Relevant bibliographies by topics / PCR-free

Academic literature on the topic 'PCR-free'

Author: Grafiati
Published: 10 March 2023

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Journal articles on the topic "PCR-free"

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Hou, Chih-Sheng Johnson, Nebojsa Milovic, Michel Godin, Peter R. Russo, Raj Chakrabarti, and Scott R. Manalis. "Label-Free Microelectronic PCR Quantification." Analytical Chemistry 78, no. 8 (April 2006): 2526–31. http://dx.doi.org/10.1021/ac0520689.

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Lee, Sang Hun, Jihwan Song, Byungrae Cho, SoonGweon Hong, Ori Hoxha, Taewook Kang, Dongchoul Kim, and Luke P. Lee. "Bubble-free rapid microfluidic PCR." Biosensors and Bioelectronics 126 (February 2019): 725–33. http://dx.doi.org/10.1016/j.bios.2018.10.005.

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Erdeniz, Naz, Uffe H. Mortensen, and Rodney Rothstein. "Cloning-Free PCR-Based Allele Replacement Methods." Genome Research 7, no. 12 (December 1, 1997): 1174–83. http://dx.doi.org/10.1101/gr.7.12.1174.

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Shao, Zhenyu, Yuexing Liu, Han Xiao, and Genxi Li. "PCR-free electrochemical assay of telomerase activity." Electrochemistry Communications 10, no. 10 (October 2008): 1502–4. http://dx.doi.org/10.1016/j.elecom.2008.07.051.

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Chen, Yuchao, and Tae Seok Seo. "PCR-free digital minisatellite tandem repeat genotyping." ELECTROPHORESIS 32, no. 12 (May 30, 2011): 1456–64. http://dx.doi.org/10.1002/elps.201100073.

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Jafari, Hamed Mazhab, Karim Abdelhalim, Leyla Soleymani, Edward H. Sargent, Shana O. Kelley, and Roman Genov. "Nanostructured CMOS Wireless Ultra-Wideband Label-Free PCR-Free DNA Analysis SoC." IEEE Journal of Solid-State Circuits 49, no. 5 (May 2014): 1223–41. http://dx.doi.org/10.1109/jssc.2014.2312571.

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Brouard, D., O. Ratelle, J. Perreault, D. Boudreau, and M. St-Louis. "PCR-free blood group genotyping using a nanobiosensor." Vox Sanguinis 108, no. 2 (December 3, 2014): 197–204. http://dx.doi.org/10.1111/vox.12207.

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Senchyna, Fiona, Rajiv L. Gaur, Saurabh Gombar, Cynthia Y. Truong, Lee F. Schroeder, and Niaz Banaei. "Clostridium difficile PCR Cycle Threshold Predicts Free Toxin." Journal of Clinical Microbiology 55, no. 9 (June 14, 2017): 2651–60. http://dx.doi.org/10.1128/jcm.00563-17.

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ABSTRACTThere is no stand-aloneClostridium difficilediagnostic that can sensitively and rapidly detect fecal free toxins. We investigated the performance of theC. difficilePCR cycle threshold (CT) for predicting free toxin status. Consecutive stool samples (n= 312) positive for toxigenicC. difficileby the GeneXpertC. difficile/EpitcdBPCR assay were tested with the rapid membrane C. Diff Quik Chek Complete immunoassay (RMEIA). RMEIA toxin-negative samples were tested with the cell cytotoxicity neutralization assay (CCNA) and tgcBIOMICS enzyme-linked immunosorbent assay (ELISA). Using RMEIA alone or in combination with CCNA and/or ELISA as the reference method, the accuracy ofCTwas measured at differentCTcutoffs. Using RMEIA as the reference method, aCTcutoff of 26.35 detected toxin-positive samples with a sensitivity, specificity, positive predictive value, and negative predictive value of 96.0% (95% confidence interval [CI], 90.2% to 98.9%), 65.9% (95% CI, 59.0% to 72.2%), 57.4% (95% CI, 52.7% to 62%), and 97.1% (95% CI, 92.8% to 98.9), respectively. Inclusion of CCNA in the reference method improvedCTspecificity to 78.0% (95% CI, 70.7% to 84.2%). Intercartridge lotCTvariability measured as the average coefficient of variation was 2.8% (95% CI, 1.2% to 3.2%). Standardizing the input stool volume did not improveCTtoxin specificity. The medianCTvalues were not significantly different between stool samples with Bristol scores of 5, 6, and 7, between pediatric and adult samples, or between presumptive 027 and non-027 strains. In addition to sensitively detecting toxigenicC. difficilein stool, on-demand PCR may also be used to accurately predict toxin-negative stool samples, thus providing additional results in PCR-positive stool samples to guide therapy.
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Kraytsberg, Yevgenya, and Konstantin Khrapko. "Single-molecule PCR: an artifact-free PCR approach for the analysis of somatic mutations." Expert Review of Molecular Diagnostics 5, no. 5 (September 2005): 809–15. http://dx.doi.org/10.1586/14737159.5.5.809.

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Hsieh, Shen-Yuan, Mohammad A. Tariq, Andrea Telatin, Rebecca Ansorge, Evelien M. Adriaenssens, George M. Savva, Catherine Booth, Tom Wileman, Lesley Hoyles, and Simon R. Carding. "Comparison of PCR versus PCR-Free DNA Library Preparation for Characterising the Human Faecal Virome." Viruses 13, no. 10 (October 18, 2021): 2093. http://dx.doi.org/10.3390/v13102093.

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The human intestinal microbiota is abundant in viruses, comprising mainly bacteriophages, occasionally outnumbering bacteria 10:1 and is termed the virome. Due to their high genetic diversity and the lack of suitable tools and reference databases, the virome remains poorly characterised and is often referred to as “viral dark matter”. However, the choice of sequencing platforms, read lengths and library preparation make study design challenging with respect to the virome. Here we have compared the use of PCR and PCR-free methods for sequence-library construction on the Illumina sequencing platform for characterising the human faecal virome. Viral DNA was extracted from faecal samples of three healthy donors and sequenced. Our analysis shows that most variation was reflecting the individually specific faecal virome. However, we observed differences between PCR and PCR-free library preparation that affected the recovery of low-abundance viral genomes. Using three faecal samples in this study, the PCR library preparation samples led to a loss of lower-abundance vOTUs evident in their PCR-free pairs (vOTUs 128, 6202 and 8364) and decreased the alpha-diversity indices (Chao1 p-value = 0.045 and Simpson p-value = 0.044). Thus, differences between PCR and PCR-free methods are important to consider when investigating “rare” members of the gut virome, with these biases likely negligible when investigating moderately and highly abundant viruses.
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Dissertations / Theses on the topic "PCR-free"

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White, James David dvm. "Real-Time Quantitative PCR of tet (C), in 2 Swine Populations: Antibiotic Free versus Conventionally Reared." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1437046111.

