The use of polymerase chain reaction (PCR) has revolutionized DNA typing in forensic laboratories. Producing a deoxyribonucleic acid (DNA) profile now requires less time and less DNA than before. However, not all evidence samples can be reliably profiled, particularly those with low masses of DNA. These samples often exhibit stochastic effects such as allele dropout, elevated stutter and peak height imbalance, which are challenging to separate from true donor alleles. Several scholarly articles have documented these difficulties and suggest that these stochastic effects are due to uneven amplification of heterozygous alleles in early PCR. However, in early PCR all reaction components are at their maximum concentrations and should be able to amplify all alleles in a sample proportionate to their original concentrations. If both alleles are present in the sample at equal concentrations prior to PCR, both alleles should theoretically be amplified with the same efficiency; the fact that this is not the case suggests that there may already be variation within the sample. One possible reason is that pre-PCR sampling error from pipetting and sample transfers results in an uneven number of allele copies in the sample prior to amplification. Thus, it may not be PCR chemistry alone that contributes to stochastic effects, but also sampling error, which creates unequal allele concentrations prior to PCR. In order to separate and study these possibilities, a single-tube DNA extraction method was developed. The forensicGEM™ Saliva kit developed by ZyGEM provides an extraction method that utilizes a thermostable proteinase found in a proprietary Bacillus species to lyse the cell and destroy nucleases without inhibiting downstream amplification. Combining this extraction protocol with the McCrone and Associates, Inc. cell transfer method allowed for the addition of cells directly to the PCR tube, giving an approximate DNA mass without quantitation. These samples should show the effects of PCR chemistry alone, with pipetting and tube transfer steps prior to amplification removed. For comparison, samples of bulk DNA extracted with forensicGEM™ Saliva were diluted down to a comparable concentration and subjected to multiple transfer steps in an effort to identify both pre-PCR sampling error and any error due to PCR chemistry. Results show that the single-tube extraction method gives reliable results, with forensicGEM™ Saliva showing comparable peak heights (PH) and peak height ratios (PHR) to the Qiagen QIAmp DNA Investigator kit and the cell transfer method providing accurate DNA concentrations with minimal PCR inhibition. Comparison of the cell transfer-generated samples to the diluted bulk DNA samples showed that the cell transfer samples had higher average PHRs at 0.0625 ng of target DNA when amplified with Identifiler® Plus, but showed no significant difference between the sample types at 0.125 ng of target DNA. The cell transfer samples were also shown to have lower overall PHs at both concentrations and a higher occurrence of allelic dropout, but only when amplified with the Identifiler® kit; when amplified with Identifiler® Plus, the occurrence of dropout was low for cell transfer and bulk DNA samples at both concentrations. These results suggest that as DNA mass decreases, pre-PCR sampling error may contribute to the development of stochastic effects; however, the vast majority of stochastic effects are due to the PCR chemistry itself. As the PCR chemistry improves and the prevalence of stochastic effects decreases, the importance of pre-PCR sampling error may increase.

Three genomic clones encompassing human DNA segments (designated LhX-3, LhX-4, and LhX5) were isolated from an X chromosome-specific library and subjected to analysis by physical mapping and DNA sequencing. It was found that these three clones are very rich in repetitive DNA sequence elements and retropseudogenes.

