Dissertations / Theses on the topic 'Species specific primers'

What is the genetic relatedness between and among populations of turkey vultures? By determining genetic relatedness, foraging and roosting behaviors of vultures may be better understood. Also as a result of this research, a system of determining genetic relationship will be developed ultimately allowing evolutionary behaviors of vulture populations including altruism and/or group selection to possibly be uncovered. The purpose of this research was to obtain sequence information in order to design species-specific primers for future comparisons of minisatellite variation among and between populations of turkey vultures. Two different methods for DNA isolation from blood were compared for their ability to produce high quantities of amplifiable DNA. The Rapid Method (Lahiri et al., 1993) yielded 5.6 ug of DNA from 500 ul ofblood with a purity ratio [A260/A2S0] of 0.926, while the protocol using IsocodeTM Stixyielded 4.3 ug DNA from 15 ul of blood and had a higher purity ratio of 1.365. Although both methods yielded amplifiable DNA, better amplification was attained using the IsocodeTM Stix, which was used for the rest of the project. The polymerase chain reaction, using RAPD (Random Amplified Polymorphic DNA) primers (Operon Technologies, Alameda, CA), was performed to obtain DNA regions containing minisatellites. Fragments generated by the OPB 08 primer hybridized to a pool of labeled minisatellite core sequences by Southern hybridization. This minisatellitecontaining fragment (800 bp) was excised from a gel and cloned into a plasmid vector (pCR®2.1-TOPO) producing a recombinant plasmid. The recombinant plasmids werereplicated in E. coli, plasmid DNA was isolated, and the cloned fragment was sequenced for determination of the flanking sequences around the minisatellite core. Multiple colonies (pTpvul 1-4) were picked from the cloning/transformation stages but only one brightly hybridizing colony was chosen for sequencing (pTpvul 1). Sequencing and sequencing analysis proved difficult and no minisatellite core sequences could be located. This could be attributed to extensive secondary structure in the DNA sequence or to recombination within the fragment when grown in E. coli. These flanking sequences, thought to be identical at each locus of the minisatellite in a genome, were to be used as species-specific primers in future minisatelhte-PCR DNA fingerprinting.
Department of Biology