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Sillence, Kelly. "Cell-free fetal DNA (cffDNA) enrichment for non-invasive prenatal testing (NIPT) : a comparison of molecular techniques." Thesis, University of Plymouth, 2016. http://hdl.handle.net/10026.1/5319.

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Prenatal assessment of fetal health is routinely offered throughout pregnancy to ensure that the most effective management can be provided to maintain fetal and maternal well-being. Currently, invasive testing is used for definitive diagnosis of fetal aneuploidy, which is associated with a 1% risk of iatrogenic fetal loss. Developing non-invasive prenatal testing (NIPT) is a key area of research and methods to increase the level of cell-free fetal DNA (cffDNA) within the maternal circulation have been discussed to improve accuracy of such tests. In this study, three strategies; co-amplification at lower denaturation temperature polymerase chain reaction (COLD-PCR), inverse-PCR and Pippin Prep™ gel electrophoresis, were analysed to identify a novel approach to selectively enrich shorter cffDNA fragments from larger maternal cell-free DNA (cfDNA). The sensitivity of droplet digital PCR (ddPCR) against real-time PCR (qPCR) was compared for fetal sex and RHD genotyping. In addition RHD zygosity testing was carried out for non-maternal samples. Consequently, Pippin Prep™ gel electrophoresis was combined with ddPCR analysis for the NIPD of Down Syndrome (DS) in pseudo-maternal samples. The results revealed that the Pippin Prep™ gel electrophoresis enrichment approach successfully demonstrated 2-fold to 5-fold increases in the cffDNA fraction. However, further optimisation assays of COLD-PCR and inverse-PCR using actual maternal samples were required. The spike experiments for DS detection revealed that with the present assay IV overrepresentation of the chromosome 21 target could be significantly detected for samples with ≥15% ‘cffDNA fraction’. In conjunction with the Pippin Prep™ enrichment method, this would have enabled assessment of all 10 maternal samples. Alternatively, fetal sex and RHD genotyping results determined that ddPCR provides a more sensitive platform compared to qPCR approaches, particularly for samples that express low cffDNA fractions (<2%). The ddPCR platform also proved to be a rapid and accurate system for the determination of RHD zygosity. This study highlights that ddPCR could be used as opposed to qPCR for accurate determination of fetal sex and RHD status. While sequencing approaches currently provide the most sensitive platforms for NIPT of fetal aneuploidy, high costs (>£400) prevent universal application. The combination of cffDNA enrichment with ddPCR analysis could provide a cheaper and more widely available platform for NIPD. However, further large scale validation studies using actual maternal samples are required.
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Miran, Tara. "Enrichment of minority DNA in admixes of DNA samples : potential use in non-invasive prenatal diagnosis (NIPD) of Down syndrome." Thesis, University of Plymouth, 2012. http://hdl.handle.net/10026.1/1190.

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Non-invasive prenatal diagnosis (NIPD) is a promising approach that is currently being developed. The principle is that fetal material can be detected in maternal plasma and potentially enable women to pursue reliable and timely prenatal diagnosis, whilst eliminating the risk of miscarriage associated with chorionic villus sampling or amniocentesis. However NIPD research has been restricted up until now for the diagnosis of Down syndrome due to the low concentration of free fetal DNA (ffDNA) in maternal plasma. Various methods have been developed in an attempt to increase the concentration of ffDNA. This study uses COLD-PCR (co-amplification at lower denaturation temperature PCR) to analyse potential enrichment of ffDNA over maternal DNA through optimization of the critical denaturation temperature (Td), using Real Time-PCR in an attempt to selectively enrich smaller fetal DNA fragments. Fake fetal DNA was created in two different spike experiments to imitate the natural environment of viable ffDNA. One spike experiment used 5% of fake fetal DNA in a 95% maternal background to represent levels of ffDNA during early pregnancy. The other spike experiment utilized 10% of fake fetal DNA in 90% maternal background to denote late pregnancy. Before running COLD-PCR, various adjustments took place to find the critical Td at which one could run the spike experiment by COLD-PCR. Products of spike experiment were analysed on a genetic analyser for fragment analysis. Melt curve analysis was also performed for the spike experiment to identify the specificity of each sample at each denaturation temperatures. A critical Td (80°C) was identified for the D21S1890 region of chromosome 21 by COLD-PCR. This temperature does allow enrichment of fetal DNA, as fake maternal DNA was undetermined by RT-PCR compared to fake fetal DNA. The spike experiments clearly showed amplification of fake fetal DNA from the mixture of fake fetal and fake maternal DNA at the critical Td of 80°C. Running same samples of spike experiment on genetic analyser identified peaks from all samples at a Td of 95°C, while at a critical Td of 80°C the result showed decreased numbers of maternal peaks, regardless of stutter peaks formation. Melt curve analysis results clearly identified heteroduplex formation in the samples at the critical Td of 80°C. The results represent a good indication for using COLD-PCR in enriching ffDNA for detection by RT-PCR. However, as each individual has only two alleles, the observed results of multiple peaks for fragment analysis were not expected. Further research needs to focus on both eliminating heteroduplex formation and stutter peaks. COLD-PCR has the potential to open a new gateway in NIPD for aneuploidy detection. This method could be particularly useful in the detection of genetic abnormalities in the fetus, in particular Down syndrome and other aneuploidies.
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Batista, Ribrio Ivan Tavares Pereira. "Efeito do ácido linoléico conjugado TRANS-10, CIS-12 na regulação do acúmulo de lípides e expressão gênica em embriões produzidos in vitro." Universidade Federal de Juiz de Fora (UFJF), 2010. https://repositorio.ufjf.br/jspui/handle/ufjf/2507.