Ho, Ka Chai. / Thesis submitted in: November 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 127-145). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese) --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Tables --- p.ix / List of Figures --- p.x / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Molecular phylogeny of Metanephrops --- p.1 / Chapter 1.2 --- Identification of Nephropidae using DNA barcodes --- p.3 / Chapter Chapter 2 --- Literature Review --- p.5 / Chapter 2.1 --- Molecular phylogenetic studies of crustaceans --- p.5 / Chapter 2.1.1 --- Molecular phylogeny and reasons of using molecular markers in phylogenetic studies --- p.5 / Chapter 2.1.2 --- Characteristics of animal mitochondrial genome --- p.7 / Chapter 2.1.3 --- Examples of crustacean phylogenetic studies derived from mitochondrial DNA --- p.8 / Chapter 2.2 --- Identification of species based on DNA barcode --- p.17 / Chapter 2.2.1 --- Traditional taxonomy and its current practice --- p.17 / Chapter 2.2.2 --- Needs for DNA barcode --- p.18 / Chapter 2.2.3 --- Molecular identification based on DNA barcodes --- p.21 / Chapter 2.3 --- Taxonomy of Nephropidae --- p.28 / Chapter 2.3.1 --- Classification and phylogenetic relationship of Nephropidae --- p.28 / Chapter 2.3.2 --- Classification and distribution of Metanephrops --- p.31 / Chapter 2.3.3 --- Evolutionary history of Metanephrops --- p.36 / Chapter Chapter 3 --- Molecular Phylogeny of Metanephrops --- p.38 / Chapter 3.1 --- Introduction --- p.38 / Chapter 3.2.1 --- Species studied and sample collection --- p.41 / Chapter 3.2.2 --- DNA extraction --- p.43 / Chapter 3.2.3 --- Amplification of mitochondrial genes --- p.43 / Chapter 3.2.4 --- Nucleotide sequencing --- p.46 / Chapter 3.2.4.1 --- Asymmetric PCR --- p.46 / Chapter 3.2.4.2 --- Purification of asymmetric PCR products --- p.47 / Chapter 3.2.5 --- Sequence alignment --- p.47 / Chapter 3.2.6 --- Phylogenetic analyses --- p.48 / Chapter 3.3 --- Results --- p.50 / Chapter 3.3.1 --- PCR products of 16S rRNA and COI genes --- p.50 / Chapter 3.3.2 --- Nucleotide composition of 16S rRNA gene alignments --- p.52 / Chapter 3.3.3 --- Nucleotide composition of COI gene alignments --- p.54 / Chapter 3.3.4 --- Intraspecific and interspecific genetic variation --- p.56 / Chapter 3.3.5 --- Phylogenetic analysis based on 16S rRNA gene sequences --- p.61 / Chapter 3.3.6 --- Phylogenetic analysis based on COI gene sequences --- p.68 / Chapter 3.3.7 --- Phylogenetic analysis based on combined data set --- p.74 / Chapter 3.4 --- Discussion --- p.80 / Chapter 3.4.1 --- Interspecific genetic divergence --- p.80 / Chapter 3.4.2 --- Monophyly of the four species groups --- p.81 / Chapter 3.4.3 --- Phylogenetic relationship in Metanephrops --- p.84 / Chapter 3.4.4 --- Evolutionary history of Metanephrops --- p.90 / Chapter Chapter 4 --- Molecular Identification of Nephropidae --- p.92 / Chapter 4.1 --- Introduction --- p.92 / Chapter 4.2 --- Materials and methods --- p.93 / Chapter 4.2.1 --- Species studied and sample collection --- p.93 / Chapter 4.2.2 --- DNA extraction --- p.95 / Chapter 4.2.3 --- Amplification of genes --- p.95 / Chapter 4.2.4 --- PCR profiles for mitochondrial genes --- p.97 / Chapter 4.2.5 --- Nucleotide sequencing --- p.97 / Chapter 4.2.6 --- Purification of asymmetric PCR products --- p.97 / Chapter 4.2.7 --- Sequence alignment --- p.97 / Chapter 4.2.8 --- Cluster analysis --- p.97 / Chapter 4.2.9 --- Graphical summary of species similarity --- p.98 / Chapter 4.2.10 --- Testing of molecular identification system in Nephropidae --- p.98 / Chapter 4.3 --- Results --- p.100 / Chapter 4.3.1 --- PCR products and sequence alignments of 16S rRNA and COI genes --- p.100 / Chapter 4.3.2 --- Species identification for clawed lobsters --- p.100 / Chapter 4.3.2.1 --- 16S rRNA profile --- p.100 / Chapter 4.3.2.2 --- COI profile --- p.108 / Chapter 4.4 --- Discussion --- p.116 / General Conclusion --- p.124 / Literature Cited --- p.127 / Appendices --- p.146

