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J Biomol Tech. 2007 February; 18(1): 18.
PMCID: PMC2291987

P52-S Characterization of the Number of CAG repeats in the Huntingtin Gene of a Huntington Disease (HD) Mouse Strain by Fragment Analysis and DNA Sequencing

Abstract

Mutations in the human Huntingtin gene cause the neurological disorder Huntington’s disease (HD). Specifically, the expansion of a trinucleotide repeat (CAG) in excess of 39 leads to a disease state in individuals. Therefore, determining the number of repeats in subjects is important, since the number of repeats has an effect on the severity of the pathology and the number of repeats shows a high degree of instability between generations. The present study utilizes fragment analysis and DNA sequencing to confirm the exact number of repeats present in exon 1 of the human Huntingtin gene in the transgenic mice used in a drug study. Since genotyping with an automated capillary electrophoresis instrument can be very precise, but not necessarily accurate as to the exact number of bases in the fragment, some of the PCR products were also sequenced. By the use of a 3730 DNA Analyzer, the actual fragment sizes as determined by DNA sequencing were found to be in close agreement with the predicted sizes based upon the fragment analysis—for example, 506 and 501 bases, respectively. The use of a mixture of sequencing kits, BigDye Terminator v3.1 and dGTP BigDye Terminator v3.0, without additives was found to be the best method to sequence over 100 trinucleotide repeats.


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