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A suplementação do ácido linoléico conjugado trans-10, cis-10 no meio de cultivo, representa uma importante alternativa para aumento da sobrevivência dos embriões após a criopreservação. No entanto este isômero de CLA no cultivo in vitro sem antioxidante pode aumentar a taxa apoptótica. O objetivo do presente estudo foi avaliar o efeito da adição CLA trans-10, cis-12 no cultivo in vitro de embriões sem antioxidante. Zigotos bovinos (n = 1.694) foram divididos em dois tratamentos: (T1) grupo controle, zigotos cultivados no meio CR2aa suplementado com soro fetal (n = 815); (T2) zigotos cultivados no meio CR2aa suplementado com soro fetal mais 100 µM CLA trans-10, cis-12. Os embriões foram avaliados quanto a desenvolvimento, quantidade de lípides e criotolerância. Transcritos dos RNA mensageiros (RNAm) dos genes selecionados foram mensurados pelo Real Time PCR. Suplementação de CLA trans-10, cis-12 não afeta significativamente a taxa de blastocisto (31,8% e 34,1% T1 e T2, respectivamente, p = 0,20) e nível dos RNAm dos genes relacionados com stress celular, apoptose e síntese de novo de ácido graxo. Quantidade de lípides e transcritos do RNAm do gene 1-acilglicerol-3-fosfato oaciltransferase 1 enzima relacionado a síntese de triglicérides foram significativamente reduzidos nos embriões cultivados na presença de CLA trans-10, cis-12 em comparação com o grupo controle. Teve aumento significativo na taxa de re-expansão dos blastocistos cultivados na presença de CLA trans-10, cis-12, após o descongelamento (34.4 e 56.3% para T1 e T2, respectivamente p = 0,002). Essa diferença não persistiu na taxa de eclosão (14,0% e 16,5% para T1 e T2, respectivamente, P = 0,62). Esses resultados mostram que o CLA trans-10, cis-12 reduz o acúmulo de lípides nos embriões pela redução nos níveis dos transcritos do gene 1-acilglicerol-3-fosfato o-aciltransferase 1 sem afetar a qualidade do embrião. Adicionalmente, este ácido graxo aumenta a taxa de re-expansão, no entanto, não melhora a taxa de eclosão.
Supplementation of conjugated linoleic acid trans-10, cis-10 in the culture medium, represents an important alternative to increasing the survival of embryos after cryopreservation. However the addition of culture media with CLA trans-10, cis-12 without antioxidant may increase the apoptotic rate. The aim of this study was to evaluate the effect of adding CLA trans-10, cis-12 in vitro culture of embryos without antioxidant. Bovine zygotes (n = 1,694) were divided into two treatments: (T1) control group, zygotes cultured in CR2aa medium supplemented with fetal calf serum (n = 815), (T2) zygotes cultured in CR2aa medium supplemented with fetal calf serum plus 100 µM CLA trans-10, cis-12. Embryos were evaluated for development, amount of lipids and cryotolerance. Transcripts of messenger RNA (mRNA) of selected genes were measured by real time PCR. Supplementation of CLA trans-10, cis-12 did not significantly affect the blastocyst rate (31.8% and 34.1% T1 and T2, respectively, p = 0,20) and the mRNA level of genes related to cell stress, apoptosis and de novo synthesis of fatty acid . Lipids and transcripts of the mRNA of the gene 1-acilglicerol-3-phosphate o-acyltransferase 1 enzyme related to the synthesis of triglycerides were significantly reduced in embryos cultured in the presence of CLA trans-10, cis-12 in comparison the control group. Had increased rate re-expansion of blastocysts cultured in the presence of CLA trans-10, cis-12, after thawing (34.4 and 56.3% for T1 and T2, respectively p = 0,002). This difference did not persist in the hatching rate (14.0 and 16.5% for T1 and T2, respectively, P = 0,62). These results show that the CLA trans-10, cis-12 reduces the accumulation of lipids in the embryos by reducing the levels of gene transcripts acilglicerol-1-3-phosphate oacyltransferase 1 without affecting the quality of the embryo. Additionally, this fatty acid increases the rate re-expansion, but does not improve the hatching rate.
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Gul, Fatma. "Development Of An Oligonucleotide Based Sandwich Array Platform For The Detection Of Transgenic Elements From Plant Sources Using Label-free Pcr Products." Master's thesis, METU, 2010. http://etd.lib.metu.edu.tr/upload/12612659/index.pdf.

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Advances in DNA micro and macroarray technologies made these high-throughput systems good candidates for the development of cheaper, faster and easier qualitative and quantitative detection methods. In this study, a simple and cost effective sandwich hybridization-based method has been developed for the rapid and sensitive detection of various unmodified recombinant elements in transgenic plants. Attention was first focused on the optimization of conditions such as time, concentration and temperature using commercial ssDNA, which in turn could be used for real sample detection. In this sandwich-type DNA chip platform, capture probes complementary to the first half of recombinant element (target adapter) were immobilized onto poly-L-lysine covered conventional microscope slides. PCR-amplified un-purified target adapter and biotin labeled detection probe, which is complementary to the second half of target adapter, were hybridized in solution-phase to complementary capture probes to create a sandwiched tripartite complex. Later, hybridization signal was visualized by the attachment of streptavidin conjugated Quantum Dot to the sandwiched complex under UV illumination. Sandwich based array system that has been developed in this study allows multiplex screening of GMO events on a single DNA chip platform. 35S promoter, NOS terminator, CRY1Ab and BAR target sequences were successfully detected on the same DNA chip platform. The platform was able to detect unlabeled PCR amplified DNA fragments of CaMV 35S promoter sequence and NOS terminator and BAR transgene sequences from transgenic potato plants and NK603 Certified GMO Reference material, respectively. The DNA-chip platform developed in this study will allow multiple detection of label-free PCR-amplified transgenic elements from real GMO samples on a single slide via a cost effective, fast, reliable and sensitive sandwich hybridization assay.
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Herrmann, Simon [Verfasser], and Matthias [Akademischer Betreuer] Ebert. "Multiplex-PCR and deep sequencing for mutation detection in circulating cell-free DNA of colorectal cancer patients / Simon Herrmann ; Betreuer: Matthias Ebert." Heidelberg : Universitätsbibliothek Heidelberg, 2021. http://d-nb.info/1234987856/34.

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Berg, Emily Katherine. "Thermodynamics of λ-PCR Primer Design and Effective Ribosome Binding Sites." Thesis, Virginia Tech, 2019. http://hdl.handle.net/10919/89900.

