>Magister Scientiae - MSc / The discovery of cell free foetal DNA (cffDNA) in 1997 allows for the combination of accuracy as well as non-invasiveness for prenatal diagnosis. This non-invasive genetic test requires only a maternal blood sample from which the cffDNA can be isolated and analysed. In this work cffDNA was isolated from a maternal blood sample using a micro-fluidic device which was fabricated using hot embossing and laser ablation techniques. The DNA sample was first pre-concentration by electrokinetic trapping (EKT) and then isotachophoresis (ITP). The concentrated sample was then separated by size using capillary electrophoresis (CE), all in a single device. All parameters and processes concerned with the micro-fluidic device were optimised sequentially. These parameters include both the chemical components as well as the physical processes which occur. The DNA used for the optimisation protocol was analysed using fluorescence spectroscopy, agarose gel electrophoresis as well as an Agilent Bioanalyser. The optimised protocol included a 9% acrylamide/pDMA matrix, 3 M N,N-dimethylurea as a denaturing agent, with tris based buffers for pre-concentration steps and 1X TBE (tris/borate/EDTA) buffer for capillary electrophoresis. The applied voltage of ITP was 300 V and CE was carried out at 180 V. The timing at which DNA was extracted from the device was kept at time = 60 s intervals. The optimised protocol was then used for real sample analysis and these samples were obtained from mothers pregnant with male foetuses. The DNA extracted from the micro-fluidic device was then analysed using real time PCR (RT-PCR) in order to distinguish which was maternal and which was foetal. This was carried out by amplification of male and general (present in male and female) genes respectively. RT-PCR results confirmed that only the male specific gene was amplified in initial samples exiting the device and it was thus successful in isolating cffDNA from a maternal plasma sample.

The experiences of pregnant teenagers as related to ante-natal care were researched. The primary objective of the study was to explore and describe the experiences pregnant teenagers had of their ante-natal care. The researcher recommended measures based on the findings of the study to midwives to encourage ante-natal care clinic attendance amongst pregnant teenagers. The recommendations were such that even nurses at Primary Health Care clinics could utilise them as they also come into contact with pregnant teenagers. An increasing number of pregnant teenagers in the Nelson Mandela Metropolitan Municipality area are admitted to referral hospitals with complications during pregnancy and labour. Some of these teenagers are un-booked or had only a few ante-natal clinic visits. The researcher therefore wanted to obtain first hand information related to the reluctance of pregnant teenagers to attend ante-natal care clinics. Attendance at ante-natal care clinics plays an important part in enabling the staff to screen pregnant mothers for health problems and so limit complications. Hence the concern and need to develop measures to motivate pregnant teenagers to attend ante-natal clinics.

A non‐mosaic trisomy 20 was discovered in all cells in two separate cultures from an age‐related genetic amniocentesis. Karyotypes of cells obtained via amniocentésis at the time of termination and of cells cultured from the placenta gave the same unambiguous results. However, the fetus, under macro‐ and microscopic analysis, showed only two minor anomalies: left simian crease and low‐set ears. These findings are more suggestive of a normal or at most mosaic trisomy 20 state. The significance of this finding for prenatal diagnosis is discussed.

No description available.