Molecular DNA markers have become widely used in all areas of genetic research. The objectives of this thesis were to develop polymorphic markers in sunflower and utilize the markers for genetic and candidate gene analyses. Amplified fragment length polymorphism (AFLP) markers were used to estimate genetic similarities and assess the genetic diversity among 24 public oilseed inbred lines of sunflower (Helianthus annuus L.). A total of 359 AFLP markers were scored by using six AFLP primer combinations. Genetic similarities ranged from 0.70 to 0.91, polymorphism rate ranged from 7 to 24%, and polymorphic information contents (PICs) ranged from 0.0 to 0.5. Principal coordinate and cluster analysis separated the lines into two groups, B-lines and R-lines, illustrating breeding history, basic heterotic pattern and the widespread practice of using each group to develop new lines. ��9 stearoyl-ACP desaturase (SAD) and ��l2 oleate desaturase (OLD) cDNAs were cloned and sequenced. DNA fragment length polymorphism (DFLP), single strand conformational polymorphism (SSCP), and simple sequence repeat (SSR) markers were developed for the SAD6 and SAD17 genes among eight elite inbred lines. PICs for DFLP, SSCP, and SSR markers were 0.18, 0.37, and 0.30, respectively. Length variants were due to long monomeric repeats, insertions, and deletions in intron sequences, thereby producing polymorphic markers. OLD desaturates 18:1-PC (oleoyl phosphatidylcholine) to 18:2-PC, thereby converting oleic to linoleic acid. It is a likely candidate gene to be causing the high oleic phenotype in mutant sunflower. The expression of OLD7 in developing seeds was greatly reduced in mutant as opposed to wildtype backcross-derived lines. The restriction fragment length polymorphism (RFLP) patterns suggest that OLD7 is duplicated and rearranged in mutant lines. Utilizing sunflower SAD gene sequences and 27 inbred lines, intron fragment length polymorphism (IFLP) markers were developed for automated genotyping. These IFLP markers with ~470 to ~850 bp in length had a mean PIC score of 0.414, versus 0.336 for DFLP markers, and 0.582 for SSCP markers. One and two nucleotide length polymorphisms were reliably detected in PCR fragments up to ~150 and ~680 bp, respectively. / Graduation date: 1998

A sophisticated computer program is developed to analyze flow linear dichroism data on nucleic acids for individual base inclinations. Measured absorption and linear dichroism data for synthetic AT and GC polymers and natural DNAs are analyzed. The reliability of the program is tested on data for the synthetic polymers, and the results are similar to earlier, more straightforward analyses. For the first time, specific base inclinations are derived for all bases individually from the linear dichroism data for natural deoxyribonucleic acids. For B-form DNA in aqueous solution at moderate salt concentrations, the inclinations from perpendicular are as follows: d(A)=16.1 �� 0.5; d(T)=25.0 �� 0.9; d(G)=18.0 �� 0.6; d(C)=25.1 �� 0.8 deg. Our results indicate that the bases in synthetic and natural DNAs are not perpendicular to the helix axis, even in the B form. The mathematical bases and numerical analyses are presented in detail since both are the keys for successful spectral decompositions in this study, and could be applied to nonlinear optimization problems encountered in other types of biochemistry and biophysics measurements. The interplay between computer programming and scientific measurements can not be overemphasized for modern research. / Graduation date: 1994

published_or_final_version / abstract / toc / Medicine / Doctoral / Doctor of Philosophy

No description available.

No description available.

M.Tech. (Biomedical Technology) / TB continues to be one of the major causes of morbidity and mortality in developed and developing countries, (Mauch, 1993), despite the development of drugs and vaccines. Today, TB is one of the most serious health problems not only in South Africa, but worldwide. The transmission rate for TB for the population of Cape Town is 3% per year, while the transmission rate in gold mineworkers is estimated at 10% per year (Churchyard and Corbett, 2001). Tubercle bacilli have the ability of evading the immune system by entering a dormant phase while in the human host, and are able to reactivate at a later stage. Little is known about the mechanisms of this reactivation. TB remains a global emergency because of our lack of understanding of the details of its pathogenesis (Rook and Zumla, 2001). Since radioactive minerals are found in mines, it was postulated that radioactivity may be the reason for pulmonary cancers, a fact which is now well established. The biologic effects of radiation have been shown to produce irreparable deoxyribonucleic acid double-strand breaks or singlestrand breaks, or create structural changes by damaging the nucleus. Although no studies have shown toxic effects resulting from long-term, low-dose radiation exposure, risks are still assumed, (Herscovici and Sanders, 2000), and research concerning the mutagenic affects of lowdose radiation exposure is necessary. All the risk factors for pulmonary tuberculosis (PTB) in mineworkers are not known, although many have been identified, such as age and mining occupation. This study aimed to determine if long-term, low-dose exposure to ionizing radiation has an effect on the reactivation of dormant tubercle....

polyhomeotic is a gene of the Polycomb-group required for proper segment determination in Drosophila. Genetic and molecular analysis has shown that ph has a repetetive structure. The DNA sequence presented here shows that ph consists of a direct tandem duplication with very high sequence conservation. Analysis of the sequence has revealed several conserved open reading frames and splice junctions, putative transcriptional promoter and terminator sequences, polyadenylation signals and translational start signals. In addition, the DNA sequence shows that ph contains a zinc finger sequence in each repeat. This suggests that ph may encode a DNA-binding protein. / Science, Faculty of / Zoology, Department of / Graduate