925 resultados para DNA Sequencing
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Context: Genes from the ovarian bone morphogenetic signaling pathway (GDF9 and BMP15) are critical for normal human fertility. We previously identified a deletion mutation in GDF9 in sisters with spontaneous dizygotic (DZ) twins, but the prevalence of rare GDF9 variants in twinning families is unknown. Objective: The objective was to evaluate the frequency of rare variants in GDF9 in families with a history of DZ twinning. Design and Subjects: We recruited 3450 individuals from 915 DZ twinning families (1693 mothers of twins) and 1512 controls of Caucasian origin. One mother of DZ twins was selected from 279 of the 915 families, and a DNA sample was screened for rare variants in GDF9 using denaturant HPLC. Variants were confirmed by DNA sequencing and genotyped in the entire sample by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. Results: We found two novel insertion/deletions (c.392-393insT, c.1268-1269delAA) and four missense alterations in the GDF9 sequence in mothers of twins. Two of the missense variants (c.307C > T, p.Pro103Ser and c.362C > T, p.Thr121Leu) were located in the proregion of GDF9 and two (c.1121C > T, p.Pro374Leu and c.1360C > T, p.Arg454Cys) in the mature protein region. For each variant, the frequencies were higher in cases compared with controls. The proportion of mothers of DZ twins carrying any variant (4.12%) was significantly higher (P < 0.0001) than the proportion of carriers in controls (2.29%). Conclusion: We describe new variants in the GDF9 gene that are significantly more common in mothers of DZ twins than controls, suggesting that rare GDF9 variants contribute to the likelihood of DZ twinning.
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Prostate-specific antigen (PSA) is important in tumour detection, monitoring disease progression and tumour recurrence. however, PSA is not a cancerspecific marker as levels can also be elevated in benign prostatic disease. A number of different mRNA transcripts of PSA have also been identified in prostatic tissue, but have not been fully characterized (PSA 424, PSA 525, Schulz transcript). Tissue specimens from transurethral resection of the prostate (TURP) or radical prostatectomy were obtained from 17 men with BPH and 15 men with prostate cancer. Total RNA was extracted, and reverse-transcriptionpolymerase chain reaction (RT-PCR) and Southern analysis carried out using transcript-specific primers and probes to determine which mRNA PSA transcripts were expressed. Real-time PCR was performed to determine transcript levels between the two groups using transcript-specific primers and SYBR green fluorescence. Values obtained were normalized to a standard housekeeping gene, B2-microglobulin. Transcripts amplified by RT-PCR and real-time PCR were confirmed by DNA sequencing. Our results show that the transcripts were present in some, but not all, BPH and cancer samples indicating that they are not specific to either BPH or cancer. Analysis of real-time PCR normalized values using a Student’s t -test, shows that there is a significant difference between the two groups for PSA 424, but not wild-type PSA, PSA 525 or the Schulz transcript. Although a larger cohort of samples is needed to further confirm these results, these findings suggest that mRNA levels of PSA 424 may have some utility as a diagnostic or prognostic marker in prostate cancer detection.
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Objective. Infective endocarditis (IE) is diagnosed by the Duke criteria, which can be inconclusive particularly when blood cultures are negative. This study investigated the application of polymerase chain reaction (PCR) to identify bacterial DNA in excised valvular tissue, and its role in establishing the diagnosis of IE. Methods. Ninety-eight patients undergoing valve replacement surgery were studied. Twenty-eight patients were confirmed as definite for endocarditis by the Duke criteria; nine were considered as possible and 61 had no known or previous microbial infection of the endocardium. A broad-range PCR technique was used to amplify prokaryotic 16S rRNA genes present within homogenised heart valve tissue. Subsequent DNA sequencing of the PCR amplicon allowed identification of the infecting microorganism. Results. PCR results demonstrated the presence of bacterial DNA in the heart valves obtained from 14 out of 20 (70%) definite IE patients with positive blood cultures preoperatively. The causative microorganism for one patient with definite culture negative endocarditis was identified by PCR. Two out of nine (22%) of the valves from possible endocarditis patients also had bacterial DNA present converting them into the definite criteria whereas in the valves of seven out of nine (78%) of these patients no bacterial DNA was detected. Conclusion. The application of PCR to the explanted valves in patients with possible or confirmed diagnosis can augment the Duke criteria thereby improving post-surgical antimicrobial therapeutic options. © 2003 The British Infection Society. Published by Elsevier Ltd. All rights reserved.
