Dissertations / Theses on the topic 'Single Strand Conformation Polymorphism'

Orientadores : Maria de Fatima Sonati, Monica Barbosa de Melo
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
Made available in DSpace on 2018-08-03T14:43:34Z (GMT). No. of bitstreams: 1 Jorge_SimoneBordignonde_M.pdf: 1672707 bytes, checksum: 1ca1b039159c022f6091792558490b00 (MD5) Previous issue date: 2002
Resumo: Mutações de ponto e pequenas inserções ou deleções nos genes da globina a humana podem produzir variantes estruturais de cadeia a e a-talassemia. As mutações podem ser identificadas tanto pelo sequenciamento total dos genes, como por métodos de triagem, os quais selecionam o exon mutado para, então, ser seqüenciado. Embora sejam pequenos (cerca de 1Kb, com 3 exons e 2 introns), os genes da globina a são duplicados (a2 and a1) e extremamente ricos em ligações G-C, o que torna difícil a desnaturação e reduz a eficiência do sequenciamento, causando freqüentes artefatos e necessidade de repetições. Como o sequenciamento é ainda um método demorado e de custo elevado para muitos laboratórios, nós modificamos algumas condições do Single Strand Conformation Polymorphism (SSCP) com o intuito de otimizar a triagem de mutações nos genes da globina a humana
Abstract: Point mutations and small insertions or deletions in the human a-globin genes may produce a-chain structural variants and a-thalassemia. Mutations can be detected either by direct sequencing of the whole gene or by screening methods, which select the mutated exon for sequencing. Although small (around 1 Kb, 3 exons and 2 introns), the a-globin genes are duplicate (a2 and a1) and extremely G-C rich, what makes them difficult to denature and reduces sequencing efficiency, causing frequent artifacts and the need of repetitions. As DNA sequencing is still a time-consuming and expensive method for many laboratories, we modified some conditions of the Single Strand Conformation Polymorphism (SSCP) in order to optimize mutation detection in the a-globin genes
Mestrado
Ciencias Biomedicas
Mestre em Ciências Médicas

La microcouche de surface (SML) correspond au premier millimètre de la colonne d’eau. Située à l’interface air-eau, la SML est un environnement unique, riche en microorganismes, en matières organiques, en hydrocarbures et présentant des conditions extrêmes par l’impact des radiations UV et les fortes concentrations de polluants. Les échantillons ont été prélevés dans la SML et dans l’eau sous-jacente (UW) à 7 stations de la Baie de Guanabara, Brésil. Cette étude a permis de caractériser l’abondance et la diversité des bactéries et des hydrocarbures présents dans la SML de la baie. L’origine des hydrocarbures a été analysée par des marqueurs chimiques par chromatographie en phase gazeuse. Sur le plan microbiologique, 43 souches bactériennes ont été isolées et identifiées. Leur résistance aux radiations UV a été évaluée à l’aide d’un simulateur solaire. La SML de la baie est bien définie par rapport à l’UW et enrichie en hydrocarbures et en bactéries. Le bactérioneuston est plus diversifié que le bactérioplancton. La plupart des souches testées sont très résistantes aux UV et sont des espèces potentiellement intéressantes sur le plan biotechnologique.

Le but de ce travail est de relier les dynamiques microbiennes de présence et d'activité suivies par analyse Single Strand Conformation Polymorphism au cours de la fabrication du fromage d'AOC Salers aux caractéristiques sensorielles du fromage affiné. Des régions des ADNr et ARNr 16S extraits des fromages ont été amplifiées par PCR et RT-PCR et analysées par SSCP. Les profils microbiens ont été reliés aux données sensorielles par régression Partial Least Square. Des séquences correspondant à des bactéries lactiques, entérobactéries, bactéries Gram positif à bas et haut GC% ont été identifiées. La comparaison des profils issus des ARN et des ADN montre des dynamiques bactériennes et des niveaux d'activité différents. Les corrélations avec les variables sensorielles sont complexes. Chaque descripteur est expliqué par de nombreux groupes microbiens. Cela montre l'intérêt de prendre en compte les dynamiques bactériennes de présence et d'activité lors de la fabrication des fromages

Includes bibliographical references.
This thesis reports the development of Single-Strand Conformation Polymorphism (SSCP) technique to assess sequence level variation within the Major Histocompatibility Complex (MHC) DRB1 gene in four South African buffalo populations. MHC gene products are involved in the immune response, and so variation within these genes provides information on the immunological fitness of the population under study. The aims of this study were: (i) to develop the SSCP technique; (ii) to investigate the level of genetic variation at the peptide binding region (PBR) of the DRB1 gene in four South African buffalo populations. (iii) This data was then compared to data generated previously in a study on the same populations using microsatellite DNA, (iv) the statistical comparisons were used to assess the appropriateness of SSCP data for population genetic analysis. Levels of heterozygosity, allelic diversity and population differentiation were quantified using MHC DRB1 gene. The amplified region (Exon 2 of the DRB1 gene) showed high levels of variability, with 77 alleles found in the 84 individuals examined using SSCP analyses.

