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A Unified Rapid PCR Method for Detection of Normal and Expanded Trinucleotide Alleles of CAG Repeats in Huntington Chorea and CGG Repeats in Fragile X Syndrome

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Abstract

We report on a unified rapid betaine-based-PCR protocol for amplification of the (CAG)n region in Huntington disease (HD) and the (CGG)n region in Fragile X syndrome (FXS), followed by an electrophoretic separation on automated sequencer for precise determination of the triplet numbers. The high betaine concentration (2.5 M betaine) permits precise amplification of the CAG and CGG repeats. Ten HD affected patients and 10 healthy individuals from HD families were re-evaluated. For FXS the CGG region in normal individuals and premutations of about 100 repeats were precisely amplified by this protocol. Ten unrelated FXS premutation carriers and 24 mentally retarded non-FXS affected boys were re-examined by this method. The results totally coincided with the previous ones. This protocol is a good choice as a fast screening test. Within 24 h we can have preliminary information on the patient’s genetic status. Normal individuals, CGG premutation carriers up to 100 repeats, as well as HD patients carrying an expansion up to 50 CAG repeats can be easily clarified. This accounts for a relatively large proportion (about 90%) of the suspected HD and FXS patients, referred to our laboratory for genetic analysis. The calculation of the repeat’s number is more accurate for the correct interpretation of the results, screening tests and genetic counselling.

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Acknowledgments

The fellowship from Alexander von Humboldt Foundation to Dr. Albena Todorova is gratefully acknowledged. The study was supported by the Grants No 23/2008 and No 32/2009, Medical University Sofia, Bulgaria.

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Authors and Affiliations

  1. Department of Medical Chemistry and Biochemistry, Medical University Sofia, 2 “Zdrave” street, 1431, Sofia, Bulgaria

    Tihomir Todorov, Albena Todorova, Bilyana Georgieva & Vanyo Mitev

Authors
  1. Tihomir Todorov
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  2. Albena Todorova
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  3. Bilyana Georgieva
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  4. Vanyo Mitev
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Corresponding author

Correspondence to Tihomir Todorov.

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Tihomir Todorov and Albena Todorova contributed equally to the present study.

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Todorov, T., Todorova, A., Georgieva, B. et al. A Unified Rapid PCR Method for Detection of Normal and Expanded Trinucleotide Alleles of CAG Repeats in Huntington Chorea and CGG Repeats in Fragile X Syndrome. Mol Biotechnol 45, 150–154 (2010). https://doi.org/10.1007/s12033-010-9260-y

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  • DOI: https://doi.org/10.1007/s12033-010-9260-y

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