Interleukin (IL)-2 and IL-4, together, down-regulated the expression and functions of CCR9 in T-ALL CD4+ T cells including chemotaxis and adhesion. It was also demonstrated that IL-2 and IL-4, together, internalized CCR9 on T-ALL CD4+ T cells and subsequently inhibited functions of CCR9 in these cells. Thymus-expressed chemokine mRNA was highly expressed in CD4+ T cells, involving lymph node and skin in T-ALL patients, and was expressed at moderate levels in lymph node and skin tissues in T-CLL patients. Our findings may provide new clues to understanding various aspects of T-ALL CD4+ T cells, such as functional expression of CCR9-thymus-expressed chemokine receptor-ligand pairs as well as the effects of IL-2 and IL-4, which may be especially important in cytokine/chemokine environment for the pathophysiological events of T-ALL CD4+ T-cell trafficking.

Adolescent↗
PubMed1997

A common prothrombin variant (20210 G to A) increases the risk of myocardial infarction in young women.

Using specimens from a population-based case control study among women ages 18 to 44 years in western Washington, we assessed the relationship between carriership of a genetic clotting factor II variant (20210 G-->A) and myocardial infarction (MI). The factor II variant was previously shown to be present in 1% to 2% of the population, to increase the levels of factor II, and to be associated with venous thrombotic disease. Personal interviews and blood samples were obtained from 79 women with a first myocardial infarction and 381 control women identified through random-digit telephone dialing. Polymerase chain reaction (PCR) method was used to determine the factor II genotypes. The factor II 20210 G to A transition was present more often in women with MI (5.1%) than among control women (1.6%). The age-adjusted odds ratio for MI was 4.0 (95% confidence interval 1.1 to 15.1). The relative risk was high when another major cardiovascular risk factor was also present, such as smoking (odds ratio 43.3, 95% confidence interval 6.7 to 281), and the risk seemed limited to those with other risk factors. These results, in which the effect of major coronary risk factors is enhanced fourfold to sixfold by the prothrombin variant, are similar to those previously reported for another genetic clotting abnormality, factor V Leiden. We conclude that factor II 20210 G to A increases the risk of myocardial infarction in young women, especially in the women with other major risk factors for coronary heart disease.

Adolescent↗
PubMed1996

Rapid detection of thymidylate synthase gene expression levels by semi-quantitative competitive reverse transcriptase polymerase chain reaction followed by quantitative digital image analysis.

We describe a simplified and reliable polymerase chain reaction (PCR) method to quantify thymidylate synthase (TS) gene expression levels from clinical human tumor biopsy samples as small as 100 mg using the beta-actin housekeeping gene as a reference standard. The semiquantitative RT-PCR is carried out by the coamplification of the target template and an external competitor using primer pairs common to both templates in the same reaction vessel. Quantitative digital image analysis is performed directly after electrophoresis, thus mRNA quantification is done quickly and without the use of radioactive substances. The observed relative TS gene expression levels varied between 3- and 40-fold, but most of the values were grouped within a 10-fold range. There is an observed 89% correlation between TS mRNA expression and protein levels. These findings suggest that preliminary experiments used to determine the linear range of RT-PCR amplification in non-competitive semiquantitative PCR experiments, and the use of radioactive substances to quantify PCR products may be unnecessary.

Base Sequence↗
PubMed2025

Methylation-based droplet digital polymerase chain reaction shows high concordance with chronic lymphocytic leukemia IGHV somatic mutation status.

OBJECTIVE: Somatic hypermutation at immunoglobulin heavy chain variable (IGHV) genes, an established prognostic and predictive biomarker for chronic lymphocytic leukemia (CLL), is assessed by gene sequencing. We developed a single methylation-specific droplet digital polymerase chain reaction (methyl-ddPCR) to predict IGHV status in patients with CLL. METHODS: The CLL methylation array and IGHV data from the International Cancer Genome Consortium (ICGC) were used for biomarker discovery. Top-ranked candidate regions were manually screened for PCR primer and probe binding sites. A single methyl-ddPCR was evaluated on an internal cohort of CLLs with mutated (M), unmutated (U), and inconclusive IGHV results originally determined by next-generation sequencing (NGS). RESULTS: Analysis of ICGC data identified array probe cg23844018 as a candidate for the PCR. The corresponding CpG site showed high methylation levels in U-CLL and lower levels in M-CLL. On the internal cohort, a single optimal cutoff correctly classified 104 of 115 U- and M-CLLs (90.4%; area under the curve&#x2005;=&#x2005;0.96). The PCR data correlated with some prognostic fluorescence in situ hybridization and CLL subset groupings. Limited analysis suggests that the PCR may be able to stratify some patients with CLL who have inconclusive results on IGHV NGS testing. CONCLUSIONS: The methyl-ddPCR showed high concordance with CLL IGHV status in an internal cohort.

Humans↗
PubMed1993

Minisatellite variant repeat mapping: application to DNA typing and mutation analysis.

Most DNA typing systems assay allele length variation at tandemly repeated loci such as minisatellites and microsatellites. Allele length measurements are approximate, which impedes the use of such loci in forensic analysis and in studies of allelic variability at hypervariable loci. We now review progress in the development of alternative DNA typing systems based on allelic variation in the interspersion patterns of variant repeat units along minisatellite alleles. Minisatellite variant repeat mapping by PCR (MVR-PCR) not only provides a powerful new digital approach to DNA typing, but also for the first time allows investigation of the true level of allelic variability at minisatellite loci and of the mutational mechanisms that generate ultravariability.

Alleles↗