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Title: Application of nested PCR, whole genome amplification and comparative genomic hybridisation for single cell genetic analysis
Author: Chiang, Sheng
ISNI:       0000 0001 3590 5975
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2001
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Single cell genetic analysis is a necessity in the field of preimplantation genetic diagnosis (PGD), non-invasive prenatal diagnosis and can be applied to isolated tumour cells in the blood. Current techniques used in PGD and non-invasive prenatal diagnosis are mainly based on either the polymerase chain reaction (PCR) or fluorescence in situ hybridisation (FISH). However, the genetic analysis at a specific locus by PCR has the drawback of sacrificing further detection of common chromosomal aberrations if there is only one cell available. The limitation of FISH using specific probes is that it provides information on only one or a few loci at a time. Comparative genomic hybridisation (CGH), a molecular cytogenetic technique, has the advantage over FISH in permitting a comprehensive analysis of chromosomal imbalances across the whole genome. Combining degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) and CGH techniques, it is possible to study minute quantities of DNA prepared from a very few cells. The aim of this project is to verify the feasibility of applying these techniques to a single cell and investigate the prospect of a novel strategy using whole genome amplification (WGA), nested PCR and CGH to increase the scope and capacity of single cell genetic analysis. The work started first to prove the amplification power of DOP-PCR from a single cell. Various protocols aiming at whole genome amplification (WGA) have different efficiencies in terms of yield and genomic coverage. DOP- PCR is superior to primer extension preamplification (PEP) in producing more quantity of DNA from a single cell. The amplification power of DOP- PCR from a single cell (5 pg) resulted in 10 mug yield with the bulk of DOP- PCR products between 200-2000 bp. DOP-PCR is designed to faithfully amplify the genome, and provide sufficient DNA template. Thus, numerous specific loci and the imbalance of every chromosome can be assessed in a single cell. To investigate the feasibility of locus detection from WGA products, two sets of nested PCR aiming at sex determination or CF ?F508 detection were optimised and tested on a range of DNA sources. The results proved that a strategy of nested PCR could be used to determine the sex and CF status on DOP-PCR-amplified DNA derived from single cells. Nested PCR was also used in the genetic analysis directly on a single cell. In the study of single blastomeres, the sex could be determined in 55.6% of cases (10/18) when nested PCR was used directly on single blastomeres. The feasibility of single cell CGH in the diagnosis of major chromosome abnormalities such as sex chromosome anomaly, trisomy 18 and 21 was established in this study. To produce successful single cell CGH experiments, the normal reference DNA could be made from either amplified DNA or non-amplified DNA. This study demonstrated that a reliable non-amplified reference DNA could increase the success rate of single cell CGH and help to identify the underlying causes of failed CGH experiments. For quantitative analysis of CGH experiments, the fluorescence ratio cutoff value was usually set at 1.2 and 0.8 to represent chromosomal gains and chromosomal losses, respectively. However, the fluorescent ratio profiles using stringent cut-off values would reduce the false positives with the risk of failing to detect the true abnormalities. In this study, the average number of false positives was 5 when threshold of 1.2/0.8 was used. Apart from the objective quantitative analysis, single cell CGH diagnosis of trisomy 21, 18 and sex could also be qualitatively verified from CGH images. After the DOP-PCR/CGH techniques had been proved feasible from a single cell, the sensitivity and reliability of CGH was further tested on 10-20 stained cells scraped from slides in order to simulate the current strategy used in the non-invasive prenatal diagnosis. This part also involved firstly, the correct diagnosis of trisomy 18 and 21 and secondly, several coded samples of known sex were tested for the performance of XXI single cell CGH. The results showed that chromosomal aberrations such as isochromosome X and segmental aneuploidy could be reliably detected. However, a small deletion at single band level could not be detected by single cell CGH. Thirdly, a manufactured mosaicism designed by mixing trisomy 18 male cells with normal female cells was tested to see if at 50-70% mosaic level the trisomy could be detected by single cell CGH. The results illustrated that trisomy 18 could not be detected at the 70% mosaic level using the current protocol. This may illustrate that accurate identification of cells of fetal origin is mandatory if current DOP- PCR/CGH techniques are to be applied in the field of non-invasive prenatal diagnosis. The final part involved using CGH on blastomeres and tested the possibility of employing a shortened protocol for single cell CGH. The preliminary results confirm that chromosome abnormalities may be a common phenomenon in the early embryonic cells. The success of overnight CGH illustrates that further reduction of time is possible. This may allow an expansion of its future application in PGD. Overall, this study demonstrates that DOP-PCR/nested PCR/CGH has the potential to serve as a powerful supplement to the present genetic analysis from a single cell. Concomitant detailed chromosome analysis and specific locus detection may become feasible in the field of PGD and non-invasive prenatal diagnosis in the near future.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Preimplantation gentic diagnosis; Prenatal