“Each year at the season of the falling of the waters, the people living in the vicinity of the Golden Basin, the home of the Pla Buk [Mekong giant catfish, Pangasianodon gigas], join together for the purpose of catching these fish…..The ceremonies connected with the taking of these fish are ancient and have been performed from time immemorial, and carried out once a year.” - F.H. Giles, 1935 This thesis was undertaken to test the potential of the relatively new survey technique of analyzing water samples which contain DNA that has been shed by organisms (environmental DNA) for the study of diversity and distributions of fishes inhabiting tropical and subtropical rivers. The purpose was to gain information on species richness, assemblages, and distributions; with the ultimate aim of improving conservation management of rare and endangered freshwater fishes. The limitations of conventional (observation-based) survey methods to study rare and threatened fishes in large tropical rivers motivated the choice of testing environmental DNA (eDNA) analysis approaches. Specifically, challenges encountered during prior work to conserve the Mekong giant catfish and other rare species fostered the goal of testing eDNA analysis methods. The Mekong giant catfish was once part of a fishery with substantial cultural and socioeconomic import, as described by Giles (1935). However; today the fishery and ceremony surrounding it no longer exists, as the species has become so rare that the capture of wild individuals is banned and it has been assessed as Critically Endangered by the IUCN Red List. The case of the Mekong River and giant catfish represents an archetypical example of the challenges and limitations of using conventional methods to study and conserve rare fishes in large tropical rivers. These rivers host the highest species diversity of any freshwater environment, yet are facing the highest rates of extinction. This dire situation is partially due to the difficulty of gaining access to data on spatiotemporal distributions that are needed to inform effective conservation actions. The relatively new technique of using eDNA to access data on species richness, assemblages and distributions may enable access to data from tropical rivers that are not accessible with conventional methods. This thesis compares the benefits and limitations of conventional survey methods with eDNA techniques, within the context of rare species in large rivers. It reviews the development and application of eDNA to study aquatic macroorganisms, specifically fishes. It describes the rapid growth in application of the technique through an analysis of published literature, and finds the majority of studies are focused on developing the methodology. This technique was tested on captive populations of Mekong giant catfish (Pangasianodon gigas) in Thailand, and then used to detect wild giant catfish in the Mekong River at locations where it has been captured in the past. The giant catfish was detected in one of six survey locations, and only one sample from the river. Given the developmental stage of the eDNA technique, the method was further tested in two smaller subtropical rivers with well-known fish species composition. Three different clade-specific (also termed ‘universal’ or ‘generic’) fish primers were used to see if they returned congruent results. Clade-specific primers are designed to amplify all species within a given group, such as bony fishes, without amplifying species outside that group, such as other vertebrates. It was found that each primer set had different biases and that eDNA samples did not cluster either by primer set or by location. A subsequent study analyzed whether such variance was related to the choice of sampling location within the river. Given the highly dynamic and heterogeneous nature of rivers, it is reasonable to assume that eDNA may not be equally distributed along a cross-section within a single hydraulic unit. High variation was found among samples collected at three different points along a cross-section in a pool and riffle in the Brisbane River, Queensland, Australia when using universal primers and eDNA metabarcoding to determine taxa assemblage. Finally, the method was tested on sediment cores collected in a subtropical embayment at the outflow of the Brisbane River to determine whether a chronology of catchment-scale assemblage of freshwater fish communities could be identified. The results were inconclusive, as primarily bacterial sequences were identified from high-throughput sequencing. These results could indicate that eDNA of macroorganisms is not preserved well enough for detection in subtropical sediment cores. This is consistent with the endosymbiont theory that states that mitochondria are descendent from bacteria that have been assimilated into another cell ( Sagan, 1967). This has implications for the design of eDNA experiments that target extremely low copy number template, because eDNA studies often target mitochondria rather than nuclear DNA. The conservation implications of the overall findings of this thesis highlight the fact that all survey methods face limitations when the target organisms are rare aquatic species. Conservation practitioners and resource managers must be creative and forward-thinking in order to gain data on the distribution of threatened species that are needed to inform effective conservation actions. The lack of such data is a contributing factor to the high rates of decline faced by freshwater biodiversity around the globe. The potential of eDNA tools as described in the literature’s early stages may have been somewhat overstated, given that the limitations of the method hadn’t yet been exhaustively tested. There is still a great deal to learn about how useful this method can be. The tool seems to work best when using species-specific primers, which amplify a single species, rather than clade-specific primers which can assess total species richness or assemblage through metabarcoding. The method needs further refinement, yet is currently most useful when applied in tandem with conventional sampling methods to survey areas that are difficult to access or for the case of large tropical rivers that have high spatial extent and little knowledge currently available regarding distributions of threatened species.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
Full Text

Albert Einstein published the English translation of Relativity: The Special and General Theory in the midst of two big events in 1920: the confirmation of the two theories of relativity and spacetime in 1919 and the Nobel prize in physics in 1921. The new global celebrity wanted to make the theories intelligible and readable for an international English-speaking audience, an audience that also included antagonistic scientists and even anti-Semites. The aim of this thesis is to do a rhetorical analysis of Einstein’s character, his ethos, in Relativity, with a specific focus on creation of credibility in regard to his historical context: scientific ideals, values and norms as well as the political and cultural tendencies in Europe during the early 20th century. This was done firstly by identifying the implied auditor. Secondly, based on the material, I have identified three stereotypes or characters – the professional idealist, the mentor and the internationalist –  which emphases different features and capacities that are crucial for the credibility of the text. Thirdly, by using these stereotypes and in regard to the specific historical context, I investigated how Einstein developed his primary ethos into a secondary ethos in the text. The rhetorical analysis of Einstein’s Relativity shows that his ethos stands in relation to the social and cultural perception of the virtuous epistemic scientist; to fight prejudices regarding being a Jewish-German theoretical physicist; and, noteworthy, a way to produce a well-needed international space – a crucial alternative to continue the positivistic knowledge production counter to the nationalistic project.

