Made available in DSpace on 2014-06-12T15:04:52Z (GMT). No. of bitstreams: 2 arquivo4510_1.pdf: 1312401 bytes, checksum: 103b70bf03d8851fef2766e8bd0290f9 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2004 / Paracoccidioides brasiliensis é um fungo dimórfico e agente etiológico da paracoccidioidomicose, uma micose sistêmica de evolução aguda ou crônica que se não diagnosticada e tratada a tempo pode ser fatal. Um método molecular para caracterização e detecção de P. brasiliensis foi desenvolvido a partir da clonagem e do sequenciamento de um fragmento de DNA de ~750 pb, obtido por RAPD (Random Amplified Polymorphic DNA), presente em isolados virulentos e ausente em isolados avirulentos deste fungo. Uma região interna do fragmento de DNA seqüenciado foi usada para desenhar primers que posteriormente foram utilizados em uma reação de hemi-nested PCR em tubo único. A reação de PCR específica foi capaz de amplificar DNA de três isolados de P. brasiliensis reconhecidamente virulentos e três isolados recentemente obtidos de pacientes com paracoccidioidomicose. A especificidade desta PCR foi confirmada pela ausência de produtos amplificados com DNA genômico de isolados de Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Sporothrix schenckii, Cryptococcus neoformans, Candida albicans, Aspergillus fumigatus, Mycobacterium tuberculosis, Leishmania braziliensis, Trypanosoma cruzi, Schistosoma mansoni, DNA genômico humano (leucócitos) e de isolados de P. brasiliensis reconhecidamente avirulentas. A amplificação de cDNA de um isolado virulento sugere tratar-se de um gene expresso. A detecção específica de isolados virulentos de P. brasiliensis sugere ser este um candidato a marcador de virulência para este fungo. O potencial diagnóstico da PCR específica foi verificado com DNA extraído de aspirado de linfonodo de um paciente com paracoccidioidomicose

The three South African crane species, namely, the Wattled Crane (Bugeranus carunculatus), the Blue Crane (Anthropoides paradisea) and the Grey Crowned Crane (Balearica regulorum regulorum) are all threatened. South African legislation protects the cranes, however eggs and/or fledglings are sometimes illegally collected from the wild. These are then sold, often by registered breeders, who falsely claim them as the offspring of their captive breeding pair. DNA fingerprinting is one method to detect this crime. Fifteen RAPD primers were screened for polymorphism in the three species. Seven primers produced polymorphic profiles in the Blue Crane and eight each in the Grey Crowned Crane and Wattled Crane, with an average of 14.57, 12.38 and 5.88 scorable loci per primer, respectively. The Band Sharing Coefficient for unrelated individuals was found to be 0.665, 0.745 and 0.736 for the Blue, Grey Crowned and Wattled Crane respectively. Five microsatellite primers, originally developed for use in Whooping Cranes (Grus american), had previously been shown to be polymorphic in the Wattled Crane. This was also the case in this study with an average of 3.6 alleles per primer. Although all primers cross amplified, only a single primer each showed polymorphism in the Blue Crane (showing 6 alleles) and the Grey Crowned Crane (showing 5 alleles). The RAPDs were found to be irreproducible, show high numbers of novel bands and had parent: offspring BSC values that were not significantly higher than those of unrelated individuals. Statistics showed that, in the Blue Crane, the probability that misassigned parents would be detected was low whilst there was an almost certainty that true parents would be incorrectly excluded. The five microsatellite primers examined gave exclusionary powers of 0.869 and 0.641 where one or two parents were unknown in the Wattled Crane. The exclusionary powers for the Blue Crane and Grey Crowned Crane calculated at only one locus were much lower. It was concluded that RAPDs were totally inappropriate for parentage analyses, however, microsatellites are a suitable technique and recommendations are made that other microsatellites, developed for other species of crane, should be examined for their potential in this respect. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