La microcouche de surface (SML) correspond au premier millimètre de la colonne d'eau. Située à l'interface air-eau, la SML est un environnement unique, riche en microorganismes, en matières organiques, en hydrocarbures et présentant des conditions extrêmes par l'impact des radiations UV et les fortes concentrations de polluants. Les échantillons ont été prélevés dans la SML et dans l'eau sous-jacente (UW) à 7 stations de la Baie de Guanabara, Brésil. Cette étude a permis de caractériser l'abondance et la diversité des bactéries et des hydrocarbures présents dans la SML de la baie. L'origine des hydrocarbures a été analysée par des marqueurs chimiques par chromatographie en phase gazeuse. Sur le plan microbiologique, 43 souches bactériennes ont été isolées et identifiées. Leur résistance aux radiations UV a été évaluée à l'aide d'un simulateur solaire. La SML de la baie est bien définie par rapport à l'UW et enrichie en hydrocarbures et en bactéries. Le bactérioneuston est plus diversifié que le bactérioplancton. La plupart des souches testées sont très résistantes aux UV et sont des espèces potentiellement intéressantes sur le plan biotechnologique.

Initially, a pilot study was carried out in order to reconstruct the major paternal and maternal lineages of the Muslim population living in the Cape metropolitan area. The Study has shown the ability of molecular genetic tools to give insight into the origins and history of local communities. The study was also used as a point of reference for the Strand Muslim Community project. Genetic variations of the Y-chromosome and mitochondrial DNA for the pilot study were analyzed using the RFLP technique. The SNaPshot mini-sequencing technique was used to genotype single nucleotide polymorphisms (SNP) on the Y-chromosome and mitochondrial DNA in 115 males from the Strand Muslim community.

Mycoplasma synoviae (Ms) causes respiratory infection and synovitis in chickens and turkeys and is an economically important pathogen of poultry worldwide. It is critically important to characterize Ms strains, especially in countries in which poultry flocks are vaccinated with the live attenuated Ms strain MS-H. Vaccination with this vaccine may cause sero-conversion and persistence of the vaccine strain in the respiratory tract and will frequently result in positive Ms cultures and PCR results. Vaccination of flocks therefore complicates the diagnosis of Ms by the presence of detectable antibodies in the blood. Many diagnostic techniques cannot distinguish between the vaccine strain and wild type strain. Single stranded conformation polymorphism (SSCP) and real-time PCR with high melting curve (HRM) analysis can discriminate between the different Ms strains obtained from the field and also distinguish them from the live vaccinestrains. These techniques provide effective tools for the further study of the epidemiology and spread of Ms strains in chickens in South Africa. This project was undertaken to establish whether SSCP and HRM analyses can be used effectively to discriminate between Ms field isolates and the vaccine strain. Mycoplasma synoviae DNA was extracted from samples and conventional PCR was performed using oligonucleotide primers complementary to the single-copy conserved 5’ end of the variable lipoprotein and haemagglutinin encoding gene (vlhA). Twenty six samples were separated by agarose gel electrophoresis and prepared for SSCP and real-time PCR and HRM curve analysis. Results obtained from SSCP were compared to real-time PCR/HRM. Differences obtained by SSCP and melting curve analysis between different isolates were confirmed by sequencing. Results obtained from the different techniques differentiated the strains from the vaccine strain (isolate Ms10), which had a different melting temperature to the others and a different band pattern on the SSCP gel. These results confirmed that HRM and SSCP methods can be used to detect and discriminate between Mycoplasma synoviae field isolates and the vaccine strain.
Dissertation (MSc)--University of Pretoria, 2012.
Veterinary Tropical Diseases
unrestricted

