Capillary PCR Research Articles

Cell-specific protein profiling in Arabidopsis thaliana trichomes: identification of trichome-located proteins involved in sulfur metabolism and detoxification

Metabolite, protein, and transcript analysis at the cellular level gives unparalleled insight into the complex roles tissues play in the plant system. However, while capillary electrophoresis and PCR amplification strategies make the profiling of metabolites and transcripts in specific cell types possible, the profiling of proteins in small samples represents a bottleneck. Here for the first time protein profiling has been achieved in a specific plant cell type: The application of specific cell sampling and shotgun peptide sequencing (nano LC/MS/MS) resulted in the identification of 63 unique proteins from pooled Arabidopsis trichome cells. A complete S-adenosylmethionine pathway cluster, two S-adenosylmethionine synthase isoforms, a glutathione S-conjugate translocator and other proteins involved in sulfur metabolism and detoxification are shown to be present in these cells, in agreement with previous work done at the level of trichome transcript analysis. The technology described here brings the simultaneous identification and localization of physiologically relevant cellular proteins within reach.

Endotoxin inhibitor blocks heat exposure-induced expression of brain cytokine mRNA in aged rats

To investigate the age-related changes in the expression of interleukin-1β (IL-1β) and its related substances in the brain during heat stress, we measured amounts of mRNAs for IL-1β, cyclooxygenase-2 (COX-2), and an inhibitor of nuclear factor (NF)-κB-β (IκB-β) that is known to reflect an activation of NF-κB, in the cortex, cerebellum, and hippocampus using a quantitative real-time capillary PCR method. The basal levels of IL-1β mRNA in aged rats (108–110 weeks old) was significantly higher than those in young animals (10–11 weeks old) in these brain regions. Heat exposure (33 °C) for 1 h enhanced the expression of IL-1β and COX-2 mRNAs in aged rats but not in young ones. The amount of lipopolysaccharide (LPS) assessed by its bioactivity in the cortex increased by heat exposure only in aged rats. To further examine an involvement of LPS in the increase in mRNAs, an endotoxin inhibitor (EI), a synthetic peptide that detoxifies LPS by binding to the toxic component of LPS, lipid A, was intraperitoneally injected before heat exposure in aged rats. An intraperitoneal injection of EI significantly attenuated the heat exposure-induced increases in mRNAs for IL-1β, COX-2, IκB-β, and the LPS activity. Administration of EI also debilitated the heat exposure-induced hyperthermia and responses of plasma ACTH and catecholamines. These findings, taken together, suggest that the bacterial translocation is involved in the mechanisms of the responses to heat exposure in aged rats including the increased expression of mRNAs for IL-1β and its related substances in the brain.

Myeloperoxidase (MPO) genotype and lung cancer histologic types: the MPO -463 A allele is associated with reduced risk for small cell lung cancer in smokers.

MPO participates in the metabolic activation of tobacco carcinogens such as PAHs. A frequent MPO -463 G-->A polymorphism in the promoter region reduces MPO transcription and has been correlated with >4-fold lower benzo[a]pyrene-DNA adduct levels in the skin of coal tar-treated patients. Four of 7 case-control studies found significantly reduced lung cancer risk associated with the A allele. Due to their different etiologies, we examined whether the MPO genotype affects histologic lung cancer types differentially. A case-control study was conducted in 625 ever-smoking lung cancer patients, including 228 adenocarcinomas, 224 SCCs, 135 SCLCs and 340 ever-smoking hospital controls. MPO genotyping was performed by capillary PCR followed by fluorescence-based melting curve analysis. Combining the MPO -463 (G/A+A/A) genotypes, a protective effect approaching significance (OR = 0.75, 95% CI 0.55-1.01) was observed when comparing all lung cancer cases to controls. Among histologic types of lung cancer, a weak protective effect was found for both adenocarcinoma (OR = 0.81, CI 0.55-1.19) and SCC (OR = 0.82, CI 0.56-1.21); a stronger and significant effect was found for SCLC (OR = 0.58, CI 0.36-0.95; p = 0.029). Our results also suggest that the MPO genotype varies among inflammatory nonmalignant lung diseases. In conclusion, our results emphasize the need for a separate analysis of lung cancer histologic types and an adjustment for inflammatory nonmalignant lung diseases in future MPO-related studies. We confirm that the MPO -463 A variant affords a protective effect against lung cancer risk in smokers, which was strongest for SCLC patients.