Maize (Zea mays L.) is an important agronomic crop with the maize industry forming an important component of the South African economy. Considerable effort has been directed towards the genetic improvement of maize through both conventional breeding and biotechnology. Genotype identification by DNA fingerprinting is becoming an important activity in plant breeding. A widely used molecular based and relatively inexpensive method for DNA fingerprinting is the randomly amplified polymorphic DNA (RAPD) technique. The RAPD technique was tested in this study for its potential use in maize breeding programmes. Initial results using the technique showed a low degree of reproducibility, therefore both the DNA isolation and RAPD protocols were extensively optimised. DNA quality and quantity, and choice of Taq polymerase buffer were three of the variables found to be influential in ensuring reproducibility. The ability of the RAPD technique to characterise seven maize genotypes was evaluated. Sixty random oligonucleotide primers were screened. Forty two primers scored a total of 233 fragments (an average of 5.5 per primer), but not all primers gave reproducible profiles. Eighteen primers scored a total of 110 loci for the presence (1) and absence (0) of DNA fragments. RAPD markers were able to distinguish between all seven genotypes with five primers producing specific fragments for four genotypes. Genetic similarity matrices were calculated using two software programmes i.e. Genstat 5™ release 4.1 (1993) and PAUP (Phylogenetic Analysis Using Parsimony) 4.0 beta version (Swafford, 1998). Cluster analysis was used to generate dendrograms to visualise the genetic relationships of the seven maize genotypes (only minor differences were observed between the Genstat or PAUP method of analysis). Genetic diversity ranged from 0.62 to 0.96. The estimation of genetic relationship was in accordance with the presumed pedigree of the genotypes showing that the RAPD technique demonstrates potential for genome analysis of maize. The applicability of the technique for marker assisted selection was also evaluated. Near-isogenic lines (NILs) for leaf blight (Helminthosporium spp.) were screened for polymorphisms using a total of 120 primers. Ten primers identified polymorphisms between the NILs. Four primers produced five polymorphic fragments present in the resistant inbred K0315Y and absent in the susceptible inbred D0940Y. A small F2 population of 14 individuals was produced by selfing the F1 of a cross between K0315Y and D0940Y. To speed up the generation time, the F1 and F2 plants were cultured by embryo rescue from 18d old harvested seed. One fragment of 627 base pairs produced by primer OPB-01 (5' GTTTCGCTCC 3') showed a 3: 1 segregation in the small F2 population and was considered putatively linked to the HtN gene for leaf blight resistance. This study shows that the RAPD technique does have application in maize breeding programmes. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

A instabilidade genômica tem sido amplamente usada para caracterizar células cancerosas. Alterações genéticas em queratose actínica (QA) e carcinoma espinocelular (CEC) foram investigadas pelo método de random amplified polymorphic DNA (RAPD) e análise de microssatélites com o objetivo de encontrar marcadores moleculares para auxiliar o prognóstico e o diagnóstico médico. O DNA foi obtido de pacientes brasileiros cirurgiados e tratados no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, totalizando oito QAs, 24 CECs, e tecidos normais e/ou leucócitos correspondentes. Os microssatélites estudados foram D6S251, D6S252, D9S15, D9S50, D9S52, D9S180, D9S196, D9S280 e D9S287, tendo em vista a detecção de instabilidade genômica representada por perda de heterozigosidade (LOH) e instabilidade de microssatélites (MSI). Os \"primers\" usados para comparar os padrões de RAPD foram OPA-2, OPA-7, OPA-13, OPA-17, OPB-8, OPB-13, OPB-17 e OPB-19. Foi obtida correlação significativa na progressão de QA (1/8) para CEC (5/22) referente ao microssatélite D6S251. As diferenças nos padrões de DNA obtidos pelo método RAPD comparados aos controles foram maiores em lesões com maior grau de severidade segundo critério histológico. O mesmo padrão RAPD foi observado no controle e no tumor em 27% QA, 24% CEC I, 9% CEC II e 0% CEC III. Estes resultados mostram que o microssatélite D6S251 e o método de RAPD são informativos, podendo ser potenciais candidatos para auxílio no diagnóstico e prognóstico de QA e CEC. / Genomic instability has been widely used to characterize cancer cells. Genetic alterations in human actinic