Search results for "Pyrosequencing"
showing 10 items of 58 documents
Plasma cell-free DNA in diagnostic KRAS mutation testing
2015
Epidermaalisen kasvutekijän reseptorin (EGFR) monoklonaalisia vasta-aineita (mAb) käytetään apuna metastaattisen kolorektaalisyövän (mCRC) hoidossa. Aktivoivat mutaatiot KRAS- ja NRAS-onkogeeneissä tekevät syöpäsolut vastustuskykyisiksi anti-EGFR-vasta-aineille, ja tästä syystä tuumorit tulee genotyypittää ennen hoidon aloittamista. Kudosbiopsian ottaminen on kuitenkin invasiivinen toimenpide, eikä näyte välttämättä edusta kattavasti syövän kaikkien solupopulaatioiden geneettisiä muutoksia. Verenkierron solunulkoisen DNA:n (cfDNA) on havaittu sisältävän maligneista soluista lähtöisin olevia onkogeenisiä mutaatioita, ja se voi olla hyvä vaihtoehtoinen tuumoriperäisen DNA:n lähde. Kehitimme j…
Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.
2012
Natural environments represent an incredible source of microbial genetic diversity. Discovery of novel biomolecules involves biotechnological methods that often require the design and implementation of biochemical assays to screen clone libraries. However, when an assay is applied to thousands of clones, one may eventually end up with very few positive clones which, in most of the cases, have to be "domesticated" for downstream characterization and application, and this makes screening both laborious and expensive. The negative clones, which are not considered by the selected assay, may also have biotechnological potential; however, unfortunately they would remain unexplored. Knowledge of t…
Analysis of RET promoter CpG island methylation using methylation-specific PCR (MSP), pyrosequencing, and methylation-sensitive high-resolution melti…
2016
Background Already since the 1990s, promoter CpG island methylation markers have been considered promising diagnostic, prognostic, and predictive cancer biomarkers. However, so far, only a limited number of DNA methylation markers have been introduced into clinical practice. One reason why the vast majority of methylation markers do not translate into clinical applications is lack of independent validation of methylation markers, often caused by differences in methylation analysis techniques. We recently described RET promoter CpG island methylation as a potential prognostic marker in stage II colorectal cancer (CRC) patients of two independent series. Methods In the current study, we analy…
Functional Metagenomics of the Bronchial Microbiome in COPD
2015
Altres ajuts: Sociedad Catalana de Neumología; Fundació Catalana de Neumología; Fundació Parc Tauli; Marató de TV3; Sociedad Española de Neumología y Cirugía Torácica; Fundación Menarini; Generalitat Valenciana (Spain) [Prometeo/2009/092] i Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES) The course of chronic obstructive pulmonary disease (COPD) is frequently aggravated by exacerbations, and changes in the composition and activity of the microbiome may be implicated in their appearance. The aim of this study was to analyse the composition and the gene content of the microbial community in bronchial secretions of COPD patients in both stability and exacerbati…
Concordance of assays designed for the quantification of JAK2V617F: a multicenter study
2008
Background Many different techniques have been designed for the quantification of JAK2 V617F allelic burden, sometimes producing discrepant results. Design and Methods JAK2 V617F quantification techniques were compared among 16 centers using 11 assays based on quantitative polymerase chain reaction (with mutation-specific primers or probes, or fluorescent resonance energy transfer/melting curve analysis), allele-specific polymerase chain reaction, conventional sequencing or pyrosequencing. Results A first series of blinded samples (granulocyte DNA, n=29) was analyzed. Seven assays (12 centers) reported values inside the mean±2SD; the mean coefficient of variation was 31%. Sequencing techniq…
The gut microbiota of larvae of Rhynchophorus ferrugineus Oliver (Coleoptera: Curculionidae)
2014
Background: The red palm weevil (RPW) Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) is one of the major pests of palms. The larvae bore into the palm trunk and feed on the palm tender tissues and sap, leading the host tree to death. The gut microbiota of insects plays a remarkable role in the host life and understanding the relationship dynamics between insects and their microbiota may improve the biological control of insect pests. The purpose of this study was to analyse the diversity of the gut microbiota of field-caught RPW larvae sampled in Sicily (Italy). Results: The 16S rRNA gene-based Temporal Thermal Gradient Gel Electrophoresis (TTGE) of the gut microbiota of RPW …
Functional gene pyrosequencing reveals core proteobacterial denitrifiers in boreal lakes
2015
Denitrification is an important microbial process in aquatic ecosystems that can reduce the effects of eutrophication. Here, quantification and pyrosequencing of nirS, nirK, and nosZ genes encoding for nitrite and nitrous oxide reductases was performed in sediment samples from four boreal lakes to determine the structure and seasonal stability of denitrifying microbial populations. Sediment quality and nitrate concentrations were linked to the quantity and diversity of denitrification genes, the abundance of denitrifying populations (nirS and nosZ genes) correlated with coupled nitrificationdenitrification (Dn), and the denitrification of the overlying water NO3 − (Dw) correlated with the n…
METAGENOMIC ANALYSIS OF PHYTOPHTHORA DIVERSITY IN NURSERIES OF POTTED ORNAMENTAL SPECIES.
2015
The genetic diversity of Phytophthora was investigated in soil and root samples of potted ornamental and fruit tree plants collected in nurseries located in Apulia and Calabria (Southern Italy) using metagenomic approaches based on Phytophthora genus specific primers. PCR amplicons containing the ITS1 region of the rDNA were sequenced using both a conventional cloning and Sanger sequencing approach and a 454 pyrosequencing protocol. All sequences were accurately analyzed with an appropriate software and used as barcode for species identification utilizing a validated ITS database. The cloning/Sanger sequencing approach enabled the identification of nine different Phytophthora taxa (P. nicot…
De Novo Assembly and Functional Annotation of the Olive (Olea europaea) Transcriptome
2013
Olive breeding programmes are focused on selecting for traits as short juvenile period, plant architecture suited for mechanical harvest, or oil characteristics, including fatty acid composition, phenolic, and volatile compounds to suit new markets. Understanding the molecular basis of these characteristics and improving the efficiency of such breeding programmes require the development of genomic information and tools. However, despite its economic relevance, genomic information on olive or closely related species is still scarce. We have applied Sanger and 454 pyrosequencing technologies to generate close to 2 million reads from 12 cDNA libraries obtained from the Picual, Arbequina, and L…