In the first part of this thesis, two chromosome Y specific genes ( SRYand TSPY) were chosen as the molecular targets to investigate the characteristics of fetal-specific DNA fragments in maternal plasma. By employing the touch down ligation-mediated PCR coupled with cloning and sequencing, the end property and the fragment species of fetal DNA were studied. / Noninvasive prenatal detection of fetal chromosomal aneuploidies is a much sought-after goal in fetomaternal medicine. The discovery of fetal DNA in the plasma of pregnant women has offered new opportunities for this purpose. However, the fact that fetal DNA amounts to just a minor fraction of all DNA in maternal plasma makes it challenging for locus-specific DNA assays to detect the small increase in sequences derived from a trisomic chromosome. On the other hand, although the clinical applications of plasma DNA for prenatal diagnosis are expanding rapidly, the biological properties of circulating DNA in plasma remain unclear. Recently, next-generation sequencing technologies have transformed the landscape of biomedical research through the ultra-high-throughput sequence information generated in a single run. Massively parallel sequencing allows us to study plasma DNA at an unprecedented resolution and also precisely detect fetal chromosomal aneuploidies in a locus-independent way. / Our group has demonstrated the use of massively parallel sequencing to quantify maternal plasma DNA sequences for the noninvasive prenatal detection of fetal trisomy 21. In the second part of this thesis, the clinical utility of this new sequencing approach was extended to the prenatal detection of fetal trisomy 18 and 13. A region-selection method was developed to minimize the effects of GC content on the diagnostic sensitivity and precision for the prenatal diagnosis of trisomy 13. To facilitate the next-generation sequencing-based maternal plasma DNA analysis for clinical implementation, two measures, i.e., lowering the starting volume of maternal plasma and barcoding multiple maternal plasma samples, were investigated. / Taken together, the results presented in this thesis have demonstrated the clinical utility of massively parallel sequencing of maternal plasma DNA and have also provided us a better understanding of the biology of circulating DNA molecules. / The third part of this thesis focuses on the massively parallel paired-end sequencing of plasma DNA. By analyzing millions of sequenced DNA fragments, the biological properties of maternal plasma DNA were elucidated, such as the size distribution of fetal-derived and maternally-contributed DNA molecules and the potential effect of epigenetic modification on DNA fragmentation. Moreover, the plasma DNA from hematopoietic stem cell transplant patients was characterized by paired-end sequencing approach. These sequencing data not only confirmed the predominant hematopoietic origin of cell-free DNA but also revealed the size difference between hematologically-derived and other tissue-derived DNA molecules in plasma. / Zheng, Wenli. / Adviser: Lo Yu Ming Dennis. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 261-275). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

1997年，胎兒DNA被首次證實存在於母體血漿中。這一發現促進了無創產前診斷技術的發展。由於孕婦血漿中含大量來自母體的背景DNA，這給針對胎兒特異性DNA序列以外的產前診斷變得有挑戰性。近期開發的大規模平行測序技術把DNA定量精度提升到了一個空前的水平。本團隊已證實這一技術可應用於對胎兒染色體非整倍體和對胎兒全基因組的檢測。由於目前平行測序的費用仍相當昂貴，目標性測序技術可提高目標區域的數據比例從而降低測序成本。 / 在論文第一部分，本人論述了目標性測序在母體血漿DNA應用的可行性。本實驗採用雜交型富集技術對X染色體的外顯子進行富集。我們用平行測序比較了經由和未經富集處理的樣本。對比發現，經富集處理的樣本在目標區域的平均測序深度是未經富集處理樣本的213倍。目標區域的母體和胎兒DNA分子的富集程度相當。經富集處理後，目標區域的胎兒特異性等位基因的檢測率從3.5%提升至95.9%。 / 在論文第二部分，本人論述了目標性測序對胎兒21三體無創產前診斷的應用價值。我們對7，13，18和21號染色體上的單核苷酸多態性位點進行目標性測序。目標性測序數據顯示，在父源性21三體的樣本中，21號染色體上的胎兒特異性等位基因與共有性等位基因的比值上升約2倍。而在母源性21三體的樣本中，這一比值則下降約11%。本人採用電腦模擬實驗探討胎兒DNA濃度，有效等位基因數量和測序深度對檢測準確率的影響。 / 在論文第三部分，本人論述了目標性測序對胎兒單基因疾病無創產前診斷的應用。針對兩個需進行β地中海貧血產前診斷的家庭，我們對其β球蛋白基因進行目標性測序。我們用數字PCR技術推導了父母親β球蛋白基因區域的單倍型。經過相對性單倍型劑量分析，兩個胎兒的β地中海貧血遺傳狀況均得到了正確的推斷。其中一對夫婦位於致病區域的單倍型結構相近。 / 鑒於目標性測序技術可降低測序成本和提高目標序列的通量，其在血漿DNA的應用將有助於平行測序技術在無創性產前診斷、癌症診斷和移植監控等分子診斷學領域的發展。 / The presence of fetal DNA in the cell-free plasma of pregnant women was first reported in 1997. This discovery has facilitated the development of noninvasive prenatal diagnosis. The coexistence in maternal plasma of a minor population of fetal DNA among a major background of maternal DNA has posed challenges for extending noninvasive prenatal diagnostic applications that require analytical information beyond the detection of fetus-specific DNA sequences. The recent availability of massively parallel sequencing has enhanced the precision of DNA quantification to an unprecedented level. Our group has demonstrated the application of massively parallel sequencing in noninvasive prenatal diagnosis of chromosomal aneuploidies, as well as genome-wide fetal whole genome sequencing and mutational profiling. While the current costs of massively parallel sequencing are relatively expensive, targeted massively parallel sequencing may enhance the cost-effectiveness compared with the non-targeted approach because it increases the proportion of informative data from the regions-of-interest. / In the first part of this thesis, I have demonstrated the feasibility of targeted massively parallel sequencing in maternal plasma DNA. In this proof-of-principle study, hybridization-based target enrichment was used to enrich exons on chromosome X. Plasma DNA libraries with and without target enrichment were analyzed by massively parallel sequencing. For the targeted regions, the mean sequencing depth of the enriched samples was 213-fold higher than that of the non-enriched samples. Maternal and fetal DNA molecules were enriched to similar extents within the targeted regions. With target enrichment, the detection rate of fetus-specific alleles within the targeted regions increased from 3.5% to 95.9%. / In the second part of this thesis, I have demonstrated the potential application of targeted massively parallel sequencing of plasma DNA for noninvasive prenatal diagnosis of trisomy 21 using an allelic ratio approach. Targeted sequencing was used to enrich single nucleotide polymorphism loci on chr7, chr13, chr18 and chr21. The targeted sequencing data showed that the ratio between fetus-specific and shared alleles increased by approximately 2-fold on chr21 in a paternally-derived trisomy 21 case, and decreased by approximately 11% on chr21 for maternally-derived trisomy 21 cases. I have also used computer simulation to determine the impact of fractional fetal DNA concentration, number of informative alleles and sequencing depth on the detection accuracy. / In the third part of this thesis, I have demonstrated the feasibility of targeted massively parallel sequencing of maternal plasma DNA for noninvasive prenatal diagnosis of monogenic diseases. Targeted sequencing was used to enrich the β-globin gene region in two families undergoing prenatal diagnosis for β-thalassemia. Parental haplotypes of the β-globin gene region were deduced via digital polymerase chain reaction. Relative haplotype dosage analysis was performed successfully to determine the β-thalassemic status for the fetuses, including one family in which the parents had similar haplotype structures in the disease-causing region. / Because of its potential to save costs and increase throughput, targeted sequencing may catalyse the translation of massively parallel sequencing based molecular diagnostics into many fields, including noninvasive prenatal diagnosis, cancer diagnostics and transplantation monitoring. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liao, Jiawei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 147-155). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / ABSTRACT --- p.i / ACKNOWLEDGEMENTS --- p.vi / PUBLICATIONS --- p.vii / CONTRIBUTORS --- p.viii / TABLE OF CONTENTS --- p.ix / LIST OF TABLES --- p.xiii / LIST OF FIGURES --- p.xiv / LIST OF ABBREVIATIONS --- p.xvi / Chapter SECTION I : --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- Cell-free fetal DNA and targeted massively parallel sequencing --- p.2 / Chapter 1.1 --- Cell-free fetal DNA in maternal plasma --- p.2 / Chapter 1.2 --- NIPD by qualitative analysis --- p.3 / Chapter 1.2.1. --- Fetal sex assessment --- p.4 / Chapter 1.2.2. --- RHD genotyping --- p.5 / Chapter 1.2.3. --- Other implementations --- p.5 / Chapter 1.3 --- NIPD by quantitative analysis --- p.6 / Chapter 1.3.1. --- NIPD of chromosomal aneuploidies --- p.6 / Chapter 1.3.2. --- NIPD of autosomal recessive monogenic diseases --- p.8 / Chapter 1.4 --- Massively parallel sequencing of maternal plasma --- p.9 / Chapter 1.4.1. --- Massively parallel sequencing --- p.9 / Chapter 1.4.2. --- MPS-based NIPD of chromosomal aneuploidies --- p.11 / Chapter 1.4.3. --- MPS-based prenatal fetal whole-genome scanning --- p.15 / Chapter 1.5 --- Targeted massively parallel sequencing of maternal plasma --- p.19 / Chapter 1.5.1. --- Microdroplet-based PCR --- p.19 / Chapter 1.5.2. --- Molecular inversion probe --- p.20 / Chapter 1.5.3. --- On-array capture --- p.21 / Chapter 1.5.4. --- In-solution capture --- p.21 / Chapter 1.5.5. --- Implementation on plasma DNA --- p.22 / Chapter 1.6 --- Aims of this thesis --- p.29 / Chapter SECTION II : --- Feasibility of targeted MPS in maternal plasma DNA --- p.30 / Chapter CHAPTER 2: --- Targeted MPS of maternal plasma DNA permits efficient and unbiased detection of fetal alleles --- p.31 / Chapter 2.1 --- Introduction --- p.31 / Chapter 2.2 --- Methods --- p.34 / Chapter 2.2.1 --- Study participants and sample collection --- p.34 / Chapter 2.2.2 --- Sample processing and DNA extraction --- p.34 / Chapter 2.2.3 --- DNA quantification --- p.36 / Chapter 2.2.4 --- Microarray genotyping --- p.39 / Chapter 2.2.5 --- Plasma DNA library preparation --- p.39 / Chapter 2.2.6 --- Plasma DNA library validation --- p.40 / Chapter 2.2.7 --- Target enrichment --- p.44 / Chapter 2.2.8 --- Massively parallel sequencing and alignment --- p.45 / Chapter 2.3 --- Results --- p.48 / Chapter 2.3.1 --- Efficiency of target enrichment --- p.48 / Chapter 2.3.2 --- Sequence coverage within the targeted region --- p.53 / Chapter 2.3.3 --- Fetus-specific allele detection --- p.59 / Chapter 2.3.4 --- Fractional fetal DNA concentrations before and after enrichment --- p.63 / Chapter 2.4 --- Discussion --- p.66 / Chapter SECTION III : --- NIPD of trisomy 21 by targeted MPS of maternal plasma DNA --- p.71 / Chapter CHAPTER 3: --- NIPD of fetal trisomy 21 by allelic ratio analysis using targeted MPS of maternal plasma DNA --- p.72 / Chapter 3.1 --- Introduction --- p.72 / Chapter 3.2 --- Methods --- p.74 / Chapter 3.2.1 --- Study participants and sample collection --- p.74 / Chapter 3.2.2 --- Sample processing and DNA extraction --- p.74 / Chapter 3.2.3 --- Targeted MPS of plasma DNA libraries --- p.74 / Chapter 3.2.4 --- F-S ratio calculation --- p.76 / Chapter 3.2.5 --- Microarray genotyping --- p.78 / Chapter 3.2.6 --- Computer simulation --- p.78 / Chapter 3.3 --- Results --- p.80 / Chapter 3.3.1 --- Efficiency of target enrichment --- p.80 / Chapter 3.3.2 --- Estimation of fractional fetal DNA concentrations --- p.83 / Chapter 3.3.3 --- F-S ratio calculation using non-targeted sequencing data --- p.83 / Chapter 3.3.4 --- F-S ratio calculation using targeted sequencing data --- p.85 / Chapter 3.3.5 --- Computer simulation --- p.85 / Chapter 3.4 --- Discussion --- p.90 / Chapter SECTION IV : --- NIPD of monogenic diseases by targeted MPS of maternal plasma DNA --- p.94 / Chapter CHAPTER 4: --- NIPD of monogenic diseases by targeted MPS of maternal plasma: application to Beta-thalassemia --- p.95 / Chapter 4.1 --- Introduction --- p.95 / Chapter 4.2 --- Methods --- p.98 / Chapter 4.2.1 --- Sample collection and DNA extraction --- p.98 / Chapter 4.2.2 --- Microarray-based genotyping --- p.100 / Chapter 4.2.3 --- Targeted MPS of plasma DNA libraries --- p.100 / Chapter 4.2.4 --- Genotyping by Sanger sequencing --- p.103 / Chapter 4.2.5 --- Haplotyping by digital PCR --- p.105 / Chapter 4.2.6 --- RHDO analysis --- p.105 / Chapter 4.3 --- Results --- p.110 / Chapter 4.3.1 --- Effectiveness of targeted sequencing --- p.110 / Chapter 4.3.2 --- Determination of fetal HBB genotype in the first family --- p.112 / Chapter 4.3.3 --- Determination of fetal HBB genotype in the second family --- p.113 / Chapter 4.4 --- Discussion --- p.115 / Chapter SECTION V : --- CONCLUDING REMARKS --- p.120 / Chapter CHAPTER 5: --- Conclusion and future perspectives --- p.121 / Chapter 5.1 --- Targeted MPS in plasma DNA --- p.121 / Chapter 5.2 --- Targeted MPS in NIPD of chromosomal aneuploidies --- p.124 / Chapter 5.3 --- Targeted MPS in NIPD of monogenic diseases --- p.126 / Chapter Appendix I: --- Primer names and sequences for genotyping and haplotyping of βeta-globin gene cluster region --- p.128 / Chapter Appendix II: --- Primers used in PCRs and sequencing for the parents in the first family --- p.132 / Chapter Appendix III: --- Primers used in PCRs and sequencing for the parents in the second family --- p.138 / Chapter Appendix IV: --- RHDO analysis on maternal plasma DNA in the first family --- p.140 / Chapter Appendix V: --- RHDO analysis on maternal plasma DNA in the second family --- p.145 / References --- p.147