Two species of Cryptosporidium are routinely found in pigs: Cryptosporidium suis and Cryptosporidium pig genotype II. Identification of Cryptosporidium species and genotypes currently relies on molecular methods such as polymerase chain reaction (PCR) followed by restriction fragment lenght polymorphism (RFLP) or gene sequencing. However their applications are limited in identification of mixed infections. To overcome this problem, novel species specific primers were developed in this study. A total of 457 pig fecal samples were collected and examined using microscopy and molecular tools including PCR-RFLP, species or genus specific nested PCR and sequencing. Of these, 12.8 % were microscopicaly positive for oocysts presence and 36.5 % using molecular methods. While PCR-RFLP with genus specific primers revealed 1 case of C. suis and Cryptosporidium pig genotype II mixed infection only, nested PCR with species specific primers identified 41 cases of mixed infections. Our results showed that C. suis is infectious for all age categories of pigs and Cryptosporidium pig genotype II has been found in animals older than 6 weeks of age. Morphometric analysis proved oocyst size difference between both pig specific Cryptosporidium spp. Histological examination revealed that Cryptosporidium pig genotype II infects epithelia of both small and large intestine.

Bibliography: leaves 138-160. The 152 species of mycorrhizal fungi can be difficult to identify and quantify because the taxonomy of these fungi is based on the description of spores, which is time consuming, requires considerable expertise and cannot be assumed to reflect the situation within the root. Few attempts have been made to identify the species which are present in roots. Several approaches have been identified in previous work and the development of sensitive molecular methods for identification and quantification of two species of arbuscular mycorrhizal (AM) fungi are described in this study. Mycorrhizal fungal communities were sampled in both natural and agricultural ecosystems at two sites in South Australia. The combination of spore identification from trap culture and field-collected soil promises to be an effective means to study diversity of AM fungi in a particular system. PCR primers for Glomus mosseae and Gigaspora margarita were designed from the internal transcribed spacer (ITS) sequences of field-collected spores, with the aim of providing tools for field diagnosis.

碩士
國立中興大學
動物科學系所
101
Approximately 60% of avian species are sexually monomorphic, and most of the parrots could not be sexed by feather colors or any other morphological characteristics in appearances. It has been reported that specific DNA sequences could be amplified by using polymerase chain reaction (PCR) with the specific primers of chromodomain helicase DNA binding protein 1 (CHD-1) gene. In birds, there are two genes related to CHD-1, CHD-W and CHD-Z, which are located in chromosome W and Z, respectively. Since these two genes are evolutionarily conserved, and their sequences vary sex specific manner, application of the differences between the nucleotide sequences could be used for sex identification. Multiple primers, including P1/P2/P3, P4/P5, P2/P8, 2550F/2718R, primer1/2 and 1272H/1237L have been designed according to the sequence of introns or exons in CHD-1 gene. By using different combinations of these specific primers for PCR, 19 species of examined parrots could be successfully determined in this study. Amplification and sequencing fragments of mitochondrial DNA (mtDNA) genes, such as cytochrome oxidese I (COI), cytochrome b (Cytb), 12S ribosomal RNA (12S) and 16S ribosomal RNA (16S) are widely used for species identification. In this study, Cytb and 12S were tried to identify different species of parrots. Comparisons of sequences were conducted by using the BLASTn portal within the National Centre for Biotechnology Information (NCBI) database. The threshold value for sequence similarities of Cytb or 12S rRNA genes between analyzed samples and database is 98%. Twenty-six species of parrots were analyzed in this study. Results showed that the sequences of Cytb gene in 19 species of examined parrots could be found in NCBI. Furthermore, 13 species of analyzed parrots have the high similarity in 12S gene published in database. However, 2 species were found neither the sequences of cyt b nor 12S rRNA published in NCBI. Hence, in addition by comparison of mtDNA to identify parrot species is an accurate method, uploading the unpublished sequences would help the integrity of database. In addition to use mitochondrial DNA for species identification, random amplified polymorphic DNA (RAPD) were used to identify 17 different parrot species. In this study, 40 random primers were employed. One of these primer, OPH-17, was successfully amplified a specific band in Orthopsittaca manilata. After analyzing 8 sequences amplified from the same individual, there are no significant correlations among these 8 sequences. Thus, it is not possible to find species specific sequence using the 40 random primers in this study. More stricted conditions way are further tried to inprove reproducibility.