Thirty-Cycle Temperature Optimization of a Closed-Cycle Capillary PCR Machine

Thirty-Cycle Temperature Optimization of a Closed-Cycle Capillary PCR Machine

Development of a molecular diagnosis assay based on electrohybridization at plastic electrodes and subsequent PCR

Technologies enabling specific recognition of medically relevant nucleic acid sequences will play a pivotal role in future medical diagnosis. Whereas many approaches to molecular diagnosis systems include DNA microarrays on chips and fluorometric detection, the basis of our approach is the use of inexpensive components like plastic or metal thin film electrodes with low multiplexing and an electrochemical detection unit. To increase the sensitivity, PCR can be used as an intermediate step. For selective enrichment, specific nucleic acid probes were covalently attached at their 5′-ends to conducting polycarbonate/carbon fiber electrodes. Complementary oligonucleotides were enriched at the electrodes by cyclic inversion of an electrochemical potential, transferred into a PCR vial and thermally or electrochemically desorbed. The analysis of the PCR product shows the efficiency and selectivity of the electrochemical enrichment. Hybridization of DNA was shown by electrochemical methods, in this work especially by differential pulse voltammetry (DPV) using the single strand specific hybridization redox indicator osmium(VIII)-tetroxide, and potentiometric stripping analysis (PSA). This combination of experimental methods is the basis for a molecular diagnosis system including a disposable nucleic acid modified working electrode for specific enrichment, detection and quantification, and an optional capillary PCR module for fast amplification.

Ｎｏｎ‐ＲＩ法を用いた遺伝子解析 キャピラリーＰＣＲ法による遺伝子の定量および変異解析

Ｎｏｎ‐ＲＩ法を用いた遺伝子解析 キャピラリーＰＣＲ法による遺伝子の定量および変異解析

Quantitative detection of Chlamydia spp. by fluorescent PCR in the LightCycler.

Quantitative detection of intracellular bacteria of the genus Chlamydia by the standard cell culture method is cumbersome and operator dependent. As an alternative, we adapted hot-start PCR to the glass capillary quantitative PCR format of the LightCycler. The optimized PCR was consistently more efficient than commercially available pre-assembled PCRs. Detection by quantitative PCR of as few as single copies of DNA of Chlamydia spp. was accomplished by SYBR Green fluorescence of the dsDNA product and by fluorescence resonance energy transfer (FRET) hybridization probes. The PCRs were 15-fold more sensitive than the cell culture quantitative assay of C. psittaci B577 infectious stock. The number of chlamydial genomes detected by C. psittaci B577 FRET PCR correlated well with cell culture determination of inclusion forming units (IFUs) (r = 0.96, P < 0.0008). When infected tissue samples were analyzed by cell culture and PCR, the correlation coefficient between IFUs and chlamydial genomes was higher with C. psittaci B577 FRET PCR (r = 0.90, P < 0.0004) than with Chlamydia omp1 SYBR Green PCR (r = 0.85, P < 0.002).

Clinical use of capillary PCR to diagnose Mycoplasma pneumonia.

In the present study, serologic data were compared with data obtained by capillary PCR to establish the efficacy of capillary PCR for the determination of Mycoplasma infection in samples obtained from throat swabs, bronchoalveolar lavage fluids (BALF), and sputum of patients with Mycoplasma pneumonia. We performed PCR analysis for Mycoplasma DNA on a total of 325 samples from 197 patients with community-acquired pneumonia and in whom Mycoplasma pneumonia was suspected. There were 68 PCR-positive specimens. Review of the differences in PCR positivity rates based on the site of specimen collection showed the highest rate of detection (28.6%) from throat swabs. From among the 31 patients with significantly elevated titers of serum Mycoplasma antibodies, the PCR results were positive for 25 patients. Thus, capillary PCR had a sensitivity of 80.6% (25 of 31). Five of the six false-negative results were from throat swab specimens. Moreover, testing (PCR) had been performed only once for these five patients with false-negative results. From among the PCR-positive findings from BALF specimens, there were no false-positive results. BALF specimens were very useful, except for the technical procedures and increased patient burden required to obtain these specimens. We suggest that the use of throat swab specimens in capillary PCR is much more suitable for diagnosing Mycoplasma pneumonia in routine clinical practice; however, careful throat swab specimen collection and an increase in the number of times that the PCR is performed are necessary to reduce the rate of false-negative results.

Identifying mutations in Drosophila genes by direct sequencing of PCR products.