Circulating RNA was previously demonstrated through the identification of tumour-derived transcripts in the plasma and serum of cancer patients. This finding inspired the detection of cell-free fetal mRNA in maternal plasma which in turn facilitated the development of promising non-invasive prenatal assessment strategies applicable to pregnancies regardless of fetal sex. / Finally, in the last study, I implemented what I have learnt from the analysis of circulating fetal RNA into the development of brain-derived RNA transcripts for detection in the plasma of patients who had sustained brain injuries. A systematic approach based on gene expression microarray analysis was adopted to search for circulating brain-specific mRNA markers. Transcripts showing high expression levels in brain tissues but low expression levels in peripheral blood were identified. However, the detectability of these brain-derived mRNA markers in both peripheral and jugular plasma was found to be low. Instead, concentrations of these mRNA markers were found to be higher in cerebral spinal fluid (CSF) from brain-injured than non-brain-injured patients. / In section IV of this thesis, I reviewed and modified the blood sample processing and plasma RNA extraction protocols that were previously practised, in an attempt to enrich circulating fetal RNA in maternal plasma. Besides mRNA, extraction protocols for microRNAs (miRNAs), a new class of circulating nucleic acid markers, were also evaluated. The modifications in the protocols that I introduced led to significant improvements in RNA yield and enhanced the accuracy and reliability of circulating RNA detection in plasma, especially for those marginally detectable transcripts. / Recently, in addition to maternal plasma, there have been studies by other research groups reporting the presence of placental/fetal mRNA in maternal whole blood. In the first part of this thesis, I studied a list of previously identified placental mRNA transcripts, including chorionic somatomammotropin hormone 1 (CSH1), KiSS-1 metastasis-suppressor (KISS1), placenta-specific 4 (PLAC4) and placenta-specific 1 (PLAC1) in maternal whole blood and determined if this whole blood-based approach offered advantages over maternal plasma analysis. The presence of KISS1, PLAC4 and PLAC1 in non-pregnant and post-partum blood samples as well as the confirmed maternal contribution of PLAC4 mRNA in maternal blood proved that most of the detected 'placental' mRNA molecules in maternal whole blood were of maternal origin. To explore if more pregnancy-associated circulating mRNA markers could be developed for maternal whole blood analysis, candidates were mined after performing gene expression microarray comparison of whole blood samples from pregnant and non-pregnant individuals. The pregnancy-specificity of the identified gene candidates was further investigated. However, their presence in non-pregnant whole blood and lack of clearance after pregnancy indicated that they were not "pregnancy-specific" markers. These data suggested that pregnancy specific transcripts could be more readily identified from maternal plasma than whole blood. / The results presented in this thesis have not only provided a foundation facilitating the precise and accurate detection of fetal-specific RNA markers but have also improved the current understanding of the biology of circulating RNA. / Heung, May Sze. / Advisers: Dennis Lo; Rossa Wk Chiu. / Source: Dissertation Abstracts International, Volume: 72-11, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 212-239). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Lo, Yin Wai Wyatt. / "December 2010." / Thesis (M.