keratosis (AK) and squamous cell carcinomas (SCC) were investigated by the random amplified polymorphic DNA (RAPD) method, and microsatellite analysis. DNA was obtained from Brazilian patients diagnosed and treated in the School of Medicine of University of Sao Paulo out Clinics Hospital. Eight AKs, 24 SCCs, and 4 BCCs, matched to normal skin tissue and/or leukocytes were studied. Microsatellite patterns were obtained with primers specific to amplify D6S251, D6S252, D9S15, D9S50, D9S52, D9S180, D9S196, D9S280, and D9S287, in search of detection Loss of heterozygosity (LOH) and Microsatellite instability (MSI). The RAPD primers were: OPA-2, OPA-7, OPA-13, OPA-17, OPB-8, OPB-13, OPB-17, and OPB-19. A significant correlation was obtained regarding the progress of AK (1/8) to SCC (5/22) detected with the D6S251 microsatellite. DNA fingerprint obtained with RAPD primers were altered in increasing number of samples, according to their histological degree of differentiation. Similar RAPD patterns were observed in tumor and control in 27% AK, 24% SCC I, 9% SCC II, and zero SCC III. These results suggest microsatellite D6S251 and RAPD method to be potential tools in diagnosis and prognosis of AK and SCC.

The characterization of the banana germplasm collection from Rubona - Rwanda was investigated using morphological and cytological characteristics of the genomic groups. Genetic diversity was assessed using Random Amplified Polymorphic DNA analysis. The survey was conducted to evaluate the distribution of banana cultivars in the four major growing regions of Rwanda. A total of 90 accessions from the National Banana Germplasm Collection at Rubona Rwanda were characterized and six characters of the fingers (length, width, weight, green life, post green life and length/width ratio) were subjected to principal component analysis (PCA). The cooking and beer clones were separated. The cooking clones were further grouped into three clone sets: Musakala, Nakabululu, and one that constitutes Nakitembe and Nfuuka clone sets. The AAB genomic group was separated from AAA, AB and ABB genomic groups. The results from the survey showed that East African Highland bananas are the most important genotype group in the four major banana growing regions of Rwanda ranging between 60 - 90% of banana mats counted. Several new Highland banana cultivars were recorded, such as 'Intokatoke', 'Igihuna', 'Ingenge', 'Ingaju', 'Icyerwa', 'Mitoki', 'Madamu', 'Inkokobora', 'Intokekazi', 'Bugoyi', 'Ishoki'. Amongst these cultivars, some were classified as cooking and others as brewing bananas. However, in the National Banana Germplasm Collection at Rubona - Rwanda, the uses of these cultivars are recorded differently therefore increasing the need for agro-morphological characterization. The assessment of ploidy level of accessions from the National Banana Germplasm Collection at Rubona - Rwanda, by flow cytometry showed misclassification of some accessions such as 'Pomme', 'Kamaramasenge', 'Gisubi kayinja', 'Gisubi kagongo', and 'Dibis' which were classified as diploid, diploid, triploid, and tetraploid respectively. They IV were found to be triploid, triploid, triploid, diploid and triploid. All these bananas were recently introduced into Rwanda, while the endemic Highland bananas were triploid. The genomic group and genetic similarities of 49 accessions were investigated using Random Amplified Polymorphic DNA markers. The genomic group of bananas assessed were established using OPA-18 (PILLAY et al., 2000) and OPG-17 primers. These primers showed bands 441 and 443 base pairs (bp) respectively for the accessions having only the B genome. Whilst they were absent for the accessions " having an A genome. The genetic similarity was estimated via a Simple Matching coefficient which showed the lowest value 0.46 measured between 'Ingumba' and 'Ishika 'and the highest value of 0.85 between 'Kirayenda' and 'Inyabukuwe'. The data of matrix of coefficient of similarity was subjected to cluster analysis with unweighted pair group method with arithmetic average (UPGMA). Each accession was clearly separated demonstrating the usefulness of RAPDs in analysis of genetic diversity. The results of this study are very important to the Curator of the banana germplasm collection in Eastern Central Africa and for the future breeding of this crop. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2006.

