The aim of the present study was to evaluate the power of discrimination and genetic (diversity) parameters in the Y chromosome extended haploytpe markers in populations of Tanzania for forensic and populations studies. Eleven Y chromosome extended haplotype markers were selected for this study, these includes Minimal haplotypes markers i.e. DYS19, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS389I/II and two additional markers DYS438 and DYS439. Six populations of Tanzania were investigated under this study. These populations were selected based on the language family categories / Niger Congo (Kuria and Sukuma), Nilo Saharan (Luo and Maasai) and Afro Asiatic (Iraqw and Alagwa).

Nucleic acid technology has assumed an essential role invarious areas ofin vitrodiagnostics ranging from infectious diseasediagnosis to human genetics. An important requirement of suchmolecular methods is that they achieve high sensitivity andspecificity with a fast turnaround time in a cost-effectivemanner. To this end, in this thesis we have focused on thedevelopment of sensitive nucleic acid strategies thatfacilitate automation and high-throughput analysis. The success of nucleic acid diagnostics in the clinicalsetting depends heavily on the method used for purification ofthe nucleic acid target from biological samples. Here we havefocused on developing strategies for hybridisation capture ofsuch templates. Using biosensor technology we observed that thehybridisation efficiency could be improved using contiguousoligonucleotide probes which acted co-operatively. Byimmobilising one of the probes and annealing the second probein solution, we achieved a marked increase in target capturedue to a base stacking effect between nicked oligonucleotidesand/or due to the opening up of secondary structure. Suchco-operatively interacting modular probes were then combinedwith bio-magnetic bead technology to develop a capture systemfor the extraction of hepatitis C RNA from serum. Viral capturewith such co-operatively interacting probes extracted 2-foldmore target as capture with only a single probe achieving asimilar sensitivity to the conventional extraction protocol. Ananalogous strategy was designed to enrich for sequencingproducts prior to gel electrophoresis removing sequencingreagents and template DNA which interfere with the separationand detection of sequencing ladders, especially in the case ofcapillary gel electrophoresis. This protocol facilitates highthroughput clean-up of cycle sequencing reactions resulting inaccurate sequence data at a low cost, which is a pre-requisitefor large-scale genome sequencing products. Currently, a large effort is directed towards differentialsequencing to identify mutations or polymorphisms both in theclinical laboratory and in medical genetics. Inexpensive, highthroughput methods are therefore required to rapidly screen atarget nucleic acid for sequence based changes. In the clinicalsetting, sequence analysis of human immunodeficiency virus(HIV-1) is used to determine the presence of drug resistancemutations. Here we describe a bioluminometric pyrosequencingapproach to rapidly screen for the presence of drug resistancemutations in the protease gene of HIV-1. This sequencingstrategy can analyse the protease gene of HIV-1 from eightpatients in less than an hour and such non-gel based approachesshould be useful in the future in a clinical setting for rapid,robust mutation detection. Microarray technology facilitates large-scalemutation/polymorphism detection and here we developed amicroarray based single nucleotide polymorphism (SNP)genotyping strategy based on apyrase mediated allele specificextension (AMASE). AMASE exploits the fact that mismatchedprimers exhibit slower reaction kinetics than perfectly matchedprimers by including a nucleotide degrading enzyme (apyrase)which results in degradation of the nucleotides before themismatched primer can be extended. We have successfully typed200 genotypes (14% were incorrect without apyrase) by AMASEwhich cluster into three distinct groups representing the threepossible genotypes. In the future, AMASE on DNA microarraysshould facilitate association studies where an accuracy&gt;99%is required. <b>Keywords:</b>nucleic acid capture, modular probes,biosensor, bio-magnetic separation, hepatitis C, sequencing,pyrosequencing, mutation detection, HIV-1, drug resistance,SNP, allele-specific extension, apyrase, genotyping.