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 186-206). / Abstracts in English and Chinese. / ABSTRACT --- p.II / 摘要 --- p.VII / ACKNOWLEDGEMENTS --- p.X / PUBLICATIONS --- p.XI / TABLE OF CONTENTS --- p.XII / LIST OF TABLES --- p.XVIII / LIST OF FIGURES --- p.XXI / LIST OF ABBREVIATIONS --- p.XXIV / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- PRENATALTESTNG --- p.2 / Chapter 1.1. --- THE AIM --- p.2 / Chapter 1.2. --- INVASIVE PRENATAL DIAGNOSIS --- p.4 / Chapter 1.3. --- NONINVASIVE PRENATAL SCREENING --- p.6 / Chapter CHAPTER 2: --- NONINVASIVE PRENATAL DIAGNOSIS --- p.10 / Chapter 2.1. --- CIRCULATING FETAL CELLS IN PRENATAL DIAGNOSIS --- p.10 / Chapter 2.2. --- CIRCULATING FETAL NUCLEIC ACIDS IN PRENATAL DIAGNOSIS --- p.12 / Chapter 2.3.1. --- Biology of circulating fetal DNA . --- p.14 / Chapter 2.3.2. --- Clinical applications of circulating fetal DNA --- p.15 / Chapter 2.3.2.1. --- Qualitative analysis of fetal DNA in maternal plasma --- p.16 / Chapter 2.3.2.2. --- Quantitative analysis of fetal DNA in maternal plasma --- p.17 / Chapter 2.4. --- CIRCULATING FETAL RNA IN MATERNAL PLASMA --- p.20 / Chapter 2.4.1. --- Biology of circulating fetal RNA --- p.20 / Chapter 2.4.2. --- Clinical applications of circulating fetal RNA --- p.22 / Chapter 2.4.2.1. --- Quantitative analysis of fetal RNA in maternal plasma --- p.22 / Chapter CHAPTER 3: --- TECHNICAL CHALLENGES IN ANALYZING CIRCULATING FETAL NUCLEIC ACIDS --- p.26 / Chapter 3.1. --- INTRODUCTION --- p.26 / Chapter 3.2. --- "PREANALYTICAL ISSUES IN MATERNAL PLASMA NUCLEIC ACID ANALYSE"";" --- p.28 / Chapter 3.2.1. --- Low abundance of cell-free fetal nucleic acids in maternal plasma --- p.28 / Chapter 3.2.2. --- High level of maternal background in maternal plasma --- p.30 / Chapter 3.3. --- ANALYTICAL ISSUES IN MATERNAL PLASMA NUCLEIC ACID ANALYS --- p.IS / Chapter 3.3.1. --- Imprecise measurement of fetal nucleic acid quantity --- p.33 / Chapter 3.3.2. --- Coexistence of fetal nucleic acids and maternal nucleic acid background in maternal plasma --- p.36 / Chapter 3.4. --- AIMS OF THIS THESIS --- p.41 / Chapter SECTION II: --- MATERIALS AND METHODS --- p.42 / Chapter CHAPTER 4: --- QUANTITATIVE AND QUALITATIVE ANALYSIS OF NUCLEIC ACIDS --- p.43 / Chapter 4.1. --- SAMPLE COLLECTION AND PROCESSING --- p.43 / Chapter 4.1.1. --- Preparation of plasma and blood cells --- p.43 / Chapter 4.1.2. --- Preparation of placental tissues --- p.44 / Chapter 4.2. --- NUCLEIC ACID EXTRACTION --- p.45 / Chapter 4.2.1. --- Extraction of total RNA --- p.45 / Chapter 4.2.1.1. --- Plasma samples --- p.45 / Chapter 4.2.1.2. --- Placental tissue samples --- p.46 / Chapter 4.2.2. --- Extraction of genomic DNA --- p.48 / Chapter 4.2.2.1. --- Plasma samples --- p.48 / Chapter 4.2.2.2. --- Blood cell samples --- p.48 / Chapter 4.3. --- CONVENTIONAL REAL-TIME PCR ANALYSIS OF NUCLEIC ACIDS --- p.50 / Chapter 4.3.1. --- Principles of real-time polymerase chain reaction --- p.50 / Chapter 4.3.2. --- Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) --- p.52 / Chapter 4.3.3. --- Quantitative polymerase chain reaction (qPCR) --- p.53 / Chapter 4.4. --- DIGITAL REAL-TIME PCR ANALYSIS OF NUCLEIC ACIDS --- p.55 / Chapter 4.4.1. --- Principles of digital PCR (dPCR) --- p.55 / Chapter 4.4.2. --- 384-reaction well dPCR v --- p.56 / Chapter 4.4.3. --- Microfluidics dPCR --- p.57 / Chapter 4.5. --- MATRIX-ASSISTED LASER DESORPTIONIONIZATION/TIME-OF-FLIGHT MASS SPECTROMETRY (MALDI-TOF MS) ANALYSIS OF NUCLEIC ACIDS --- p.59 / Chapter 4.5.1. --- Principles of MALDI-TOF MS --- p.59 / Chapter 4.5.2. --- DNA genotyping analysis by MassArray Homogenous MassExtend (hME) assay --- p.60 / Chapter 4.6. --- CLONING AND DNA SEQUENCING --- p.63 / Chapter SECTION III: --- IMPROVEMENTS ON MATERNAL PLASMA ANALYSIS OF CIRCULATING RNA --- p.65 / Chapter CHAPTER 5: --- ENRICHMENT OF PLACENTA EXPRESSED MRNA MARKERS BY WHOLE TRANSCRIPTOME PREAMPLIFICATION --- p.66 / Chapter 5.1. --- INTRODUCTION --- p.66 / Chapter 5.2. --- MATERIALS AND METHODS --- p.69 / Chapter 5.2.1. --- Study design --- p.69 / Chapter 5.2.2. --- Subjects and sample collection --- p.71 / Chapter 5.2.3. --- RNA