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Recombinant DNA technology has been commonly used in a number of fields to synthesize new products or generate products with a new pathway. Conventional cloning methods are expensive and require significant time and labor; λ-PCR, a new cloning method developed in the Senger lab, has a number of advantages compared to other cloning processes due to its employment of relatively inexpensive and widely available materials and time-efficiency. While the amount of lab work required for the cloning process is minimal, the importance of accurate primer design cannot be overstated. The target of this study was to create an effective procedure for λ-PCR primer design that ensures accurate cloning reactions. Additionally, synthetic ribosome binding sites (RBS) were included in the primer designs to test heterologous protein expression of the cyan fluorescent reporter with different RBS strengths. These RBS sequences were designed with an online tool, the RBS Calculator. A chimeric primer design procedure for λ-PCR was developed and shown to effectively create primers used for accurate cloning with λ-PCR; this method was used to design primers for CFP cloning in addition to two enzymes cloned in the Senger lab. A total of five strains of BL21(DE3) with pET28a + CFP were constructed, each with the same cyan fluorescent protein (CFP) reporter but different RBS sequences located directly upstream of the start codon of the CFP gene. Expression of the protein was measured using both whole-cell and cell-free systems to determine which system yields higher protein concentrations. A number of other factors were tested to optimize conditions for high protein expression, including: induction time, IPTG concentration, temperature, and media (for the cell-free experiments only). Additionally, expression for each synthetic RBS sequence was investigated to determine an accurate method for predicting protein translation. NUPACK and the Salis Lab RBS Calculator were both used to evaluate the effects of these different synthetic RBS sequences. The results of the plate reader experiments with the 5 CFP strains revealed a number of factors to be statistically significant when predicting protein expression, including: IPTG concentration, induction time, and in the cell-free experiments, type of media. The whole-cell system consistently produced higher amounts of protein than the cell-free system. Lastly, contrasts between the CFP strains showed each strain's performance did not match the predictions from the RBS Calculator. Consequently, a new method for improving protein expression with synthetic RBS sequences was developed using relationships between Gibbs free energy of the RBS-rRNA complex and expression levels obtained through experimentation. Additionally, secondary structure present at the RBS in the mRNA transcript was modeled with strain expression since these structures cause deviations in the relationship between Gibbs free energy of the mRNA-rRNA complex and CFP expression.
Master of Science
Recombinant DNA technology has been used to genetically enhance organisms to produce greater amounts of a product already made by the organism or to make an organism synthesize a new product. Genes are commonly modified in organisms using cloning practices which typically involves inserting a target gene into a plasmid and transforming the plasmid into the organism of interest. A new cloning process developed in the Senger lab, λ-PCR, improves the cloning process compared to other methods due to its use of relatively inexpensive materials and high efficiency. A primary goal of this study was to develop a procedure for λ-PCR primer design that allows for accurate use of the cloning method. Additionally, this study investigated the use of synthetic ribosome binding sites to control and improve expression of proteins cloned into an organism. Ribosome binding sites are sequences located upstream of the gene that increase the molecule’s affinity for the rRNA sequence on the ribosome, bind to the ribosome just upstream of the beginning of the gene, and initiate expression of the gene. Tools have been developed that create synthetic ribosome binding sites designed to produce specific amounts of protein. For example, the tools can increase or decrease expression of a gene depending on the application. These tools, the Salis Lab RBS Calculator and NUPACK, were used to design and evaluate the effects of the synthetic ribosome binding sites. Additionally, a new method was created to design synthetic ribosome binding sites since the methods used during the design process yielded inaccuracies. Each strain of E. coli contained the same gene, a cyan fluorescent protein (CFP), but had different RBS sequences located upstream of the gene. Expression of CFP was controlled via induction, meaning the addition of a particular molecule, IPTG in this system, triggered expression of CFP. Each of the CFP strains were tested with a variety of v conditions in order to find the conditions most suitable for protein expression; the variables tested include: induction time, IPTG (inducer) concentration, and temperature. Media was also tested for the cell-free systems, meaning the strains were grown overnight for 18 hours and lysed, a process where the cell membrane is broken in order to utilize the cell’s components for protein expression; the cell lysate was resuspended in new media for the experiments. ANOVA and multiple linear regression revealed IPTG concentration, induction time, and media to be significant factors impacting protein expression. This analysis also showed each CFP strain did not perform as the RBS Calculator predicted. Modeling each strain’s CFP expression using the RBS-rRNA binding strengths and secondary structures present in the RBS allowed for the creation of a new model for predicting and designing RBS sequences.
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Tanniche, Imen. "New Methods of DNA Assembly, Gene Regulation with a Synthetic sRNA, and Cyanobacterium Phenotype Monitoring with Raman Spectroscopy." Diss., Virginia Tech, 2019. http://hdl.handle.net/10919/100954.

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Metabolic engineering has enabled studying microorganisms by the modification of their genetic material and analysis of their metabolism for the isolation of microbial strains capable of producing high yields of high value chemicals and biofuels. In this research, novel tools were developed to improve genetic engineering of microbial cells. In this matter, λ-PCR (lambda-PCR) was developed enabling the construction of plasmid DNA. This technique allows DNA assembly and manipulation (insertion, substitution and/or deletion) at any location of a vector. λ-PCR addresses the need for an easy, highly-efficient, rapid and inexpensive tool for genetic engineering and overcoming limitations encountered with traditional techniques. Then, novel synthetic small RNA (sRNA) regulators were designed in a cell-free-system (in vitro) in order to modulate protein expression in biosynthetic pathways. The ability of the sRNAs to regulate mRNA expression with statistical significance was demonstrated. Up to 70% decrease in protein expression level was achieved by targeting specific secondary structures of the mRNA with antisense binding regions of the sRNA. Most importantly, a sRNA was identified capable of protein overexpression by up to 65%. An understanding of its mechanism showed that its mRNA target region(s) likely lead to occlusion of RNase E binding. This mechanism was translated for expression of a diaphorase enzyme, which has relevance to synthetic biology and metabolic engineering in in vitro systems. Results were successful, showing a greater than 75% increase in diaphorase expression in a cell-free protein synthesis reaction. Next, Raman spectroscopy was employed as a near real-time method for microbial phenotyping. Here, Raman spectroscopy was used in combination with chemometric analysis methods through RametrixTM Toolboxes to study the effects of environmental conditions (i.e. illumination, glucose, nitrate deprivation, acetate, sodium chloride and magnesium sulfate) on the phenotypic response of the cyanobacterium Synechocystis sp. PCC6803. The RametrixTM LITE Toolbox for MATLAB® enabled processing of Raman spectra and application of principal component analysis (PCA) and discriminant analysis of principal components (DAPC). Two studies were performed. PCA and DAPC produces distinct clustering of Raman spectra, representing multiple Synechocystis phenotypes, based on the (i) presence of glucose in the growth medium, (ii) illumination, (iii) nitrate limitation, and (iv) throughout a circadian rhythm growth cycle, in the first study. The second study focused on the phenotypic response based on (i) growth in presence of acetate, (ii) presence of high concentrations of sodium chloride and (iii) magnesium sulfate starvation. RametrixTM PRO was applied for the validation of the DAPC models through leave-one-out method that allowed calculation of prediction accuracy, sensitivity and selectivity for an unkown Raman spectrum. Statistical tests (ANOVA and pairwise comparison) were performed on Raman spectra to identify statistically relevant changes in Synechocystis phenotypes. Next, comparison between Raman data and standardized analytical methods (GF-FID, UPLC, spectrometric assays) was established. Overall, good correlation were obtained (R > 0.7). Finally, genomic DNA libraries were enriched to isolate a deoxynivalenol detoxifying enzyme. To do this, library fragments from microorganisms was generated through oligonucleotide primed polymerase chain reaction (DOP-PCR) and transformed in a DON-sensitive yeast strain. Rounds of subculture were performed in the presence of DON and ferulic acid in order to isolate a strain capable of enzymatic degradation of DON.
Doctor of Philosophy
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Periyannan, Rajeswari Prem Kumar. "Droplet microfluidics for single cell and nucleic acid analysis." Doctoral thesis, KTH, Proteomik och nanobioteknologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-192668.