Developments within the field of genetic analysis have during the last decade become enormous. Advances in DNA sequencing technology have increased throughput from a thousand bases to over a billion bases in a day and decreased the cost thousandfold per base. Nevertheless, to sequence complex genomes like the human is still very expensive and efforts to attain even higher throughputs for less money are undertaken by researchers and companies. Genotyping systems for single nucleotide polymorphism (SNP) analysis with whole genome coverage have also been developed, with low cost per SNP. There is, however, a need for genotyping assays that are more cost efficient per sample with considerably higher accuracy. This thesis is focusing on a technology, based on competitive allele-specific extension and microarray detection, for genetic analysis. To increase specificity in allele-specific extension (ASE), a nucleotide degrading enzyme, apyrase, was introduced to compete with the polymerase, only allowing the fast, perfect matched primer extension to occur. The aim was to develop a method for analysis of around twenty loci in hundreds of samples in a high-throughput microarray format. A genotyping method for human papillomavirus has been developed, based on a combination of multiplex competitive hybridization (MUCH) and apyrase-mediated allele-specific extension (AMASE). Human papillomavirus (HPV), which is the causative agent in cervical cancer, exists in over a hundred different types. These types need to be determined in clinical samples. The developed assay can detect the twenty-three most common high risk types, as well as semi-quantifying multiple infections, which was demonstrated by analysis of ninety-two HPV-positive clinical samples. More stringent conditions can be obtained by increased reaction temperature. To further improve the genotyping assay, a thermostable enzyme, protease, was introduced into the allele-specific extension reaction, denoted PrASE. Increased sensitivity was achieved with an automated magnetic system that facilitates washing. The PrASE genotyping of thirteen SNPs yielded higher conversion rates, as well as more robust genotype scoring, compared to ASE. Furthermore, a comparison with pyrosequencing, where 99.8 % of the 4,420 analyzed genotypes were in concordance, indicates high accuracy and robustness of the PrASE technology. Single cells have also been analyzed by the PrASE assay to investigate loss of alleles during skin differentiation. Single cell analysis is very demanding due to the limited amounts of DNA. The multiplex PCR and the PrASE assay were optimized for single cell analysis. Twenty-four SNPs were genotyped and an increased loss of genetic material was seen in cells from the more differentiated suprabasal layers compared to the basal layer. / QC 20100714

Background. Familial Amyloidosis with Polyneuropathy (FAP) is an autosomal dominantly inherited systemic amyloid disease. The disease is caused by mutations in the transthyretin (TTR) gene, where close to 100 different amyloidogenic mutations have been identified. FAP is found worldwide, but endemic areas with a high frequency of patients are found in Portugal, Japan and northern Sweden. Cases from these endemic areas all share the same TTR c.148G&gt;A, p.V50M ("V30M") mutation, but the phenotype of the disease varies between the areas, and also within the endemic areas. The mean onset of the disease is two decades earlier in Portugal and Japan compared to Sweden, but late as well as early age at onset cases occur within all the populations. Interestingly, the different populations all display a maternal anticipation, where an earlier onset is observed for those individuals who inherit the trait from their mother. Since substantial variation in the phenotype is observed for different populations, epigenetic/genetic and/or environmental factors must exert a significant impact on the penetrance of the disease. Amyloid formation is caused by conformational changes of proteins, which facilitates their assembly into fibrils, amyloid. Oxidative stress can mediate conformational changes of proteins and since the mitochondria regulate oxidative processes within the cell, mitochondrial function may affect amyloid formation. The mitochondrial DNA is a non-nuclear DNA, which is entirely maternally inherited, and therefore could be related to the observed maternal anticipation of the disease. In addition, differences within the surrounding regions of the TTR gene may have an impact on the transcription of the gene and thereby on the expression of the different alleles. Material and methods. DNA from early and late onset V30M cases and from non-carriers (the latter utilised as controls) from Swedish, French, Japanese and Portuguese populations were analysed. In addition, DNA from healthy Swedish V30M carriers was analysed. Conventional analytical methods were employed, such as PCR, sequencing and genotyping. Conventional statistical methods used were t-test, Chi-squared test and maximum likelihood. Results. The study of V30M carrier frequency in two counties (Lycksele and Skellefteå) within the Swedish endemic area revealed a carrier frequency of 2.14% and 2.54%, respectively. The mitochondrial haplogroup analysis showed that in populations with generally late onset (French and Swedish), the haplogroup distribution of late onset cases resembled that of the controls derived from the same area, whereas haplogroup distribution for early onset patients was significantly different. The most pronounced difference was for the rare haplogroup K, of which early onset cases had a higher frequency than the controls. Analysis of the Portuguese population, with predominantly early onset, showed that haplogroup distribution for early onset cases were similar to the Portuguese control group, which had a different distribution than the Swedish control group. By analysis of pedigrees from Swedish and Portuguese patients it could be shown that mitochondrial genetic variation entirely could explain maternal anticipation in the Portuguese patients, whereas for Swedish patients, an additional parent of origin effect is present. Our analysis of the TTR gene disclosed a polymorphism (rs62093482) in the 3'UTR region of the Swedish patients. This polymorphism was found in all V30M carriers, irrespective of symptoms. In addition, homozygous TTR V30M carriers were homozygous also for the polymorphism. Since Swedish patients share a common founder this polymorphism thus is localised on the V30M allele. This polymorphism was found in only 4% of the Swedish controls. French controls showed the same frequency, but none of the French V30M patients displayed the polymorphism. In the Japanese population the polymorphism was not present at all. Interestingly, this polymorphism generates a potential binding site for microRNA and thereby possibly could down-regulate the expression of the mutated TTR allele. Conclusions. The carrier frequency in the endemic area is remarkably high, above 2% in the Lycksele and Skellefteå areas. The prevailing haplogroup distributions in the different endemic areas are consistent between the general population and the patient group with the predominant phenotype of that area. Mitochondrial genetic differences may explain maternal anticipation in Portuguese patients, and have an influence in Swedish patients. A polymorphism in the 3'UTR regulatory region of the mutated TTR allele is found in all Swedish patients. This polymorphism may down-regulate TTR V30M expression and thereby contribute to the late onset of the disease noted in the Swedish population.