Bibliography: leaves 138-160.
xii, 160 leaves : ill. ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
The 152 species of mycorrhizal fungi can be difficult to identify and quantify because the taxonomy of these fungi is based on the description of spores, which is time consuming, requires considerable expertise and cannot be assumed to reflect the situation within the root. Few attempts have been made to identify the species which are present in roots. Several approaches have been identified in previous work and the development of sensitive molecular methods for identification and quantification of two species of arbuscular mycorrhizal (AM) fungi are described in this study. Mycorrhizal fungal communities were sampled in both natural and agricultural ecosystems at two sites in South Australia. The combination of spore identification from trap culture and field-collected soil promises to be an effective means to study diversity of AM fungi in a particular system. PCR primers for Glomus mosseae and Gigaspora margarita were designed from the internal transcribed spacer (ITS) sequences of field-collected spores, with the aim of providing tools for field diagnosis.
Thesis (Ph.D.)--University of Adelaide, Dept. of Soil and Water, 2000?

Bibliography: leaves 138-160.
xii, 160 leaves : ill. ; 30 cm.
The 152 species of mycorrhizal fungi can be difficult to identify and quantify because the taxonomy of these fungi is based on the description of spores, which is time consuming, requires considerable expertise and cannot be assumed to reflect the situation within the root. Few attempts have been made to identify the species which are present in roots. Several approaches have been identified in previous work and the development of sensitive molecular methods for identification and quantification of two species of arbuscular mycorrhizal (AM) fungi are described in this study. Mycorrhizal fungal communities were sampled in both natural and agricultural ecosystems at two sites in South Australia. The combination of spore identification from trap culture and field-collected soil promises to be an effective means to study diversity of AM fungi in a particular system. PCR primers for Glomus mosseae and Gigaspora margarita were designed from the internal transcribed spacer (ITS) sequences of field-collected spores, with the aim of providing tools for field diagnosis.
Thesis (Ph.D.)--University of Adelaide, Dept. of Soil and Water, 2000?

碩士
國防醫學院
牙醫學系
84
Previous of studies indicated that oligonuclotide probes combined with polymerase chain reaction for identification of periodontal pathogens, Actinobacillus actinomycetemcomitans (A.a.), Prevotella intermedia (P.i.) and Porphyromonas gingivalis (P.g.) exhibited species-specificity and were more better than traditional culture methods; The sensitivity of polymerase chain reaction is nicer than oligonucleotide probe. However, all of these oligonucleotide probes had been published with patent and thus can not conveniently used for identification of these pathogens clinically without authorization. The purpose of this study was to develop species-specific primer pairs for the detection of periodontal pathogen, B.f. The analysis of 16S rDNA under Pc-Gene software were performed and produced the Dendrogram of Phylogen Tree. The hypervariable region of B.f. 16S rDNA were screened by Oligo-software with high G+C content (50%-60%), Tm (60℃), lack of dimer and secondary structure fragments, and 20-22 bps oligonucleotide pairs were selected as primer, Then these sequence were used to compare the Pc-Gene genBank published Procaryotic sequence whether it is complementary with others or not. At first, this study indicated that the reference strain of B.f. is gram negative, strictly anaerobic and rod shaped as well as nonmotile under darkfield microscopy. Ultrastructural studies of B.f. confirmed that the cells revealed a well-defined inner membrane, outer membrane and no distinct peptidoglycan layer between both membranes with external layer. Primer pairs, consisting of 22 bps amplified to 527-bp regions of 16S ribosomal DNA genes of this organism. The primers for B.f. did not show to cross-react with other known oral organisms. It implied that the specific 16S rDNA sequence (B.f. paired primers) can be powerful and useful aid in the detection of the periodontal pathogens. Clinically, subgingival specimens were obtained by Gracey's curette from tooth sites with deep periodontal pockets (> 5mm) and bleeding upon probing in 9 patients. Under one-tube method , PCR reaction was performed in a PTC-100 thermaller, included an initial denaturation at 93℃ for 3min, followed by 30 cycles of a denaturation step at 93℃ for 30", a primer annealing step at 60℃ for 90", a primer extension step at 72℃ for 90" and a final step 72℃ for lOmin. A species-specific 0.5 kb PCR product for B.f. were detected in 1.2% agarose gel (containing ethidium bromide) with UV-light. ThThis PCR product did not show cross reaction with other species (Aa, P.g., P.i.). The B.f. (ATCC43037) 0.5kb PCR product were cloned to TA vector. The minus strain sequence was confirmed via T7 primer by Ganger's method. Direct sequence analysis was performed with the Applied Biosystems of Dyedeoxy(TM) terminator Cycle Sequencing Kit. Pre-reduced medium and ddH2O served as negative control, A species specific cut-sited was proved with double check under specific enzyme. It was concluded that the SS-PCR with one tube would permit the accurate detection of some periodontal pathogens in subgingival specimens qualitatively and quantitatively.