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Droplet microfluidics is an emerging technology for analysis of single cells and biomolecules at high throughput. The controlled encapsulation of particles along with the surrounding microenvironment in discrete droplets, which acts as miniaturized reaction vessels, allows millions of particles to be screened in parallel. By utilizing the unit operations developed to generate, manipulate and analyze droplets, this technology platform has been used to miniaturize a wide range of complex biological assays including, but not limited to, directed evolution, rare cell detection, single cell transcriptomics, rare mutation detection and drug screening. The aim of this thesis is to develop droplet microfluidics based methods for analysis of single cells and nucleic acids. In Paper I, a method for time-series analysis of mammalian cells, using automated fluorescence microscopy and image analysis technique is presented. The cell-containing droplets were trapped on-chip and imaged continuously to assess the viability of hundreds of isolated individual cells over time. This method can be used for studying the dynamic behavior of cells. In Paper II, the influence of droplet size on cell division and viability of mammalian cell factories during cultivation in droplets is presented. The ability to achieve continuous cell division in droplets will enable development of mammalian cell factory screening assays in droplets. In Paper III, a workflow for detecting the outcome of droplet PCR assay using fluorescently color-coded beads is presented. This workflow was used to detect the presence of DNA biomarkers associated with poultry pathogens in a sample. The use of color-coded detection beads will help to improve the scalability of the detection panel, to detect multiple targets in a sample. In Paper IV, a novel unit operation for label-free enrichment of particles in droplets using acoustophoresis is presented. This technique will be useful for developing droplet-based assays that require label-free enrichment of cells/particles and removal of droplet content. In general, droplet microfluidics has proven to be a versatile tool for biological analysis. In the years to come, droplet microfluidics could potentially be used to improve clinical diagnostics and bio-based production processes.

QC 20160926

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Wuttig, Daniela. "Identifizierung metastasierungsassoziierter molekularer Faktoren durch genomweite Expressionsanalysen an pulmonalen Metastasen und Primärtumoren des klarzelligen Nierenzellkarzinoms." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2010. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-63743.

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Aufgrund ihres sehr hohen Metastasierungsrisikos weisen Patienten mit klarzelligem Nierenzellkarzinom (kzNZK) eine sehr hohe Sterblichkeit auf. Mit den zurzeit zur Verfügung stehenden klinischen Parametern kann der Krankheitsverlauf der Patienten nach der operativen Entfernung des Primärtumors nur unzureichend vorhergesagt werden. Um das Nachsorge- und Therapieregime der Patienten zu optimieren, muss die Vorhersagegenauigkeit der bestehenden Prognosemodelle durch molekulare Marker erhöht werden. Um geeignete Gene für eine Abschätzung von Metastasierungsrisiko und krankheitsfreiem Überleben (DFS) zu identifizieren, wurden genomweite Expressionsanalysen sowohl an Lungenmetastasen (n = 24) als auch an Primärtumoren (n = 24) des kzNZK vorgenommen. Durch Vergleich von Metastasensubgruppen, die sich nach unterschiedlich langen DFS entwickelt hatten bzw. Primärtumoren, die nach unterschiedlich langen DFS Metastasen bedingten, wurden tumorintrinsische DFS-assoziierte Expressionsmuster identifiziert. Weiterhin wurden Gene identifiziert, deren Expression sich zwischen Primärtumoren unterschied, die im Krankheitsverlauf manifeste Metastasen bedingten und solchen, die dies nicht taten. Die differenzielle Expression funktionell interessanter, teilweise auch in anderen publizierten Microarraystudien an kzNZK bestätigter Gene wurde im Folgenden mittels quantitativer Polymerasekettenreaktion (qPCR) validiert. Anschließend wurde die Assoziation ausgewählter Gene mit klinischen Parametern und dem Überleben der Patienten untersucht. Ein von klinischen Parametern unabhängiger Einfluss auf den Krankheitsverlauf der Patienten wurde dabei für EDNRB und PECAM1 auf Expressionsebene (qPCR; n = 86) sowie für TSPAN7 auf Proteinebene (Immunhistochemie an „Tissue Microarrays“; n = 106) belegt. EDNRB und PECAM1 waren signifikant höher exprimiert in Primärtumoren mit günstigen klinischen Parametern (TNMI/II, G1/2, V0, N0/M0). TSPAN7 war vorwiegend in den Gefäßen der primären kzNZK nachweisbar; eine signifikant höhere Zahl TSPAN7-positiver Gefäße war ebenfalls in Tumoren mit günstigen klinischen Parametern zu verzeichnen (pT1/2, TNMI/II, N0). Überlebensanalysen zeigten ein signifikant längeres DFS für Patienten mit einer hohen im Vergleich zu solchen mit einer geringen EDNRB-Expression und für Patienten, die in beiden untersuchten Gewebestanzen der „Tissue Microarrays“ TSPAN7-positive Gefäße aufwiesen im Vergleich zu Patienten mit nur einer oder keiner TSPAN7-gefäßpositiven Stanze. Für Patienten mit einer hohen im Vergleich zu solchen mit einer geringen EDNRB- bzw. PECAM1-Expression oder mit zwei im Vergleich zu keiner oder einer TSPAN7-gefäßpositiven Gewebestanze war zudem ein signifikant längeres tumorspezifisches Überleben (TSS) zu verzeichnen. Mit Hilfe multivariater Cox-Regressionsanalysen wurde eine unabhängige günstige prognostische Relevanz für EDNRB auf das DFS sowie für EDNRB, PECAM1 und TSPAN7 auf das TSS nachgewiesen. Somit sind diese molekularen Faktoren geeignet, um die Genauigkeit der bestehenden und ausschließlich auf klinischen Parametern basierenden Prognosemodelle zu erhöhen. Für eine Abschätzung von DFS und Metastasierungsrisiko erscheint dabei insbesondere EDNRB geeignet
Patients with clear cell renal cell carcinoma (ccRCC) have an extremely poor prognosis due to their high risk of metastases. Currently used clinico-patological parameters are insufficient for reliable prediction of metastatic risk and disease free survival (DFS) after surgical resection of the primary tumor. Molecular markers are strongly needed to improve outcome prediction, and thus to optimize the follow up and treatment schedule for patients with ccRCC. To identify genes which are suitable for the prediction of metastatic risk and DFS, genome-wide expression analyses were performed on pulmonary metastases (n = 24) and primary tumors (n = 24) obtained from patients with ccRCC. Tumor-intrinsic DFS-associated expression patterns were observed by comparing subgroups of metastases, which had developed within different DFS as well as primary tumors, which had caused metastases after different DFS. Furthermore, genes differentially expressed in primary tumors, which caused macroscopic metastases and tumors, which did not were identified. The differential expression of genes with a potential function in metastatic spread, which has in part been identified in independent published microarray studies as well, were validated by quantitative polymerase chain reaction (qPCR). Moreover, an independent prognostic impact on the survival of ccRCC patients was observed for the EDNRB und the PECAM1 gene expression (qPCR; n = 86) as well as for the TSPAN7 protein level (immunohistochemistry on tissue microarrays; n = 106). Primary tumors of patients with favourable clinico-pathological parameters (TNMI/II, G1/2, V0, N0/M0) showed a significantly higher EDNRB und PECAM1 gene expression than those with unfavorable parameters. TSPAN7 was predominantly detected in blood vessels of ccRCC tissues. In patients with favourable clinico-pathological parameters (pT1/2, TNMI/II, N0) a significantly higher number of TSPAN7-positive vessels was observed. Using survival analyses, a significantly longer DFS was observed for patients with a high compared to those with a low EDNRB expression as well as for patients with TSPAN7-positive vessels in both cores compared to no or one of the both cores investigated on tissue microarrays. A significantly longer TSS was observed for patients with a high EDNRB or PECAM1 expression as well as for patients with TSPAN7-positive vessles in both tissue cores investigated. Furthermore, EDNRB was an independent prognostic factor for the DFS of the patients; EDNRB, PECAM and TSPAN7 had an independent prognostic impact on the TSS. Therefore, these molecular markers are suitable to improve the accuracy of outcome prediction based on clinico-pathological parameters in ccRCC. For the prediction of DFS and metastatic risk EDNRB is particularly interesting
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Book chapters on the topic "PCR-free"