A instabilidade genômica tem sido amplamente usada para caracterizar células cancerosas. Alterações genéticas em queratose actínica (QA) e carcinoma espinocelular (CEC) foram investigadas pelo método de random amplified polymorphic DNA (RAPD) e análise de microssatélites com o objetivo de encontrar marcadores moleculares para auxiliar o prognóstico e o diagnóstico médico. O DNA foi obtido de pacientes brasileiros cirurgiados e tratados no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, totalizando oito QAs, 24 CECs, e tecidos normais e/ou leucócitos correspondentes. Os microssatélites estudados foram D6S251, D6S252, D9S15, D9S50, D9S52, D9S180, D9S196, D9S280 e D9S287, tendo em vista a detecção de instabilidade genômica representada por perda de heterozigosidade (LOH) e instabilidade de microssatélites (MSI). Os \"primers\" usados para comparar os padrões de RAPD foram OPA-2, OPA-7, OPA-13, OPA-17, OPB-8, OPB-13, OPB-17 e OPB-19. Foi obtida correlação significativa na progressão de QA (1/8) para CEC (5/22) referente ao microssatélite D6S251. As diferenças nos padrões de DNA obtidos pelo método RAPD comparados aos controles foram maiores em lesões com maior grau de severidade segundo critério histológico. O mesmo padrão RAPD foi observado no controle e no tumor em 27% QA, 24% CEC I, 9% CEC II e 0% CEC III. Estes resultados mostram que o microssatélite D6S251 e o método de RAPD são informativos, podendo ser potenciais candidatos para auxílio no diagnóstico e prognóstico de QA e CEC. / Genomic instability has been widely used to characterize cancer cells. Genetic alterations in human actinic keratosis (AK) and squamous cell carcinomas (SCC) were investigated by the random amplified polymorphic DNA (RAPD) method, and microsatellite analysis. DNA was obtained from Brazilian patients diagnosed and treated in the School of Medicine of University of Sao Paulo out Clinics Hospital. Eight AKs, 24 SCCs, and 4 BCCs, matched to normal skin tissue and/or leukocytes were studied. Microsatellite patterns were obtained with primers specific to amplify D6S251, D6S252, D9S15, D9S50, D9S52, D9S180, D9S196, D9S280, and D9S287, in search of detection Loss of heterozygosity (LOH) and Microsatellite instability (MSI). The RAPD primers were: OPA-2, OPA-7, OPA-13, OPA-17, OPB-8, OPB-13, OPB-17, and OPB-19. A significant correlation was obtained regarding the progress of AK (1/8) to SCC (5/22) detected with the D6S251 microsatellite. DNA fingerprint obtained with RAPD primers were altered in increasing number of samples, according to their histological degree of differentiation. Similar RAPD patterns were observed in tumor and control in 27% AK, 24% SCC I, 9% SCC II, and zero SCC III. These results suggest microsatellite D6S251 and RAPD method to be potential tools in diagnosis and prognosis of AK and SCC.