碩士
國立嘉義大學
動物科學系研究所
106
Most birds can’t be identified sex by appearance, and sex must be correctly identified by other methods for mating, especially on endangered birds. Nowadays, molecular technique is a common way for sex identification in commercial, and Chelex 100 is a wide range of methods for DNA extraction, the extracted DNA can be used effectively for a variety of PCR experiments. This experiment intends to explore the optimum treatment conditions for the heating temperature and time of using 5 % Chelex 100 to extract feathers DNA and use different PCR specific primers to distinguish parrot species. The first experiment explored the optimal treatment conditions for the heating temperature and time of feather DNA extracted with 5 % Chelex 100. The experiment was divided into three parts. The first part was heated at 58℃for 4 hours, heated at 60℃for 2 hours and heated at 62℃for 1 hour to investigate the optimal temperature conditions for extracting feather DNA. The results showed that the extraction time could be shortened when the temperature increased to 62℃ to 1 hour. The second part is the use of 62℃ heating 1 to 6 hours to explore the extraction of feather DNA optimal heating time, the results show that the longer the heating time, DNA extraction concentration and purity increased significantly. The third part is to simulate the hot summer conditions. The fresh Columba livia feathers were stored at 37℃ for 30 days. DNA was extracted after 3 days and stored at 37℃ to investigate the extraction concentration and purity of feather DNA. The second experiment explored the use of different sex-specific primers to distinguish parrot species. The avians were identified using nine primer sets (P1, P2, P4, P5, P9, P11, P23, P25 and P28). The results showed that P1 primer can be distinguished by PCR amplification Myiopsitta monachus (male and female common band 700 bp), Pionus (male and female common band 680 bp), Megalaima nuchalis and Forpus coelestis (male and female common band 1100 bp), Cygnus atratus (female specific band 590 bp), Polytelis swainsonii and Polytelis alexandrae (female specific band 320 bp), Pyrrhura perlata (male and female common band 650 bp and 550 bp), Rhyacornis fuliginosa (female specific band 450 bp), Columba livia(male and female common band 680 bp,650 bp and female specific band 450 bp),Polyplectron napoleonis and Pavo cristatus (male and female common band 590 bp and female specific band 450 bp), 18s ribosomal gene primer was added during PCR, whereas 18s ribosomal gene primer was added as control in Psittacula krameri, Psittacula eupatria, Psittacula cyanocephala, Psittacula alexandri, Eclectus roratus, Tanygnathus megalorynchos and Neophema splendida (female specific band 430 bp). The P2 primer pair distinguishes Gracula religiosa (male and female common band 350 bp and female specific band 380 bp). P5 primer pairs distinguish Myiopsitta monachus (female specific band slightly lower). P4 primer pair according to the ring position can be resolved Psittacula krameri, Psittacula eupatria (female specific band 520 bp, 690 bp), Psittacula cyanocephala (female specific band 520 bp), Psittacula alexandri (female specific band 490 bp, 520 bp and 590 bp). The P9 primer pair distinguishes the difference between the bands of Agapornis roseicollis, Agapornis personatus (male and female common band 250 bp and female specific band 420 bp) and Cygnus atratus (female specific band 810 bp). The P23 primer pair is a Cygnus atratus specific fragment (male and female common band 600 bp, 350 bp and female specific band 810 bp). The P11 primer pair is a specific fragment of Columba livia (male and female common band 250 bp and female specific band 730 bp). The P25 and P28 primer pairs were Passeridae specific fragments. The P25 primer pair identified Passeridae (male and female common band 300 bp and female specific band330 bp), as well as Zoothera citrina and Rhyacornis fuliginosa (two bands slightly higher). In summary, the optimal conditions for feather DNA extraction with 5 % Chelex 100 were 62℃for 1 hour. When the heating time is longer, DNA extraction concentration and purity increased significantly. Feather was stored at 37℃ for 15 days and the DNA extraction concentration was significantly reduced. By the 30th day, enough DNA was not extracted for PCR amplification. 9 sex identification primer, the P1 primer pair has the wide range of distinguishable birds. P2 primers differ only for Gracula religiosa. The P5, P4, P9, P11, P23, P25 and P28 primer pairs are specific fragments. Different species of birds can be identified outside the band location, which can distinguish between bird species.

