Elsevier

Molecular Diagnosis

Volume 2, Issue 4, December 1997, Pages 259-269
Molecular Diagnosis

Original Research
Automated detection of trinucleotide repeats in fragile X syndrome*

https://doi.org/10.1016/S1084-8592(97)80037-9Get rights and content

Background:

The conventional method for diagnosis of fragile X syndrome has been amplification of the trinucleotide repeat region of the FMR-1 gene by polymerase chain reaction (PCR) and Southern blot analysis to detect full expansion and hypermethylation. “Stuttering” resulting from incomplete amplification is still observed in the PCR products despite the use of reagents that reduce the secondary structure of the GC-rich template. In addition, PCR products can be detected by autoradiography only after 1 to 2 days of exposure. By combination of a recently reported amplification protocol with fluorescence detection of PCR products in an automated DNA sequencer, the PCR protocol for amplification of trinucleotide repeats was simplified. This modified protocol is highly reproducible, more accurate, and less costly than the conventional protocol because of the elimination of radioisotopes from the PCR.

Methods and Results:

PCRs were conducted with betaine and Pfu DNA polymerase. This improved PCR protocol allowed immediate detection of PCR products in agarose gels containing ethidium bromide. Stuttering was completely climinated and fragments of up to 1 kb (∼250 repeats) were visible in agarose gels. PCR products were automatically detected by laser fluorescence in an automated DNA sequencer by inclusion of a fluorescently-labeled primer in the PCR reaction. A short electrophoresis run of 100 minutes in denaturing acrylamide gels was sufficient to give high resolution of fragments with higher accuracy and sensitivity than conventional detection by authoradiography.

Conclusions:

A simple, nonradioactive protocol that is more rapid and less expensive than the conventional PCR protocol for the detection of trinucleotide repeats has been developed. By use of this detection protocol, fragment sizes containing up to 100 repeats could be detected, alleles differing by one trinucleotide repeat were clearly resolved, and heterogeneous repeat patterns such as those present in mosaics could be discriminated. This protocol has been adapted to the amplification and detection of at least two other classes of trinucleotide repeats [(CAG)n and (CTG)n], suggesting that it may be a universal protocol for PCR amplification and detection of trinucleotide repeats.

References (20)

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Cited by (20)

  • An enhanced polymerase chain reaction assay to detect pre- and full mutation alleles of the fragile X mental retardation 1 gene

    2005, Journal of Molecular Diagnostics
    Citation Excerpt :

    The presence of 7-deaza-2′-GTP greatly reduces the detection of stained PCR products with ethidium bromide;15 PCR-based methods using this analog thus require an additional detection method such as silver staining,11 inclusion of an α-32P-labeled dNTP in the reaction, or hybridization with a radioactive or chemiluminescent probe. The use of betaine instead of DMSO to reduce secondary structures or the Expand Long Template PCR system (Roche Diagnostics) has been independently proposed in the analysis of repeat ex-pansions,12, 13, 14 often in conjunction with fluorescence PCR-based assays. Here, we describe a simple and robust procedure that can be used to screen for FMR1 expansions, which can be detected directly on agarose or acrylamide gels using ethidium bromide staining.

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Supported by a grant from Pharmacia Biotech.

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