Die Frage nach adäquaten mikrobiologischen und molekulargenetischen Methoden, um die Kolonisation des Respirationstrakts von Mukoviszidose-Patienten mit Pseudomonas aeruginosa nachzuweisen und zu charakterisieren, wird kontrovers diskutiert. Von 38 klinisch stabilen Patienten mit cystischer Fibrose (CF) wurden sequentiell im Abstand von 18 Monaten Proben aus Rachenabstrich, Sputum und Bronchiallavage (BAL) entnommen und bezüglich Pseudomonas-Nachweis untersucht. Die Pseudomonas-Stämme wurden mittels Random Amplified Polymorphic DNA (RAPD)-Analyse und Pulsfeld-Gelelektrophorese (PFGE) von DNA-Makrorestriktionsfragmenten typisiert und bezüglich der Frage nach genetisch divergierenden Isolaten innerhalb des selben Individuums sowie nach möglichen longitudinalen genetischen Veränderungen evaluiert. Sensitivität, negative und positive prädiktive Werte und Spezifität, um eine P. aeruginosa-Besiedlung zu erkennen, waren 36%, 74%, 83% und 96% im Falle der Kulturen aus dem Oropharynx von nicht-expektorierenden Patienten und 92%, 94%, 100% und 100% für Sputumkulturen von expektorierenden Probanden. RAPD-Analyse und PFGE waren in der Lage, zwischen unterschiedlichen Pseudomonas-Stämmen zu diskriminieren, wobei nur die DNA-Makrorestriktion zwischen Subtypen unterscheiden konnte. Die Genotypen der Pseudomonas-Isolate aus Rachenabstrich und Sputum divergierten in 55% und 40% zu den Isolaten der BAL. Longitudinale Variationen des Genotyps wurden in 62% der Fälle beobachtet, die Hälfte davon war nur mittels bronchoskopisch gewonnener Proben erkennbar. Zusammengefasst besitzen Sputumproben bezüglich des Pseudomonas-Nachweises dieselbe Wertigkeit wie Kulturen aus der BAL, während Rachenabstriche in einer frühen Krankheitsphase für die Charakterisierung der bakteriellen Flora des unteren Respirationstrakts wenig geeignet sind. Die Methode der DNA-Makrorestriktion kann als zuverlässige Technik für epidemiologische Untersuchungen empfohlen werden. Unterschiedliche Genotypen innerhalb desselben Individuums und longitudinale genetische Alterationen sind häufig, jedoch unter Umständen nur bronchoskopisch nachweisbar. / There is controversy about adequate specimen to detect and characterise colonisation of cystic fibrosis (CF) airways by Pseudomonas aeruginosa. Oropharyngeal, sputum and bronchoalveolar lavage (BAL) samples were evaluated sequentially from 38 stable CF patients for the detection of P. aeruginosa. Pseudomonas strains were typed by random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. The occurrence of genetically different isolates within the same host and longitudinal variations in the genotype during repeated examinations was assessed. Sensitivity, negative and positive predictive values and specificity to detect P. aeruginosa were 36%, 74%, 83% and 96% for oropharyngeal cultures in non-expectorating patients and 92%, 94%, 100% and 100% for sputum cultures from expectorating patients, respectively. RAPD analysis and PFGE were suitable to characterize P. aeruginosa CF isolates, although only DNA macrorestriction was able to distinguish between identical and closely related strains. Genotypes of Pseudomonas isolates recovered from oropharyngeal swabs and sputum differed to the strains recovered by bronchoscopy in 55% and 40%, respectively. In 62% longitudinal variations in the genotype occurred. Half of these alterations were only detectable from bronchoscopically obtained samples. In conclusion, sputum samples have the same value as specimens from BAL to detect P. aeruginosa colonisation, whereas cultures from the oropharynx are not suitable for characterising the bacterial conditions in the CF lungs in an early disease state. DNA macrorestriction is recommended as an excellent tool for epidemiological investigations. Different genotypes within the same host and longitudinal genetic alterations are common and may be detectable in the BAL fluid exclusively.