extraction and sample dilution --- p.71 / Chapter 5.2.4. --- Preamplification --- p.73 / Chapter 5.2.5. --- qPCR analysis --- p.74 / Chapter 5.3. --- RESULTS --- p.83 / Chapter 5.3.1. --- Comparison of mRNA expression profiles in placental tissues with and without preamplification --- p.83 / Chapter 5.3.1.1. --- Undiluted placental tissue RNA --- p.84 / Chapter 5.3.1.2. --- Diluted placental tissue RNA --- p.88 / Chapter 5.3.2. --- The effect of RNA input on the degree of amplification --- p.92 / Chapter 5.3.2.1. --- Correlation between RNA input and RNA output --- p.94 / Chapter 5.3.2.2. --- Correlation between RNA input and output/input ratio --- p.96 / Chapter 5.3.3. --- Preamplification of maternal plasma RNA --- p.98 / Chapter 5.3.3.1. --- Concentrations of placenta expressed mRNA in third trimester maternal plasma --- p.98 / Chapter 5.3.3.2. --- Concentrations of placenta expressed mRNA in first trimester maternal plasma --- p.100 / Chapter 5.4. --- DISCUSSION --- p.102 / Chapter SECTION IV: --- IMPROVEMENTS ON MATERNAL PLASMA ANALYSIS OF CIRCULATING DNA --- p.105 / Chapter CHAPTER 6: --- ACCURATE GENE DOSAGE ANALYSIS BY MULTIPLEX QPCR --- p.106 / Chapter 6.1. --- INTRODUCTION --- p.106 / Chapter 6.2. --- MATERIALS AND METHODS --- p.110 / Chapter 6.2.1. --- Study design --- p.110 / Chapter 6.2.2. --- Subjects and sample collection --- p.112 / Chapter 6.2.3. --- DNA extraction and sample dilution --- p.113 / Chapter 6.2.4. --- qPCR analysis --- p.113 / Chapter 6.2.4.1. --- Monoplex assays --- p.114 / Chapter 6.2.4.2. --- Multiplex assays --- p.114 / Chapter 6.2.5. --- Microfluidics dPCR assay --- p.122 / Chapter 6.2.6. --- Gene Dosage Comparison --- p.122 / Chapter 6.2.6.1. --- In adult male samples --- p.123 / Chapter 6.2.6.2. --- In maternal plasma samples --- p.123 / Chapter 6.3. --- RESULTS --- p.125 / Chapter 6.3.1. --- The influence of using the same and different sets of primers for amplifying different chromosomal loci --- p.125 / Chapter 6.3.2. --- Effects of using monoplex and multiplex real-time PCR formulations --- p.130 / Chapter 6.3.3. --- Effects of incorporating calibration curves for template quantification in conventional qPCR --- p.135 / Chapter 6.4. --- DISCUSSION --- p.140 / Chapter CHAPTER 7: --- DPCR DETECTION OF PATERNALLY INHERITED POINT MUTATIONS --- p.144 / Chapter 7.1. --- INTRODUCTION --- p.144 / Chapter 7.2. --- MATERIALS AND METHODS --- p.153 / Chapter 7.2.1. --- Study design --- p.153 / Chapter 7.2.2. --- Subjects and sample collection --- p.157 / Chapter 7.2.3. --- DNA extraction and sample preparation --- p.158 / Chapter 7.2.4. --- MassArray hME assays --- p.159 / Chapter 7.2.5. --- dPCR assay --- p.159 / Chapter 7.3. --- RESULTS --- p.161 / Chapter 7.3.1. --- Validation of the digital HbE assay --- p.161 / Chapter 7.3.2. --- Determination of the minimum fetal DNA amount required for digital PCR detection --- p.165 / Chapter 7.3.3. --- Detection of paternally inherited fetal HbE mutation in maternal plasma --- p.172 / Chapter 7.4. --- DISCUSSION --- p.175 / Chapter SECTION V: --- CONCLUDING REMARKS --- p.180 / Chapter CHAPTER 8: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.181 / Chapter 8.1. --- IMPROVEMENTS ON QUANTITATIVE AND QUALITATIVE ANALYSIS OF FETAL NUCLEIC ACIDS IN MATERNAL PLASMA --- p.181 / Chapter 8.2. --- PERSPECTIVES FOR FUTURE WORK --- p.184 / REFERENCES --- p.186

Chan, Ka Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 133-150). / Abstract also in Chinese. / ABSTRACT --- p.i / 摘要 --- p.iii / ACKNOWLEDGEMENTS --- p.iv / CONTRIBUTORS --- p.vi / TABLE OF CONTENTS --- p.vii / LIST OF TABLES --- p.x / LIST OF FIGURES --- p.xi / LIST OF ABBREVIATIONS --- p.xiii / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- PRENATAL DIAGNOSIS OF FETAL TRISOMY 21 --- p.2 / Chapter 1.1 --- Down syndrome --- p.2 / Chapter 1.2 --- Current methods of prenatal diagnosis of fetal trisomy 21 --- p.3 / Chapter 1.2.1 --- Non-invasive procedures --- p.3 / Chapter 1.2.2 --- Invasive procedures --- p.5 / Chapter 1.3 --- Alternative methods for the prenatal diagnosis of fetal trisomy 21 --- p.7 / Chapter CHAPTER 2: --- CELL-FREE FETAL NUCLEIC ACIDS IN