碩士
國立澎湖科技大學
食品科學研究所
103
Ivory shell（Babylonia areolata) and Coral trout (Plectropomus leopardus) are high-valued seafood in Taiwan,and this study use ISSR (Inter-simple sequence repeat)、DGGE (Denaturing gradient gel electrophoresis) and SSCP (Single strand conformation polymorphism) of molecular marker technique to analyze genetic diversity of B. areolata and P. leopardus .The samples were collected from Penghu Marine Biology Research Center.All samples were confirmed to the species and extracted DNAs were analyzed for COXI gene、16S rRNA gene and ITS gene sequences,and than 59 primers of ISSR,and use DGGE and SSCP were used to identify the genetic diversity for all samples. Results showed that COXI and 16S rRNA gene sequences be undifferentiated of B. areolata ; in ISSR analysis, 59 primers were selected in this study. A total of 3 primers, ISSR3, ISSR7 and ISSR13, yielded polymorphic bands and had a good distinguishing ability; DGGE analysis had banding patterns being different in each sample, this results also showed which patterns and clusters were similar to COXI sequencing analysis, thus DGGE method could discriminate Babylonia samples between and within species; SSCP result this method could discriminate Babylonia samples between. In this study, results showed that COXI could be amplified the target fragment 710 bp,and ITS gene could be amplified the target fragment 543 bp,but this sequences be undifferentiated of Plectropomus leopardus ;and than used DGGE and SSCP method, SSCP result can't analysis Plectropomus leopardus but DGGE method could discriminate Plectropomus leopardus and Serranidae fish samples between and within species.In this study,the DNA moleculer marker were developed and could be provide information for variety identification, clonal propagation, and genetic improvement of species-specific in aqultues.

碩士
東海大學
化學系
101
The methylation of the promoter region of DNA is an important regulatory mechanism for the downstream gene expression, and the extent of methylation has been linked to cancer formation. In this thesis a simple and cost-effective method was proposed to rapid screening of the DNA methylation by single-strand conformation polymorphism (SSCP) and capillary electrophoresis with laser-induced fluorescence (CE-LIF). In this study, DNA sample was heated at 95 ºC for the denaturation following by cooled at 0 ºC to forms single strand DNA. The different conformation of DNA fragments were then separated by capillary electrophoresis using 1.5% poly(ethylene) oxide (Mave, 8,000,000 Da) in the presence of electroosmotic flow. The electropherograms demonstrated the bisulfite treated, PCR amplified DNA standards (methylated and unmethylated) could be fully separated before 20 minutes at 5 kV. Otherwise, our results also demonstrated that the four hepatocellular carcinoma cells displayed different heterogeneity of DNA methylation. These data also corresponded to the results that obtained from combined bisulfite restriction analysis and DNA sequencing. Therefore, combined SSCP and CE-LIF provided a simple, method for rapid screening of heterogeneity of DNA methylation and may potentially useful for cancer diagnosis or prognosis mornitoring.

