Whole genome amplification for PGD and PND; molecular and a-CGH diagnosis.
Doctoral thesis, UCL (University College London).
Whole genome amplification amplifies the entire genome in a few hours from samples of minimal DNA quantities, even from single cells. This may have many applications, especially in prenatal diagnosis, PGD and PGS. The hypothesis for chapter 3 was: Can multiple displacement amplification (MDA) be used as a universal step prior to molecular analysis for PGD? WGA using MDA (Qiagen) was used on single cells in order to overcome the problem of limited DNA in PGD. MDA allows the diagnosis through haplotyping or a combination of direct and indirect mutation analysis. Different cell types, including buccal cells, lymphocytes, fibroblasts and blastomeres were examined. A modification on the cell lysis buffer was also tested in order to achieve more accurate results. PGD seems to benefit from MDA when multiple tests are performed for direct and indirect analysis. The modified lysis buffer (exclusion of DTT) produced better results than the other lysis buffers and buccal cells do not produce as accurate results as other cell types. The hypothesis was met as the amount of DNA produced by MDA can be used for direct and indirect testing and haplotyping. The hypothesis for chapter 4 was: Is it possible to accurately assess the chromosomes of a single cell by a-CGH? WGA was achieved by MDA and GenomePlex (Sigma) on single lymphocytes, fibroblasts and blastomeres prior to a-CGH analysis. The difficulty of this technique was the high background noise that was produced by WGA that makes interpretation difficult. Different lysis buffers, modifications of the WGA reaction and analysis software were examined for better results. A-CGH slides from different companies and institutions were used. The results showed that GenomePlex produced less background noise compared to MDA but the amplification efficiency of the technique was less reliable. The BlueGnome Cytochip arrays produced the best compared to arrays from any other companies or institutions. More experiments would be necessary to determine if the hypothesis was met as a number of chromosomal abnormalities detected were not always confirmed by other experiments. The hypothesis for chapter 5 was: Can aneuploidy be detected in coelomic fluid using a-CGH? The possibility of using WGA and a-CGH on coelomic fluid was tested as this could be used as an early form of prenatal diagnosis. Coelomic fluid was collected between the 5th and 11th week of pregnancy from women undergoing termination of pregnancy. MDA and GenomePlex were used to amplify the DNA prior to a-CGH analysis. Both genomic (high resolution) and constitutional (low resolution) arrays were tested. The results showed that aneuploidy can be detected by a-CGH. BlueGnome Cytochip slides produced the best results. A triploid sample was detected as normal. The hypothesis was met and even higher resolution could be achieved with the use of GenomePlex and BlueGnome Cytochip arrays. WGA may be very important for downstream genetic tests when the DNA is from very low quality and quantity. Further optimisation of the technique is needed in order to achieve similar results to those of good quality genomic DNA. Arrays from different companies or institutions may produce very different results. In conclusion, the results showed that WGA can benefit PGD and PND, and a-CGH gives great potential to PGS and coelomic fluid diagnosis.
|Title:||Whole genome amplification for PGD and PND; molecular and a-CGH diagnosis|
|Open access status:||An open access version is available from UCL Discovery|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Population Health Sciences > Institute for Women's Health|
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