MATERNAL PLASMA --- p.13 / Chapter 2.1 --- Circulating fetal cells --- p.15 / Chapter 2.2 --- Circulating cell-free fetal nucleic acids --- p.15 / Chapter 2.3 --- Diagnostic applications of cell-free fetal nucleic acids in maternal plasma --- p.17 / Chapter 2.4 --- Digital relative chromosome dosage approach --- p.20 / Chapter 2.5 --- Validation of digital RCD approach on artificial DNA mixtures --- p.22 / Chapter SECTION II --- : MATERIALS AND METHODS --- p.25 / Chapter CHAPTER 3: --- QUANTITATIVE ANALYSIS OF NUCLEIC ACIDS --- p.26 / Chapter 3.1 --- Subject recruitment and sample collection --- p.26 / Chapter 3.2 --- Sample processing --- p.26 / Chapter 3.3 --- Nucleic acid extraction --- p.27 / Chapter 3.3.1 --- Extraction of DNA from placental tissues --- p.27 / Chapter 3.3.2 --- Extraction of DNA from maternal blood cells --- p.27 / Chapter 3.4 --- Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) --- p.28 / Chapter 3.5 --- Paralogous sequence assays optimisation workflow --- p.31 / Chapter 3.5.1 --- Monoplex paralogous sequence assays --- p.31 / Chapter 3.5.2 --- Multiplex paralogous sequence assay --- p.38 / Chapter 3.6 --- Digital PCR --- p.42 / Chapter 3.6.1 --- Principle --- p.42 / Chapter 3.6.2 --- Digital multiplex paralogous sequence assay --- p.42 / Chapter 3.7 --- Statistical analysis --- p.46 / Chapter 3.7.1 --- Disease classification of samples --- p.46 / Chapter 3.7.2 --- Poisson distribution --- p.46 / Chapter 3.7.3 --- Data analysis --- p.48 / Chapter 3.7.4 --- Sequential probability ratio test (SPRT) analysis --- p.49 / Chapter SECTION III: --- ASSAY DEVELOPMENT --- p.53 / Chapter CHAPTER 4: --- TESTING OF ASSAY SPECIFICITY WITH CORIELL CELL LINES --- p.54 / Chapter 4.1 --- Coriell cell lines --- p.54 / Chapter 4.2 --- Specificity of initial PCR primers --- p.56 / Chapter 4.2.1 --- Principle --- p.56 / Chapter 4.2.2 --- Materials and methods --- p.56 / Chapter 4.2.3 --- Results --- p.60 / Chapter 4.2.4 --- Conclusion --- p.63 / Chapter 4.3 --- Specificity of the iPLEX® Gold extension primers --- p.63 / Chapter 4.3.1 --- Principle --- p.63 / Chapter 4.3.2 --- Materials and methods --- p.64 / Chapter 4.3.3 --- Results --- p.65 / Chapter 4.4 --- Further analysis on the specificity of PV2107a initial PCR primers --- p.67 / Chapter 4.5 --- Conclusion --- p.71 / Chapter CHAPTER 5: --- ASSAY OPTIMISATION --- p.72 / Chapter 5.1 --- Introduction --- p.72 / Chapter 5.2 --- Optimisation of initial PCRs with AmpliTaq Gold® DNA polymerase followed by homogeneous MassEXTEN´DёØ (hME) assays (Sequenom) --- p.72 / Chapter 5.2.1 --- Optimisation of initial PCR reactions --- p.72 / Chapter 5.2.2 --- Principle of homogeneous MassEXTEN´DёØ assays (Sequenom)… --- p.75 / Chapter 5.2.3 --- Homogeneous MassEXTEN´DёØ assays (Sequenom) on euploid and T21 samples --- p.76 / Chapter 5.3 --- Assay selection by iPLEX® Gold single base primer extension reactions (Sequenom) --- p.82 / Chapter 5.4 --- Optimisation of multiplex PCR with AmpliTaq Gold® DNA polymerase --- p.88 / Chapter 5.5 --- Optimisation of multiplex iPLEX® Gold single base primer extension reaction --- p.93 / Chapter 5.6 --- Single molecule detection test for the multiplex paralogous sequence assays … --- p.103 / Chapter SECTION IV: --- ANALYSIS OF CLINICAL SAMPLES --- p.107 / Chapter CHAPTER 6: --- DISEASE CLASSIFICATION OF EUPLOID AND TRISOMY SAMPLES WITH MULTIPLEX PARALOGOUS SEQUENCE ASSAY --- p.108 / Chapter 6.1 --- Introduction --- p.108 / Chapter 6.2 --- Materials and methods --- p.109 / Chapter 6.2.1 --- Sample collection --- p.109 / Chapter 6.2.2 --- Experimental design --- p.110 / Chapter 6.3 --- Results --- p.111 / Chapter 6.4 --- Discussion --- p.114 / Chapter SECTION V: --- CONCLUDING REMARKS --- p.122 / Chapter CHAPTER 7: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.123 / Chapter 7.1 --- Conclusion --- p.123 / Chapter 7.2 --- Future perspectives --- p.124 / Appendix 1 --- p.126 / Appendix II --- p.127 / REFERENCE --- p.133

Thesis (doctoral)--Universiteit Leiden, 2005. / Title from e-book title screen (viewed Aug. 10, 2009). Description based on print version record. Includes bibliographical references and index.