碩士
國立成功大學
藥理學研究所
85
p53是一種癌症抑制基因(tumor supressor gene)，在人類常見的癌症當 中，可發現p53基因的等位基因缺失(allelic deletion)或點突變(point mutation)所造成的功能喪失。當DNA受到了損傷之後，p53蛋白的量在細 胞內急遽增加，隨之使細胞週期停留在G1期；細胞若包含突變的p53或缺 乏p53則不能誘導此種細胞週期停止。此外，突變的p53基因使細胞進行計 畫性細胞自殺(apoptosis)的能力降低，因此癌細胞內的p53基因若突變則 其對化學療法或放射線療法將具有抵抗性。在與其他種致癌基因比較之下 ，p53基因的突變或缺失是在乳房癌中最常見的基因變化。因此，若能建 立p53基因與乳房癌惡化程度之間的關係，將有助於診斷乳房癌或者評估 其在治療後復發的可能性。本實驗的目的在於偵測不同病期的乳房癌中其 p53基因突變的情形，藉此來推斷乳房癌的預後(prognosis)。我們首先利 用去氧核醣核酸單股結構多型性(single-stand conformation polymorphism)，偵測涵蓋了p53之exons 5~9的部分，篩選出具有突變的 片段，再據此進行直接循環去氧核糖核酸定序(direct-cyclic DNA sequencing)以建立p53基因在乳房癌中突變的圖譜。經篩選及定序之後， 我們發現了三個病人在exon 5的位置產生了突變。編號33的病人，其p53 基因的第175個密碼子(codon)由CGC變為TGC，即胺基酸由原本的arginine 變為cysteine。編號36的病人其p53基因第175個密碼子由原本的CGC變為 CAC，即胺基酸由正常的arginine變為histidine。編號54的病人p53基因 的第138個密碼子則由原本的GCC變為CCC，即胺基酸由正常的alanine變為 proline，而以上三者均屬於錯譯突變(missense mutation)，並且其第一 個等位基因發生突變後伴隨著第二個等位基因的缺失，此現象稱之為loss of heterozygosity，是p53基因常被發現的現象之一。若想根據以上三個 p53突變之病人，來找出其病程與預後之關係，則尚無法確切地下結論。 其原因為此三人目前仍繼續在門診，其中雖有一人有癌症轉移之現象，但 代表性不夠，故仍需進一步的觀察。 p53 is a tumor supressor gene whose inactivation, either by allelic deletion or point mutation, is one of the most common genetic changes in human malignancies. The P53 protein level is strongly increased after experimental DNA damage and this is followed by a specific arrest of the cell cycle in G1 phase. Cells containing mutated p53 or lacking P53 are unable to induce this cell cycle arrest; furthermore, these cells would have a reduced capacity to induce apoptosis after DNA damage incurred by ionizing radiotherapy or some therapeutic drugs, suggesting that cancer cells containing mutated p53 are more resistant to radiotherapy or chemotherapy. Compared with the other oncogenes, p53 gene mutations and deletions are the most frequently observed genetic change in breast cancer. Therefore, p53 gene mutations coupled with the disease status could provide an invaluable tumor marker for diagnosis and/or prognosis of breast neoplasm. This research project aims at detecting p53 gene mutations from breast tumor samples of various neoplastic stages and with these information to infer the prognosis of breast cancer. We first obtained genomic DNA from archival and frozen-breast cancer tissues, then amplified exons 5,6,7,8 and 9 of p53 gene by the method of polymerase chain reaction (PCR). We used single-strand conformation polymorphism (SSCP) as a means to prescreen mutations in some specific exon fragments. Eleven variant fragments was identified by their band shifting following polyacrylamide gel electrophoresis (PAGE). Those variants are subjected to direct-cyclic sequencing so as to establish the mutation spectrum of p53 gene. Out of 11 SSCP variants, 3 showed nucleotide mutations upon sequencing. In patient number 33, codon 175 was changed from CGC to TGC, and patient number 36 has the same mutation site in codon 175: CGC ->CAC. Patient number 54 has mutation in codon 138: GCC->CCC. In agreement with other studies, these point mutations are believed to lead to changes in the conformation of p53 protein and loss of its normal functions.