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Purpose: SCN1A is the most clinically relevant epilepsy gene, most mutations lead to severe myoclonic epilepsy of infancy (SMEI) and generalized epilepsy with febrile seizures plus (GEFS+). We studied 132 patients with epilepsy syndromes with seizures precipitated by fever, and performed phenotype-genotype correlations with SCN1A alterations. Methods: We included patients with SMEI including borderline SMEI (SMEB), GEFS+, febrile seizures (FS), or other seizure types precipitated by fever. We performed a clinical and genetic study focusing on SCN1A, using dHPLC, gene sequencing, and MLPA to detect genomic deletions/duplications on SMEI/SMEB patients. Results: We classified patients as: SMEI/SMEB = 55; GEFS+ = 26; and other phenotypes = 51. SCN1A analysis by dHPLC/sequencing revealed 40 mutations in 37 SMEI/SMEB (67%) and 3 GEFS+ (11.5%) probands. MLPA showed genomic deletions in 2 of 18 SMEI/SMEB. Most mutations were de novo (82%). SMEB patients carrying mutations (8) were more likely to have missense mutations (62.5%), conversely SMEI patients (31) had more truncating, splice site or genomic alterations (64.5%). SMEI/SMEB with truncating, splice site or genomic alterations had a significantly earlier age of onset of FS compared to those with missense mutations and without mutations (p = 0.00007, ANOVA test). None of the remaining patients with seizures precipitated by fever carried SCN1A mutations. Conclusion: We obtained a frequency of 71% SCN1A abnormalities in SMEI/SMEB and of 11.5% in GEFS+ probands. MLPA complements DNA sequencing of SCN1A increasing the mutation detection rate. SMEI/SMEB with truncating, splice site or genomic alterations had a significantly earlier age of onset of FS. This study confirms the high sensitivity of SCN1A for SMEI/SMEB phenotypes. © 2007 International League Against Epilepsy.
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Fusarium oxysporum is a diverse, asexual fungal species composed of both saprophytic and pathogenic members. The destructive phytopathogens are classified into formae speciales based on the host species and into vegetative compatibility groups (VCGs) based on the ability of two individuals to form heterokaryons. Parasexuality, a non-sexual mode of genetic exchange unique to some fungi has been demonstrated in the laboratory in Fusarium oxysporum f. sp. cubense (FOC). The goals of this dissertation were threefold: to ascertain whether mitochondrial (mt) markers can distinguish race differences in FOC; to determine genetic relatedness of VCGs in FOC based on a mt marker; and to discover the mode of mt inheritance during a parasexual cycle.^ Band patterns produced by electrophoresis of Hae III digested genomic DNA indicated that VCG differences, not race, could be discerned by mtDNA analysis. Primers were designed to amplify a mt intergenic locus which served as a molecular marker to screen 55 strains of FOC in 16 VCGs using both single strand conformational polymorphism and DNA sequencing. Based on homogeneity of the locus, strains were assigned to seven mitotypes, a classification unit which I introduced and found informative for grouping related VCGs.^ To determine the mode of mt inheritance during a parasexual cycle, strains in different mitotypes were paired. Mitochondrial inheritance in all hybrid progeny was found to be uniparental. I speculated that if a parasexual cycle occurs in nature there would be greater variation in the nuclear genome than the mt. This could produce multiple VCGs within a mitotype, a phenomenon observed in FOC. Based on these data, I concluded that parasexuality in nature may contribute to the diversity observed in Fusarium oxysporum. ^
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Metagenomics is the culture-independent study of genetic material obtained directly from environmental samples. It has become a realistic approach to understanding microbial communities thanks to advances in high-throughput DNA sequencing technologies over the past decade. Current research has shown that different sites of the human body house varied bacterial communities. There is a strong correlation between an individual’s microbial community profile at a given site and disease. Metagenomics is being applied more often as a means of comparing microbial profiles in biomedical studies. The analysis of the data collected using metagenomics can be quite challenging and there exist a plethora of tools for interpreting the results. An automatic analytical workflow for metagenomic analyses has been implemented and tested using synthetic datasets of varying quality. It is able to accurately classify bacteria by taxa and correctly estimate the richness and diversity of each set. The workflow was then applied to the study of the airways microbiome in Chronic Obstructive Pulmonary Disease (COPD). COPD is a progressive lung disease resulting in narrowing of the airways and restricted airflow. Despite being the third leading cause of death in the United States, little is known about the differences in the lung microbial community profiles of healthy individuals and COPD patients. Bronchoalveolar lavage (BAL) samples were collected from COPD patients, active or ex-smokers, and never smokers and sequenced by 454 pyrosequencing. A total of 56 individuals were recruited for the study. Substantial colonization of the lungs was found in all subjects and differentially abundant genera in each group were identified. These discoveries are promising and may further our understanding of how the structure of the lung microbiome is modified as COPD progresses. It is also anticipated that the results will eventually lead to improved treatments for COPD.
