Non-invasive prenatal diagnosis (NIPD) is a promising approach that is currently being developed. The principle is that fetal material can be detected in maternal plasma and potentially enable women to pursue reliable and timely prenatal diagnosis, whilst eliminating the risk of miscarriage associated with chorionic villus sampling or amniocentesis. However NIPD research has been restricted up until now for the diagnosis of Down syndrome due to the low concentration of free fetal DNA (ffDNA) in maternal plasma. Various methods have been developed in an attempt to increase the concentration of ffDNA. This study uses COLD-PCR (co-amplification at lower denaturation temperature PCR) to analyse potential enrichment of ffDNA over maternal DNA through optimization of the critical denaturation temperature (Td), using Real Time-PCR in an attempt to selectively enrich smaller fetal DNA fragments. Fake fetal DNA was created in two different spike experiments to imitate the natural environment of viable ffDNA. One spike experiment used 5% of fake fetal DNA in a 95% maternal background to represent levels of ffDNA during early pregnancy. The other spike experiment utilized 10% of fake fetal DNA in 90% maternal background to denote late pregnancy. Before running COLD-PCR, various adjustments took place to find the critical Td at which one could run the spike experiment by COLD-PCR. Products of spike experiment were analysed on a genetic analyser for fragment analysis. Melt curve analysis was also performed for the spike experiment to identify the specificity of each sample at each denaturation temperatures. A critical Td (80°C) was identified for the D21S1890 region of chromosome 21 by COLD-PCR. This temperature does allow enrichment of fetal DNA, as fake maternal DNA was undetermined by RT-PCR compared to fake fetal DNA. The spike experiments clearly showed amplification of fake fetal DNA from the mixture of fake fetal and fake maternal DNA at the critical Td of 80°C. Running same samples of spike experiment on genetic analyser identified peaks from all samples at a Td of 95°C, while at a critical Td of 80°C the result showed decreased numbers of maternal peaks, regardless of stutter peaks formation. Melt curve analysis results clearly identified heteroduplex formation in the samples at the critical Td of 80°C. The results represent a good indication for using COLD-PCR in enriching ffDNA for detection by RT-PCR. However, as each individual has only two alleles, the observed results of multiple peaks for fragment analysis were not expected. Further research needs to focus on both eliminating heteroduplex formation and stutter peaks. COLD-PCR has the potential to open a new gateway in NIPD for aneuploidy detection. This method could be particularly useful in the detection of genetic abnormalities in the fetus, in particular Down syndrome and other aneuploidies.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:577029 |
| Date | January 2012 |
| Creators | Miran, Tara |
| Contributors | Avent, Neil |
| Publisher | University of Plymouth |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10026.1/1190 |
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