Wong Ka Lok. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 121-129). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / Table A --- p.v / Table B --- p.vi / Table of Contents --- p.vii / Abbreviations --- p.xi / Chapter Chapter 1 --- Molecular authentication of endangered crocodiles and snakes / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Traditional method of snake and crocodile identification / Chapter 1.2.1 --- Morphology --- p.7 / Chapter 1.2.2 --- Chemical Analysis --- p.9 / Chapter 1.3 --- Molecular Technology in Authentication / Chapter 1.3.1 --- Polymerase Chain Reactions (PCRs) --- p.11 / Chapter 1.3.2 --- Random-primed amplification reaction --- p.12 / Chapter 1.3.3 --- Sequence Characterized Amplified Region (SCAR) --- p.13 / Chapter 1.3.4 --- PCR-RFLP --- p.13 / Chapter 1.3.5 --- DNA sequencing --- p.14 / Chapter 1.4 --- Objectives and strategies of the study --- p.15 / Chapter Chapter 2 --- Materials and General Methods / Chapter 2.1 --- Reagents and Buffers / Chapter 2.1.1 --- Buffers for Total DNA Extraction --- p.17 / Chapter 2.1.2 --- Reagents for Agarose Gel Electrophoresis --- p.17 / Chapter 2.1.3 --- Reagents for Plasmid DNA Preparation --- p.18 / Chapter 2.1.4 --- Medium for Bacterial Culture --- p.18 / Chapter 2.1.5 --- Reagents for Preparation of Competent Cells --- p.19 / Chapter 2.2 --- DNA Isolation / Chapter 2.2.1 --- Extraction of DNA from meats --- p.20 / Chapter 2.2.2 --- Extraction of DNA from blood --- p.20 / Chapter 2.3 --- Phenol/Chloroform Extraction --- p.21 / Chapter 2.4 --- Ethanol Precipitation --- p.22 / Chapter 2.5 --- DNA Concentration/Purity Estimation --- p.22 / Chapter 2.6 --- Mitochondrial DNA amplification --- p.23 / Chapter 2.7 --- Random-Primed Polymerase Chain Reactions --- p.24 / Chapter 2.8 --- SCAR for Snake samples --- p.24 / Chapter 2.9 --- SCAR for Crocodile samples --- p.25 / Chapter 2.10 --- Restriction fragment length polymorphism analysis --- p.25 / Chapter 2.11 --- Agarose Gel Electrophoresis of DNA --- p.26 / Chapter 2.12 --- Purification of PCR product --- p.26 / Chapter 2.13 --- Preparation of Escherichia coli Competent Cells --- p.27 / Chapter 2.14 --- Ligation and transformation of E. coli --- p.27 / Chapter 2.15 --- Plasmid preparation --- p.28 / Chapter 2.16 --- Screening of Plasmid DNA by Restriction Digestion --- p.29 / Chapter Chapter 3 --- DNA sequencing of snakes & construction of snake database / Chapter 3.1 --- Introduction --- p.30 / Chapter 3.2 --- Materials and methods / Chapter 3.2.1 --- Snake samples --- p.32 / Chapter 3.2.2 --- "DNA Extraction, mitochondrial gene amplification and DNA sequencing" --- p.33 / Chapter 3.2.3 --- Construction of database --- p.33 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Cytochrome b gene amplification and sequencing --- p.34 / Chapter 3.3.2 --- Gene amplification and sequencing of 16S rRNA --- p.42 / Chapter 3.3.3 --- Cytochrome b sequence database --- p.50 / Chapter 3.3.4 --- 16S rRNA sequence database --- p.53 / Chapter 3.4 --- Discussion / Chapter 3.4.1 --- Cytochrome b and 16S rRNA genes of snake species --- p.55 / Chapter 3.4.2 --- Cytochrome b and 16S rRNA databases --- p.55 / Chapter Chapter 4 --- Application of PCR-RFLP and SCAR in snake species identification / Chapter 4.1 --- Introduction --- p.57 / Chapter 4.2 --- Material and Methods / Chapter 4.2.1 --- DNA extraction and PCR-RFLP --- p.58 / Chapter 4.2.2 --- RAPD and SCAR --- p.58 / Chapter 4.3 --- Results / Chapter 4.3.1 --- PCR-RFLP of cytochrome b genes of snakes --- p.59 / Chapter 4.3.2 --- PCR-RFLP of 16S rDNA --- p.61 / Chapter 4.3.3 --- RAPD & SCAR analysis --- p.67 / Chapter 4.4 --- Discussion --- p.72 / Chapter Chapter 5 --- "Application of DNA sequencing, PCR-RFLP and SCAR to identify crocodile species" / Chapter 5.1 --- Introduction --- p.74 / Chapter 5.2 --- Materials and methods / Chapter 5.2.1 --- "Crocodile, human and four animal samples" --- p.75 / Chapter 5.2.2 --- "DNA Extraction, mitochondrial gene amplification and DNA sequencing" --- p.75 / Chapter 5.2.3 --- PCR-RFLP and SCAR --- p.76 / Chapter 5.3 --- Results / Chapter 5.3.1 --- Isolation of crocodiles DNA --- p.77 / Chapter 5.3.2 --- Isolation of DNA from Human and four animal species --- p.78 / Chapter 5.3.3 --- Cytochrome b gene amplification and sequencing --- p.78 / Chapter 5.3.4 --- 16S rRNA gene