BioTechniquesVol. 27, No. 2 BenchmarksOpen AccessIdentifying Mutations in Drosophila Genes by Direct Sequencing of PCR ProductsErin M. Schlag & David A. WassarmanErin M. Schlag*Address correspondence to Erin M. Schlag, Cell Biology and Metabolism Br., National Institute of Child Health and Human Dev., Bldg. 18T, Rm. 101, National Institutes of Health, Bethesda, MD 20892, USA. Internet: E-mail Address: eschlag@helix.nih.govNational Institute of Child Health and Human Development, NIH, Bethesda, MD, USASearch for more papers by this author & David A. WassarmanNational Institute of Child Health and Human Development, NIH, Bethesda, MD, USASearch for more papers by this authorPublished Online:22 Aug 2018https://doi.org/10.2144/99272bm09AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail "Identifying Mutations in Drosophila Genes by Direct Sequencing of PCR Products." , 27(2), pp. 262–264FiguresReferencesRelatedDetailsCited ByInter-individual and intragenomic variations in the ITS region of Clonorchis sinensis (Trematoda: Opisthorchiidae) from Russia and VietnamInfection, Genetics and Evolution, Vol. 55Characterising functionally important and ecologically meaningful genetic diversity using a candidate gene approach21 September 2008 | Genetica, Vol. 138, No. 4echinoid mutants exhibit neurogenic phenotypes and show synergistic interactions with the Notch signaling pathwayDevelopment, Vol. 130, No. 25MASK, a large ankyrin repeat and KH domain-containing protein involved in Drosophila receptor tyrosine kinase signalingDevelopment, Vol. 129, No. 1The RNA-binding protein Tsunagi interacts with Mago Nashi to establish polarity and localize oskar mRNA during Drosophila oogenesis1 November 2001 | Genes & Development, Vol. 15, No. 21Developmental and Transcriptional Consequences of Mutations in Drosophila TAFII60Molecular and Cellular Biology, Vol. 21, No. 20Clinical Use of Capillary PCR To Diagnose Mycoplasma PneumoniaJournal of Clinical Microbiology, Vol. 38, No. 4TAF250 is required for multiple developmental events in Drosophila1 February 2000 | Proceedings of the National Academy of Sciences, Vol. 97, No. 3 Vol. 27, No. 2 Follow us on social media for the latest updates Metrics Downloaded 114 times History Published online 22 August 2018 Published in print August 1999 Information© 2018 Author(s)PDF download

キャピラリーＰＣＲ その原理と応用

キャピラリーＰＣＲ その原理と応用

A colony based confirmation assay for Legionella and Legionella pneumophila employing the EnviroAmp Legionella system and seroagglutination.

Modifications to the EnviroAmp Legionella detection system are described which permit the rapid analysis of bacterial colonies taken from Legionella selective media. Capillary PCR permitted twice the number of samples to be analysed with a single kit. When PCR was positive for Leg. pneumophila, this result was confirmed by seroagglutination. The reverse dot blot hybridization assay was only used where PCR indicated a Legionella sp. other than Leg. pneumophila, permitting further savings on detection system components. This technique and standard confirmation procedures were applied to 133 isolates arising from 63 water samples plated to Legionella isolation media. Results agreed except for two isolates which gave a positive result for Legionella spp. by PCR and hybridization but were negative using standard procedures. Raising the annealing/extension temperature of the PCR by 2 degrees C eliminated the false positive result with these two isolates but did not adversely effect the sensitivity of the assay, as determined by re-testing of 68 environmental isolates and testing of 69 new environmental isolates and 12 Legionella reference species. The modified technique provides a convenient and cost effective alternative to standard confirmation procedures.

Rapid and sensitive diagnosis of cytomegalovirus and Pneumocystis carinii pneumonia in patients with haematological neoplasia by using capillary polymerase chain reaction.

We attempted the simultaneous detection of cytomegalovirus DNA (CMV-DNA) and Pneumocystis carinii (carinii-DNA) in sputum samples obtained from 20 patients with haematological neoplasm with pneumonia, using rapid cycle DNA amplification (capillary PCR). We used a thermal cycler for capillary PCR which featured recirculation of hot air for rapid temperature control of 10 microliters reaction samples in thin glass capillary tubes. We extracted DNA from patients' sputa using a simple method. A comparison of the results obtained using the phenol extraction-ethanol precipitation method and those obtained using our simple method was made, and demonstrated complete agreement between the two. For detection of CMV-DNA and P. carinii-DNA with capillary PCR it was not necessary to vary temperature setting based on the primers used. Therefore, capillary PCR was used for the simultaneous detection of CMV and P. carinii. After amplification, the total time required for which was 20 min, amplified products were electrophoresed on agarose gels and visualized with ethidium bromide. Product sensitivity was higher with capillary PCR than with conventional PCR. We conclude that capillary PCR amplification is a valuable tool for rapid and simple diagnosis of CMV and P. carinii pneumonias.

