Analysis of short tandem repeat markers by capillary array electrophoresis.

The chapter describes the use of capillary array electrophoresis (CAE) for the detection of triplet repeat expansion at the huntingtin locus associated with autosomal dominant Huntington disease (HD), an adult-onset neuro-degenerative disorder. The region of this gene that expands to disease-causing mutations consists of two adjacent tandemly repeated polymorphic triplet repeats: a diagnostic (CAG)n repeat immediately followed by a (CCG)n region. Early polymerase chain reaction (PCR)-based methods used to genotype disease-causing mutations amplified through both of the repeat regions. More accurate diagnostic risk assessment for expansion is obtained when the (CAG)n and the (CCG)n repeats are amplified separately, as well as together in a single reaction. However, these tests require three separate PCR amplifications to be run using conventional methods. The use of multicolor fluorescence detection and CAE can simplify this improved diagnostic test. The CAE method described here makes use of reverse-strand primers labeled with two different fluorescent dyes that are strategically positioned to amplify the (CAG)n region separately from the (CCG)n region. In this study, DNA samples previously analyzed by conventional slab-gel electrophoresis with autoradiographic detection are analyzed by CAE with multicolor fluorescence detection in a blinded manner. Results from the two methods are compared and a consistent diagnosis of all disease-causing mutations is obtained. The sizing precision of CAE permits the length of certain elongated HD alleles to be more accurately measured, since each capillary contains a coelectrophoresing DNA sizing standard and the intensities of each fragment is reported. We generated an allelic ladder standard from a mixture of both patient and control DNA samples to further improve the diagnostic identification of intermediate length alleles. The successful resolution of the normal, intermediate, and disease-causing alleles demonstrates the feasibility of detection of HD using CAE methods. Similar CAE protocols have been applied to the genotyping CA-repeat markers commonly used in linkage analysis studies.