amplification and sequencing --- p.84 / Chapter 5.3.5 --- PCR-RFLP of cytochrome b --- p.89 / Chapter 5.3.6 --- PCR-RFLP of 16S rRNA --- p.91 / Chapter 5.3.7 --- SCAR primers for four crocodile species --- p.93 / Chapter 5.4 --- Discussion --- p.97 / Chapter Chapter 6 --- A case report - authentication of animal samples using DNA sequencing / Chapter 6.1 --- Introduction --- p.99 / Chapter 6.2 --- Material and methods / Chapter 6.2.1 --- Materials --- p.101 / Chapter 6.2.2 --- DNA Extraction and sequencing --- p.101 / Chapter 6.3 --- Result and discussion / Chapter 6.3.1 --- Cytochrome b gene sequencing --- p.102 / Chapter 6.3.2 --- Sequence homology among samples and meats obtained from the market --- p.111 / Chapter 6.3.3 --- Identity of samples B & D --- p.113 / Chapter Chapter 7 --- General Discussion / Chapter 7.1 --- Advantages and weakness of DNA technology --- p.116 / Chapter 7.2 --- Choosing appropriate molecular markers --- p.118 / Chapter 7.3 --- Further suggested work --- p.119 / Chapter 7.4 --- Conclusion --- p.119 / References --- p.121 / Appendix --- p.130

Black wattle (Acacia mearnsii), which belongs to the genus Acacia, is one of the many species of trees or hardwoods grown commercially in South Africa. Black wattle is a species indigenous to Australia and was introduced into South Africa by the van der Plank brothers in 1864. These trees are grown in South Africa because of its tannin-rich bark, the extract of which is used by the leather tanning industry. Black wattle is also grown for its timber, timber products and pulp. The introduction and cultivation history of black wattle suggests that the South African plantations contain limited genetic variation with relatedness amongst groups estimated to be high, thus implying a narrow genetic base in the South African black wattle population. In this investigation, Random Amplified Polymorphic DNA (RAPD) was used to estimate the genetic variation between seven different black wattle groups. A total number of 34 individuals obtained from different areas in South Africa were examined; Piet Retief (group 47 and 50: half-sibs), Kumbula (group 85: unrelated individuals), Howick (group 400: unrelated individuals) and an unknown area (groups 88, 89, 91: full-sibs). As this investigation was the first of its kind, a DNA isolation method as well as a PCR-RAPD protocol had to be modified. Total genomic DNA was successfully extracted using the CTAB DNA extraction method. This method removed large amounts of tannin present in the cells of the black wattle leaves and extracted high quality DNA to conduct between 50-100 RAPD reactions. The DNA purities ranged from 0.1 to 1.8, with an average of 1.46. A total of fourteen 10-mer RAPD primer sequences were randomly selected from the Operon Technologies primer list A, and tested in this investigation. Of the 14 primers used, only nine primers produced clear, single and repeatable bands. Therefore nine primers were selected for subsequent analyses. Ninety one loci that generated bands ranging from 300-3050 base pairs were produced. Seven to 13 loci per primer were generated. A total of 95.6 % of the loci were polymorphic. The overall expected mean heterozygosity (H = 0.3) obtained in this study was high in comparison to other studies conducted on acacias. The high levels of genetic variation were attributed to mating systems, dissortative mating and geographic distribution. The statistical packages POPGENE and ARLEQUIN were used to analyse the RAPD fingerprints. The genetic measures, Nei's diversity and Shannon's Information Index, showed that there was greater diversity exhibited (Nei's gene diversity = 32.09 % and Shannon's = 48.31 %), in the whole population than in each of the groups (with average of Nei's gene diversity = 20.33 % and Shannon's = 34.64 %). With regards to individual group analyses, low levels of genetic variation was obtained in group 400 (unrelated), from the Howick region, and group 85 (unrelated), from the Kumbula region, (mean 0.14 and 0.17 respectively). The low genetic values were attributed to limited gene exchange occurring in these two areas, bottlenecks and selection pressures. Groups 88, 89 and 91, from the unknown region (full-sib groups), were the most variable in comparison to the other groups, with means of (0.27,0.24 and 0.18 respectively). These