The diploma thesis is focused on providing support to pupils with specific behavioral disorders at ordinary primary schools. Its aim is to find out how support is provided to pupils with specific behavioral disorders at ordinary elementary schools, what are the potentialities for providing optimal support to these pupils and suggest possible measures and procedures to improve them. The thesis consists of theoretical and research part. The theoretical part characterizes specific behavioral disorders, education and support for pupils with these disorders. The research part of the thesis focuses on the implementation of support measures for pupils with specific behavioral disorders at selected elementary schools. This problem is solved in two levels. The first plane is qualitative, it is a case study of the school (author's workplace). The basis for the preparation of case report are interviews with school special pedagogues and unstructured observations. The second part is aimed at supporting pupils with specific behavioral disorders on a wider scale. The basis for the data is the questionnaire survey, which was attended by teachers and teacher assistants from other schools. According to the results of the research, the pedagogical staff focuses mainly on the attentiveness and behavior of pupils with...

The thesis is aimed to development of communication of students at special primary school. Special primary school is creating unique conditions for education of students with lower intellectual competences. The aim of education is equip students by knowledges and skills for integration into the society. The theoretical part of thesis consists of four chapters. The first of them specifies the term mental disability, classifies type of mental retardation, characterizes specific characteristics of person with mental disability and presents possibilities primary education mental disabilited students. The second and the third chapter compare psycho-motion development and ontogenezis of speech to mental handicapped children with children without disability. The fourth chapter characterizes supporting communication methods used in educational process of students with mental disability. The fifth chapter is applied to study. It characterizes chosen school institution and special pedagogical attitude to students and application augmentative and alternative aids. At the end of chapter are summarized knowledges from the study. The main goal of the study is presentation of methods using for development of communication of mental disabilited students and characterization supporting communication methods which...

Title: The learning aids for educational therapy of dysortographia of primary school pupils pupils Thesis "The learning aids for educational therapy of dysortographia of primary school pupils" aims at assembling a list of method of reducing dysortographia - a specific grammar disability - and assessing their effectiveness for treating this disability. The theoretical part of the thesis covers terminology, symptoms and ethiology of specific learning disabilities. Next, the topic of specific learning disabilities is discussed in the context of primary school (ages 7 - 12). Diagnostics, methods of educational therapy and learning aids are described in relation to dysortographia. The reserch part of the thesis describes the methods used in special pedagogical intervention and, based on analysis of input and output diagnostics, assesses the effect of used methods on management of dysortographia in a chosen sample of pupils, compared to the control group which hasn't been treated with these methods. Key words: Powered by TCPDF (www.tcpdf.org)