Piyamongkol, Wirawit; (2001) Molecular genetic diagnosis of single gene disorders at the single cell level. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Diagnosis of single gene disorders in isolated single cells can be accomplished after DNA amplification using the polymerase chain reaction (PCR). This has allowed preimplantation genetic diagnosis (PGD) to be performed for a range of inherited diseases. Essentially PGD is an early form of prenatal diagnosis, carried out on 1-2 cells biopsied from 8-10 cell embryos generated using in vitro fertilization (IVF) techniques. During the course of this study PGD protocols were designed for myotonic dystrophy (DM) and alpha and beta-thalassaemias. The PGD protocols developed utilized multiplex-PCR amplification of a fragment encompassing the mutation site (within DM, β-globin, or α-globin) and a highly polymorphic marker. Microsatellite polymorphisms, deletions and insertions were analysed on a fluorescent sequencing apparatus. While nested-PCR and single stranded conformation polymorphism (SSCP) detected base-pair substitutions. Other analytical techniques investigated included long template amplification of trinucleotide repeat expansions, single cell sequencing and minisequencing. The amplification of a hypervariable polymorphism, allows the generation of a very basic DNA fingerprint, assisting in the detection of contaminating DNA. Contamination is one of the most important problems facing single cell PCR. If the marker used is linked to the mutation site, it can also provide confirmatory diagnostic information, which is useful in cases of allele dropout (ADO). ADO is a phenomenon unique to single cell PCR in which one of the alleles in a heterozygous cell fails to amplify. Factors such as ADO were investigated using over 3,000 single cells, isolated by micromanipulation. This provided important information that might lead to the development of more reliable PGD protocols in the future. Three cycles were carried out using the preimplantation diagnosis strategy for DM, resulting in two pregnancies; a singleton for one family and twins for the second. All three babies have been confirmed free of DM by CVS and were born healthy.

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