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Biomedical subjects

P M Stapleton

Publications and source records attributed to P M Stapleton.

7 recordsLinked to original sources
PubMed1999

Comparative semi-automated analysis of (CAG) repeats in the Huntington disease gene: use of internal standards.

Huntington disease (HD) belongs to the group of neurodegenerative disorders characterized by unstable expanded trinucleotide repeats. In the case of HD, the expansion of a CAG repeat occurs in the IT15 gene. The detection of the expanded CAG repeats has usually involved the electrophoretic separation of polymerase chain reaction (PCR) amplification products using conventional agarose and acrylamide gel electrophoresis. We have undertaken the comparative analysis of sizing CAG repeats of the IT15 gene using radioactive and fluorescent PCR amplification, and the subsequent separation of these products by slab gel and capillary electrophoresis. The assays have been performed on both cloned and sequenced CAG repeats, as well as genomic DNA from HD patients with a wide range of repeat lengths. The mobility of the CAG repeat amplification products of the IT15 gene is greater using capillary electrophoresis compared to slab gel electrophoresis. The analysis of 40 DNA samples from HD patients indicates that the mobility difference increases with the length of the repeat. However, we have devised an allele ladder for sizing the CAG repeats. This ladder provides a mandatory internal calibration system for diagnostic purposes and enables the confident use of either capillary or slab gel electrophoresis for sizing HD alleles.

Automation↗
PubMed1996

Molecular analysis of the Huntington's disease gene in New Zealand.

AIMS: To establish and validate a polymerase chain reaction (PCR)-based diagnostic test in New Zealand, which enables the number of CAG repeats present in the Huntington's disease (HD) gene to be determined with speed and accuracy. To develop procedures for reporting and counselling probands and families. METHODS: The analysis of the CAG repeat region in Huntington's disease and normal chromosomes involved PCR amplification of genomic DNA using either the incorporation of radioactive deoxynucleotides or fluorescent oligonucleotide primers. RESULTS: The molecular analysis of the CAG repeat sequence in the Huntington's disease gene of over 100 New Zealand individuals has been performed. Huntington's disease chromosomes contained 37-70 (median 44) repeats whereas normal chromosomes contained 9-27 (median 18) repeats. Six individuals from three families had an allele in the intermediate range (30-36 repeats). Instability of the CAG repeat upon transmission from generation to generation was also observed. A comparison of the results obtained using radioactive and fluorescent assays indicates that while both methods are reliable, the latter method is more rapid and allows for automation to be incorporated in the scoring of allele sizes. CONCLUSIONS: Our analysis of Huntington's disease alleles has shown a profile of CAG repeat lengths that is consistent with those reported internationally. In addition, reporting and counselling procedures have been established for presymptomatic testing of Huntington's disease in New Zealand.

DNA↗
PubMed1995

Molecular characterization of further dystrophin gene microsatellites.

Microsatellites of the dystrophin gene have been used extensively in the genetic analysis of Duchenne and Becker muscular dystrophy families. The microsatellites that have been reported to date are clustered within disparate regions of the dystrophin gene, specifically at the 5'-end and in the central rod-domain. YACs encompassing the gene were screened for further microsatellites to improve the density of available genetic markers. Four microsatellites were localized to defined regions of the dystrophin gene by the analysis of patient DNA samples, somatic cell hybrids and YACs. In addition, varying combinations of microsatellite loci were amplified in multiplex PCRs, which complement those loci that have been studied to date.

Base Sequence↗