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 H D, the expansion of a CAG repeat occurs in the IT15 gene. The detection of the expanded CAG repeats has usually involved the electrophoretic separatio n of polymerase chain reaction (PCR) amplification products using conventio nal agarose and acrylamide gel electrophoresis. We have undertaken the comp arative analysis of sizing CAG repeats of the IT15 gene using radioactive a nd fluorescent PCR amplification, and the subsequent separation of these pr oducts by slab gel and capillary electrophoresis. The assays have been perf ormed 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 r epeal amplification products of the IT15 gene is greater using capillary el ectrophoresis compared to slab gel electrophoresis. The analysis of 40 DNA samples from HD patients indicates that the mobility difference increases w ith the length of the repeat. However, we have devised an allele ladder for sizing the CAG repeats. This ladder provides a mandatory internal calibrat ion system for diagnostic purposes and enables the confident use of either capillary or slab gel electrophoresis for sizing HD alleles. (C) 1999 Acade mic Press.