An investigation into the determination of relative chromosome dosage by digital PCR.

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