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.
Williams
L C
LC
School of Biological Sciences, The University of Auckland, Auckland, New Zealand.
Hegde
M R
MR
Herrera
G
G
Stapleton
P M
PM
Love
D R
DR
D001331:Automation; D001483:Base Sequence; D017931:DNA Primers; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000235:genetics*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011506:Proteins / Q000235:genetics*; D018911:Trinucleotide Repeats
D017931:DNA Primers; C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D011506:Proteins