Management of coastal communities: A. J. Gray, in: The scientific management of temperate communities for conservation. 31st symposium of the British Ecological Society, Southampton, 1989, ed I.F. Spellerberg & others, (Blackwell Scientific, for BES), 1991, pp 227–243

Tumor necrosis factor α (TNF-α) influences the pathogenesis of lung-fibrosis and carcinogenesis in normal cells. Polymorphisms of this gene are suggested to be associated with susceptibility to lung-diseases. Additionally TNF-α is postulated to play a significant role in regulating. Transforming growth factor (TGF-β1) expression Therefore we investigated if the TNF-α or TGF-β1 gene expression level is different within the −308 TNF-α genotypes.Quantitative Real-time PCR of TNF-α and TGF-β1 was performed in 178 Germans. Calculations of expression were made with the 2−ΔΔCT method. Detection of the −308 promoter polymorphism of the TNF-α gene was performed by rapid capillary PCR with melting curve analysis.The relative TNF-α mRNA expression revealed significant differences between the TNF-α −308 homozygote wild-type G/G (0.00079 ± 0.00011; n = 113) and the heterozygote genotype G/A (0.0005 ± 0.00008; n = 52; p = 0.030) as well as between homozygote wild-type G/G and the homozygote mutant A/A (0.00029 ± 0.00009; n = 5; p = 0.004). The relative TGF-β mRNA expression showed, similar to TNF-α, the highest mRNA expression was seen within the TNF-α −308 homozygote wild-types, while the lowest mRNA expression lay within the homozygote mutant-types.Our findings suggest that the G-allele of TNF-α −308 is associated with a significantly higher TNF-α mRNA expression compared to the A-allele and that this also reflects in TGF-β expression. Therefore we support the thesis that TGF-β is regulated by TNF-α.

Analysis of polygamous systems using DNA fingerprinting: B. Amos, in: Biotechnology and the conservation of genetic diversity. Proc. symposium, London, 1990, ed H.D.M. Moore & others, (Clarendon Press, for Zoological Society of London; Symposia of the Zoological Society of London, 64), 1992, pp 151–165

Tumor necrosis factor α (TNF-α) influences the pathogenesis of lung-fibrosis and carcinogenesis in normal cells. Polymorphisms of this gene are suggested to be associated with susceptibility to lung-diseases. Additionally TNF-α is postulated to play a significant role in regulating. Transforming growth factor (TGF-β1) expression Therefore we investigated if the TNF-α or TGF-β1 gene expression level is different within the −308 TNF-α genotypes.Quantitative Real-time PCR of TNF-α and TGF-β1 was performed in 178 Germans. Calculations of expression were made with the 2−ΔΔCT method. Detection of the −308 promoter polymorphism of the TNF-α gene was performed by rapid capillary PCR with melting curve analysis.The relative TNF-α mRNA expression revealed significant differences between the TNF-α −308 homozygote wild-type G/G (0.00079 ± 0.00011; n = 113) and the heterozygote genotype G/A (0.0005 ± 0.00008; n = 52; p = 0.030) as well as between homozygote wild-type G/G and the homozygote mutant A/A (0.00029 ± 0.00009; n = 5; p = 0.004). The relative TGF-β mRNA expression showed, similar to TNF-α, the highest mRNA expression was seen within the TNF-α −308 homozygote wild-types, while the lowest mRNA expression lay within the homozygote mutant-types.Our findings suggest that the G-allele of TNF-α −308 is associated with a significantly higher TNF-α mRNA expression compared to the A-allele and that this also reflects in TGF-β expression. Therefore we support the thesis that TGF-β is regulated by TNF-α.

Short-term dynamics of a neotropical forest: why ecological research matters to tropical conservation and management: S. P. Hubbell & R. B. Foster, Oikos, 63(1), 1992, pp 48–61

Tumor necrosis factor α (TNF-α) influences the pathogenesis of lung-fibrosis and carcinogenesis in normal cells. Polymorphisms of this gene are suggested to be associated with susceptibility to lung-diseases. Additionally TNF-α is postulated to play a significant role in regulating. Transforming growth factor (TGF-β1) expression Therefore we investigated if the TNF-α or TGF-β1 gene expression level is different within the −308 TNF-α genotypes.Quantitative Real-time PCR of TNF-α and TGF-β1 was performed in 178 Germans. Calculations of expression were made with the 2−ΔΔCT method. Detection of the −308 promoter polymorphism of the TNF-α gene was performed by rapid capillary PCR with melting curve analysis.The relative TNF-α mRNA expression revealed significant differences between the TNF-α −308 homozygote wild-type G/G (0.00079 ± 0.00011; n = 113) and the heterozygote genotype G/A (0.0005 ± 0.00008; n = 52; p = 0.030) as well as between homozygote wild-type G/G and the homozygote mutant A/A (0.00029 ± 0.00009; n = 5; p = 0.004). The relative TGF-β mRNA expression showed, similar to TNF-α, the highest mRNA expression was seen within the TNF-α −308 homozygote wild-types, while the lowest mRNA expression lay within the homozygote mutant-types.Our findings suggest that the G-allele of TNF-α −308 is associated with a significantly higher TNF-α mRNA expression compared to the A-allele and that this also reflects in TGF-β expression. Therefore we support the thesis that TGF-β is regulated by TNF-α.