high genetic variation values could be due to the fact that gene migration could have occurred between these groups and others in the area. It is thought that most acacias are insect-pollinated and this could have lead to gene migration between groups or populations, thereby explaining the high mean values. The gene flow obtained for the seven groups (FST = 0.174) indicated that great genetic differentiation existed in this population of black wattle studied. This value is higher in comparison to other woody species; however it is similar to other acacia species. UPGMA cluster analysis using Nei's unbiased genetic distance, revealed four distinct clusters of groups corresponding to the distribution areas represented in this study. The Howick (group 400: unrelated) and Kumbula (group 85: unrelated) were more closely related to each other than to the other groups, since both these groups are from Natal. The Piet Retief groups (groups 47 and 50: half-sibs), branched-off together, indicating that they are distinct from the other groups. The pairwise analysis of identity showed that the relationship between the group from Howick (group 400: unrelated) and all the other groups from the other regions was the lowest, ranging from 64 % to 79 %. The relationship between all the groups beside the group from Howick (group 400: unrelated) was reasonably high, ranging from 78 % to 90 %. This distance displayed by group 400 (unrelated) from Howick in relation to the groups, is attributed to the fact that it is frost resistant and the other groups not. Genetic variation was also detected and partitioned, between and within groups, by Analysis of Molecular Variance (AMQVA). Majority of the variation existed within groups (82.65 %) but significant differentiation was recorded between groups (17.44 %). This high level of within group differentiation may be explained by many aspects, such as the species breeding system, genetic drift or genetic isolation of groups or populations. The application of RAPD fingerprinting in black wattle has provided a more in depth understanding of the genetic variation residing in the South African population. The results achieved implementing this technique has shown that significant genetic variation exists within the black wattle population in South Africa. The results obtained in this study are also important since it is contrary to the expectation that the black wattle population in South Africa has low genetic variation. This knowledge is of great value to genetically discriminate between individuals or groups, to improve the selection of superior genotypes and allowing improved quality control in breeding programmes and seed orchard management. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

A identificação de espécie de Leishmania é crucial no diagnóstico de leishmaniose cutânea (LC), para terapia e prognóstico, em áreas com diferentes espécies endêmicas. A reação em cadeia de polimerase (PCR) foi usada em amostras de biópsias estocadas em parafina e formol de pacientes com LC, do Espírito Santo, Brasil. Usando sequências de mini-exon do gênero Leishmania, 63,2% (36/57) das amostras em parafina e 46,1% (6/13) das amostras em formol foram positivas. Numa abordagem usando sequências de kDNA e RFLP, 100% (58/58) das amostras de parafina testadas foram identificadas como Leishmania (V.) braziliensis. Estas reações podem ser usadas tanto para diagnóstico como para definição da espécie infectante / The identification of Leishmania species, a crucial step in cutaneous leishmaniasis, had therapeutics and prognostics implications, specially at when several agent species are endemic. Polymerase chain reaction (PCR) was used for analysis of paraffin-embedded and formalin stored skin biopsies from patients with parasitologic confirmed cutaneous formalin stored skin biopsies from patients with parasitologic confirmed cutaneous leishmaniasis, from the Espirito Santo State, Brazil. Tested by PCR targeted to mini-exon genus specific primer, 63,2% (36/57) of parafin and 46,1% (6/13) of formalin stored samples were positive, but using a PCR for kDNA primer followed by RFLP analysis, only Leishamnia (V.) braziliensis was identified in 100% (58/58) of parafin biopsies studied. Those reactions allow either diagnosis or species identification in cutaneous leismaniasis