Use of capillary electrophoresis for accurate determination of CAG repeats causing Huntington disease. An oligonucleotide design avoiding shadow bands

Abstract

Huntington disease (HD) is a neurodegenerative disorder associated with the expansion of a polymorphic trinucleotide CAG repeat in the HD gene. We have developed an assay to accurately determine CAG repeats that combines a novel oligonucleotide design and the resolution of capillary electrophoresis. A mismatch in the second nucleotide from the 3′ end enhanced specificity by avoiding mispriming and diminishing shadow bands and artifactual PCR products. The coupling of capillary electrophoresis analysis with the assay added the advantages of accuracy, high resolution, semi‐automation, rapid analysis and low sample consumption. Analysis of 200 chromosomes in the Spanish population sample studied (control group) gave a peak frequency for 16 CAG repeats and of 7 triplets for CCG repeats. Diagnosis of HD was confirmed in 22 of 34 individuals with a range of CAG repeats from 39 to 52. Predictive testing was also carried out for 19 relatives of the HD families diagnosed at our laboratory. The method proposed in this article provides an accurate sizing of DNA repeats that can be applied to the analysis of DNA size‐related disorders.

• Capillary electrophoresis
• Huntington's disease
• molecular diagnostic techniques
• polymerase chain reaction
• trinucleotide repeat expansion

Acknowledgements

SB is recipient of a University of Granada Pre‐doctoral fellowship. MEF‐V is recipient of a grant from the Spanish Fondo de Investigación Sanitaria (FIS) del Instituto de Salud Carlos III. CGLL is recipient of a Predoctoral FIS fellowship. We are grateful to Richard Davies for his assistance with the English version.