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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Authors: | Tihomir Todorov Albena Todorova Bilyana Georgieva Vanyo Mitev |
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Institution: | (1) Department of Medical Chemistry and Biochemistry, Medical University Sofia, 2 “Zdrave” street, 1431 Sofia, Bulgaria |
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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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