<p>Nucleic acid technology has assumed an essential role invarious areas of<i>in vitro</i>diagnostics ranging from infectious diseasediagnosis to human genetics. An important requirement of suchmolecular methods is that they achieve high sensitivity andspecificity with a fast turnaround time in a cost-effectivemanner. To this end, in this thesis we have focused on thedevelopment of sensitive nucleic acid strategies thatfacilitate automation and high-throughput analysis.</p><p>The success of nucleic acid diagnostics in the clinicalsetting depends heavily on the method used for purification ofthe nucleic acid target from biological samples. Here we havefocused on developing strategies for hybridisation capture ofsuch templates. Using biosensor technology we observed that thehybridisation efficiency could be improved using contiguousoligonucleotide probes which acted co-operatively. Byimmobilising one of the probes and annealing the second probein solution, we achieved a marked increase in target capturedue to a base stacking effect between nicked oligonucleotidesand/or due to the opening up of secondary structure. Suchco-operatively interacting modular probes were then combinedwith bio-magnetic bead technology to develop a capture systemfor the extraction of hepatitis C RNA from serum. Viral capturewith such co-operatively interacting probes extracted 2-foldmore target as capture with only a single probe achieving asimilar sensitivity to the conventional extraction protocol. Ananalogous strategy was designed to enrich for sequencingproducts prior to gel electrophoresis removing sequencingreagents and template DNA which interfere with the separationand detection of sequencing ladders, especially in the case ofcapillary gel electrophoresis. This protocol facilitates highthroughput clean-up of cycle sequencing reactions resulting inaccurate sequence data at a low cost, which is a pre-requisitefor large-scale genome sequencing products.</p><p>Currently, a large effort is directed towards differentialsequencing to identify mutations or polymorphisms both in theclinical laboratory and in medical genetics. Inexpensive, highthroughput methods are therefore required to rapidly screen atarget nucleic acid for sequence based changes. In the clinicalsetting, sequence analysis of human immunodeficiency virus(HIV-1) is used to determine the presence of drug resistancemutations. Here we describe a bioluminometric pyrosequencingapproach to rapidly screen for the presence of drug resistancemutations in the protease gene of HIV-1. This sequencingstrategy can analyse the protease gene of HIV-1 from eightpatients in less than an hour and such non-gel based approachesshould be useful in the future in a clinical setting for rapid,robust mutation detection.</p><p>Microarray technology facilitates large-scalemutation/polymorphism detection and here we developed amicroarray based single nucleotide polymorphism (SNP)genotyping strategy based on apyrase mediated allele specificextension (AMASE). AMASE exploits the fact that mismatchedprimers exhibit slower reaction kinetics than perfectly matchedprimers by including a nucleotide degrading enzyme (apyrase)which results in degradation of the nucleotides before themismatched primer can be extended. We have successfully typed200 genotypes (14% were incorrect without apyrase) by AMASEwhich cluster into three distinct groups representing the threepossible genotypes. In the future, AMASE on DNA microarraysshould facilitate association studies where an accuracy>99%is required.</p><p><b>Keywords:</b>nucleic acid capture, modular probes,biosensor, bio-magnetic separation, hepatitis C, sequencing,pyrosequencing, mutation detection, HIV-1, drug resistance,SNP, allele-specific extension, apyrase, genotyping.</p>