The aetiological diagnosis of recessive non-syndromic hearing loss poses a challenge owing to marked heterogeneity and the lack of identifying clinical features. The finding that up to 50% of recessive non-syndromal genetic hearing loss among Caucasians was due to mutations in GJB2, the gene encoding Connexin 26 (Cx26) was a breakthrough, whose value as a diagnostic tool has been limited by the significant variation in the prevalence of deafness genes and loci among population groups. The significant association of the GJB6-D13S1830 deletion among individuals with one mutant GJB2 allele highlighted the need to explore population specific genetic mutations for NSHL. Although data from Sub-Saharan Africa is limited, reported studies found a high prevalence of R143W GJB2 mutation among Ghanaian, the 35delG mutation in 5 out of 139 Sudanese and a low prevalence of GJB2 variations among 385 Kenyan deaf children. The mutation spectrum of Waardenburg Syndrome (WS) in Africans has not been documented. During a visit to a School for the Deaf in the Limpopo Province of South Africa in 1997, it was noted that a high number of students came from Nzhelele sub-district. All had childhood onset hearing loss with no associated anomalies or disorders. The question arose as to whether there was a high-risk area for deafness in the Limpopo Province and what the aetiology of this hearing loss was.The main aim of this study was to investigate the role of GJB2, the GJB6-D13S1830 deletion, and the four common mitochondrial mutations, A1555G, A3243G, A7511C and A7445G, in the African hearing-impaired population of Limpopo province in South Africa, and to identify the mutation spectrum of the deafness genes found. The type and degree of hearing loss in this hearing impaired population would also be assessed. Secondly, this study sought to identify the mutations in a sibling pair with 2 clinical WS and to use the findings in a future study to establish the mutation spectrum of WS in the African population of the Limpopo province and of South Africa in general. The study was designed as a two phase study, in which phase 1 was used for hypothesis formulation and phase 2 was for hypothesis testing. While phase 1 was a descriptive retrospective case study, phase 2 was a combination of sample survey and prospective descriptive case study. In phase 1, demographic data of 361 students in two schools of the deaf in the Limpopo province was analyzed for evidence of areas of high risk populations for deafness in the province. In phase 2, a group of 182 individuals with genetic non-syndromic hearing loss (NSHL) and two siblings with clinical WS from two schools for the Deaf in the Limpopo Province of South Africa were investigated. A thorough clinical examination, audiological evaluation and urinalysis were done. Mutational screening was carried out in all 184 subjects using genomic DNA using single-strand conformation polymorphism (SSCP), multiplex polymerase chain reaction (PCR), and direct sequencing for GJB2, and Restriction Fragment-Length Polymorphism (PCR–RFLP) analysis for GJB6, and SSCP, hetero-duplex analysis, and direct sequencing of the first 8 exons of PAX3 and all of MITF for Waarenburg syndrome. Data analysis was by geographical mapping, frequency tables, tests of association with calculation of odds ratios, and binary logistic regression analysis using STATA and GIS mapping systems. The results indicate that there seem to be areas of genuine populations at risk for hearing loss in the Limpopo province of South Africa, namely Mutale and parts of Makhado and Thulamela municipalities. In Thulamela (NP343) wards 11-15, 26-30 and 31-35, and in Mutale (NP 344) wards 6-10, together accounted for 67 (18%) of participants in phase 1, and 33 (18%) of the participants in phase 2 of the study. Mutale municipality in the Vhembe 3 district gave with a projected prevalence of at least 13.14 deaf children per 100,000 African population attending the local school for the deaf. The observed hearing loss is a genetic, non-syndromic form, which is mainly severe and severe to profound, although without any clear defining configuration or shape. It is a stable, non-progressive and prelingual form of hearing loss, implying that this may be a recessive form of deafness. No identifiable environmental confounding factors or associations were identified. The deafness is not linked the common known auditory gene mutations in GJB2, the GJB6-D13S1830 deletion, or the common mitochondrial mutations A1555G, A3243G, A7511C and A7445G. Severe and profound levels of hearing loss were found in 22.8% and 75% of the cohort respectively, with the majority exhibiting flat (70.1%) or sloping (23.4%) audiograms that were commonly symmetrical (81.5%). However, as indicated, there was no clear pattern in the audiological findings overall. None of the 184 hearing impaired individuals exhibited any of the reported disease causing mutations of GJB2, including 35delG. There was, however, a high prevalence of two variants, the C>T variant at position g.3318-15 and the C>T variant at position g.3318-34, occurring in 21.4% and 46.2% of the deaf cohort respectively. The same variants were found to occur in 35% and 42.6% of a normal hearing control group (n = 63) respectively, indicating that these variations are polymorphisms. In three subjects (1.63% of the cohort), a T>A homozygous variation at position g.3318-6 was detected. Its significance in the causation of NSSNHL is yet to be determined. The GJB6-D13S1830 deletion was not detected in any of the participants. None of the four mitochondrial mutations screened for were found. 4 These results indicate that GJB2 is not a significant deafness gene in the African population of the Limpopo Province of South Africa and that significant genes for non-syndromic recessive hearing loss in this population are yet to be found. The geographical clustering of deafness found in this study, combined with the lack of identifiable common associated clinical features among the subjects of this study (excluding the WS sibling pair), suggests that these subjects have a genetic recessive non-syndromal type of hearing loss. In the context of historical and cultural evidence of consanguinity in this population, a founder effect cannot be ruled out. A rare mutation, R223X, previously identified only once out of 470 WS patients, was identified in the PAX3 gene among the WS sibling pair. A novel silent change GGG>GGT at amino acid 293, was also identified. These identical findings document, for the first time, a molecular defect in WS in an African sibling pair, and confirm WS Type I in this family, which could be found in other WS type I South Africans in the Limpopo Province of South Africa. The current study demonstrated that parents of genetically hearing impaired children in these areas are able to detect hearing loss at an early age, with over 60% suspecting their children’s hearing loss below 6 months of age. A child-centered management model encompassing all the areas relevant to childhood deafness/hearing impairment, which takes into consideration the prevailing logistical and financial constraints of the available healthcare system, is proposed. The implementation of this model requires a paradigm shift from the current fragmented model of service delivery to a cohesive patient-centered approach, based on concrete data from appropriate community based research, in which all the relevant parties communicate and share resources. 5 It would achieve the goals of early detection and intervention, as well as inclusive education for all. The relevant health and education policies are already in place and the posts funded. Equitable implementation of these policies would require appropriate community based research, as well as improved communication and consultation between the various stakeholders to ensure an efficient and affordable quality healthcare service for all hearing impaired South Africans.

Thesis (M.S.)--Georgia Southern University, 2004.
"A thesis submitted to the Graduate Faculty of Georgia Southern University in partial fulfillment of the requirements for the degree Master of Science" ETD. INDEX WORDS: Palaemonetes pugio, microphallus turgidus, host-parasite interaction, single-strand conformation polymorphism, gene flow. Includes bibliographical references (p. 26-26) and appendices.