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Gudkov, Anatoly T., and Kirill A. Martemyanov. "Direct Expression of PCR Products in Cell-Free Translation Systems." In Cell-Free Translation Systems, 61–66. Berlin, Heidelberg: Springer Berlin Heidelberg, 2002. http://dx.doi.org/10.1007/978-3-642-59379-6_5.

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Hoffmann, Thomas, Cordula Nemetz, Regina Schweizer, Wolfgang Mutter, and Manfred Watzele. "High-Level Cell-Free Protein Expression from PCR-Generated DNA Templates." In Cell-Free Translation Systems, 203–10. Berlin, Heidelberg: Springer Berlin Heidelberg, 2002. http://dx.doi.org/10.1007/978-3-642-59379-6_19.

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Wadle, Simon, Stefanie Rubenwolf, Michael Lehnert, Bernd Faltin, Manfred Weidmann, Frank Hufert, Roland Zengerle, and Felix von Stetten. "Mediator Probe PCR: Detection of Real-Time PCR by Label-Free Probes and a Universal Fluorogenic Reporter." In Methods in Molecular Biology, 55–73. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4939-0733-5_6.

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Miura, Fumihito, and Takashi Ito. "Post-Bisulfite Adaptor Tagging for PCR-Free Whole-Genome Bisulfite Sequencing." In Methods in Molecular Biology, 123–36. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-7481-8_7.

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Sigalotti, Luca, Alessia Covre, Francesca Colizzi, and Elisabetta Fratta. "Quantitative Methylation-Specific PCR: A Simple Method for Studying Epigenetic Modifications of Cell-Free DNA." In Cell-free DNA as Diagnostic Markers, 137–62. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4939-8973-7_11.

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Podlesniy, Petar, and Ramon Trullas. "Biomarkers in Cerebrospinal Fluid: Analysis of Cell-Free Circulating Mitochondrial DNA by Digital PCR." In Methods in Molecular Biology, 111–26. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4939-7778-9_7.

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Watzele, Manfred, C. Nemetz, W. Obermeier, A. Seidl, and B. Buchberger. "High-Throughput Expression PCR Used to Systematically Investigate Regulation of Translation Initiation in an E. coli Cell-Free Expression System." In Cell-Free Protein Expression, 25–34. Berlin, Heidelberg: Springer Berlin Heidelberg, 2003. http://dx.doi.org/10.1007/978-3-642-59337-6_4.

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Whale, Alexandra S., Ana Fernandez-Gonzalez, Alice Gutteridge, and Alison S. Devonshire. "Control Materials and Digital PCR Methods for Evaluation of Circulating Cell-Free DNA Extractions from Plasma." In Methods in Molecular Biology, 45–65. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4939-7778-9_4.

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Cortès, Sandra, Fatima-Ezzahra Hibti, Frydman Chiraz, and Safia Ezzine. "High-Throughput E. coli Cell-Free Expression: From PCR Product Design to Functional Validation of GPCR." In Methods in Molecular Biology, 261–79. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9624-7_12.

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Ochert, A., M. J. Slomka, J. Ellis, and C. G. Teo. "Use of Chelex 100TM in the Extraction of Viruses from Diverse Cell-Free Clinical Samples for PCR." In Methods in DNA Amplification, 47–53. Boston, MA: Springer US, 1994. http://dx.doi.org/10.1007/978-1-4615-2530-1_6.

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Conference papers on the topic "PCR-free"

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Yi-Shao Liu, Padmapriya P. Banada, Arun K. Bhunia, and Rashid Bashir. "Label free detection of PCR amplification." In 2008 IEEE Sensors. IEEE, 2008. http://dx.doi.org/10.1109/icsens.2008.4716498.

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Satsanarukkit, Penvipha, Hsiwen Lo, and Yu Chong Tai. "A free-standing and flexible parylene PCR device." In 2012 7th IEEE International Conference on Nano/Micro Engineered and Molecular Systems (NEMS). IEEE, 2012. http://dx.doi.org/10.1109/nems.2012.6196743.