<p>The objective of this study was to develop and validate a six Non-CODIS miniSTR genotyping system and to determine its suitability for forensic casework in South Africa. In Non-CODIS miniSTR genotyping systems, smaller PCR products are amplified and the primers are positioned as close as possible to the repeat region. For this reason, these systems can be valuable in a variety of scenarios including complex paternity cases, missing persons work, and mass fatality disasters.</p>

The aim of the present study was to evaluate the power of discrimination and genetic (diversity) parameters in the Y chromosome extended haploytpe markers in populations of Tanzania for forensic and populations studies. Eleven Y chromosome extended haplotype markers were selected for this study, these includes Minimal haplotypes markers i.e. DYS19, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS389I/II and two additional markers DYS438 and DYS439. Six populations of Tanzania were investigated under this study. These populations were selected based on the language family categories / Niger Congo (Kuria and Sukuma), Nilo Saharan (Luo and Maasai) and Afro Asiatic (Iraqw and Alagwa).

The objective of this study was to develop and validate a six Non-CODIS miniSTR genotyping system and to determine its suitability for forensic casework in South Africa.In Non-CODIS miniSTR genotyping systems, smaller PCR products are amplified and the primers are positioned as close as possible to the repeat region. For this reason, these systems can be valuable in a variety of scenarios including complex paternity cases,missing persons work, and mass fatality disasters. After the successful implementation of the genotyping system in the laboratory, allele size range was determined for each of the loci and allelic ladders were constructed. The entire repeat regions of the six loci under investigation were successfully sequenced.Consequently, allele repeat number, structure and observed size were determined for each locus.An internal validation study of the six Non-CODIS miniSTR genotyping system was conducted following the SWGDAM guidelines. A comprehensive population study,covering five population groups from South Africa was also carried out.The genotyping system produced consistent, accurate and precise genetic profiles for low concentrations of template DNA. When analyzing mixed DNA samples, successful differentiation of minor and major DNA components was identifiable. Amplification products were observed in non-human DNA studies but in all instances complete genotype profiles were not obtained. Allele frequencies and forensic parameters were determined for the system in five South African population groups (i.e. Afrikaner, Asian-Indian, Mixed Ancestry, Xhosa and Cape Muslim). No deviation from Hardy-Weinberg equilibrium was observed in any of the populations. Furthermore, all populations displayed a high power of discrimination and a high power of exclusion.The six Non-CODIS miniSTR genotyping system has shown a good potential to aid in the analysis of degraded DNA samples. This system can be further improved by including additional loci. Even in its current form, it can certainly provide additional discrimination in complex paternity and/or missing person cases. / >Magister Scientiae - MSc

