Kamakari, Smaragda; (1994) Physical and genetic mapping in the proximal short arm of the X chromosome. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The aim of this thesis was to further develop the techniques of Fluorescence In Situ Hybridisation (FISH) for the localisation of markers on the X chromosome and to contribute to the physical mapping of the proximal short arm with particular emphasis on the region where the gene responsible for one form of Retinitis Pigmentosa (RP2) has been mapped by genetic linkage studies. FISH technique was initially used to sublocalise markers to X chromosome regions. Two-colour FISH was subsequently used to order selected X specific markers because of their position in proximal Xp relative to reference markers DXS7 and DXS426. These markers are known to be tightly linked to the RP2 locus. On the basis of the in situ results one of the newly isolated markers was eventually mapped genetically as it appeared to map in the region of interest. Therefore, a microsatellite was isolated from this cosmid and sequenced. The unique sequences flanking the repeat were used to design Polymerase Chain Reaction (PCR) amplification primers. After establishing allele number, length and frequency of this marker, its genetic position was defined by following its inheritance in twelve X linked Retinitis Pigmentosa families. These families had been previously analysed and linkage data for a number of other proximal short arm markers were available. The new marker eventually mapped distal to the region of interest and, therefore, was not included in further physical mapping studies. An alternative approach to generating markers was to construct a Yeast Artificial Chromosome (YAC) contig of the region using the DXS426 locus as a starting point for chromosome walking. A genomic YAC library was, therefore, screened by PCR and two YACs containing the DXS426 locus were isolated. The orientation of the two YACs was defined by end-cloning. Primers of the proximal end clone were used in order to isolate an overlapping YAC. The end clones of the overlapping YAC were isolated, sequenced and PCR amplification primers were designed. The above YAC clones covered a physical distance of approximately 1 Megabase and physically linked the DXS426 locus with the OATL1 locus in the proximal short arm of X chromosome. This work has contributed to the high resolution physical and genetic linkage map of the proximal short arm of the X chromosome and has provided a resource for the identification of candidate genes involved in inherited diseases localised to this part of the chromosome.

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