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Background: Human papillomavirus (HPV) causes cervical cancer and external genital warts. The purpose of this study is to document the genotype distribution of HPV in females aged between 18 and 34 who self-referred to an STI clinic with visible external genital warts (EGW). Scrapings were taken from visible external genital warts (EGW). These scrapings were analysed by PCR for the presence of HPV DNA. Positive samples were then genotyped by means of a commercially available assay (LiPA). A comparison of genotyping results determined by the LiPA assay and direct amplicon DNA sequencing was also performed. Results: Ninety-two patients out of 105 samples (88%) had detectable levels of HPV DNA. The majority of individuals with EGW (66%) showed the presence of two or more genotypes. The most common HPV genotypes present in the study population were HPV-6, HPV-11, HPV-16, HPV-18, HPV-33 and HPV-53. Potential effects of vaccination on HPV molecular epidemiology indicate that 40% of the patients could have been protected from the high risk genotypes HPV-16 and HPV-18.Conclusion: This is the first report of the molecular epidemiology of external genital warts in women aged between 18 and 34 from Ireland based on results from a LiPA assay. The study shows that most individuals are infected with multiple genotypes including those with high oncogenic potential and that the newly available HPV vaccines could have a significant impact on prevalence of the most common HPV genotypes in this study population.
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Testing for differences within data sets is an important issue across various applications. Our work is primarily motivated by the analysis of microbiomial composition, which has been increasingly relevant and important with the rise of DNA sequencing. We first review classical frequentist tests that are commonly used in tackling such problems. We then propose a Bayesian Dirichlet-multinomial framework for modeling the metagenomic data and for testing underlying differences between the samples. A parametric Dirichlet-multinomial model uses an intuitive hierarchical structure that allows for flexibility in characterizing both the within-group variation and the cross-group difference and provides very interpretable parameters. A computational method for evaluating the marginal likelihoods under the null and alternative hypotheses is also given. Through simulations, we show that our Bayesian model performs competitively against frequentist counterparts. We illustrate the method through analyzing metagenomic applications using the Human Microbiome Project data.
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Haptophyta are predominantly planktonic and phototrophic organisms that have their main distribution in marine environments worldwide. They are a major component of the microbial ecosystem, some form massive blooms and some are toxic. Haptophytes are significant players in the global carbonate cycle through photosynthesis and calcification. They are characterized by the haptonema, a third appendage used for attachment and food handling, two similar flagella, two golden-brown chloroplasts, and organic body scales that serve in species identification. Coccolithophores have calcified scales termed coccoliths. Phylogenetically Haptophyta form a well-defined group and are divided into two classes Pavlovophyceae and Coccolithophyceae (Prymnesiophyceae). Currently, about 330 species are described. Environmental DNA sequencing shows high haptophyte diversity in the marine pico- and nanoplankton, of which many likely represent novel species and lineages. Haptophyte diversity is believed to have peaked in the past and their presence is documented in the fossil record back to the Triassic, approximately 225 million years ago. Some biomolecules of haptophyte origin are extraordinarily resistant to decay and are thus used by geologists as sedimentary proxies of past climatic conditions.