The aim of the present study was to evaluate the power of discrimination and genetic(diversity) parameters in the Y chromosome extended haploytpe markers in populations of Tanzania for forensic and populations studies. Eleven Y chromosome extended haplotype markers were selected for this study, these includes Minimal haplotypes markers i.e. DYS19, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS389I/II and two additional markers DYS438 and DYS439. Six populations of Tanzania were investigated under this study. These populations were selected based on the language family categories; Niger Congo (Kuria and Sukuma), Nilo Saharan (Luo and Maasai) and Afro Asiatic (Iraqw and Alagwa).Buccal swabs were collected from unrelated males from Mwanza province (Sukuma),Mara (Kuria and Luo), Arusha (Maasai and Iraqw) and Dodoma province (Alagwa).Samples were typed using ABI 377 Genetic Analyser (Applied Biosystem) followed by analysis using softwares Gelprocessor, GeneScan 3.0.0 (Applied Biosystems) and Genotyper 3.7 (Applied Biosystems). The data obtained were analysed by GenePop 4.0,Arlequin 3.11 and Genetix v.4.05.2 software packages. Analyses such as AMOVA, Fst population pairwise comparison, Factorial component Analysis were used to obtain Allele frequency, haplotype frequency, gene diversities among various loci and levels of gene flow between populations.For the overall individuals, the highest Gene Diversity value was 0.8251 (DYS385) and the lowest was 0.2723 (DYS392). The overall Haplotype Diversity was 0.9984 and Discrimination capacity resulted 84.27%. A total of 225 distinct haplotypes were identified in 267 individuals, 28 were shared, the most frequent haplotype was present in 5 individuals. The levels of genetic diversity for the haplotypes per group as revealed by haplotype diversities confirmed that the most diverse group being Sukuma, Kuria,Iraqw, Maasai, Luo and Alagwa being the least diverse. The Discrimination capacity of these set of markers showed the highest value in Sukuma population (100%) subsequently followed by Iraqw, Luo, Maasai, Kuria and Alagwa (78.38%) being the lowest. Analysis of Molecular Variance showed a significant differentiation among populations, 93.96% of variance was found within population and 6.04% among population. Population pairwise results between all population pairs (except Sukuma and kuria and Alagwa and Luo) showed significant results (P < 0.05). Genetic heterogeneity that was found among Tanzanian populations could not be attributed to language barriers but was largely being contributed by a limited level of gene flow between these populations due to different ethnical, social, cultural and historical backgrounds between them. All Y chromosome extended haplotype loci used in this study (except DYS392 and DYS391 which showed the lowest level of polymorphism) were found to be likely useful for forensic application in Tanzania. Furthermore the extended haplotype markers used in this study may be useful in the establishment of the National DNA database following the enactment of the Human DNA Legislation in Tanzania (http://www.parliament.go.tz). / Magister Scientiae - MSc

Malignant hyperthermia (MH) is a dominant skeletal muscle disorder caused by mutations in the ryanodine receptor skeletal muscle calcium release channel (RyR1). Allele-specific differences in RyR1 expression levels might provide insight into the observed incomplete penetrance and variations in MH phenotypes between individuals. Firstly, an H4833Y allele-specific PCR (AS-PCR) assay was designed that allowed for the relative quantification of the two RYR1 mRNA alleles in heterozygous samples. In four MHS skeletal muscle samples and two lymphoblastoid cell lines (LCLs), the wild type allele was found to be expressed at higher levels than the mutant RyR1 allele. These differences were not caused by variations in RYR1 mRNA stabilities. Secondly, high-throughput amplicon sequencing was employed for the quantification of both the T4826I and H4833Y causative MH mutations in heterozygous MHS samples. With the exception of one, all detected H4833Y and T4826I mutation frequencies were about 50%. This included a control, which was constructed and proven to have a 3:1 ratio of the wild type (H4833) versus the mutant (Y4833) RYR1 allele. This suggested that that the high-throughput amplicon sequencing approach as used here, was not suitable for accurate quantification of the two RyR1 alleles in heterozygous H4833Y MHS samples. To detect possible variations in RyR1 alleles at the protein level, the RyR1 was to be isolated from microsomes prepared from a H4833Y MHS frozen skeletal muscle tissue. Microsomes isolated from MHS skeletal muscle tissues lacked the immunoreactive band that was believed to be the full length RyR1. Poor muscle quality, due to long term storage was believed to be the main cause of RyR1 depletion. Faster and less expensive screening methodologies are required for the identification of genetic variants in MH research. Thus, in an additional project inexpensive and high-throughput high-resolution melting (HRM) assays were developed to allow screening of the RYR1 gene, for mutations associated with MH and/or central core disease (CCD).