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Jiang, Li, Zhengda Lu, Matthew Mancuso, and David Erickson. "Solar-thermally driven PCR for power-free diagnostics." In CLEO: Applications and Technology. Washington, D.C.: OSA, 2013. http://dx.doi.org/10.1364/cleo_at.2013.ath4n.5.

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Satsanarukkit, P., H. Lo, Q. Quach, and Y. C. Tai. "ON-CHIP PCR WITH FREE-STANDING PARYLENE CHANNEL." In 2010 Solid-State, Actuators, and Microsystems Workshop. San Diego: Transducer Research Foundation, 2010. http://dx.doi.org/10.31438/trf.hh2010.118.

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Huw, Ling-Yuh, Jill Spoerke, Rajesh Patel, Weiqun Liu, Rajiv Raja, Lukas Amler, Garret Hampton, Elizabeth Punnoose, and Mark Lackner. "Abstract 3180: Mutation detection in circulating free DNA using mutant-enriched PCR and digital PCR." In Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL. American Association for Cancer Research, 2011. http://dx.doi.org/10.1158/1538-7445.am2011-3180.

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Ali, Md Eaqub, Uda Hashim, Md Fazul Bari, and Thakra S. Dhahi. "Colorimetric sensor for label free detection of porcine PCR product." In 2010 International Conference on Enabling Science and Nanotechnology (ESciNano). IEEE, 2010. http://dx.doi.org/10.1109/escinano.2010.5700934.

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Sayers, Michael B., and Tara M. Dalton. "A Novel Contamination Free Two Temperature Continuous Flow Polymerase Chain Reactor." In ASME 2007 International Mechanical Engineering Congress and Exposition. ASMEDC, 2007. http://dx.doi.org/10.1115/imece2007-43055.

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Polymerase Chain Reaction (PCR) is an enzymatic process that has dramatically advanced many fields of life sciences, where it is an indispensable tool in a burgeoning range of applications, including diagnostic medicine, molecular biology, forensics and food testing. Recent increased demand for extremely high throughput PCR systems has led to the development of miniaturised continuous flow microfluidic PCR devices, which may have extremely high throughput compared to standard commercial PCR thermal cyclers. A novel continuous flow microfluidic PCR device has been designed and fabricated, consisting of two thermal zones maintained on aluminium thermal blocks providing the precise temperatures required for denaturation and annealing/extension. Polycarbonate sideplates retain the denaturation thermal block vertically above the annealing/extension thermal block while allowing for a variable air gap to be maintained between them. Heating of the denaturation thermal block is achieved using a Labview controlled Thermofoil heater, while the annealing/extension thermal block is maintained at temperature by optimised heat transfer from the denaturation block. Flow-through capillary tubing is positioned into a grooved serpentine channel machined into these thermal blocks. This serpentine channel passes through each thermal block fifty times, providing fifty PCR thermal cycles. Contamination free high throughput continuous flow PCR necessitates that the samples be encapsulated in an immiscible carrier fluid to eradicate cross contamination between samples and suppress the likelihood of the sample contacting the capillary leading to sample degradation. Encapsulation of the PCR reaction mixture is achieved upstream of the thermal cycler through segmentation of the sample into droplets entrained within an immiscible carrier fluid, which are then cycled through the thermal cycler. High throughput DNA amplification of two genes, GAPDH and LEF1, from the REH cell line has been successfully demonstrated on this microfluidic platform without any detectable contamination between samples. The PCR droplet reactors were approximately 250nl which is two orders of magnitude less than the standard sample size for most commercial PCR thermal cyclers.
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Zhang, Rong, Jian Qin, Hao Tian, Weihua Si, Taihong Wang, and Zewen Liu. "Study on DNA electrochemical behavior for label-free micro PCR application." In 2011 IEEE International Conference on Nano/Micro Engineered and Molecular Systems (NEMS). IEEE, 2011. http://dx.doi.org/10.1109/nems.2011.6017512.

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Culha, Mustafa, Omer F. Karatas, Omer Aydin, Mehmet Kahraman, Kemal Keseroğlu, Ismail Sayin, and Omer F. Bayrak. "Toward PCR-free mutation detection based on surface-enhanced Raman scattering." In SPIE BiOS: Biomedical Optics, edited by Tuan Vo-Dinh and Joseph R. Lakowicz. SPIE, 2009. http://dx.doi.org/10.1117/12.808267.

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Daly, John, and Mark Davies. "A Quantitative Free Convection DNA Amplifier." In ASME/JSME 2007 Thermal Engineering Heat Transfer Summer Conference collocated with the ASME 2007 InterPACK Conference. ASMEDC, 2007. http://dx.doi.org/10.1115/ht2007-32381.

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The Polymerase Chain Reaction (PCR) has been used extensively to amplify targeted nucleic acids for many applications in molecular biology and, increasingly, in medical diagnostics. Outlined in this paper is a PCR device which takes account of the advantages offered by free convection. The design is, in it fundamental format a time-wise isothermal well-based thermocycler. A temperature gradient induced across the well causes convection forces to circulate the sample through the required temperatures necessary for amplification. Quantitative amplification is demonstrated with real time measurements of SYBR Green I fluorescence within the free convective DNA amplifier. Amplification of an 86-bp fragment of the pGEM®-T vector (Promega) is performed in a 25μl volume in eight minutes. A 10-fold dilution series and methods for calculating effective cycle times are presented. Also detailed within this paper are PIV and thermal imaging results of the free convection cavity. This device presents an opportunity for the development of a practical and inexpensive gene-expression measurement system.
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Reports on the topic "PCR-free"

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Manulis, Shulamit, Christine D. Smart, Isaac Barash, Guido Sessa, and Harvey C. Hoch. Molecular Interactions of Clavibacter michiganensis subsp. michiganensis with Tomato. United States Department of Agriculture, January 2011. http://dx.doi.org/10.32747/2011.7697113.bard.