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Haptophyta are predominantly planktonic and phototrophic organisms that have their main distribution in marine environments worldwide. They are a major component of the microbial ecosystem, some form massive blooms and some are toxic. Haptophytes are significant players in the global carbonate cycle through photosynthesis and calcification. They are characterized by the haptonema, a third appendage used for attachment and food handling, two similar flagella, two golden-brown chloroplasts, and organic body scales that serve in species identification. Coccolithophores have calcified scales termed coccoliths. Phylogenetically Haptophyta form a well-defined group and are divided into two classes Pavlovophyceae and Coccolithophyceae (Prymnesiophyceae). Currently, about 330 species are described. Environmental DNA sequencing shows high haptophyte diversity in the marine pico- and nanoplankton, of which many likely represent novel species and lineages. Haptophyte diversity is believed to have peaked in the past and their presence is documented in the fossil record back to the Triassic, approximately 225 million years ago. Some biomolecules of haptophyte origin are extraordinarily resistant to decay and are thus used by geologists as sedimentary proxies of past climatic conditions.
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Dissertação submetida à Universidade de Lisboa, Faculdade de Ciências para a obtenção do Grau de Mestre em Microbiologia Aplicada.
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Animal-associated microbiotas form complex communities, which are suspected to play crucial functions for their host fitness. However, the biodiversity of these communities, including their differences between host species and individuals, has been scarcely studied, especially in case of skin-associated communities. In addition, the intraindividual variability (i.e. between body parts) has never been assessed to date. The objective of this study was to characterize skin bacterial communities of two teleostean fish species, namely the European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata), using a high-throughput DNA sequencing method. In order to focus on intrinsic factors of host-associated bacterial community variability, individuals of the two species were raised in controlled conditions. Bacterial diversity was assessed using a set of four complementary indices, describing the taxonomic and phylogenetic facets of biodiversity and their respective composition (based on presence/absence data) and structure (based on species relative abundances) components. Variability of bacterial diversity was quantified at the interspecific, interindividual and intraindividual scales. We demonstrated that fish surfaces host highly diverse bacterial communities, whose composition was very different from that of surrounding bacterioplankton. This high total biodiversity of skin-associated communities was supported by the important variability, between host species, individuals and the different body parts (dorsal, anal, pectoral and caudal fins).
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Tropical Australian shark fisheries target two morphologically indistinguishable blacktip sharks, the Australian blacktip (Carcharhinus tilstoni) and the common blacktip (C. limbatus). Their relative contributions to northern and eastern Australian coastal fisheries are unclear because of species identification difficulties. The two species differ in their number of precaudal vertebrae, which is difficult and time consuming to obtain in the field. But, the two species can be distinguished genetically with diagnostic mutations in their mitochondrial DNA ND4 gene. A third closely related sister species, the graceful shark C. amblyrhynchoides, can also be distinguished by species-specific mutations in this gene. DNA sequencing is an effective diagnostic tool, but is relatively expensive and time consuming. In contrast, real-time high-resolution melt (HRM) PCR assays are rapid and relatively inexpensive. These assays amplify regions of DNA with species-specific genetic mutations that result in PCR products with unique melt profiles. A real-time HRM PCR species-diagnostic assay (RT-HRM-PCR) has been developed based on the mtDNA ND4 gene for rapid typing of C. tilstoni, C. limbatus and C. amblyrhynchoides. The assay was developed using ND4 sequences from 66 C. tilstoni, 33. C. limbatus and five C. amblyrhynchoides collected from Indonesia and Australian states and territories; Western Australia, the Northern Territory, Queensland and New South Wales. The assay was shown to be 100% accurate on 160 unknown blacktip shark tissue samples by full mtDNA ND4 sequencing.
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Forty-four species of Colletotrichum are confirmed as present in Australia based on DNA sequencing analyses. Many of these species were identified directly as a result of two workshops organised by the Subcommittee on Plant Health Diagnostics in Australia in 2015 that covered morphological and molecular approaches to identification of Colletotrichum. There are several other species of Colletotrichum reported from Australia that remain to be substantiated by DNA sequence-based methods. This body of work aims to provide a basis from which to critically examine a number of isolates of Colletotrichum deposited in Australian culture collections.
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International audience