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Clavibacter michiganensis subsp. michiganensis (Cmm), the causal agent of bacterial wilt and canker of tomato, is the most destructive bacterial disease of tomato causing substantial economic losses in Israel, the U.S.A. and worldwide. The molecular strategies that allow Cmm, a Gram-positive bacterium, to develop a successful infection in tomato plants are largely unknown. The goal of the project was to elucidate the molecular interactions between Cmmand tomato. The first objective was to analyze gene expression profiles of susceptible tomato plants infected with pathogenic and endophytic Cmmstrains. Microarray analysis identified 122 genes that were differentially expressed during early stages of infection. Cmm activated typical basal defense responses in the host including induction of defense-related genes, production of scavenging of free oxygen radicals, enhanced protein turnover and hormone synthesis. Proteomic investigation of the Cmm-tomato interaction was performed with Multi-Dimensional Protein Identification Technology (MudPIT) and mass spectroscopy. A wide range of enzymes secreted by Cmm382, including cell-wall degrading enzymes and a large group of serine proteases from different families were identified in the xylem sap of infected tomato. Based on proteomic results, the expression pattern of selected bacterial virulence genes and plant defense genes were examined by qRT-PCR. Expression of the plasmid-borne cellulase (celA), serine protease (pat-1) and serine proteases residing on the chp/tomA pathogenicity island (chpCandppaA), were significantly induced within 96 hr after inoculation. Transcription of chromosomal genes involved in cell wall degradation (i.e., pelA1, celB, xysA and xysB) was also induced in early infection stages. The second objective was to identify by VIGS technology host genes affecting Cmm multiplication and appearance of disease symptoms in plant. VIGS screening showed that out of 160 tomato genes, which could be involved in defense-related signaling, suppression of 14 genes led to increase host susceptibility. Noteworthy are the genes Snakin-2 (inhibitor of Cmm growth) and extensin-like protein (ELP) involved in cell wall fortification. To further test the significance of Snakin -2 and ELP in resistance towards Cmm, transgenic tomato plants over-expressing the two genes were generated. These plants showed partial resistance to Cmm resulting in a significant delay of the wilt symptoms and reduction in size of canker lesion compared to control. Furthermore, colonization of the transgenic plants was significantly lower. The third objective was to assess the involvement of ethylene (ET), jasmonate (JA) and salicylic acid (SA) in Cmm infection. Microarray and proteomic studies showed the induction of enzymes involved in ET and JA biosynthesis. Cmm promoted ET production 8 days after inoculation and SIACO, a key enzyme of ET biosynthesis, was upregulated. Inoculation of the tomato mutants Never ripe (Nr) impaired in ET perception and transgenic plants with reduced ET synthesis significantly delayed wilt symptoms as compared to the wild-type plants. The retarded wilting in Nr plants was shown to be a specific effect of ET insensitivity and was not due to altered expression of defense related genes, reduced bacterial population or decrease in ethylene biosynthesis . In contrast, infection of various tomato mutants impaired in JA biosynthesis (e.g., def1, acx1) and JA insensitive mutant (jai1) yielded unequivocal results. The fourth objective was to determine the role of cell wall degrading enzymes produced by Cmm in xylem colonization and symptoms development. A significance increase (2 to 7 fold) in expression of cellulases (CelA, CelB), pectate lyases (PelA1, PelA2), polygalacturonase and xylanases (XylA, XylB) was detected by qRT-PCR and by proteomic analysis of the xylem sap. However, with the exception of CelA, whose inactivation led to reduced wilt symptoms, inactivation of any of the other cell wall degrading enzymes did not lead to reduced virulence. Results achieved emphasized the complexity involved in Cmm-tomato interactions. Nevertheless they provide the basis for additional research which will unravel the mechanism of Cmm pathogenicity and formulating disease control measures.
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Jordan, Ramon L., Abed Gera, Hei-Ti Hsu, Andre Franck, and Gad Loebenstein. Detection and Diagnosis of Virus Diseases of Pelargonium. United States Department of Agriculture, July 1994. http://dx.doi.org/10.32747/1994.7568793.bard.

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Pelargonium (Geranium) is the number one pot plant in many areas of the United States and Europe. Israel and the U.S. send to Europe rooted cuttings, foundation stocks and finished plants to supply a certain share of the market. Geraniums are propagated mainly vegetatively from cuttings. Consequently, viral diseases have been and remain a major threat to the production and quality of the crop. Among the viruses isolated from naturally infected geraniums, 11 are not specific to Pelargonium and occur in other crops while 6 other viruses seem to be limited to geranium. However, several of these viruses are not sufficiently characterized to conclude that they are distinct agents and their nomenclature and taxonomy are confusing. The ability to separate, distinguish and detect the different viruses in geranium will overcome obstacles te developing effective detection and certification schemes. Our focus was to further characterize some of these viruses and develop better methods for their detection and control. These viruses include: isolates of pelargonium line pattern virus (PLPV), pelargonium ringspot virus (PelRSV), pelargonium flower break virus (PFBV), pelargonium leaf curl (PLCV), and tomato ringspot virus (TomRSV). Twelve hybridoma cell lines secreting monoclonal antibodies specific to a geranium isolate of TomRSV were produced. These antibodies are currently being characterized and will be tested for the ability to detect TomRSV in infected geraniums. The biological, biochemical and serological properties of four isometric viruses - PLPV, PelRSV, and PFBV (and a PelRSV-like isolate from Italy called GR57) isolated from geraniums exhibiting line and ring pattern or flower break symptoms - and an isolate ol elderbeny latent virus (ELV; which the literature indicates is the same as PelRSV) have been determined Cloned cDNA copies of the genomic RNAs of these viruses were sequenced and the sizes and locations of predicted viral proteins deduced. A portion of the putative replicase genes was also sequenced from cloned RT-PCR fragments. We have shown that, when compared to the published biochemical and serological properties, and sequences and genome organizations of other small isometric plant viruses, all of these viruses should each be considered new, distinct members of the Carmovirus group of the family Tombusviridae. Hybridization assays using recombinant DNA probes also demonstrated that PLPV, PelRSV, and ELV produce only one subgenomic RNA in infected plants. This unusual property of the gene expression of these three viruses suggests that they are unique among the Carmoviruses. The development of new technologies for the detection of these viruses in geranium was also demonstrated. Hybridization probes developed to PFBV (radioactively-labeled cRNA riboprobes) and to PLPV (non-radioactive digoxigenin-labeled cDNAs) were generally shown to be no more sensitive for the detection of virus in infected plants than the standard ELISA serology-based assays. However, a reverse transcriptase-polymerase chain reaction assay was shown to be over 1000 times more sensitive in detecting PFBV in leaf extracts of infected geranium than was ELISA. This research has lead to a better understanding of the identity of the viruses infecting pelargonium and to the development of new tools that can be used in an improved scheme of providing virus-indexed pelargonium plants. The sequence information, and the serological and cloned DNA probes generated from this work, will allow the application of these new tools for virus detection, which will be useful in domestic and international indexing programs which are essential for the production of virus-free germplasm both for domestic markets and the international exchange of plant material.
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